GF10

Second International Symposium on

Gluten-Free Cereal Products and Beverages

June 811, 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 worldwide. 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

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 Mki, University of Tampere, Finland
Dr. Katri Kaukinen, University of Tampere, Finland
Professor Frits Koning, University of Leiden, The Netherlands
Professor Michael Gnzle, University of Alberta, Canada
Professor Peter Khler, German Research Center for Food Chemistry, Germany
Dr. Gerard Downey, Teagasc, Ireland
Dr. Juan Pablo Albar, Centro Nacional de Biotecnologa / 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
Pivi 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: 09:009:20
Location: Conference room Rondo, Tampere Hall
Session 1: Coeliac disease and the triggering molecules
Time: 09:2010:40
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 Mki, University of Tampere, Finland
09:50 THE TRIGGERING PROTEINS AND PEPTIDES IN COELIAC DISEASE
Peter Khler, 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: 10:4011:10
Session 2: Gluten analysis and safety testing
Time: 11:1014:30
Location: Conference room Rondo
Chair: Peter Khler, German Research Centre for Food Chemistry, Germany; Jussi Loponen,
University of Helsinki, Finland
11:10 IMPROVING ACCURACY IN DETECTING GLUTEN
Pivi 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: 12:0513:20

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: 14:3015:30
Location: Winter Garden, Tampere Hall
Session 3: Coeliac food market and nutritional requirements, Gluten-free cereals and pseudocereals
Time: 15:3017:40
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 Kk, Abant Izzet Baysal University, Turkey

IV

Thursday, June 10th, 2010

Sesion 4: Gluten detoxification
Time: 08:3010:00
Location: Conference room Rondo
Chair: Michael Gnzle, University of Alberta, Canada; Pivi 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 Garca-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: 10:0010:30
Session 5: Gluten-free baking and processing I
Time: 10:3012:00
Location: Conference room Rondo
Chair: Nanna Mossberg, Fria Brd, 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 Gnzle, 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: 12:0013:00

Thursday, June 10th, 2010

Session 6: Gluten-free baking and processing II
Time: 13:0014:55
Location: Conference room Rondo
Chair: Markku Mikola, Sennet, Finland; Eimear Gallagher, Teagasc, Ireland
13:00 FRIA BRD COMPANY PRESENTATION
Nanna Mossberg, Fria Brd, 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 Mnchen, 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: 14:5515:55
Location: Winter Garden
Session 7: Gluten-free pasta, beer, and industrial perspectives
Time: 15:5517:25
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, Universit 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 Mnchen, Germany
16:35 GLUTEN-FREE BEER FROM BARLEY
Saara Pyri, Oy Sinebrychoff Ab, Kerava, Finland
16:45 GLUTEN-FREE WHEAT STARCH SAFETY AND FUNCTIONALITY ON A
NATURAL WAY
Maren Wiese, Hermann Krner 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 Bcker GmbH & Co. KG, Germany

Friday, June 11th, 2010

Session 8: Coeliac disease and gluten-free foods update Joint session with the Coeliac Fair
Time: 08:4513:00
Location: Small Auditorium, Tampere Hall
Chair: Markku Mki, 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 Brd AB, Sweden
Coffee break
Time: 10:3011:15
11:15 OATS IN GLUTEN-FREE DIET CONSIDERATIONS OF A PRAGMATIC POLICY
Hannu Salovaara, Pivi 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 Mki, University of Tampere, Finland
Closing ceremonies of GF10 Symposium
Time: 13:0013:15
Location: Small Auditorium
Lunch
Time: 13:1514:30

VII

VIII

Table of Contents
Wednesday, June 9th , 2010
Session 1: Coeliac disease and the triggering molecules
CURRENT PREVALENCE, DIAGNOSIS AND TREATMENT OF COELIAC DISEASE . . . . . . . . . . . . . . . . . . . . .
Markku Maki, University of Tampere, Finland

THE TRIGGERING PROTEINS AND PEPTIDES IN COELIAC DISEASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

THREE DAYS ORAL GLUTEN CHALLENGE RESPONSE IN GLUTEN SENSITIVE INDIVIDUALS WITH
AND WITHOUT CD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Margit Brottveit, Oslo University Hospital, Norway; Stig Tollefsen, National Veterinary Institute, Norway; Melinda
Raki, Ann-Christin R. Beitnes, Frode L. Jahnsen, Centre for Immune Regulation, Norway; Jorunn Bratlie, Oslo
University 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 HAVE
COELIAC DISEASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Jessica Biesiekierski, Evan Newnham, Peter Irving, Jacqueline Barrett, Melissa Haines, Monash University, Australia; James Doecke, CSIRO, Australia; Susan Shepherd, Jane Muir and Peter Gibson, Monash University, Australia

DIVERSITY OF AVENIN GENES IN OAT (AVENA SATIVA L.) AND CONTENT OF CELIAC DISEASE
EPITOPES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Diana Londono, Wageningen UR, Netherlands

ACUTE ADMINISTRATION OF TRITICUM MONOCOCCUM SSP MONOCOCCUM, CULTIVAR MONLIS:A PHASE II, SINGLE BLIND, CROSS-OVER STUDY IN PATIENTS WITH COELIAC DISEASE. . . . . .
Beatrice Petroboni, Barbara Zanini, University of Brescia, Italy; Tarciso Not, University of Trieste, Italy; Norberto
Pogna, Research Unit for Cereals, Italy; and Alberto Lanzini, University of Brescia, Italy

DOWN-REGULATION OF -GLIADINS IN BREAD WHEAT BY RNA INTERFERENCE (RNAI) . . . . . . . . . .

Javier Gil-Humanes, IAS. CSIC, Spain; Fernando Piston, Universidad de Cordoba, Spain; and Francisco Barro,
IAS. CSIC, Spain

11

GLUTEN EPITOPES IN FINNISH PATIENTS WITH DQ2+ CELIAC DISEASE . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Andrea de Kauwe, University of Helsinki, Finland; Katri Kaukinen, University of Tampere and Tampere University
Hospital, 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

13

IX

Session 2: Gluten analysis and safety testing

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

15

PRELIMINARY RESULTS OF THE COLLABORATIVE STUDY TO VALIDATE THE CHARACTERISTICS

OF THE GLUTEN-TEC ELISA KIT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Jorge Raul Mujico Fernandez and Frits Koning, Leiden University Medical Center, Netherlands

17

SECOND GENERATION TESTING FOR GLIADIN IN COMPLIANCE WITH CODEX ALIMENTARIUS

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

19

DETECTION OF TOXIC FRAGMENTS FROM GLUTEN USING A NEW MONOCLONAL ANTIBODYBASED TEST . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Richard Fielder, Romer Labs UK, United Kingdom; and Elisabeth Halbmayr, Romer Labs Division Holding,
Austria

21

GENOMICS APPROACHES TO ANALYSE CD-TOXICITY FOR THE PRODUCTION OF CD-SAFE WHEAT

Luud Gilissen, Hetty C. Van den Broeck, Elma Salentijn, Ingrid M. Van der Meer and Marinus J.M. Smulders,
Plant Research International, Netherlands

23

PRESUMPTIVE SAFETY FOR CELIAC PATIENTS OF BAKED GOODS MADE OF SOURDOUGH FERMENTED WHEAT FLOUR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Marco Gobbetti, University of Bari, Italy

25

TESTING SAFETY OF LOW GLUTEN FOOD PRODUCTS IN A MOUSE MODEL OF CELIAC DISEASE . .
Tobias 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

27

GENERAL DISCUSSION ON: SAFETY AND TOLERANCE - ANALYSIS AND REGULATORY STANDARDS - TECHNOLOGY AND VARIETY OF PRODUCTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hannu Salovaara, Paivi Kanerva, Jussi Loponen and Tuula Sontag-Strohm, University of Helsinki, Finland

29

Posters of Session 2: Gluten analysis and safety testing

DETERMINATION OF THE IMMUNOTOXIC POTENTIAL OF OATS FOR THE SELECTION OF VARIETIES
POSSIBLY SAFE FOR COELIAC PATIENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Isabel Comino, Universidad de Sevilla, Spain

31

RECOGNITION OF GLIADIN AND GLUTENIN FRACTIONS IN FOUR COMMERCIAL GLUTEN ASSAYS

Laura Allred, ELISA Technologies, Inc., United States

33

NEW APPROACHES IN GLUTEN ANALYSIS OF PRODUCTS FOR CELIACS BY PROTEOMICS COMBINED WITH THE R5 ELISA TECHNIQUES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Maria Carmen Mena, Manuel Lombarda, Alberto Hernando and Juan Pablo Albar, Centro Nacional de Biotecnologia, CSIC, Spain

35

EFFECTS OF HEATING, REDUCING AND ALCOHOL CONCENTRATION ON PROLAMIN EXTRACTION

Paivi Kanerva, Tuula Sontag-Strohm, Outi Brinck and Hannu Salovaara, University of Helsinki, Finland

37

DEAMIDATION OF GLUTEN PROTEINS DRASTICALLY INFLUENCES THE QUANTITATIVE GLUTEN

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

39

Session 3: Coeliac food market and nutritional requirements, Gluten-free cereals and
pseudo-cereals
NUTRITIONAL REQUIREMENTS FOR GLUTEN-FREE FOODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tricia Thompson, The Gluten-Free Dietitian, MA, USA

41

GLUTEN-FREE FOOD MARKET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Markku Mikola, Sennet Oy, Finland; and Esa Wrang, Finpro, Finland

43

FOLATES IN GLUTEN-FREE DIETS AND FOODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Vieno Piironen, Maija Kinni, Jussi Loponen and Susanna Kariluoto, University of Helsinki, Finland

45

CEREALS AND PSEUDOCEREALS FOR GLUTEN-FREE FOODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Regine Schoenlechner and Emmerich Berghofer, University of Natural Resources and Applied Life Sciences, Austria

47

HEALTHY GRAINS FOR ENHANCED GLUTEN-FREE BREADS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Eimear Gallagher, Ashtown Food Research Centre, Teagasc, Ireland

49

Posters of Session 3: Coeliac food market and nutritional requirements, Gluten-free

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

51

NUTRITIONAL QUALITY OF LINSEED AND OIL HEMP VARIETIES CULTIVATED IN FINLAND WITH
SPECIAL ATTENTION TO LIGNAN AND CADMIUM CONTENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Marketta Saastamoinen, Satafood Development Association, Huittinen, Finland; Juha-Matti Pihlava and Merja
Eurola, MTT, Chemical Laboratory, Jokioinen, Finland

53

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

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

55

A SYSTEMATIC LITERATURE REVIEW ON THE NUTRITIONAL ADEQUACY OF A TYPICAL GLUTENFREE DIET WITH PARTICULAR REFERENCE TO IRON, CALCIUM, FOLATE AND B VITAMINS . . . . . . .
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 Manchester
and Manchester Childrens University Hospital, United Kingdom

57

TARTARY BUCKWHEAT AS A GLUTEN FREE SOURCE FOR FUNCTIONAL . . . . . . . . . . . . . . . . . . . . . . . . . . .

Mateja Germ and Ivan Kreft, University of Ljubljana, Slovenia

59

TEFF (ERAGROSTIS TEF) SUPPLEMENTED GLUTEN-FREE BREADS AS A POTENTIAL PREVENTION

OF IRON-DEFICIENCY ANAEMIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ebtesam Ben-Fayed, Valentina Stojceska and Paul Ainsworth, Manchester Metropolitan University, United Kingdom

61

OAT BETA-GLUCAN AFFECTS THE VISCOELASTIC PROPERTIES OF GASTRIC MUCIN AT PH CONDITIONS OF SMALL INTESTINE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Reetta Kivela, Sami Hietala, Tuula Sontag-Strohm, University of Helsinki, Finland; Bradley Turner, Harvard
Medical School, USA; and Rama Bansil, Boston University, Department of Physics, USA

63

XI

Thursday, June 10th , 2010

Session 4: Gluten detoxification
PROLYLENDOPROTEASES FOR GLUTEN DETOXIFICATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Frits Koning, Leiden University Medical Centre, Netherlands

65

MANY FACES OF PROLYL OLIGOPEPTIDASE WHAT WE IGNORE IN MAMMALS AND WHAT WE

KNOW IN PLANTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
J. Arturo Garca-Horsman, University of Helsinki, Finland

67

ENDOGENOUS CEREAL ENZYMES IN THE ELIMINATION OF PROLAMINS . . . . . . . . . . . . . . . . . . . . . . . . . .

Jussi Loponen, Paivi Kanerva, University of Helsinki, Finland; Michael Ganzle, University of Alberta, Canada;
Tuula Sontag-Strohm and Hannu Salovaara, University of Helsinki, Finland

69

SYNTHETIC BLOCKING PEPTIDES WITH HIGH AFFINITY TO GLIADIN REDUCE TISSUE TRANSGLUTAMINASE ACTIVITY ON WHEAT GLIADIN IN VITRO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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-Sofie
Sandberg, Chalmers University of Technology, Sweden

71

Posters of Session 4: Gluten detoxification

DEGRADATION OF GLIADIN PEPTIDES TOXIC FOR COELIAC DISEASE PATIENTS BY PROLYL ENDOPEPTIDASE SYNTHESIZED BY LACTOBACILLUS ACIDOPHILUS 5E2 AND ASPERGILLUS NIGER
Bartosz Brzozowski, Wlodzimierz Bednarski, University Of Warmia And Mazury, Poland; and Barbara Wroblewska, Polish Academy of Science, Poland

73

DETOXIFICATION OF GLUTEN BY GERMINATING CEREAL ENZYMES: IMPLICATIONS FOR NEW

TREATMENT OF COELIAC DISEASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Satumarja Stenman, University of Tampere, Finland

75

DEGRADATION OF IMMUNOGENIC GLUTEN EPITOPES BY PROBIOTIC LACTOBACILLI . . . . . . . . . . . . .

