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8th GLUTEN WORKSHOP Program and abstracts 8-10 September 2003 Viterbo (Italy) Sponsors of the 8th Gluten Workshop Comune di Viterbo Provincia di Viterbo Assoseme Consorzio” Gian Pietro Ballatore” SOCIAL PROGRAM SUNDAY, SEPTEMBER 7, 2003 20.00 Dinner at the Restaurant “L’antico casale”, San Martino al Cimino, Viterbo MONDAY, SEPTEMBER 8, 2003 20.00-21.15 21.30-22.30 Dinner at the Balletti Park Hotel, San Martino al Cimino, Viterbo Concert, Abbey of San Martino al Cimino, Viterbo TUESDAY, SEPTEMBER 9, 2003 17.55-20.00 20.00 Visit to Viterbo (starting at the Congress Venue) Social dinner at the Restaurant “Parco dei Cimini”, Soriano al Cimino, Viterbo 8th “Gluten Workshop”, Viterbo 8-10 settembre 2003 SCIENTIFIC PROGRAM SUNDAY, SEPTEMBER 7, 2003 19.00-20.00 Registration at the Restaurant “L’antico casale”, San Martino al Cimino, Viterbo MONDAY, SEPTEMBER 8, 2003 8.00-9.00 09.00-09.20 Registration at the Congress Venue and poster set up Opening Session 1: Biotechnology, Transcriptomics and Proteomics (Chairpersons O.A Anderson and P.R Shewry) 9.20-9.40 P.R Shewry, Harpenden, UK The use of biotechnology to study wheat endosperm development and improve end use quality A.E Blechl, P Bregitzer, K O’Brien, J Lin, S Nguyen, O.D Anderson, Albany, CA, USA Agronomic, biochemical and quality characteristics of wheats containing HMWglutenin transgenes S Masci, R D’Ovidio, F Scossa, C Patacchini, D Lafiandra, O.D Anderson, A.E Blechl, Viterbo, Italy Characterization of glutenin polymers in a transgenic bread wheat line overexpressing a LMW-GS H.D Jones, G Pastori, J Goodwin, P.R Shewry, Harpenden, UK The potential of biotechnology to produce novel gluten phenotypes 9.40-10.00 10.00-10.20 10.20-10.40 10.40-11.10 Coffee break 11.10-11.30 12.50-13.05 O.D Anderson, D Laudencia-Chinguanco, S Chao, G Lazo, Albany, CA, USA The use of ESTs to analyze the spectrum of wheat seed proteins P Tosi, R D’Ovidio, F Békés, J.A Napier, P.R Shewry, Harpenden, UK Transgenic expression of epitope-tagged LMW glutenin subunits in durum wheat: effect on polymer size distribution and dough mixing properties P Della Cristina, A Orsi, F Sparvoli, A Ceriotti, Milan, Italy Folding and assembly of a low molecular weight glutenin subunit G Branlard, J Dumur, E Bancel, M Merlino, M Dardevet, Clermont-Ferrand, France Proteomic analysis of wheat storage proteins: a promising approach to understand the genetic and molecular bases of gluten components V Cunsolo, S Foti, V Muccilli, R Saletti, D Lafiandra, S Masci, Catania, Italy Mass spectrometric approaches for the characterization of high- and low-molecular weight glutenin subunits General discussion 13.05-14.30 Lunch 11.30-11.50 11.50-12.10 12.10-12.30 12.30-12.50 8th “Gluten Workshop”, Viterbo 8-10 settembre 2003 Session 2: Environmental effects (Chaipersons G Branlard and W.J Hurkman) 14.30-14.50 W.J Hurkman, C.K Tanaka, W.H Vensel, J.H Wong, Y Balmer, B.B Buchanan, Albany, CA, USA Analysis of wheat endosperm proteins during grain development and in response to high temperature using proteomics T Majoul, E Bancel, E Triboi, J Ben Hamida, G Branlard, Clermont-Ferrand, France Analysis of the effect of heat stress on wheat storage proteins using the proteomic approach C Don, G Lookhart, A.H Naeem, F MacRitchie, R.J Hamer, Wageningen, the Netherlands Glutenin particles are affected by growing conditions E Johansson, M.L Prieto-Linde, R Kuktaite, A Andersson, H Larsson, Alnarp, Sweden Grain protein polymer formation: influences of cultivar, environment and dough treatment 14.50-15.10 15.10-15.30 15.30-15.50 15.50-16.20 Coffee break 16.20-16.40 17.00-17.15 J.M Carrillo, M Ruiz, M.C Martinez, M Rodrìguez-Quijano, J.F Vàzquez, Madrid, Spain Relationship between some prolamin variants and quality parameters at different levels of nitrogenous fertilizer in durum wheat P Koehler, H Wieser, R Gutser, S von Tucher, Garching, Germany Influence of sulphur fertilisation on the quantitative composition of gluten protein types in wheat flour General discussion 17.15-18.30 Poster session 16.40-17.00 TUESDAY, SEPTEMBER 9, 2003 Session 3: Gluten rheology and functionality (Chairpersons P Belton and F MacRitchie) 8.15-8.35 P Belton, Norwich, UK What makes a good theory of gluten viscoelasticity? J Lefebvre, C Rousseau, Y