Maria De Angelis, Raffaella Di cagno, Francesca Gagliardi, Carlo Giuseppe Rizzello, Ruggero Francavilla and
Marco Gobbetti, University of Bari, Italy

77

CAN PROLYL ENDOPROTEASE ENZYME TREATMENT MITIGATE THE TOXIC EFFECT OF GLUTEN
IN COELIAC PATIENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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 University Medical Centre, Netherlands; Gerrit Meijer, Mary von Blomberg, Marco Schreurs, VU University Medical
Centre, 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

79

AUTO-PROTEOLYTIC AND PHYSICAL ELIMINATION OF PROLAMINS IN ACIDIC SUSPENSIONS OF

MALTED WHEAT, BARLEY, AND RYE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Jussi Loponen, Outi Brinck, Zhongqing Jiang and Hannu Salovaara, University of Helsinki, Finland

81

HYDROLYTIC POTENTIAL OF MALTS PREPARED OF THREE RYE VARIETIES AND IMPACT ON PROLAMIN BREAKDOWN DURING ACIDIFICATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Emma Laivisto, University of Helsinki, Finland; Annika Wilhelmson, Arvi Wilpola, VTT Technical Research Centre
of Finland, Finland; J. Arturo Garcia-Horsman, Hannu Salovaara and Jussi Loponen, University of Helsinki,
Finland

83

XII

Session 5: Gluten-free baking and processing I

OVERVIEW ON THE NEW DEVELOPMENTS IN THE AREA OF GLUTEN FREE FOODS AND BEVERAGES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Elke K. Arendt, University Collage Cork, Ireland

85

GLUTEN FREE BAKED PRODUCTS: SOME QUALITY SOLUTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

87

FORMATION AND MODIFICATION OF BIOACTIVE COMPOUNDS IN GLUTEN FREE SOURDOUGHS .

Michael Ganzle, Andreas Schieber, Louise Svensson, Januana Teixeira and Victoria McNeill, University of Alberta,
Canada

89

ENZYMATIC PROCESSING OF GLUTEN-FREE FLOURS: A PROMISING TOOL TO IMPROVE THEIR

BREAD-MAKING FUNCTIONALITY? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stefano Renzetti, TNO Quality of Life, Netherlands; and Elke K. Arendt, University College Cork, Ireland

91

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

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

93

DEVELOPMENT OF GLUTEN FREE MUFFIN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Marios Pogiatzis, Weili Li, Russell Ramsden and Charles Brennan, Manchester Metrpolitan University, United
Kingdom

95

GLUTEN-FREE SORYZ COOKIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Cosciug Lidia, Technical University of Moldova, Moldova

97

METABOLISM AND COMPETITIVENESS OF LACTOBACILLUS SANFRANCISCENSIS AND TING ISOLATES IN WHEAT AND SORGHUM SOURDOUGHS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bonno Sekwati-Monang and Michael Ganzle, University of Alberta, Canada

99

OBTAINING AND CHARACTERIZATION OF GLUTEN FREE FLOUR PRODUCTS ENRICHED WITH

DRIED FRUIT AND HIPPOPHAE RHAMNOIDES EXTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ersilia Alexa, Banats University of Agricultural Science, Romania

101

OPTIMIZATION OF GLUTEN-FREE FRENCH-STYLE BREAD FORMULATION SUITABLE FOR FROZEN

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

103

RHEOLOGICAL PROPERTIES OF GLUTEN-FREE BREAD FORMULATIONS USING CHESTNUT AND

RICE FLOUR COMBINATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Ilkem Demirkesen, Behic Mert, Gulum Sumnu and Serpil Sahin, Middle East Technical University, Turkey

105

USAGE OF CHUFA FLOUR IN GLUTEN-FREE CAKES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Elif Turabi, Gulum Sumnu, Serpil Sahin, Middle East Technical University, Turkey; and Mehmet Musa Ozcan,
Selcuk University, Turkey

107

HETEROPOLYSACCHARIDE PRODUCTION FROM LACTIC ACID BACTERIA IN WHEAT AND

SORGHUM SOURDOUGH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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

109

PROMOTING OF STRUCTURE FORMATION BY HIGH PRESSURE IN GLUTEN-FREE FLOURS . . . . . . . . .

Katleen. J. R. Vallons, Liam A. M. Ryan and Elke K. Arendt, University College Cork, Ireland

111

XIII

IDENTIFICATION OF LACTIC ACID BACTERIA ISOLATED FROM OAT SOURDOUGHS AND INVESTIGATION OF THEIR POTENTIAL FOR THE IMPROVEMENT OF OAT BREAD QUALITY . . . . . . . . . . . . . . . . .
Edith K. Huttner, Fabio Dal Bello, Emanuele Zannini and Elke K. Arendt, University College Cork, Ireland

113

PILOT SCALE MANUFACTURE OF HIGHLY CONCENTRATED PROTEIN INGREDIENTS FROM OATS

USING DRY FRACTIONATION TECHNOLOGY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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, VTT
Technical Research Centre of Finland, Finland

115

Session 6: Gluten-free baking and processing II

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 Munchen, Germany

117

EXOPOLYSACCHARIDE WEISSELLA STRAINS AS STARTER CULTURES FOR SORGHUM AND

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

119

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

BREADS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Anna-Sophie Hager, Liam A. M. Ryan, University College Cork, Ireland; John V. ODoherty, University College
Dublin, Ireland; and Elke K. Arendt, University College Cork, Ireland

121

EFFECTS OF TWO-STEP TRANSAMIDATION OF WHEAT FLOUR AND SEMOLINA ON THE TECHNOLOGICAL PROPERTIES OF GLUTEN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Federica Capobianco, Salvatore Moscaritolo, IPALC Research & Development Laboratories, Italy; and Mauro
Rossi, Institute of Food Sciences, NRC, Italy

123

THE EFFECT OF DELETION LINES OF BREAD WHEAT CHINESE SPRING ON CELIAC DISEASE
STIMULATING EPITOPES AND TECHNOLOGICAL PROPERTIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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 International, 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

125

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

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

127

EFFECTS OF BASIC PROCESS PARAMETERS ON QUALITY OF GLUTEN-FREE RICE BREADS . . . . . . . .

Gina Jaspers and Markus Brandt, Ernst Bocker GmbH & Co. KG, Germany

129

CASEIN NETWORK FORMATION IN GLUTEN FREE BREAD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Sheila Kenny, Teagasc, Ireland

131

EFFECT OF MICROBIAL HOMOPOLYSACCHARIDES ON THE STRUCTURE OF GLUTEN-FREE

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

133

XIV

FERMENTED WHEY-BASED DIARY DESSERT STABILIZED WITH STARCH FROM GLUTEN-FREE

SOURCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bulgaru Viorica, Technical University of Moldova, Moldova

135

DEVELOPMENT OF A NEW GLUTEN-FREE BROWN BREAD FLOUR MIX RICH IN FIBER AND HIGH
IN NUTRITIONAL CONTENT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Virna Lucia Cerne, Dr. Schar GmbH, Italy

137

SENSORY AND TEXTURAL PROPERTIES OF GLUTEN-FREE BREAD BASED ON RICE/BUCKWHEAT

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

139

THE POSITIVE EFFECT OF AMARANTH SOURDOUGH ADDITION IN GLUTEN FREE BREAD QUALITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Andreas Houben, TU Munchen, Germany; Martin Mitzscherling, University of Hohenheim, Germany; and Thomas
Becker, TU Munchen, Germany

141

DISCRIMINATION BETWEEN GLUTEN-FREE BREAD FORMULATIONS USING NEAR INFRARED

IMAGING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Gerard Downey, Teagasc, Ireland

143

EVALUATION OF PHYSICALLY-CHEMICAL PARAMETERS OF GLUTEN-FREE DUMPLINGS . . . . . . . . . .

Tatjana Rakcejeva, Ilze Gramatina and Anastasija Fjodorova, Latvia University of Agriculture, Faculty of Food
technology, Latvia

145

NOVELTY FORMULA OF FREE GLUTEN POCKET TYPE FLAT ARABIC BREAD . . . . . . . . . . . . . . . . . . . . . . .

Hanee Al-Dmoor, Al- Balqa Applied University, Jordan

147

A GLUTEN-FREE BREAD WITH VISCOUS JAPANESE YAM INSTEAD OF WHEAT FLOUR . . . . . . . . . . . . .

Masaharu Seguchi, Kobe Womens University, Japan

149

EFFECT OF LEGUME FLOURS ON BAKING CHARACTERISTICS OF GLUTEN FREE BREADS . . . . . . . . .

Begona Minarro, Universidad Autonoma de Barcelona, Spain

151

EFFECTS OF HIGH PRESSURE AND TEMPERATURE ON BUCKWHEAT STARCH CHARACTERISTICS

Katleen. J. R. Vallons, Liam A. M. Ryan and Elke K. Arendt, University College Cork, Ireland

153

RHEOLOGICAL PROPERTIES AND BREAD MAKING PERFORMANCE OF COMMERCIAL WHOLEGRAIN OAT FLOURS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Edith K. Huttner, Fabio Dal Bello and Elke K. Arendt, University College Cork, Ireland

155

GLUTEN-FREE BREAD SUPPLEMENTED WITH CALCIUM THE IMPROVEMENT OF QUALITY AND

TEXTURE PROPERTIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Agnieszka Troszynska and Jadwiga

Ursula Krupa-Kozak, Malgorzata Wronkowska, Maria Soral- Smietana,
Sadowska, Polish Academy of Science, Poland

157

XV

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

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

159

PROSO MILLET (PANICUM MILIACEUM L.) A SUSTAINABLE RAW MATERIAL FOR THE MALTING
AND BREWING PROCESS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Martin Zarnkow, Thomas Becker, TU Munchen, Germany; and Elke K. Arendt, University College Cork, Ireland

161

GLUTEN-FREE BEER FROM BARLEY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Saara Poyri, Oy Sinebrychoff Ab, Kerava, Finland; Markku Maki, University of Tampere, Finland; Alberto Hernando, Maria Carmen Mena, Manuel Lombardia, Centro Nacional de Biotecnologia, CSIC, Spain; Pekka Lehtonen, Alko Oy, Finland; P. Soininen-Tengvall, E. Pajunen, Oy Sinebrychoff Ab, Kerava, Finland; and E. Mendez,
Centro Nacional de Biotecnologia, CSIC, Spain

163

GLUTEN-FREE WHEAT STARCH - SAFETY AND FUNCTIONALITY ON A NATURAL WAY . . . . . . . . . . . .

Maren Wiese, Hermann Kroner GmbH, Germany

164

IMPROVING THE TEXTURE AND NUTRITIONAL PROFILE OF GLUTEN FREE BAKED GOODS BY FORMULATION SCIENCE AND SPECIALITY FLOUR TECHNOLOGY: MUFFINS . . . . . . . . . . . . . . . . . . . . . . . . . . .
Despina Ioannides, Alejandro Perez and Yadunandan Dar, National Starch Food Innovation, United States

165

DEVELOPMENT OF HIGH-QUALITY GLUTEN-FREE BREADS FOR THE EUROPEAN MARKET . . . . . . .

Valentina Stojceska and Paul Ainsworth, Manchester Metropolitan University, United Kingdom

167

IMITATED RYE FLOUR - EVALUATION OF PENTOSAN SOURCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Markus Brandt and Gina Jaspers, Ernst Bocker GmbH & Co. KG, Germany

169

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

CHANGES IN BIO-ACTIVE COMPOUNDS IN BUCKWHEAT DEPENDING ON GERMINATION CONDITIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Florian Hubner and Elke K. Arendt, University College Cork, Ireland

171

PROTEIN CHANGES IN BUCKWHEAT DEPENDING ON THE MALTING CONDITIONS . . . . . . . . . . . . . . . . .

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

173

PROTEIN CHANGES DURING MALTING AND BREWING WITH OATS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Christina Klose and Elke K. Arendt, University College Cork, Ireland

175

OPTIMIZATION OF RHEOLOGICAL PROPERTIES OF GLUTEN-FREE PASTA USING MIXTURE DESIGN

Virginia Larrosa, Gabriel Lorenzo, Noem Zaritzky and Alicia Califano, CIDCA, Argentina

177

CHARACTERIZING SORGHUM-BASED PASTA: A MULTI-DISCIPLINARY APPROACH . . . . . . . . . . . . . . . . .

Maria 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

179

READY-TO-EAT MEALS BASED ON RICE PASTAS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Virtucio Luis, Pavan, Italy

181

STARCH CHARACTERIZATION OF RICE PASTA: COMPARISON BETWEEN EXTRUSION-COOKING

AND CONVENTIONAL PASTA-MAKING PROCESS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Alessandra Marti, University of Milan, Italy; Rosita Caramanico, CRA-SCV, Italy; Koushik Seetharaman, University of Guelph, Canada; and Maria Ambrogina Pagani, University of Milan, Italy

185

GLUTEN IN OAT-BASED BEVERAGES AND OATMEAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Ylva Sjogren, Birgitta Kruse, Monica Ferm, Martin Sandberg and Ingrid Malmheden Yman, National Food Administration, Sweden

187

XVI

Friday, June 11th , 2010

Session: 8 Coeliac disease and gluten-free foods update - Joint session with the Coeliac Fair
GLUTEN-FREE AND VERY LOW GLUTEN FOODS ENFORCEMENT OF COMMON EU LEGISLATION .
Annika Nurttila, Finnish Food Safety Authority EVIRA, Finland

189

WHEAT STARCH IN GLUTEN-FREE DIET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Katri Kaukinen, University of Tampere and Tampere University Hospital, Finland

190

WHEAT STARCH IN GLUTEN FREE BREAD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Nanna Mossberg, Fria Brod AB, Sweden

191

OATS IN GLUTEN-FREE DIET CONSIDERATIONS OF A PRAGMATIC POLICY . . . . . . . . . . . . . . . . . . . . . . . . .

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

193

TIPS FOR USING OATS IN THE GLUTEN-FREE DIET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Sanna Arnala, The Finnish Coeliac Society, Finland

195

PURE OATS PRODUCTION-COMPANY PRESENTATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Pirjo Alho-Lehto, Raisio Group, Food Division, Finland

197

QUO VADIS, COELIAC DISEASE? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Markku Maki, University of Tampere, Finland

199

Author Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

201

XVII

XVIII

Current Prevalence, Diagnosis and Treatment of

Coeliac Disease
Markku Mki
Coeliac Disease Study Group, University of Tampere, Finland
corresponding email: Markku.Maki@uta.fi

Notes
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________

GLUTEN
FREE

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 (/-, -, -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-, -, -40k-, -75k-secalins) and barley (D-, C-, B-, -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 155) of /-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 -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 (206217) showed toxic effects in two CD patients in
remission and no effect in the control group. In vivo challenges using three synthetic peptides
from /-gliadins showed that peptide (3149) caused significant histological changes in
four patients, whereas peptides (202220) and (321) were weakly active or inactive,
respectively. Ala-substituted variants of peptide (3149) 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
(3143) and (4455). Challenge tests with peptide (5675) and a negative control peptide
from -casein showed that the gliadin peptide caused intestinal damage, while the casein
peptide produced no response. Corresponding repetitive sequences of -type and -type

gliadins, secalins, and hordeins have not yet been tested by instillation or organ culture tests,
but fit well into the potentially toxic sequences of /-gliadins.
Table 1. Origin and amino acid sequences of gliadin peptides tested for CD-toxicity.
Origin (Position)
Sequencea
(1 - 30)
VRVPVPQLQPQNPSQQQPQEQVPLVQQF
(3 - 21)
VPVPQLQPQNPSQQQPQEQ
(3 - 24)
VPVPQLQPQNPSQQQPQEQVPL
(25 - 55)
VQQQFPGQQQPFPPQQPYPQPQPFPSQQPY
(31 - 43)
LGQQQPFPPQQPY
(31 - 49)
LGQQQPFPPQQPYPQPQPF
(31 - 49, A31)
AGQQQPFPPQQPYPQPQPF
(31 - 49, A36)
LGQQQAFPPQQPYPQPQPF
(31 - 49, A38)
LGQQQPFAPQQPYPQPQPF
(31 - 49, A39)
LGQQQPFPAQQPYPQPQPF
(31 - 49, A42)
LGQQQPFPPQQAYPQPQPF
(31 - 55)
LGQQQPFPPQQPYPQPQPFPSQQPY
(44 - 55)
PQPQPFPSQQPY
(51 - 70)
SQQPYLQLQPFPQPQLPYSQ
(56 - 68)
LQLQPFPQPQLPY
(56 - 75)
LQLQPFPQPQLPYPQPQLPY
(202 - 220)
QQYPLGQGSFRPSQQNPQA
(206 - 217)
LGQGSFRPSQQN
(247 - 266)
CNVYIAPYCTIAPFGIFGTN
a
One-letter-code for amino acids; b OC, organ culture test; IN, instillation test

Toxicityb (Test)
+ (OC)
- (IN, OC)
+ (OC)
+ (OC)
+ (IN, OC)
+ (IN, OC)
+ (OC)
+ (OC)
- (OC)
- (OC)
- (OC)
+ (OC)
+ (IN, OC)
+ (OC)
- (IN, OC)
+ (IN)
- (IN, OC)
+ (IN)
- (OC)

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 /-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 , 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.

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*
1

Oslo University Hospital, 2University of Oslo, 3National Veterinary Institute, Oslo,

Centre 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: GSRSIBS (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 (Rki 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.

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
Diagnosis

Before

During

General Health Complaints: SHC

After

Diagnosis
Non-CD,
n=35
CD, n=13

Non-CD, n=35

22

37

34

CD, n=13

24

29

27

GSRS-IBS: Total score 13-91

Before

During

After

16

12

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.
Rki 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.

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 (2959 y, four men) completed the study per-protocol. 56% had HLADQ2 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=00001; 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*

0047

0016

0031

0053

0024

0120

0001

Using the mixed effects model accounting for time, age, sex, and BMI, the severity scores of
pain (p=002), satisfaction with stool consistency (p=003), and tiredness (p=0001) 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.

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
1

Plant Research International, Wageningen UR, Wageningen, The Netherlands

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

Email addresses:
DL Diana.londono@wur.nl
IMM Ingrid.vandermeer@wur.nl
LJWJG luud.gilissen@wur.nl

WPCW wendy.vantwestende@wur.nl
EMJS elma.salentijn@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 -gliadins, -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.

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*,
1
University and Spedali Civili of Brescia, Department of Gastroenterology, Brescia, Italy
2
University of Trieste, Department of Pediatric Gastroenterology, Burlo Garofolo, Trieste,
Italy
3
CRA, 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.

0.10

L/R ratio

0.08
0.06
0.04
0.02

Ta

R
ic

Tm

0.00

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

Table 1: Gastrointestinal adverse events reported in the three different study days
Rice
Tm
Ta
Abdominal Pain
1
2
7
Bloating
2
1
11
Costipation
Diarrhoea
2
Flatus
Taste impairing
1
2
Nausea
5
5
9
Vomiting
2
Heartburn
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, DOdorico 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

10

Down-regulation of -gliadins in bread wheat by RNA

interference (RNAi)
Javier Gil-Humanes1, Fernando Piston2*, Racha Aouni3, Francisco Barro1
1Instituto de Agricultura Sostenible, CSIC, E-14080, Crdoba, Spain
2Departamento de Gentica, Universidad de Crdoba, E-14071, Crdoba, 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 - and -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 -gliadins. We used an inverted repeat construct (Figure 1) to target -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 (RPHPLC).
Results. Gliadin content estimated by R5 ELISA assay did not show significant differences
between transgenic and control lines. However, the RP-HPLC showed that -gliadins were
reduced in all transgenic lines by about 72-98.8%, depending of line (Table 1). Other
gliadin fractions (/- and -gliadins) did not show significant difference relative to
control.
Figure 1. The sequence of a -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).

11

Table 1. Transgenic plants and gliadin content estimated by R5 enzyme-linked

immunosorbent assay (ELISA) and RP-HPLC. , -gliadins; /, /-gliadins; , 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.

12

Gluten epitopes in Finnish patients with DQ2+ celiac

disease
Andrea de Kauwe1, Katri Kaukinen2, Jason Tye-din3, Jessica Stewart3, Kalle Kurppa2,
Lotta Koskinen1 Markku Mki2, Robert Anderson3 & Pivi Saavalainen1*
1

University of Helsinki, Department of Medical Genetics, Helsinki, Finland

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
2

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.

13

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

Responders (%)

Weak responders (%)

Non-responders (%)

Total (F,M)

Wheat
Rye
Barley
Combined

8 (45%)
7 (50%)
7 (47%)
6 (55%)

6 (33%)
1 (7%)
0 (0%
0 (0%)

4 (22%)
6 (43%)
8 (53%
5 (45%)

18 (13F,5M)
14 (11F,3M)
15 (11F,4M)
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.

14

Improving accuracy in detecting gluten

Pivi 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 100C for 2h. After acid treatment the sample was neutralised with 1M NaOH to pH 8

15

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% 1propanol 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 prolaminspecific 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 , Mcadam SN, Krner 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.

16

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 20-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 (2-8C). 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.

17

Table 1. 20-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).
Sample

Matrix

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17

Rice baby food

Rice baby food
Rice baby food
Rice baby food
Rice baby food
Rice baby food
Rice baby food
Rice baby food
Rice baby food
Rice baby food
Rice baby food
Rice baby food
Maize bread
Maize bread
Maize bread
Maize bread
Chocolate cake mix

Spiked / Non-spiked

Extraction

20-gliadin
Mean (ppb)
<
<
>
>
706
>
444
659
255
397
131
226
239
367
170
211
65

RSD (%)
Non-spiked
EtOH

Non-spiked
DTT

Spiked 0.4% wheat baby food

EtOH

Spiked 0.4% wheat baby food

DTT

Spiked 0.2% wheat baby food

EtOH
9
Spiked 0.2% wheat baby food
DTT

Spiked 0.1% wheat baby food

EtOH
23
Spiked 0.1% wheat baby food
DTT
11
Spiked 0.05% wheat baby food
EtOH
32
Spiked 0.05% wheat baby food
DTT
21
Spiked 0.025% wheat baby food EtOH
40
Spiked 0.025% wheat baby food DTT
29
Spiked 44.2 ppm of gliadina
EtOH
36
Spiked 44.2 ppm of gliadina
DTT
27
Spiked 22.1 ppm of gliadina
EtOH
42
Spiked 22.1 ppm of gliadina
DTT
29
Non-spiked
EtOH+Gel+PVP
51
Spiked 0.25% gluten-containing
18
Chocolate cake mix
EtOH+Gel+PVP 428
18
chocolate cake mix
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 (-, - and -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.

18

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. Khler (Khler 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.

19

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, Geendorfer 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)

20

Detection of toxic fragments from gluten using a new

monoclonal antibody-based test
Richard Fielder1*, Elisabeth Halbmayr2, Michael Z. Zheng3, Donna Houchins4
1

Romer Labs UK ltd, North Wales Business Park, Building Unit 5325 Abergele, LL22 8LJ
North Wales, United Kingdom
2
Romer Labs Division Holding GmbH, Technopark 1, 3430 Tulln, Austria
3
Romer Labs Singapore Pte. Ltd., 3791 Jalan Bukit Merah #08-08, e-Centre@redhill
building, Singapore, 159471
4
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 worlds
population, primarily adults. Immunotoxic gluten peptides, such as the fragment called 33mer, 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 (Morn 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 (Morn
2008a, 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.

21

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
Corn starch
Sugar+milk
BBQ Spices
Paprika
Wheat starch
Strawberry flavour
Pudding
Ham flavour
Glucose
Rice milk
Sausage 1
Sausage 2
Cured loin of pork
Hamburger
Cake
Aperitive snacks
Baby food
Biscuit
Bread
Chocolate
Ice cream
Cream

Results AgraStrip
>10 ppm
>10 ppm
>10 ppm
<10 ppm
>10 ppm
>10 ppm
>10 ppm
<10 ppm
>10 ppm
>10 ppm
>10 ppm
>10 ppm
<10 ppm
>10 ppm
<10 ppm
<10 ppm
>10 ppm
<10 ppm
<10 ppm
<10 ppm
<10 ppm
<10 ppm

Results ELISA
18.8 ppm
191 ppm
23.3 ppm
<3 ppm
166 ppm
12 ppm
36 ppm
<3 ppm
256 ppm
80 ppm
113 ppm
>100 ppm
<3 ppm
96 ppm
<3 ppm
<3 ppm
96 ppm
<3 ppm
<3 ppm
<3 ppm
<3 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 , 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.
Morn B, Bethune MT, Comino I, Manyani H, Ferragud M, Lpez 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.
Morn B, Cebolla A, Manyani H, Alvarez-Maqueda M, Megas M, Thomas Mdel C, Lpez
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.

22

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 -gliadin genes , which
group of gluten is considered to be the most toxic to CD patients, have been analyzed to
23

determine whether they encode for peptides potentially involved in CD. The results showed
that the -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 -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 -gliadins contain CD-toxic peptides with
comparable impact to those of the -gliadins; -I is the major epitope. About 1100 -gliadin
sequences have been analysed and related to their genome of origin, being A, B or D. The
highest frequency of expressed -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-9 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-9. 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, M 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

24

Notes
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25

Notes
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26

Testing Safety of Low Gluten Food Products in a Mouse

Model of Celiac Disease
Tobias Freitag1,3, *, Yvonne Junker3, Pivi 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 antigliadin 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.

27

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 nave 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.

28

General Discussion on
Safety and tolerance
Analysis and regulatory standards
Technology and variety of products
Hannu Salovaara, Pivi 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) *

Ciclitira et al. (1984)

Catassi et al. (1993)
Collin et al. (2004)

>2.44.8 mg/d
<200mg gluten/d
30 mg/d

Ciclitira et al. (2005)

Hischenhuber et al.
(2006)
Biagi et al. (2004)
Catassi et al. (2007)

50 mg/d
>10 mg/d but
< 100 mg/d
< 1mg/d
<50 mg/d
>10mg/d
<10 mg/d

Suggested max
gluten conc.
(ppm or mg/kg)
>1224 mg/kg
100 mg/kg
if 300g flour/d
<100mg/kg
for wheat starch
(3520 mg/kg)

Comments and complementary

information
Bread, wheat starch, 1 wk , children
Children, minimal histol changes, 4wks
not to jeopardise the GF diet with
inconvenient restrictions
a consensus suggests
A review.

1 patient 1 communion wafer/d,no recover

Amount tolerated varies among
individuals with CD.
Akobeng & Thomas
(20 mg/kg)
20 mg/kg means 6 mg/d , which is less
(2008) . Review.
likely to induce mucosal damage
* Intake of the Triticeae prolamins by a healthy average European is 2030 g/d (20 00030 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. 2433%1
Defect: poor in detecting barley prolamins
R5 Mendez
ELISA Sandwich
Commercial kits; rept. 1820%; repd. 3032%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
1
rept. = 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, Mki 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;19:127783.
Kanerva PM, Sontag-Strohm TS, Ryppy 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.

30

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,
ngel Cebolla5 and Carolina Sousa1*
1
Departamento de Microbiologa y Parasitologa, Facultad de Farmacia, Universidad de
Sevilla, Sevilla, Spain
2
Present address: Centro Nacional de Biotecnologa (CNB - CSIC), Departamento de Gentica
Molecular de Plantas, Madrid, Spain.
3
Departamento de Biologa Vegetal y Ecologa, Divisin de Fisiologa Vegetal, Facultad de
Ciencias, Universidad de Crdoba, Crdoba, Spain
4
Departamento de Biologa Experimental, Jan, 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
(Morn 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 Morn et al. (2008).

31

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 Morn 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.
Morn B, Cebolla A, Manyani H, lvarez-Maqueda M, Megas M, Thomas MC, Lpez 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 , 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.

32

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

33

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

34

New Approaches in Gluten Analysis of Products for

Celiacs by Proteomics Combined with The R5 ELISA
Techniques.
Mara Carmen Mena1*, Manuel Lombarda1, Alberto Hernando1, Juan Pablo Albar1
1
Proteomics Facility/Gluten Unit, Centro Nacional de Biotecnologa, 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-LCMS/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.