Popineau, Nantes, France Viscoelastic and flow behaviour of doughs from transgenic wheat lines differing in HMW glutenin subunits E.L Sliwinski, P Kolster, T van Vliet, Wagenigen, the Netherlands Large deformation properties of wheat flour and gluten dough in uni- and biaxial extension G Mann, F Békés, M Morell, Canberra, Australia Extensional rheology measurements as predictors of wheat quality R Haraszi, F Békés, M.L Bason, J.M.C Dang, J.L Blakeney, Canberra, Australia Dough mixing studies on the micro Z-arm mixer 8.35-8.55 8.55-9.15 9.15-9.35 9.35-9.55 9.55-10.15 C Don, J Plijter, W Lichtendonk, R.J Hamer, Zeist, the Netherlands Aggregation kinetics of GMP particles affects dough rheology 10.15-10.45 Coffee break 10.45-11.05 12.45-13.00 F MacRitchie, H Singh, Manhattan, KS, USA Polymer concepts applied to gluten behaviour in dough B.J Dobraszczyk, J.D Schofield, J Smewing, M Albertini, G Maesmans, Reading, UK Rheological mechanisms of stability of bubble expansion in breadmaking doughs R Kieffer, H Wieser, Garching, Germany Effect of temperature and high-pressure on the functional and chemical properties of gluten M.I.P Kovacs, B.X Fu, S.M Woods, C Wang, Winnipeg, Manitoba, Canada Carbon atom and the thermal stability of wheat gluten proteins: effect on dough properties and noodle texture M Pommet, A Redl, S Domenek, S Guilbert, M.H Morel, Montpellier, France Why dough inflation should have to modify the rate of gluten protein thermosetting H Larsson, H Hedlund, Lund, Sweden On the swelling properties of gluten in the presence of salts and reduced pH General discussion 13.00-14.30 Lunch Session 4: Genetics and quality (Chaipersons J.M Carrillo and D Lafiandra) 14.30-14.50 M.C Gianibelli, O.R Larroque, E De Ambrogio, D Lafiandra, Canberra, Australia Effect of the introduction of novel HMW-GS on quality of durum wheat semolina B.J Butow, K.R Gale, W Ma, O Larroque, M.K Morell, F Békés, Canberra, Australia Use of segregating double haploid populations to investigate the effects of Glu-B1 and Glu-D1 alleles on dough strength A Juhàsz, O Larroque, H Allen, J Oliver, M.C Gianibelli, Canberra, Australia Biochemical and molecular characterisation of gluten-forming proteins in a double haploid population of bread wheat and their relation to dough extensibility H Nakamura, Tsukuba, Japan The transmission route through which the common wheat (Triticum aestivum L.) has reached Far-East Japan 11.05-11.25 11.25-11.45 11.45-12.05 12.05-12.25 12.25-12.45 14.50-15.10 15.10-15.30 15.30-15.50 15.50-16.20 Coffee break 16.20-16.40 L Li, H Zhonghu, R.J Pena, Beijing, China Presence of 1B/1R, Glu-1 and Glu-3 and their effect on breadmaking quality in Chinese bread wheat T.M Ikeda, T Nagamine, H Yano, Fukuyama, Japan Characterization of LMW-GS genes and the corresponding proteins in common wheats 16.40-17.00 8th “Gluten Workshop”, Viterbo 8-10 settembre 2003 17.00-17.20 17.20-17.40 17.40-17.55 M.T Labuschagne, E Koen, T Dessalegn, Bloemfontein, South Africa The use of SE-HPLC for wheat quality prediction in two African countries O.R Larroque, M.C Gianibelli, F Bekes, P Sharp, D Lafiandra, Canberra, Australia Biochemical and functional studies of wheat isogenic lines containing single type high molecular weight glutenin subunits (HMW-GS) General discussion WEDNESDAY, SEPTEMBER 10, 2003 Session 5: Gluten polymers and tools to investigate their structure (Chairpersons R.J Hamer and K.R Preston) 8.30-8.50 10.10-10.25 C Don, G Mann, F Békés, R.J Hamer, Wageningen, the Netherlands Linking glutenin particle size to HMW glutenin subunit composition R Kuktaite, H Larsson, S Marttila, K Brismar, M Prieto-Linde, E Johansson, Alnarp, Sweden Gluten macropolymer in wheat flour doughs: structure and function for wheat quality B Schurgers, W.S Veraverbeke, J.A Delcour, Leuven, Belgium Critical factors governing gluten protein agglomeration on a micro-scale K.R Preston, S.G Stevenson, S You, M.S Izydorczyc, Winnipeg, Manitoba, Canada Application of flow field-flow fractionation and multiangle laser light scattering for size determination of polymeric wheat proteins W Li, P.J Wilde, B.J Dobraszczyk, Reading, UK Confocal laser scan microscopy of protein, lipid and starch in breadmaking dough General discussion 10.25-10.55 Coffee break Session 6: Gluten interactions and non-food uses (Chairpersons: M.H Morel and Y Popineau) 10.55-11.15 L Day, M Augustin, I.L Batey, C.W Wrigley, Werribee, Australia Association of non-protein components in wheat gluten with its quality D Every, L.D Simmons, K.H Sutton, Christchurch, New Zealand Ascorbate improver effects, dough mixing properties and bread quality interdependence on flour protein composition M Pommet, A Redl, M.H Morel, S Guilbert, Montpellier, France A way to improve the water resistance of gluten-based biomaterials: plasticization with fatty acids S Guilbert, S Domenek, M.