35

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

Wheat starch A (6 ppm gluten)
Protein
gliadin/avenin-like seed protein [Triticum aestivum]

Mass
1288.66
1288.62

Peptide
R.QLAQIPEQFR.C
R.QPSQIPEQFR.C

Mass
845.44
1180.66
1815.84

Peptide
R.TPFPQTR.G
R.QLVQIPEQAR.C
R.QQCCQPLAQISEQAR.C

1424.72
1228.66

R.CQAIHNVVESIR.Q
R.QLAQIPEQFR.C

1228.62

R.QPSQIPEQFR.C

2059.03
859.49

K.VFLQQQCSPVAMPQSLAR.S
R.VNVPLYR.T

Mass
1180.66
1949.93
2650.18
1186.64
2855.61
2393.03
1815.84
1424.72
1288.66
2082.04
1949.93
2650.18
1244.65
1228.62

Peptide
R.QLVQIPEQAR.C
R.DALLQQCSPVADMSFLR.S
R.SQAVQPRSCLVMWEQCCQQLK.A
R.APFASIVAGIGGQ.R.RPLFQLVQGQGIIQPQQPAQLEVIR.S
R.SDCQVMQQQCCQQLAQIPR.Q
R.QQCCQPLAQISEQAR.C
R.CQAIHNVVESIR.Q
R.QLAQIPEQFR.C
R.QQQHHQPQQEVQLEGLR.M
R.DALLQQCSPVADMSFLR.S
R.SQVVQHSSCLVMWEQCCQQLK.A
R.QLSQIPEQFR.C
R.QPSQIPEQFR.C

1320.60

R.ELQESSLEACR.Q

2059.09
859.42

K.VFLQQQCSPVAMPQSLAR.S
R.VNVPLYR.T

1732.89

K.VFLQQQCIPVAMQR.C

2059.09
859.42

K.VFLQQQCSPVAMPQSLAR.S
R.VNVPLYR.T

Wheat starch B (25 ppm gluten)

Protein
alpha-gliadin [Triticum turgidum subsp. durum]
gamma gliadin [Triticum monococcum]
gliadin/avenin-like seed protein [Triticum aestivum]
low molecular weight glutenin subunit
[Triticum aestivum]
s-type low molecular weight glutenin L4-55
[Triticum aestivum]
Wheat starch C (110 ppm gluten)
Protein
alpha-gliadin [Triticum turgidum subsp. durum]
gamma gliadin [Triticum aestivum]

gamma gliadin [Triticum monococcum]

gliadin/avenin-like seed protein [Triticum aestivum]

high-molecular-weight glutenin subunit

[Triticum aestivum]
low molecular weight glutenin subunit
[Triticum aestivum]
low-molecular-weight glutenin subunit group 11
[Triticum aestivum]
s-type low molecular weight glutenin L4-55
[Triticum aestivum]

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, Lombarda 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.

36

Effects of heating, reducing and alcohol concentration on

prolamin extraction
Pivi 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 21C or 50C
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 21C to 50C 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, 2propanol extracted more efficiently high molecular weight rye proteins.

37

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 glutenfree products with unknown contamination source.
References
Kanerva PM, Sontag-Strohm TS, Ryppy PH, Alho-Lehto P, Salovaara HO. Analysis of barley
contamination in oats using R5 and omega -gliadin antibodies. J Cereal Sci 2006;44:347352.

2
38

Deamidation of gluten proteins drastically influences the

quantitative gluten analysis
Pivi 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 100C 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.

40

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 wheatbased 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 glutenfree 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 glutenfree 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 Associations 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 belowrecommended 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 adults 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.

42

Gluten-Free Food Market

Markku Mikola1*; Esa Wrang2
Consultant, Sennet Oy, Finland,
2
Leading consultant, Finpro ry, Finland
*corresponding email:markku.mikola@alumni.helsinki.fi
1

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 g/100 g fw (wheat starch based mix) and 10 g/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 g/100 g (wheat flour with low ash content) to 45 g/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 nonfortified 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 g/100 g fw. In different buckwheat flours the range of the total folate contents was 31
54 g/100 g fw and that in other buckwheat milling products 2456 g/100 g fw. The total folate
contents of rice, maize and potato based flakes and flours were 2355 g/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 g/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 g.
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 g/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; 8:134139.
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, Jgerstad 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. Masters thesis. 2007. EKT-series 1393.

46

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 glutenfree 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

47

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

48

Healthy Grains for Enhanced Gluten-free Breads

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

Introduction: Gluten-free breads, as well as being safe for coeliacs, should have a nutritional
value 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 been
reported 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 using
refined flours and/or starches and, in contrast to their wheat-containing counterparts, glutenfree products are not normally fortified. Pseudocereals do not belong to the grass family but
still produce flours and seeds which can be used as flour. They are naturally gluten-free, and
are characterised by high protein, fibre and micronutrient contents. In the present study, a
number of nutritional and bioactive properties of pseudocereal flours were studied, along with
their suitability as ingredients in gluten-free breads.
Methods: Buckwheat, amaranth and quinoa flours replaced potato starch in a control glutenfree bread formulation (C), based on rice flour and potato starch. Standard baking tests were
conducted on the resulting breads. Macronutrient and mineral analyses were completed on the
flours (pre-baking) and breads (post-baking). Total antioxidant capacity of the pseudocereal
grains and breads was determined by the DPPH method and the FRAP assay. Total phenol
content 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 found
for breads containing amaranth. The crust colour of the loaves was significantly darkened
following the inclusion of all pseudocereal flours (P<0.01). Crumb texture was significantly
improved, with all pseudocereal-containing breads having a softer (P<0.05) and more
cohesive (P<0.01) texture in comparison with the control. The C-Cell image analysis system
revealed that the crumb grain of the breads with pseudocereals was improved (Figure. 1). In
particular, 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 of
all pseudocereal breads was observed. In particular, the protein, fibre and Mg levels
significantly increased (P<0.05). Levels of total antioxidants in the buckwheat flour were
significantly higher than the other samples. Antioxidant levels were significantly reduced
(although still present for buckwheat breads) after baking, whereas those present in wheat
breads were too low to measure. Similarly, significantly higher levels of total phenols were
found in buckwheat flours and breads. An example of some of these results is seen in Table 1.

49

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

Total phenol as
gallic 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

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

Grains

Breads

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 in
the formulation of gluten-free breads with good baking properties, sensory scores and crumb
texture. 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. Glutenfree Food Science and Technology. Oxford: Wiley-Blackwell; 2009. p. 42-51.
50

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

Gluten?
Blanca Esteban, Manuela Mrquez, 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

51

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.

85%

5%
10%

Persistence
iron
Persistence Low
Low
iron Persistent
Persistence
ofor
positive
of
symptoms
symptoms
antibody levels
increased
of
antibodies
Figure
1. Main reasons for diet revision.
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.

52

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 Piikki in South-western Finland in 2009. The
linseed varieties in the trial were Helmi, Helj, Laser, Abacus and Sunrise and the fibre flax
varieties Martta and Belinka. Oil and protein and conte nts 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.

53

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. Helj 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

Linseed

Farm

Linseed

Trial

Flax
Oil hemp

Farm

Variety Year

Helmi
Helmi

2007
2008
mean
Laser 2007
Laser 2008
mean
Helmi 2009
Helj 2009
Laser 2009
Abacus 2009
Sunrise 2009
Martta 2009
Belin ka 2009
Finola 2005
2007
2009

Nu mber
of samples
n

Oil
content
% d. m.

3
3

41,3
42.9
42.1
44.4
47.2
45.8
45.2
48.7
48.4
48.8
50.3
41.4
41.8
34.7
35.4
35.8

8
3
1
1
1
1
1
1
1
1
1
2

Protein Cad miu m Lead

content
content content
% d. m
mg/kg
mg/kg

SDG
lignan
mg/kg

0.73
1.11
0.92
0.43
0.36
0.39
0.61
0.61
0.48
0.46
0.54
0.57
0.42
0.023
0.017

7400
7550
7470
4990
4840
4920
9070
8910
5320
7120
7690
9560
8190

22.6
22.6
22.2
22.2
22.3
21.7
18.5
19.4
20.8
24.8
25.0

0.051
0.028
0.040
0.052
0.055
0.054
0.009
< 0.007
0.008
0.010
0.016
0.007
0.007
0.011
0.027

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 Gu idance and Guarantee Fund
through the Centres for Economic Development, Transport and Environ ment 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.

54

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,
1
Plant Research International, Wageningen UR, PO Box 16, 6700 AA Wageningen, The
Netherlands
2
Food 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 polyunsaturated 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 polyunsaturated 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.

55

Valiant

Sang

Markant

Gigant

Freddy

Firth

Dominik

Mustang

Powys

Gambo

Panache de Roye

Ascot

Leanda

Astor

Zandster

Zwarte President

Wodan

Troshaver uit Besel

Ouderwetse Zeeuwse partij

MansholtIII

Gele van Timmermans

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.
fibres vit E M+PUFA bread yield
Cultivar antig/g % of fat quality (t/ha) glucan
oxidant %
mg/g
81
3.3
3.8
2
9.7
1150 10
1
114
4.5
2
11.4 5.1
10.3
967
2
162
4.7
1
11.3 5.1
1053 10
3
113
3.9
6.2
2
9.1
10.5
939
4
150
5.1
5
6.1
8.9
9.2
1129
5
192
3.6
10
6.6
7.3
1243 10.8
6
90
3.2
7.2
8
3.6
1403 12.3
7
89
4.0
6.5
8
9.1
1229 11.2
8
81
5.4
6.1
5
7.3
1180 10.3
9
87
5.9
10
6.9
8.7
1463 10.3
10
144
4.0
6.4
5
8.1
1592 10.4
11
98
4.3
12
6.8
8.7
1109 10.8
12
184
3.3
13
6.7
6.1
1214 11.7
13
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.

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 Robins2, Anthony Akobeng3
1

Coeliac UK, Diet and Health, High Wycombe, UK

2
York Hospitals NHS Foundation Trust, UK
3
Central Manchester and Manchester Childrens 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

Collins et al

1986

Dietary history and nutritional state in treated coeliac patients

Dickey et al

2008

Grehn et al

2001

Hallert et al

2002

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.
Dietary habits of Swedish adult coeliac patient treated by a gluten-free diet
for 10 years
Evidence of poor vitamin status in coeliac patients on a gluten-free diet for
10 years

Hopman et al

2006

Nutritional management of the gluten-free diet in young people with

celiac disease in the Netherlands

Kemppainen et al

1995

Intakes of nutrients and nutritional status in coeliac patients.

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

McFarlane

1995

Robins et al

2008

10

Storsrud

2003

11

Thompson et al

2005

Subclinical nutritional deficiency in treated coeliac disease and nutritional

content of the gluten-free diet.
Coeliac patients on a gluten-free diet consume a disproportionate amount
of milk intrinsic sugars
Beneficial effects of oats in the gluten-free diet of adults with special
reference to nutrient status, symptoms and subjective experiences.
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*
1

University of Ljubljana, Biotechnical Faculty, Department of Biology, Jamnikarjeva 101,

SI-1000 Ljubljana, Slovenia
2
University of Ljubljana, Biotechnical Faculty, Department of Agronomy, Vena 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,
monik, is a soft floury dish traditionally made from buckwheat flour. Monik was
traditionally perceived as a food of the poor. Yet milky buckwheat monik has an attractive
smell, its taste is good and pleasing. As buckwheat is believed to keep the body warm, milky
buckwheat monik 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 SpectraPhysics (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).
Bran of tartary buckwheat
Bran of common buckwheat
Flour of tartary buckwheat
Flour of common buckwheat

Rutin
mg/g
17.610.20
0.2800.015
11.820.24
0.2250.014

total
24.8
26.4
6.3
6.8

Dietary fibre (% dry weight)

soluble
insoluble
1.2
23.6
0.9
25.5
0.5
5.8
0.9
5.9

% sol.
4.77
3.45
8.27
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. N hrwert des Buchweizens: Gr tze - Mehl - Brot - Gem se - Tee - Essig - Honig. In:
Kreft I, Ries C, Zewen C, editors: Das Buchweizen Buch: mit Rezepten aus aller Welt.
2. berarbeitete und erweiterte Aufl. Arzfeld: Islek ohne Grenzen EWIV; 2007. p. 92104.

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-deltalactone, 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. 401408.
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 Kivel1*, 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
3
Beth Israel Deaconess Medical Center and Harvard Medical School, Division of
Gastroenterology, Boston, Massachusetts, USA
4
Boston 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, Vrbacka, 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 pH60.2 and pH20.2.
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 betaglucan 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).
pH 2
mucin

Viscosity (Pas)

10

pH 6
mucin

Native BG

Native BG

LMW BG

LMW BG

0,1

0,01
1

10

100

1000

10000

Shear rate (1/s)

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
(0.0500.0010 Pas 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 betaglucan clearly affected the association so that native and unprocessed high molar mass betaglucan 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.

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

Notes
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________

R-Biopharm AG

Gluten analysis
on surfaces and
in food

Swabbing with RIDAQUICK Gliadin is recommended to

ensure gluten-free production conditions.

For product testing the RIDASCREEN ELISAs allow gluten

quantification to ensure compliance with legal limit values:
RIDASCREEN Gliadin*
RIDASCREENFAST Gliadin
RIDASCREEN Gliadin competitive

Swabbing with
RIDAQUICK Gliadin

* Official Codex Alimentarius Method / AOAC-RI certified

R-Biopharm AG
An der neuen Bergstrae 17
64297 Darmstadt, Germany

Phone: +49 (0) 61 51 - 81 02-0

Fax: +49 (0) 61 51 - 81 02-40
info@r-biopharm.de, www.r-biopharm.com

66

Many faces of prolyl oligopeptidase What we ignore in

mammals and what we know in plants
J. Arturo Garca-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 (GarciaHorsman 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 (Myhnen 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.

67

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.

Protein aggregates
POP
POP substrate
BBB damage

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).

68

Endogenous cereal enzymes in the elimination of prolamins

Jussi Loponen1*, Pivi Kanerva1, Michael Gnzle2, Tuula Sontag-Strohm1, Hannu
Salovaara1
1

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

University 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

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

69

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).
Wheat

Barley

Rye

0h

0h

0h

6h

6h

6h

24h

24h

24h

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.
pH 3.8

Prolamin mg / kg (DM)

pH 4.1

pH 3.6

60000

60000

60000

50000

50000

50000

40000

40000

40000

30000

30000

30000

20000

20000

20000
BARLEY

BARLEY
10000

0
0h

WHEAT
RYE
24h

10000

BARLEY

10000
WHEAT

0
0h

RYE
24h

0
0h

WHEAT
RYE
24h

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 ., 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. 297:22752279.
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., Venlinen J., Salovaara H. 2007. Prolamin hydrolysis in wheat sourdoughs with differing
proteolytic activities. Journal of Agricultural and Food Chemistry, 55, 978984
Loponen J, Kanerva P, Zhang C, Sontag-Strohm T, Salovaara H, Gnzle M.G. 2009. Prolamin hydrolysis and pentosan
solubilization in germinated-rye sourdoughs determined by chromatographic and immunological methods. J Agric Food
Chem, 57, 746753

70

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
1
Chalmers University of Technology, Department of Chemical and Biological Engineering,
Food Science, Gothenburg, Sweden
2
Guangxi Academy of Agricultural Sciences, Microbiology Institute, Nanning,
Guangxi, China
3
Gothenburg 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

WHWRNPDFWYLK

P64

WHWTWLSEYPPP

P22

LETSKLPPPAFL

PC31

AYYPQNHKSNAE

71

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

32.8214.3 (P<0.001)

P22

36.2112.8 (P<0.001)

Pool

29.9517.6 (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.

72

Degradation of gliadin peptides toxic for coeliac disease

patients by prolyl endopeptidase synthesized by
Lactobacillus acidophilus 5e2 and Aspergillus niger
Bartosz Brzozowski1*, Wodzimierz Bednarski1, Barbara Wrblewska2
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 Myny
Szczepanki Sp. z o.o. (asin, Poland) using 60% ethanol after removing albumins and
globulins. The synthetic (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,
37C) and next for trypsin digestion (65,7 mM Na2HPO4, 1 M NaOH, pH 7.8, 120 min,
37 C). The activity of PEPs was expressed as mol of pNa released from Z-Gly-Pro-pNa
after 60 min, at 37 C in pH 7.0. The LB PEP and AN PEP were used to hydrolysis gliadins
and immunoreactive peptide (31-43)-gliadin at 30 C and 37 C 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 C and 37 C for AN PEP. Whereas, gliadin degradation performed
with use LB PEP showed the highest hydrolysis ratio in pH 6.0 at 30C. 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 g/mL) was

73

obtained after gliadin treatment by LB PEP in pH 6.0 at 37 C (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 37C 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 signicantly
different (P<0.05, ANOVA, Benferroni test).
Hydrolysis conditions

Peptide contents
(g/mL)

Temp.(C) Acidity (pH)

Control
LB PEP

AN PEP
Control
LB PEP

AN PEP

Absorbance (AU)

0,025

6.0

27,30,43

C,D

30

6.0

26,50,59

37

6.0

14,00,46

30

6.0

29,80,26

37

6.0

28,20,56

B,C

4.0

26,50,38

30

4.0

16,00,20

37

4.0

11,40,09

30

4.0

30,50,10

37

4.0

30,40,04

0h peptide + LB PEP
1h peptide + LB PEP
2h peptide + LB PEP
3h peptide + LB PEP

0,020
0,015

(31-43)gliadin
LB PEP

0,010
0,005
0,000
0

10

15

20

Time (min)

Figure 1. Peptide (31-43)-gliadin electropherogram after 0, 1, 2 and 3h hydrolysis with LB

PEP at 37C 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.

74

Detoxification of gluten by germinating cereal enzymes:

Implications for new treat ment of c oeliac disease
Satumarja Stenman1, Katri Lindfors1, Jarkko I Venlinen2, Anne Hautala1, Pekka T
Mnnist3, Anu Kaukovirta-Norja 4, Shuo-Wang Qiao5, Ludviq M Sollid5, Markku
Mki1,6 and Katri Kaukinen1,7
1

Medical School, University of Tampere, Finland

Department of Pharmacology and Toxicology, University of Kuopio, Finland
3
Division of Pharmacology and Toxicology, University of Helsinki, Finland
4
Technical Research Centre of Finland, Espoo, Finland
5
Rikshospitalet University Hospital and University of Oslo, Norway
6
Department of Pediatrics, Tampere University Hospital, Finland
7
Department of Gastroenterology and Alimentary Tract Surgery, Tampere University
Hospital, Finland
2

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 pretreated, 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.

75

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 + PT
Gliadin + 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.

76

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 bakers 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-) 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 9gliadin, 62-75 of A-gliadin and 134-153 of -gliadin. As shown by electrophoresis,
chromatography, mass spectrometry and immunology analyses, several peptides from
gliadins and glutenins persisted after treatment of bakers 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-, IL-2
and IL-10 at levels comparable to the negative control.

77

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, DArchivio 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.

78

Can prolyl endoprotease enzyme treatment mitigate the

toxic 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 Netherlands
2
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 to
proline/glutamine-rich gluten peptides. Previous in vitro studies showed that the Aspergillus
niger prolyl endoprotease (AN-PEP) enzyme is highly effective in degrading these immunotoxic epitopes into harmless fragments under gastric conditions (Stepniak 2006, Mitea 2008).
Methods: To assess whether AN-PEP enzyme could mitigate the immunogenic effects of
gluten in CD patients, a randomised, double-blind placebo-controlled pilot study was
performed (Figure 1).
GLUTEN + AN-PEP
GLUTEN + AN-PEP

WASHOUT
GLUTEN + Placebo

baseline week 1
Blood
Blood
Biopsy
Questionnaire
Figure 1. Study design

week 2
Blood
Biopsy
Questionnaire

week 4

week 5
Blood

Questionnaire

week 6
Blood
Biopsy
Questionnaire

All 16 subjects (18-70 yrs) consumed 5 pieces of toast (~7 g gluten) with AN-PEP topping for
2 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 mucosa
immunohistology (Marsh scores, T-cells, anti-tissue transglutaminase-IgA (tTG-IgA)
deposits), CD-specific serum T-cells and antibodies (tTG-IgA, dual anti-deamidated gliadinrelated peptides and anti-tTG IgA (DGP/tTG-IgA), anti-gliadin-IgA and IgG (AG-IgA and
AG-IgG), and anti-endomysium (EM-IgA)), self-reported CD-specific health-related quality
of life questionnaire (CDQ, subcategories; disease-related worries, emotional, social, and
gastrointestinal problems), and physician-reported health complaints.
Results: Of the 16 subjects enrolled in the study, 2 were excluded after the AN-PEP phase
because of histological deterioration of 2 Marsh grades although serum antibodies remained
negative. Fourteen subjects did not show any deterioration on gluten plus AN-PEP and
entered the randomisation phase and all completed the study. Only minor deteriorations were
observed on gluten intake in both AN-PEP and placebo group for all parameters. No
significant differences in change from baseline between the groups were observed for any of
the 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 remained
negative in all subjects during the study. AG-IgA rose to positive levels in 2 of 7 patients on
placebo and AG-IgG in 1 placebo and 1 AN-PEP patient. DGP/tTG-IgA became positive in 1
placebo patient. Mucosal IgA-tTG deposit staining increased in 4 subjects on placebo and 1
on AN-PEP as compared to baseline. Total CDQ scores and subcategory scores were
relatively high and did not significantly change in time and differ between groups. The CDQ
gastrointestinal 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 reported
without difference between groups. Posthoc analysis revealed significant negative correlations
between 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 for
analyses, the median, and sum of ranks was lower after AN-PEP than placebo treatment for
all parameters, which was significant for Marsh and tTG-IgA (Table 1).
Parameter

Median (IU/mL)
AN-PEP
Placebo

Sum of ranks
AN-PEP (n=4) Placebo (n=4)

p-value

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 or
AN-PEP

Conclusion: Little clinical deterioration was observed in CD patients consuming a daily

amount 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 to
a 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. Highly
efficient gluten degradation with a newly identified prolyl endoprotease: implications
for 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 of
gluten by a prolyl endoprotease in a gastrointestinal model: implications for coeliac
disease. 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 3.64.1 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.
Monomeric
proteins

9-mer
19-mer
33-mer

Hydrolysis
products

Polymeric
proteins

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).

Prolamin content in the soluble fraction of malt

suspensions

Prolamin content of total fraction after 24 h malt

incubation
1 600

25 000

1 400
20 000
1 200
1 000

15 000

800
10 000

600
400

5 000
200
0

0
pH 3.6
BARLEY

pH 3.8
WHEAT

pH 3.6

pH 4.1

BARLEY

RYE

pH 3.8
WHEAT

pH 4.1
RYE

Figure 2.
Prolamin contents of malt suspensions after 24 h incubation under acidic pH conditions. The Yaxis 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

Barley
Wheat
Rye

Amino nitrogen mg kg-1 h-1

pH 3.6
pH 3.8
pH 4.1
57
63
47
16
31
40
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, 40C).
Proline oligopeptidase (POP) activity was determined (pH 7.0, 37C) 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, 34C). 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

A
alfa-amylase
p-nitrophenyl maltoheptaoside (blocked)
600,0

350,0
REETTA

250,0

RIIHI

200,0
EVOLO

150,0
100,0

RIIHI
REETTA

500,0
Activity, U g-1 (dry matter)

Activity, U g-1 (dry matter)

300,0

400,0

EVOLO

300,0
200,0
100,0

50,0

0,0

0,0
After
steeping

1-day

2-day

After
steeping

4-day

proline-oligopeptidase
Z-Gly-Pro-AMC

1-day

2-day

REETTA

175,0
150,0
125,0

REETTA

14,0

RIIHI

12,0

EVOLO

10,0

100,0

4-day

proteolytic (general)
azo-casein

16,0

200,0

AU

Activity, nmol AMC min-1 g malt-1 (dry matter)

beta-amylase
p-nitrophenyl maltopentaoside

RIIHI

EVOLO

8,0
6,0

75,0

4,0

50,0

2,0

25,0

0,0

0,0
After
steeping

1-day

2-day

After
steeping

4-day

1-day

2-day

4-day

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. Gnzle, M.G. Prolamin hydrolysis and
pentosan solubilization in germinated-rye sourdoughs determined by chromatographic and immunological
methods. J Agric Food Chem 2009; 57:746753
Brijs K., Trogh I., Jones BL. Delcour JA 2002 Proteolytic enzymes in germinating rye grains. Cereal Chem.
79:423-428.

84

Overview on the new developments in the area

of gluten free foods and beverages
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.
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

Notes
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________

N a t u r a l

F l a v o u r

Natural

Sourdoughs,
Preferments &

Best Fermentations

Ernst Bcker GmbH & Co. KG

PO Box 21 70
32427 Minden / GERMANY

88

Phone +49 (571) 8 37 99-0

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

Formation and modification of bioactive compounds in

gluten free sourdoughs
Michael G. Gnzle, 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 -galactosidase positive L. reuteri LTH5448 or the -galactosidase
negative L. sanfranciscensis LTH2590. Isogenic strains lacking levansucrase activity (L.
reuteri LTH5448 ftfA and L. sanfranciscensis LTH2590 levS) 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 coumaroylferuloylglycerol 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 antinutritive 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 glutenfree cereal-based products. Trends Food Sci. Technol. 2004, 15, 143-152.
(2) Tieking, M., M.A. Ehrmann, R.F. Vogel, M.G. Gnzle. 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. Gnzle. Sucrose utilization and
impact of sucrose on glycosyltransferase expression in Lactobacillus reuteri. 2007.
System. Appl. Microbiol. 30, 433-443.

detector signal (nC)

100

verbascose
stachyose

A
melibiose
galactosylmelibiose
sucrose
digalactosylmelibiose

raffinose
melibiose
galactosylmelibiose
sucrose
digalactosylmelibiose

80

100

80

60

60

40

40

20

20
8

10

12

14

10

12

14

Elution time (min)

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 6galactosylmelibiose and
66digalactosylmelibiose 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 LTH5448ftfA 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 LTH2590levS 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).

90

Enzymatic processing of gluten-free flours: a promising

tool to improve their bread-making functionality?
S. Renzetti1* and E.K. Arendt2
1

TO Quality of Life, Zeist, The etherlands

Department 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 structurefunctionality 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, -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,

1
91

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

Transglutaminase
EC 2.3.2.13

Beneficial for

Buckwheat
Brown rice

Batter rheology

Bread properties

Increased
resistance to
deformation and
degree of
elasticity

Decreased specific
volume

Molecular effects

Cross-linking of
major protein
fractions

Improved crumb
texture

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 zein

Laccase
EC 1.10.3.2

Oats

Increased
deformability
and elasticity at
small and large
deformation (in
combination
with side glucanase
activity of
commercial
preparation)

increased loaf
volume and
softened crumb

Slight
polymerisation of
globulins

Increased
deformability
and elasticity

Higher specific
volume, softer
crumb

Proteinase
EC 3.4.11-19

Oats
Brown rice

Insoluble -glucan
degradation due to
side enzymatic
activity
Enhanced stability
of starch phase
Opening up of
macromolecular
protein complexes,
release of LMW
proteins

Enhanced
continuity and
stability starch
phase
In oats: insoluble 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.
2
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. PCRDGGE 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

93

spontaneous biota of GF flours will help developing novel starters which are adapted
and competitive for the production of GF sourdough bread.
L

start

12h

1d

2d

3d

5d

8d

b
c

b
c

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.

94

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 CCell 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
Maize
Quinoa
Tapioca
Rice
Rice-Tapioca-Quinoa
Tapioca-Rice-Quinoa
Rice-Maize-Quinoa
Maize-Rice-Quinoa
Rice-Tapioca-Maize-Quinoa
Rice-Maize-Tapioca-Quinoa
Tapioca-Rice-Maize-Quinoa
Rice-Tapioca-Maize-Potato-Soybean

531 42
112 32
852 76
309 26
1283 2.5
1073 92
610 28
536 72
305 12
400 11
334 38
520 57
493 69

49 2.5
51 3.1
43 2
52 3.9
40 3.3
42 0.9
45 0.9
43 1.7
44 1.2
46 1.7
45 0.9
43 2.6
48 2.3

155 4
177 2
125 4
156 6
130 4
130 0.8
154 1.4
144 3.3
137 6.1
145 2
145 2.5
113 2.5
131 2

2.6 0.1
2.7 0.1
2.4 0.2
2.4 0.2
1.9 0.2
2.1 0.1
2.2 0.2
2.1 0.1
1.9 0.1
2.3 0.1
2.2 0.2
1.9 0.1
1.9 0.1

1364 79.5
1692 41.1
1296 51.4
1719 25.5
1401 42.5
1579 74.7
1602 31.5
1583 79.2
1652 33.1
1664 3.6
1558 17.7
1644 82.5
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

95

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 p0.1), but there no significant differences in firmness and volume
between them (at p0.1). 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 p0.05) compared with control
muffin. Although a significant difference existed in the cohesiveness of mass attribute (p0.05), a
slight preference to the gluten free muffin was observed.
MUFFIN SENSORY

OVERALL RATING

CONTROL
GLUTEN FREE

AFTERTASTE
COHESIVENESS
MOUTHFEEL
MOISTNESS
CHEWINESS
FIRMNESS

10

HAND SPRINGINES

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

96

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 ookies
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

97

differ essentially, their values ranging respectively 24,328,3%, 13,717,9% 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
, , . .
.: ,1990.