-H Morel, Montpellier, France Wheat gluten based biomaterials: environmental performance, degradability and physical modifications Y Popineau, Nantes, France Gluten films: effect of composition and processing on properties and structure General discussion 8.50-9.10 9.10-9.30 9.30-9.50 9.50-10.10 11.15-11.35 11.35-11.55 11.55-12.15 12.15-12.35 12.35-12.50 12.50-14.20 Lunch Session 7: Intolerances and allergies (Chairpersons A Curioni and C Mills) 14.20-14.40 E.N.C Mills, J.A Jenkins, S Griffiths-Jones, P.R Shewry, Norwich, UK The structural and biological relationships of cereal proteins involved in type I allergy H Wieser, W Engel, J Ellis, J.S Fraser, P.J Ciclitira, Garching, Germany Coeliac disease-specific toxicological and immunological studies of peptides from α-gliadins G Mamone, P Ferranti, D Melck, F Tafuro, F Addeo, Avellino, Italy Mass spectrometry as a tool for probing gluten peptide modifications relevant to celiac disease 14.40-15.00 15.00-15.20 15.20-15.50 Coffee break 15.50-16.10 D.F McCarthy, E Gallagher, T.R Gormley, T.J Schober, E.K Arendt, Dublin, Ire- 16.10-16.30 16.30-16.50 16.50-17.05 17.05-17.20 land Formulation of gluten-free breads using response surface methodology C Constantin, W.D Huber, G Granditsch, M Weghofer, R Valenta, Vienna, Austria The profile of wheat antigens recognized by patients suffering from coeliac disease and IgE-mediated food allergy F Battais, J.P Douliez, D Marion, Y Popineau, G Kanny, D.A Moneret-Vautrin, S Denery, Nantes, France Lipid transfer proteins of cereals in food allergy to wheat General discussion Concluding remarks 8th “Gluten Workshop”, Viterbo 8-10 settembre 2003 stiffer These studies demonstrate for the first time that a proportion of secondary structure elements in gluten changes during deformation Future work will elaborate how such alterations relate to the current hypothesis regarding gluten elasticity Session 6: Gluten interactions (exogenous and endogenous components) 6.1 GLIADINS AND POLYSACCHARIDES INTERACTION N Guerrieri*, P Cerletti*, F Secundo° *Dipartimento Scienze Molecolari Agroalimentari, Università degli Studi di Milano, via Celoria 2, 20133 Milano and Centro per lo Studio della Celiachia, Università degli Studi di Milano, e-mail nicoletta.guerrieri@unimi.it °Istituto di Chimica del Riconoscimento Molecolare, CNR, via Mario Bianco 9, 20131 Milano The interaction gliadin-polysaccharides have been studied in a model system: gliadins (commercial and purified) and polysaccharides of different botanical origins (wheat starch, rice starch, potato starch, maize and commercial dextrin) were mix with 60% of water, the resulting dough was heated at 100 C in a close system from 30 minutes to hours A samples containing only the polysaccharide processed in the same condition was used as reference The samples were freeze dried and liophilysed The samples were analysed using different techniques: the enzyme amyloglucosidase was used as a probe to obtain information on the polysaccharides availability, the kinetics of the glucose releases versus time is useful tools to identify a modification in the polysaccharides fraction (Guerrieri et al 1997) Rice, potato, maize did not show any difference to the availability of amyloglucosidase, while wheat starch and commercial dextrin showed a reduction in the glucose release that can be due to polysaccharide-gliadin interaction The dextrin-gliadin reaction was optimised to improve the amount of modified gliadins and the reaction was followed versus time The HPLC-SE identified a gliadins-dextrin component that was characterized and partially purified from the dextrin The surface hydrophobicity of the samples with the fluorescent probe ANS (8-anilino-1-naphthalene sulphonate) showed a reducing hydrophobicity in the modified gliadins ATR-FT/IR measurements suggest a different effect of the heat treatment on the secondary structure of gliadins respect to that observed for gliadins-dextrin systems Also the intrinsic fluorescence and the Circular Dichroism (CD) indicated a different structure of modified gliadin, the spectra indicated the presence of a folded protein not denatured even though the heat treatment, probably because of the protection effect of the dextrin The ELISA test for the gliadins identification showed a reducing response to the immunoassay for the modified gliadin Guerrieri et al (1997) ‘Interaction of protein and starch studied through amyloglucosidase action’ Cereal Chem., 74, 846-850 6.2 STRUCTURE-FUNCTION RELATIONSHIPS OF PHOSPHOLIPIDS IN BREADMAKING G Helmerich, P Koehler German Research Centre of Food Chemistry, Lichtenbergstraße 4, D-85748 Garching, Germany, E-mail: Peter.Koehler@Lrz.tum.de Because of common structural elements phospholipids can act as emulsifiers and can influence the baking performance of wheat doughs Therefore, those polar lipids, e.g lecithin, which can be isolated in an industrial scale from plant sources (soybean, rapeseed), are used as part of improvers for breadmaking Lecithin improves the fermentation behaviour of yeasted doughs, the loaf volume of bread and the structure of bread crumb Commercial lecithin contains several fractions that can be subdivided into numerous indi- 8th “Gluten Workshop”, Viterbo 8-10 settembre 2003 69 vidual components The effects of the entire product as well as of lecithin fractions are well known As lecithin is a product from natural sources, the isolation of homogeneous components is extremely difficult Synthesis of individual phospholipids, the preparation of mixtures and the comparison of their improver effect with commercial lecithin was thought to be a promising way to get insight into the effect of lecithin In the first part of the study, phospholipid classes were determined qualitatively and quantitatively in commercial lecithins and flour improvers by thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) and 31P nuclear magnetic resonance spectroscopy (31P-NMR) The total amounts of phospholipids as well as the amounts of phospholipid classes in the samples were comparable, but depended on the method used for quantification Highest selectivity was provided by 31P-NMR as all phospholipids and lysophospholipids could easily be quantified By TLC only lysophosphatidylcholine could not be quantified, whereas HPLC was the method with the lowest selectivity, because lysophospholipids, except lysophosphatidylethanolamine, could not be determined Sensitivity was best for HPLC and TLC with detection limits of 20 to 170 µg/mL, with 31P-NMR these figures increased by a factor of 10 to 70 The coefficients of variation were 5.5, 6.8, and 12.8 % for the quantification by TLC, HPLC, and 31P-NMR, respectively, showing that TLC was the method with the best reproducibility Altogether, 31P-NMR can be recommended for the quantification of phospholipids, because it is easy to perform and results can be quickly obtained Since it requires minimum instrumental equipment, TLC is a good alternative to 31P-NMR In the second part of the study structure-function relationships of phospholipids in breadmaking were established Methods for the measurement of the effects of lecithin, fractions or individual compounds were dough and gluten rheology as well as baking tests, all in a small scale with 10 g of flour Firstly, the lecithin samples were fractionated by phase partitioning between solvents of different polarities and the effects of the fractions were determined Furthermore, phospholipid classes were isolated in a semi-preparative scale (1 – g) from soybean lecithin by preparative TLC and the effects of individual phospholipid classes and mixtures of them were determined Finally, phosphatidylcholine was isolated in a 10 g scale from egg, deacylated and re-acylated by using fatty acids of different chain lengths and with one or two double bonds (10:0 to 20:0, 18:1, 18:2) Altogether, the measurement of the effects of isolated fractions and classes as well as of individual components showed that optimum performance depended on the fatty acid present in phosphatidylcholine and also on the ratio of phospholipid classes in a mixture 6.3 EFFECT OF ASCORBIC ACID IN DOUGH: REACTION OF OXIDISED GLUTATHIONE WITH REACTIVE THIOL GROUPS OF WHEAT GLUTELIN P Koehler German Research Centre of Food Chemistry, Lichtenbergstraße 4, D-85748 Garching, Germany, E-mail: Peter.Koehler@Lrz.tum.de Due to the positive effects on the dough properties, ascorbic acid has been used as flour improver for a long time The mechanism of action has been established experimentally with the exception of one reaction It has been shown that in the initial phase of mixing, ascorbic acid is quickly oxidised to dehydroascorbic acid by ascorbate oxidase Dehydroascorbic acid oxidises endogenous glutathione to its disulphide In this way a thiol/disulphide interchange of endogenous glutathione with gluten proteins is inhibited A depolymerisation of gluten proteins corresponding to a weakening of the dough can therefore not take place, because disulphide bonds of gluten proteins are not involved in these reactions Firstly, different amounts of ascorbic acid were added to flour and the concentrations of low- and highmolecular weight thiols in the dough were determined For the determination of the low-molecular weight thiols glutathione, cysteine, and the corresponding disulphides, an isotope dilution assay with a [14C]labelled internal standard was used For the determination of the high-molecular weight thiols, a method was developed which involved derivatisation of dough with Ellman’s Reagent, removal of excess reagent by dialysis, micro-Osborne fractionation, release of the label by reduction, and determination of reduced Ellman’s reagent by RP-HPLC Mixing of flour without ascorbic acid led to a decrease of the glutathione and an increase of the cysteine concentration Addition of ascorbic acid reduced the concentration of both thiols to a minimum when 125 mg ascorbic acid /kg of flour were applied Furthermore, the concentrations of high-molecular weight thiols in the glutenins of flours from different wheat cultivars were determined The values ranged from 5.6 up to 8.2 µmol/kg of protein and showed a correlation between flour quality and SH-concentration Furthermore, the reactions of oxidised glutathione generated from endogenous glutathione by the addi- 70 tion of ascorbic acid prior to dough mixing on free thiol groups of gluten proteins have been investigated A small amount of [35S]-labelled glutathione was added as a tracer to identify the reaction products of GSSG and free protein thiols by radioactivity measurement Firstly, gluten was isolated from the dough, then the gliadins were extracted and residual glutenin was partially hydrolysed with thermolysin After pre-separation by gel permeation chromatography, the fractions with the highest radioactivity were separated by high-performance liquid chromatography Radioactive peptides were identified, isolated, sequenced and assigned to amino acid sequences of gluten protein components The isolated peptides contained exclusively the cysteine residues Cb* and Cx of low-molecular-weight subunits of glutenin which are supposed to be highly reactive in forming intermolecular disulphide bonds From these results it can be assumed that the cysteine residues Cb* and Cx of the low-molecular-weight subunits of glutenin are at least partly present in the thiol form in flour During dough mixing they are converted to protein-protein disulphides or glutathione-protein mixed disulphides by thiol/disulphide interchange reactions Oxidised glutathione necessary for this reaction is generated from glutathione by the action of ascorbic acid These results are in accordance with the major hypothesis about the mechanism of action of ascorbic acid 6.4 EFFECT OF PENTOSANS ON GLUTEN FORMATION AND PROPERTIES M Wang 1, 2, T van Vliet 1,3, R J Hamer 1, Department of Agrotechnology and Food Sciences, Centre for Protein Technology, Wageningen, the Netherlands 2Wuhan Polytechnic University, Wuhan, P.R China 3Wageningen Centre for Food Sciences, Wageningen, the Netherlands Pentosans have been studied for about 50 years since they were discovered as a component in the grains of wheat and other cereals Cereal scientists have markedly increased their knowledge of the structure, properties and functionality of pentosans However, the effects of pentosans on gluten formation and properties are rarely studied Several theories exist to explain the effects of pentosans