98

Metabolism and competitiveness of Lactobacillus

sanfranciscensis and ting isolates in wheat and sorghum
sourdoughs
Bonno Sekwati-Monang and Michael G. Gnzle
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 34C 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 28C or 34C, 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.

99

(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. parabuchneri

160

L. coryniformis

-1

[metabolites] (mMol kg )

160

120

120

80
40
30
20
10
0

1, 3

Pro
pa
ne
d io
l

Ac
eta

La

Eth

te

e
cta
t

Pro
pa
ne
d io
l

an
ol

1, 2

Eth

Ac
eta

La

cta
t

te

40
30
20
10
0

an
ol

Sorghum flour
Sorghum flour +
maltose
Whole wheat flour

80

Figure 1. Metabolite formation during growth of L. parabuchneri and L. coryniformis after

24h fermentation at 34C in sorghum sourdoughs, sorghum sourdoughs supplemented with
2% maltose, and whole wheat sourdoughs. Data are representative for three independent
experiments.

cell counts (cfu / mL)

Whole wheat sourdough

Sorghum sourdough

109

109

108

108

107

107
0

24

48

96 0

72

24

48

72

96

fermentation time (h)

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 34C(P) or 28C(), and
back-slopped every 24h with a 10% inoculum. Backslopping is indicated by an arrow. Data
are representative for three independent experiments.

100

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.
Methods: 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 6283-484/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).

101

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
g/g, Zn and Mn content is higher in cackes with Hippophae rhamnoides extract 9,01 g/g
Zn, respectively 7,82 g/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
Moisture (%)
Lipid (%)
Protein (%)
Ash (%)
Vitamine B1 (mg/100g)
Vitamine B2 (mg/100g)

Flour

Pasta

Cackes

9,5
1,11
6,99
0,8
0,210
0,122

9,7
3,48
6,66
0,9
0,110
0,120

3,6
9,58
5,94
1,37
0,322
0,151

Table 2. Microelements composition of gluten free products

Flour
Pasta
cackes

Cu

Cr

Fe

Zn

Mn

(g/g)
2,21
3,10
1,70

(g/g)
0,19
0,37
0,25

(g/g)
16,86
21,20
15,60

(g/g)
7,70
8,75
9,01

(g/g)
6,21
2,94
7,82

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

102

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 Graudire, 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 glutenfree 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

Water (%)
HPMC (%) (Guar gum (%))

Levels

-1
85
0 (3)

0
90
1 (2)

+1
95
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).

103

Table 2. Effects of water and HPMC levels on the gluten-free bread characteristics.
Bread Characteristics

Specific volume
Crumb hardness
Gas cells heterogeneity
Crust lightness
Bread dry matter

Effects
HPMC level




Water level





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.

104

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 bodys 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 (30C) 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 25C, 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 couldnt 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 25C 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 ( 0 ) 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 25C.
K (Pa.sn)

0 (Pa)

r2

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

Chestnut to Rice Flour Ratio

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

106

Usage of Chufa Flour in Gluten-free Cakes

Elif Turabi1, Gulum Sumnu1, Serpil Sahin1, Mehmet Musa Ozcan2
1

Middle East Technical University, Department of Food Engineering, 06531, Ankara, Turkey
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 glutenfree 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).

107

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.

108

Heteropolysaccharide production from lactic acid bacteria

in wheat and sorghum sourdough
Sandra Galle1,2,3, Clarissa Schwab1, FabioDal Bello2,3, Elke Arendt2 Michael G nzle1
1

Department 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 G 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 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

109

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, G nzle MG. Sucrose utilization and impact of
sucrose on glycosyltransferase expression in Lactobacillus reuteri. Syst. Applied
Microb.2007; 30:433-443.
Tieking M, G 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.

111

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 tan (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
tan). When treated at pressures above 200 MPa however, the pressures became more and
more elastic (decreasing tan).
Table 3: Effect of pressure on rheological parameters of flour-water suspensions at = 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 glutenfree 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 (H 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.
H 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.

112

Identification of Lactic Acid Bacteria Isolated from Oat

Sourdoughs and Investigation of their Potential for the
Improvement of Oat Bread Quality
Edith K. H ttner1, Fabio Dal Bello1, Emanuele Zannini1 and Elke K. Arendt1,*
1
Department 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 37C
(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 (28C ). In
contrast, Lactobacillus coryniformis became dominant in SD 37 (37C ).
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.

113

G* (Pa)

100000

10000
0.1

10

100

Angular frequency (1/s)

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, OSullivan K, Arendt EK. Wheat sourdough
fermentation: effects of time and acidification on fundamental rheological properties.
Cereal Chem 2004; 81:409-417

114

Pilot scale manufacture of highly concentrated protein

ingredients from oats using dry fractionation technology
Juhani Sibakov1*, Olavi Myllymki1, Michael Kuhnen2, Anu Kaukovirta-Norja1,
Kaisa Poutanen1, Pekka Lehtinen1
1

VTT Technical Research Centre of Finland, Espoo, Finland

2
Hosokawa Alpine AG, Augsburg, Germany
*corresponding email: juhani.sibakov@vtt.fi

Introduction. Oats are special among the cereal grains, because they may be included in a
gluten-free diet (Sontag-Strohm et al. 2008). In the present study we investigated the
enrichment 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 from
the oat -glucan dry fractionation technology of Kaukovirta-Norja et al. (2008). The shortage
of plant-based proteins, other than soy protein, will be a challenge in the future. The oat
protein concentrate obtained could serve as a potential alternative to soy protein.
Methods. Dehulled oat grains were obtained from Raisio plc (Kokemki, Finland). Supercritical CO2-extraction was used to remove the lipids at 2 000 kg scale at NateCO2 GmbH
(Wolnzach, Germany). The extraction temperature was 40 C, 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 air
classification using 315 ATP air classifier (rotor speed 2 200 rpm). Thereafter, the highly
concentrated protein fraction was separated from endosperm flour either by pilot scale
50 ATP air classifier or by industrial scale 200 ATP-NG air classifier. Protein concentration
was calculated as N x 6.25, where nitrogen content was analyzed using a Kjeldahl
autoanalyzer.
Results. A protein-enriched fraction was separated from endosperm, and the protein
concentration varied between 23-73 %, depending on the mass yield of the protein-enriched
fraction (Table 1). The mass yield was adjusted by changing the rotor speed and air-inlet of
the air classifier. The maximum protein concentration obtained was 73 % with a mass yield of
5.0 %, calculated from the whole oat kernels. The particle size distribution of the most
enriched fraction was in the range of 0.5-10 m, with at least 95 % of the particles between
0.5-7 m (Fig. 1).
Discussion. Protein-enriched fractions would have versatile food applications. Oat proteins
are considered more nutritious, with higher content of essential amino acids, than in other
cereals, but are used less for human consumption than e.g. wheat and corn protein (Mohamed
et al. 2009). Similar protein fractions with over 70 % of protein have been previously reported
to 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-consuming
drying operations related to wet milling and obtains remarkably higher mass yield of proteinenriched fraction than what has been reported with air classification. The process presented
here produces cereal protein suitable for a wide range of foods also in a gluten-free diet.

115

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

Oat flour, with lipid removal by CO2

100
100

17
17

Protein fraction 1 (50 ATP)

Protein fraction 2 (50 ATP)
Protein fraction 3 (50 ATP)
Protein fraction 4 (50 ATP)

47
30
21
3.0

23
34
38
65

Protein fraction 5 (200 ATP-NG)

5.0

73

*) Mass yield was calculated from the whole oat kernels

Distribution density

Sum of distribution (%)

73 % protein
concentration

Particle size ( m)

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 oat
flour enabled the separation of protein-enriched fraction from oat flour. The maximal obtained
protein concentration 73 % was reached with a relatively high mass yield of 5.0 %.
References
Kaukovirta-Norja A, Myllymki O, Aro H, Hietaniemi V, Pihlava J-M. Method for
fractionating 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 in
the celiac diet. In: Arendt EK and Dal Bello F (eds.) Gluten-Free Products and
Beverages. Elsevier, Amsterdam, the Netherlands, 2008, pp. 191-202.
Wu YV, Cluskey JE, Wall JS, Inglett GE. Oats proteins concentrate from a wet milling
process: Composition and properties. Cereal Chem 1973;50:481-488.
Wu YV and Stringfellow AC. Enriched protein and -glucan fractions from high-protein oats
by air classification. Cereal Chem 1995;72:132-134.

116

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 Universitt Mnchen, 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 gasretention 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 (Schlmhle,
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 Mnchen 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).

117

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 knstlichen Teigen, Brot und
Gebck, 1954: 8

118

Exopolysaccharide forming Weissella strains as starter

cultures for sorghum and wheat sourdoughs
1

Clarissa Schwab1, Galle, Sandra1,2,3; Elke Arendt2 and Michael G nzle1

University 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 dUrfe, 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.

119

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, G nzle MG. Formation of oligosaccharides and
polysaccharides by Lactobacillus reuteri LTH5448 and Weissella cibaria 10M in
sorghum sourdoughs. Cereal Chem 2008;85:679-684

120

Influence of Beta-glucan from Different Origins on the

Quality of Gluten Free Breads
Anna-Sophie Hager1; Liam A.M Ryan1, John V. ODoherty2, Elke K. Arendt1*
1
Department of Food and Nutritional Sciences, University College Cork, Ireland
2
School 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 betaglucan, 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 TAXT2i 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
Control
Inulin
Oat beta-glucan
Yeast beta-glucan
Algal beta-glucan

Level of dietary fibre [%]

0.0
9.0
5.6
0.6
0.6

Water level [%]

90.0
83.0
132.0
92.7
91.3

Table 1. Level of water and dietary fibre addition to the gluten free batters given in % of flour/starch base.

121

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 betaglucan from algae showed a slight increase in volume.

45

crumb hardness [N]

40
35
30
25
20
15
10
5
0

day 0

day 2

day 5

Figure 1. Crumb hardness of breads enriched with -glucan from various origins over 5-days storage period:
yeast -glucan (horizontal lines bars); algal -glucan (diagonal line bars); oat -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 -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*
IPALC Research & Development Laboratories, Frigento-AV, Italy.
2
Institute of Food Sciences, National Research Council, Avellino, Italy.
*corresponding email: mrossi@isa.cnr.it
1

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- expression of intestinal gliadinspecific 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). Foodgrade microbial transglutaminase (mTG) was from Ajinomoto Foods (Hamburg, Germany;
ACTIVAWM; 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 30C;
second step, 3 hr at 30C 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: 90C, 83% relative humidity (rh) 3 hrs; 63C, 73 % rh 1 hr; 40C, 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).

123

Table 1. Gluten concentrations of final products.

untreated wheat bread (ICC137)
one-step transamidated bread (R5-ELISA)
two-step transamidated bread (R5-ELISA)

Gluten (mg/Kg)
72000 + 1000
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 twostep 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;133:780789.
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
1
Plant 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
3
Leiden University Medical Center, PO 9600, 2300 RC Leiden, The Netherlands
4
Laboratory 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 -gliadins (Gli-1) and -gliadins (Gli-3). Most
/-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-9 and Glia-20 that recognize important -gliadin epitopes.
The deletion lines were technologically tested with respect to dough mixing properties (2gmixograph), 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-9 and Glia20 (Figure 1).

125

kDa

CS 1

116.397.4-

CS 1

CS 1

HMW-GS

66.2-

-gliadins
D-type LMW-GS

45.0B-, C-LMW-GS
/-,-gliadins
31.0-

Figure 1. Analysis of Chinese Spring deletion lines. (A) SDS-PAGE gel (10%) stained with
PageBlue. (B) Immunoblot using mAb Glia-9. (C) Immunoblot using mAb Glia-20. CS is
Chinese Spring wild type.
Deletion of the short arm of chromosome 1D, containing loci encoding -gliadins, -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 -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 -gliadins. Compensation of this loss of -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
1

Edith K. H ttner1, Fabio Dal Bello1 and Elke K. Arendt1,*

Department 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 -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 wheatfree 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

-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 -amylase, -glucanase, protease or
peroxidase. However, -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.

128

Effects of Basic Process Parameters on Quality of Gluten-free

Rice Bread
Gina Jaspers, Markus J. Brandt*
Ernst Bcker 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 31C and 85% relative
humidity, bread baking at 230C 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.
100

30

20

90

10

crumb elasticity (%)

crumb hardness (N)

95

85

80

0
0

20

40

60

80

100

20

buckwheat flour (%)

100

80

60

40

rice flour (%)

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 bakers 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).

crumb hardness (N)

14
12
10
8
6
4
2
0
190

Do

ug

3,0
2,5

200

h y 210
i el
d

2,0
1,5
1,0

220

0,5
230

an]
n th
a
X
[

0,0

n
%o

r)
fl o u

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 Bread

Sheila Kenny1*; Eimear Gallagher2, Mark A.E. Auty1, Brendan T. O Kennedy1
1
Moorepark Food Research Centre, Teagasc, Fermoy, Co. Cork, Ireland
2
Ashtown Food Research Centre, Teagasc, Ashtown, Dublin 15.
*corresponding email: sheila.kenny@teagasc.ie
Introduction
Casein and gluten form gel networks with covalent (disulphide) and co-ordination (with
calcium) links, respectively. Under the correct conditions of pH and ionic strength, casein can
form aggregated casein networks (Stathopoulos and OKennedy 2008). The aim of this work
was to investigate the conditions required to produce a casein network with properties similar
to gluten in a gluten free dough system.
Methods
Acid casein was used in gluten free dough formulations to give a protein level of at a level of
8.3% (w/w). Calcium levels were 0mg, 17mg and 34mg calcium/g casein. pH levels
investigated were 5.8, 6.2, 6.6 and 7. pH and calcium level were controlled by appropriate
addition of calcium hydroxide, calcium chloride and sodium hydroxide. Standard baking tests
were performed and confocal scanning laser microscopy was used to determine the extent of
protein network formation in bread. Dynamic oscillation tests were carried out on yeastless
doughs in the temperature range 25 to 90C .
Results
Confocal scanning laser micrographs showed no visible protein network in formulations with
0mg calcium/g casein (Fig 1a). In contrast, good protein network formation was visible with
17mg calcium/g casein and a dough pH of 6.2 (Fig 1b). The elastic modulus, G', of the 17mg
calcium pH 6.2 formulation increased in the range from 35 to 55C, indicating the presence of
a 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 range
from 35 to 55C, indicating a weakening of the protein network on heating. Formulations with
0mg calcium had lower G' than 17mg calcium formulations and G' increased on heating. 0mg
calcium doughs were sticky and difficult to handle whereas formulations with 17mg
Calcium/g casein were most cohesive.
Figure 1.
Confocal scanning laser micrographs (a) 0mg calcium/g casein, pH 6.2. (b) 17mg calcium/g
casein, pH 6.2.
(b)

(a)
Starch

Protein
network

131

Figure 2.
Elastic Modulus (G') profiles of gluten free dough formulations
1.00E+07
0mg calcium, pH 6.2
17mg calcium, pH 5.8
17mg calcium, pH 6.2
34mg calcium, pH 5.8

Elastic Modulus G' (Pa)

34mg calcium, pH 6.2

1.00E+06

1.00E+05

1.00E+04
25

35

45

55

65

75

85

Temperature (C)

Conclusions
Under optimum conditions of pH and calcium concentration, casein aggregates and forms a
protein network capable of retaining gas in gluten free dough. The formulation with 17mg
calcium and pH 6.2 had an extensive protein network and dynamic oscillation testing
indicated strengthening of this network on heating. Decreases in G' observed by reducing the
pH to 5.8 and increasing calcium level to 34mg calcium/g casein indicate that these
conditions result over-aggregation of casein and a reduction in gas retention properties of the
casein network. The G' profile of the 0mg calcium formulation indicates that sodium caseinate
acts as a stabiliser in gluten free dough.

References
Stathopoulos, C.E. and O'Kennedy, B.T. A rheological evaluation of concentrated casein
systems as replacement for gluten: calcium effects. International Journal of Dairy Technology
2008; 61: 397-402
Stathopoulos, C.E. and O'Kennedy, B.T. The effect of salt on the rheology and texture of a
casein based ingredient intended to replace gluten. International Journal of Dairy Technology
2008; 63 430-433

132

Effect of microbial homopolysaccharides on the structure

of gluten-free breads
1

Christine Rhmkorf1, Susanne Kaditzky1* and Rudi F. Vogel1

Technische Universitt Mnchen, Lehrstuhl fr 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 nondairy food systems. In: Ullrich, M. (ed.) Bacterial polysaccharides current innovations
and future trends. 313-344.

133

Notes
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________

134

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

135

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, %

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 products 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 i a produselor lactate. Lucrri de laborator i practice. Chiinu.
Civitas. 1998.

136

Development of a new gluten-free brown bread flour mix

rich in fiber and high in nutritional content
Virna Cerne and Ombretta Polenghi
Schr 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
doughs 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.

137

Notes
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________

138

Sensory and textural properties of gluten-free bread based

on rice/buckwheat flour mixtures
Aleksandra Torbica1*; Miroslav Hadnaev1, Tamara Dapevi1, Marijana Saka1
1
Institute 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 (P0.05) 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

139

Table 1. Textural properties of final gluten-free products containing husked buckwheat (HF) or
unhusked buckwheat (UBF) flour A
Type of buckwheat
flour
HBF
Area (g.sec)
UBF

10%

20%

30%

3261190a
4121348ab

4266297ab
4706403bc

4439247abc
5597823c

Values 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 cerealbased products. Trends Food Sci Tech 2004;15:143 152.
Sciarini SL, Ribotta DP, Le n EA, Prez TG. Influence of Gluten-free Flours and their Mixtures
on Batter Properties and Bread Quality. Food Bioprocess Tech 2008; DOI 10.1007/s11947-0080098-2 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.

140

The positive effect of amaranth sourdough addition in

gluten free bread quality
Andreas Houben1*; Martin Mitzscherling2, Thomas Becker1
TU Mnchen, 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
1

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 m. 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 30C 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.
1
141

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.; Mki, M.: Gluten-free
diet and qualit of life in patients with screen-detected celiac disease. Effective Clin.
Practise 2002; 5; 105-113
2
142

Discrimination between gluten-free bread formulations

using near infrared imaging
Gerard Downey1,2*; Carlos Esquerre1,2, Eimear Gallagher1, Laura Alvarez1, Colm
ODonnell2, 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

143

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., ODonnell, 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.

144

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 glutenfree 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 Ustuki
Malnas (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

145

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.
Dough
sample
Control
Gluten free

Total sugar
content, %
0.45 0.03
0.21 0.03

Total protein
content, %
8.98 0.2
8.90 0.2

Total fat
content, %
0.21 0.01
0.23 0.01

Color differences
L*
a*
76.10 3.49
-0.83 0.73
64.941.33
0.54 0.41

b*
15.51 1.91
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 glutenfree 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, Sch 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, Betr n J, Runge ECA, Corn. Origin, history, technology, and production. Wiley;
2004. p. 867-870.

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, 416419.
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, 594601.

147

Figure 1. Free gluten pocket type flat Arabic bread

148

A Gluten-Free Bread with Viscous Japanese Yam Instead

of Wheat Flour
Masaharu Seguchi
Kobe Womens University, Kobe, Japan
*corresponding email: seguchi@suma.kobe-wu.ac.jp

Introduction. We newly designed a glutenfree 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-BuOHwater=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

149

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.

150

Effect of Legume Flours on

Baking Characteristics of Gluten Free Bread
B. Miarro, E. Albanell, M.Capellas
Centre Especial de Recerca Planta de Tecnologia dels Aliments (CERPTA), CeRTA, XiT,
Departament de Cincia Animal i dels Aliments. Edifici V - Campus UAB
Universitat Autnoma 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 10.
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

151

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.

152

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 pretreated 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).

153

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.

100

Degree of gelatinization
(%)

Degree of gelatinization
(%)

100
80
60
40
20
0
0

200

400

600

Pressure (MPa)

80
60
40
20
0
0

20

40

60

80

Temperature (oC)

B
A
Figure 1. Pressure (A) and temperature (B) dependence of the buckwheat starch degree of
gelatinisation (as determined by DSC after treatment for 10 min).

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.

154

Rheological properties and bread making performance of

commercial wholegrain oat flours
1

Edith K. H ttner1, Fabio Dal Bello1 and Elke K. Arendt1,*

Department 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 -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.

155

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 -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

-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.

156

Gluten-Free Bread Supplemented with Calcium

the Improvement of Quality and Texture Properties
Urszula Krupa-Kozak, Magorzata Wronkowska, Maria Soral-mietana, Agnieszka
Troszyska, 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 glutenfree 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 semiconsumer 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-

157

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.
6

Overall quality (arbitrary units)

b
5

ab

ab

2% CaCA

2% CaCI

ab

ab

a
3

0
Control

1% CaCA/1% CaCI

1.3% CaCA/0.7% CaCI 0.7% CaCA/1.3% CaCI

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
a
c
2%CaCA
21,99 2,99
7,93 2,42
1,068a 0,39
bcd
bc
2%CaCI
16,16 2,25
8,39 2,39
0,765bc 0,26
cd
bc
1%CaCA/1%CaCI
15,72 2,28
8,42 2,60
0,784abc 0,20
1.3%CaCA/0.7%CaCI
17,04bc 2,32
10,50b 2,21
1,022ab 0,29
b
c
0.7%CaCA/1.3%CaCI
18,39 2,84
6,65 1,43
0,724c0,13
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-mietana M. (2008). Mieszanka bezglutenowa (eng.
Gluten-free formula). Polish Patent Specification No P 386253 (in Polish).

158

Gluten-free Pasta: Technology and Quality Evaluation

Manuela Mariotti, Carola Cappa, Mara Lucisano
Universit 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-ViscoAmylograph, 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,

159

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
(%)
R1
100.0
R2
112.5
R3
162.5
R4
112.2
C1
125.0
C2
117.1
C3
117.1
CS1
122.0
CS2
107.3
CS3
142.5
CS4
125.0
M1
134.1
M2
122.0
M4
120.0

Diameter increase
(%)
44.2 8.2
45.3 7.9
41.0 7.6
41.5 4.5
52.6 4.5
39.8 2.9
51.2 4.7
47.0 5.2
42.8 4.4
54.3 5.3
47.7 5.1
61.5 4.7
39.2 3.2
53.0 4.6

Cooking loss
(g/100g d.b.)
12.4 0.1
8.5 0.1
3.2 0.2
5.6 0.3
6.2 0.1
5.3 0.2
5.0 0.1
5.1 0.1
6.8 0.1
3.9 0.1
6.7 0.1
5.1 0.1
7.5 0.1
2.5 0.1

Young Modulus*
(N/mm2)
0.39 0.02
0.37 0.01
0.26 0.01
0.36 0.01
0.36 0.01
0.34 0.01
0.34 0.01
0.28 0.01
0.33 0.01
0.27 0.01
0.31 0.01
0.31 0.01
0.36 0.001
0.30 0.01

Adhesiveness*
(10-3J)
5.21 0.72
2.47 0.48
0.67 0.05
2.65 0.60
0.90 0.10
1.48 0.13
1.01 0.16
1.99 0.69
1.09 0.17
3.82 0.18
1.38 0.26
1.89 0.25
1.34 0.07
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.
13

12

R4

11

C1

9
8

C3

7
6

R3

CS3

CS1
M1

11
10

J (MPa-1 )

J (MPa-1 )

10

12

C2-R2
R1

M4

M2

8
7

CS4

CS2

4
0

20

40

60

80

100

120

140

time (s)

20

40

60

80

100

120

140

time (s)

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.

160

Proso Millet (Panicum miliaceum L.) a Sustainable Raw

Material for the Malting and Brewing Process
1

Martin Zarnkow1, 2*; Thomas Becker1, Elke K. Arendt2

Technische Universitt Mnchen, Lehrstuhl fr Brau- und Getrnketechnologie, 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
22C 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 -amylase activity, and 105
U/g -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.
-Amylase showed its highest activity in proso millet malt wort at a temperature of 60C and
at a pH of 5.0, whereas -amylase exhibited an optimum at 40C and at a pH of 5.3. The limit
dextrinase activity reached its maximum at 50C 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

161

the fermentation trials, based on the pH reduction, increase in alcohol content and sugar
conversion, conducted at 18C, 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
18C), but it was not possible to identify proso millet specific flavour compounds.
80
70

mash 1 (40 %)

pH 5.0

temperature [C]

60
50

pH 5.3
40
30

mash 2 (60 %)

20
10
0
0

20

40

60

80

100

120

time [minutes]

Figure 1: Optimal mashing program with optimal temperatures and pH adjusting with respect
to -amylase, -amylase and limit dextrinase of proso millet malt.
Table 1: Obtained attributes of aliphatic and aromatic alcohols, esters and fatty acids after
proso millet wort fermentation using different yeast strains.
[g/L] at 12C in proso
millet malt beer
Sum of aliphatic alcohols
Sum of aromatic alcohols
Sum of esters
[g/L] at 18C proso
millet malt beer
Sum of aliphatic alcohols
Sum of aromatic alcohols
Sum of esters

S 208
150
8053
1217

S 250
109
8467
402

S 262
23
6591
2543

S 290
145
4946
497

B 20
70
3840
188

barley malt lager

45120
1006930461
2101000

S 208
158
11844
1284

S 250 S 262
78
61
10653 2437
395
1068

S 290
267
8569
580

B 20
137
7685
1149

barley malt lager

45120
1006930461
2101000

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. Pyri*4, M. Mki1, A. Hernando2, M. Mena2, M. Lombardia2, P. Lehtonen3,
P. Soininen-Tengvall4, E. Pajunen4, E. Mendez2
1
University of Tampere, Tampere, Finland
2
Unidad de gluten, Centro Nacional de Biotecnologia, Madrid, Spain
3
Alko Oy, Vantaa, Finland
4
Oy 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 R5ELISA 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 WayMaren Wiese
Hermann Krner GmbH, Ibbenbren (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 KRNER-STRKE
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 CodexAlimentarius.
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 KRNERSTRKE 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 glutenfree 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 glutencontaining 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 regulargrade 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

Smooth
Smooth
Smooth
Smooth
Smooth

Gluten-free
muffins
are
Discussion:
generally perceived as drier and grainier
versus their gluten-containing counterparts.
M
M
M
M
C
C
M
C
M
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
Figure 1. Sensory differentiation
differentiation in
in gluten-free
gluten-free muffin
muffin formulations.
formulations.
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.
111
1

222
2

Dry
Dry
Dry
Dry
Crumby
Crumbly
Crumby
Crumbly
Crumby
Crumbly
Crumby

Moist
Moist
Moist
Moist
Chewy
Chewy
Chewy
Chewy

22
2
22

M
:: NSFI
M
NSFI Gluten-free
Gluten-free prototype
prototype
M
M1111:: NSFI
NSFI Gluten-free
Gluten-free prototype
prototype
M
:: NSFI
NSFI Wholegrain
Wholegrain Gluten-free
Gluten-free prototype
prototype
M
M
NSFI Wholegrain
Wholegrain Gluten-free
Gluten-free prototype
prototype
M222:: NSFI

Grainy
Grainy
Grainy
Grainy
Grainy

Commercial
gluten-containing
Commercial
gluten-containing muffin
muffin
Commercial
Commercial gluten-containing
gluten-containing muffin
muffin
Commercial
gluten-free
Commercial
gluten-free muffin
muffin
Commercial
Commercial gluten-free
gluten-free muffin
muffin

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).
35.0
% Fibre Content
30.0
25.0
20.0
15.0
10.0
5.0
0.0
Amaranth

Buckw heat

Millet

Brow n Rice

Quinoa

Sorghum

Teff

Wild Rice

Hi-maize Whole
Grain Corn flour

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.

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 VBNs 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(%)

4.1

TDF(%)

1.7

Fat(%)

0.3

3.8

Iron(mg/100)

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 glutenfree breads with desirable nutritional and sensory quality, which has opened up the
opportunities to meet the customer needs and strengthen the companys 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 Bcker 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 % bakers 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.

170

Changes in bio-active compounds in buckwheat depending

on germination conditions
Florian H 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 embryos 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
% dm

Insoluble
Dietary Fibre
% dm

Total Dietary
Fibre
% dm

22.2

Antioxidant
power
mg gallic acid/
g malt
3.55

Phenolic
compounds
mmol trolox
equ. / g malt
1.56

unmalted

2.3

19.9

Maximum
value
Minimum
value

3.9

26.4

30.3

3.84

2.12

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 H 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.

173

350

~ 22 kDa
45 kDa

60kDa

300

14 17kDa

System peak

Fluorescence Units

250

70-80kDa

5.5-9 kDa
31 41kDa

200

50-55kDa

marker
150

100

marker
50

0
10

15

20

25

30

35

40

45

50

-50

Elution time / s
48 h 10 C

48 h 20 C

144 h 10 C

144 h 20 C

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*
1

Department 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

pH
76 kDa

pH 10

pH 10

43 kDa
36 kDa
31 kDa
22 kDa

pH

76 kDa

43 kDa
36 kDa
31 kDa
22 kDa

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.
176

Optimization of rheological properties of gluten-free

pasta using mixture design
V. Larrosaa, G. Lorenzo a,b, N. Zaritzky a,b, A. Califano a, *
Centro de Investigacin y Desarrollo en Criotecnologa de Alimentos (CIDCA),
Facultad de Cs. Exactas, UNLP-CONICET. 47 y 116, La Plata (1900), Argentina.
b
rea Departamental Ingeniera Qumica, Facultad de Ingeniera, UNLP, Argentina.
*corresponding email: anc@quimica.unlp.edu.ar
a

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.
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 4C, 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 (noncooked) 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
Water (g/g)
probe (2.5cm dia) and a pastry burst rig (TA 108)
Figure 1. Simplex-centroid
determining the breaking force (N), distance (mm).
augmented design; mass
Oscillatory shear tests (storage (G) and loss (G) moduli
fractions are expressed as
vs. frequency, ()) were performed in duplicates in a
coded variables.
0.0
0

0.0

n
Xa

n(

Pro
te i

0 .0

m
gu

an

s(

0.85

0.0

)
g/g

0.80

be

0 .1
5

0 .0

0.2

t
us

l oc

0 .1
0

n+

g/g
)

th a

0.0

0 .0
5

0.90

0.0

0.95

1.00

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
(R2force =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
Figure 2. Response surface of the
breaking force as a function of dough
solver
composition shown as coded variables.
module.
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
G G (| z) formulation 5; 35.5% water
G G ( ) formulation 9; 37.5% water
greater than G in the frequency range
G G (V T) formulation 6; 39.5% water
solid lines: Maxwell generalized model
measured and the increase of the two moduli
with frequency was small. Oscillatory spectra
were satisfactorily modeled using the
Figure 3. Mechanical spectra (G)
Maxwell Generalized model as shown in
and G vs. frequency () for
Figure 3 (Ferry 1980).
formulations containing 2.5% gums.
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;48:224231.
Zhan DF, Ridout MJ, Brownsey GJ, Morris VJ. Xanthan-locust bean gum interactions
and gelation. Carbohydrate Polym 21 (1993) 53-58.
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

2
Dipartimento 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 worlds 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 Fcontaining 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, DEgidio 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: 42584267.

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 80s 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 90s, 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

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 dItalia, Marzo 2007
5. Non-Traditional Pasta, Luisito Virtucio, Tecnologie Alimentari, Novembre-Dicembre 2004

182

Notes
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183

Notes
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184

Starch Characterization of Rice Pasta: Comparison

between Extrusion-cooking and Conventional PastaMaking 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
2
University 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 -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. Sjgren, 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

17

wheat

44

whea t, ba rley, rye

Oatmeal, organic

127

wheat, barley, rye

1403

whea t, ba rley, rye

Oatmeal

1922

whea t, ba rley

n.t.

Oatmeal

46

whea t, ba rley

470

wheat

Oatmeal

11

wheat, barley #

1105

rye

Oatmeal, organic

n.d.

n.d.

n.d.

whea t, barley

Oatmeal

368

ba rley

42

barley, wheat#

Oatmeal with extra fiber

260

rye, barley

129

whea t, ba rley, rye

Oatmeal glutenfree

n.d.

n.d.

n.t.

Oatmeal

wheat, barley #

13

Oatmeal quick

n.d.

n.d.

n.t.

Oatmeal

n.d.

rye

n.t.

Oatmeal a nd quinoa , organic

wheat, barley #

n.d.

whea t, ba rley, rye

Oatgruel
(a utoclaved oat)

65

n.d.

47

n.d.

ba rely

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 oatbased 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 gluten

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
Sweden is one
of the countries with the longest experience of wheat starch based gluten free bread
bread, and today
about 90 % of all gluten free bread in Sweden is based on wheat starch.
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,
flour compared to bread baked from
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 adhere
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
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
suitable in the gluten free diet.

191

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Fria has been baking tasty and nutritious gluten-free bread since 1996, and weve had lots of positive feedback from customers all
over Europe about the quality of our great tasting products. We believe that gluten- and milk-free bread can taste just as good as
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you wont be disappointed! You can find Fria bread in the freezer section of most supermarkets. All our bread is gluten-free
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For more information, visit www.fria.se

192
www.fria.se +46 31-734 13 30 info@fria.se

Oats in Gluten-free Diet

Considerations of a Pragmatic Policy
Hannu Salovaara, Pivi 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
Codex Stand 118-1981,
amended 1983)
Draft revised standard
for GF foods (2007)
EU regulation
(EC) (41/2009)

Definition/statement/comment
a protein fraction from wheat, rye, barley, [oats] or their crossbred varieties/
the square brackets indicating the uncertainty felt about the position of oats
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.
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 200300
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
% Prolamins
% Q (glutamine)
% P (prolin)

Wheat
7080
33
17

Rye
3050
6(30)
17

Barley
3050
3(30)
20

193

Oat
10
34
10

Rice
35
20
5

Maize
60
19
10

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 1990s 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 19531974
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 Canadas (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 100200 ppm.

194

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 (Peraho 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
(Peraho 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

195

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 ones 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 Societys website
www.keliakialiitto.fi/liitto/in_english.
References
Peraho M, Collin P, Kaukinen K, Kekkonen L, Miettinen S, Mki M. Oats forms an essential
part of gluten-free diet in celiac disease and dermatitis herpetiformis. J Am Diet Assoc
2004;104:1148-50.

196

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)

197

Notes
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________

198

Quo Vadis, Coeliac Disease?

Markku Mki
Coeliac Disease Study Group, University of Tampere, Finland
corresponding email: Markku.Maki@uta.fi

199

Notes
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________
_______________________________________________________________________________

200

Authors Index
Ainsworth, Paul
Akobeng, Anthony
Al-Dmoor, Hanee
Albar, Juan Pablo
Alexa, Ersilia
Alho-Lehto, Pirjo
Allred, Laura
Alminger, Marie
Anderson, Robert
Andlid, Thomas
Arendt, Elke K.

Arnala, Sanna
Atwell, William A.
Bansil, Rama
Barrett, Jacqueline
Barro, Francisco
Becker, Thomas
Bednarski, Wlodzimierz
Beitnes, Ann-Christin R.
Ben-Fayed, Ebtesam
Berghofer, Emmerich
Biesiekierski, Jessica
Bonomi, Francesco
Bosch, Dirk
Bottega, Gabriella
Brandt, Markus
Bratlie, Jorunn
Brennan, Charles
Brinck, Outi
Brottveit, Margit
Bruins, Maaike
Brzozowski, Bartosz
Califano, Alicia
Capobianco, Federica
Cappa, Carola
Caramanico, Rosita
Casiraghi, Maria Cristina
Cerne, Virna Lucia
Chen, Tingsu
Chevallier, Sylvie
Comino, Isabel
Dal Bello, Fabio
Dar, Yadunandan
De Angelis, Maria
De Jong, Hein
De Lamballerie, Marie
Dekking, Liesbeth
Demirkesen, Ilkem
Di cagno, Raffaella
Doecke, James
Downey, Gerard
Edens, Luppo
Elkhalifa, Abd Elmoneim
Esteban, Blanca

61, 167
57
147
35
101
197
33
71
13
71
85, 91, 93, 109, 111,
113, 119, 121, 127,
153, 155, 161, 171,
173, 175
195
87
63
7
11
141, 161
73
5
61
47
7
179
125
179
129, 169
5
95
37, 81
5
79
73
177
123
159
185
179
137
71
103
31
93, 109, 113, 127, 155
165
77
125
103
125
105
77
7
143
79
179
51

201

Eurola, Merja
Ferm, Monica
Fielder, Richard
Fjodorova, Anastasija
Francavilla, Ruggero
Freitag, Tobias
Gagliardi, Francesca
Gallagher, Eimear
Galle, Sandra
Garca-Horsman, J. Arturo
Germ, Mateja

53
187
21
145
77
27
77
49
109, 119
67, 83
59

Gibson, Peter
Gil-Humanes, Javier
Gilissen, Ludovicus J.W.J.
Gilissen, Luud
Gobbetti, Marco
Gramatina, Ilze
Ganzle, Michael
Haas-Lauterbach, Sigrid
Haase, Stephan
Hadnadev, Miroslav
Hager, Anna-Sophie
Haines, Melissa
Halbmayr, Elisabeth
Hamer, Rob
Hernando, Alberto
Hietala, Sami
Hoffmann, Karolina
Houben, Andreas
Hubner, Florian
Huttner, Edith K.
Iametti, Stefania
Immer, Ulrike
Ioannides, Despina
Irving, Peter
Jahnsen, Frode L.
Jaspers, Gina
Jiang, Zhongqing
Junker, Yvonne
Kaditzky, Susanne
Kanerva, Paivi
Kariluoto, Susanna
Kaukinen, Katri
Kaukovirta-Norja, Anu
Kauwe, Andrea de
Kenny, Sheila
Kinni, Maija
Kirk, Emily
Kivela, Reetta
Klose, Christina
Koehler, Peter
Koning, Frits
Kooy-Winkelaar, Yvonne
Koskinen, Lotta

7
11
55, 125
23
25, 77
145
69, 89, 99, 109, 119
19
117
139
121
7
21
125
35, 163
63
71
141
171, 173
113, 127, 155
179
19
165
7
5
129, 169
81
27
133
15, 29, 37, 39, 69, 193
45
13, 190
115
13
131
45
57
63
175
3
17, 65, 79
79
13

Kreft, Ivan
Krupa-Kozak, Ursula
Kruse, Birgitta
Kuhnen, Michael
Kurppa, Kalle
Laivisto, Emma
Lanzini, Alberto
Larrosa, Virginia
Le Bail, Alain
Lehtinen, Pekka
Lehtonen, Pekka
Li, Weili
Lidia, Cosciug
Lombardia, Manuel
Lombarda, Manuel
Londono, Diana
Loponen, Jussi

Perez, Alejandro
Petroboni, Beatrice
Pihlava, Juha-Matti
Piironen, Vieno
Piston, Fernando
Pogiatzis, Marios
Pogna, Norberto
Poutanen, Kaisa
Poyri, Saara
Rakcejeva, Tatjana
Raki, Melinda
Ramsden, Russell
Renzetti, Stefano
Rizzello, Carlo Giuseppe
Robins, Gerry
Rossi, Mauro
Ryan, Liam A. M.

165
9
53
45
11
95
9
115
163
145
5
95
91
77
57
123
111, 121, 153

Lorenzo, Gabriel
Lucisano, Mara
Luis, Virtucio
Lundin, Knut E. A.
Malmheden Yman, Ingrid
Mariotti, Manuela
Marquez, Manuela
Marti, Alessandra

59
157
187
115
13
83
9
177
103
115
163
95
97
163
35
8
15, 29, 39, 45, 69, 81,
83, 193
177
159
181
5
187
159
51
185

Ruhmkorf, Christine
Saastamoinen, Marketta
Saavalainen, Paivi
Sadowska, Jadwiga
Sahin, Serpil
Sakac, Marijana
Salentijn, Elma
Salovaara, Hannu

McGough, Norma
McNeill, Victoria
Meijer, Gerrit
Mena, Maria Carmen
Mendez, E.
Meri, Seppo
Merrikin, Emma
Mert, Behic
Mes, Jurriaan J.
Mezaize, Sandra
Mikola, Markku
Minarro, Begona
Mitzscherling, Martin
Moroni, Alice V.
Moscaritolo, Salvatore
Mossberg, Nanna
Muir, Jane
Mujico Fernandez, Jorge Raul

57
89
79
35, 163
163
27
57
105
55
103
43
151
141
93
123
191
7
17

Sandberg, Ann-Sofie
Sandberg, Martin
Schieber, Andreas
Schoenlechner, Regine
Schreurs, Marco
Schuppan, Detlef
Schwab, Clarissa
Seetharaman, Koushik
Seguchi, Masaharu
Sekwati-Monang, Bonno
Serrano-Vela, Juan Ignacio
Shepherd, Susan
Sibakov, Juhani
Sjogren, Ylva
Smulders, Marinus J.M.
Soininen-Tengvall, P.
Sollid, Ludvig M.
Sontag-Strohm, Tuula

Mulder, Chris
Myllymaki, Olavi
Maki, Markku
Newnham, Evan
Not, Tarciso
Nurttila, Annika
Olsson, Olof
Ozcan, Mehmet Musa
ODoherty, John V.
Pagani, Maria Ambrogina
Pajunen, E.

79
115
1, 13, 163, 199
7
9
189
71
107
121
179, 185
163

Soral-Smietana, Maria
Stenman, Satumarja
Stewart, Jessica
Stojceska, Valentina
Sumnu, Gulum
Svensson, Louise
Tack, Greetje
Teixeira, Januana
Thompson, Tricia
Tollefsen, Stig
Torbica, Aleksandra

133
53
13, 27
157
105, 107
139
23
15, 29, 37, 39, 69, 81,
83, 193
71
187
89
47
79
27
109, 119
185
149
99
51
7
115
187
23, 55, 125
163
5
15, 29, 37, 39, 63, 69,
193
157
75
13
61, 167
105, 107
89
79
89
41
5
139

202

Troszynska, Agnieszka
Turabi, Elif
Turner, Bradley
Tye-din, Jason
Vallons, Katleen. J. R.
Van Bergen, Jeroen
Van de Water, Jolanda
Van den Broeck, Hetty C.
Van der Meer, Ingrid M.
Viorica, Bulgaru
Vogel, Rudi
von Blomberg, Mary
Wiese, Maren
Wieser, Herbert
Wilhelmson, Annika
Wilpola, Arvi
Wrang, Esa
Wroblewska, Barbara
Wronkowska, Malgorzata
Zanini, Barbara
Zannini, Emanuele
Zaritzky, Noem
Zarnkow, Martin

157
107
63
13
111, 153
79
79
23, 55, 125
23, 55, 125
135
133
79
164
3
83
83
43
73
157
9
113
177
161

203

204