and pentosan modifying enzymes, but some of them are disputed Nowadays, wheat pentosans are becoming more and more important in view of the increased use of ‘dark’ (high pentosans containing) flour for the production of wheat bakery and gluten products Therefore, to understand how pentosans affect gluten formation and properties is very important Our results show that both water unextractable solids (WUS) and water extractable pentosans (WEP) affect not only gluten yield, but also composition and properties This was especially clear when gluten GMP was dispersed and GMP particles were characterised in terms of size ([η],D3,2) and aggregative properties (K’) The interference of WUS or WEP with gluten formation caused an incomplete aggregation of gluten protein, the resulting gluten contained less smaller GMP particles When this interference was prevented by xylanase or ferulic acid (FA), aggregation was more complete and now also smaller GMP particles were recovered This was reflected in a smaller [η] and a larger K’ The same trend was found with three wheat cultivars of very different qualities ranging from weak (Scipion) to strong (Amazon) We observed however that Amazon was clearly less affected by addition of pentosans or xylanase than the other two cultivars The very higher protein content, the lower pentosans content , and the very high D3,2 and K’ of Amazon may explain why pentosans and xylanase have a small effect on its gluten yield and properties Clear correlations between gluten properties and GMP particle properties could help explain why also glutenrheological properties were changed upon addition of pentosans Maximum resistance against extension (Rmax) of gluten is best predicted by [η] and D3,2, while extensibility at maximum resistance against extension (E at Rmax) of gliuten is best predicted by K’ Based on our observations, we propose a possible explanation for the effect of pentosans on gluten formation and properties Both a physical effect and a chemical effect are involved The physical effect is related to viscosity and likely also depletion attraction between protein particles Viscosity is a general effect limiting aggregation rate and hence gluten yield Depletion attraction is related to the ratio in size of pentosans on the one hand and GMP particles on the other The chemical effect is related to pentosan bound FA and ‘controls’ K’ and hence also gluten yield We assume K’ reflects the Van der Waals attraction between GMP particles Session 7: Nutritional aspects, intolerances and allergies 8th “Gluten Workshop”, Viterbo 8-10 settembre 2003 71 7.1 PROPERTIES OF PROTEINS IN IMMATURE WHEAT GRAINS RELEVANT FOR THEIR TRASFORMATION F Bonomi 1, S Iametti 1, M.A Pagani2, M Zardi2 M.G D’Egidio3 Dipartimento di Scienze Molecolari Agroalimentari and 2Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, University of Milan, Via Celoria 2, I-20133 Milan, Italy; 3Istituto Sperimentale per la Cerealicoltura, via Cassia 176, I-00191 Roma, Italy Phone:0039 02 50316658; Fax: 0039 02 50316672; e-mail: ambrogina.pagani@unimi.it Some properties of immature durum wheat grains suggest that they may represent a valuable ingredient for the production of special foods, as also indicated by technological tests Immature grains were found to have a high content of fructo-oligosaccarides, fructose-rich polymers with important biological functions These compounds are no more present in the ripe grains The total content and composition of protein fraction vary during grain ripening Of particular interest is the development of the gliadin and glutenin fractions involved in the formation of the gluten network during bread and pasta-making processes, and responsible of gluten intolerance Biochemical studies on the protein components during the different ripening phases have been limited to coarse chemical studies and to approaches based on protein fractionation In this work we have determined the properties of proteins in immature grain by detecting the number of accessible thiols and the surface hydrophobicity properties on ground immature wheat grains at different times after flowering Biochemical analyses were carried out on whole meals obtained from two durum wheat cultivars without prior protein fractionation, and in conditions (presence or absence of denaturants) apt at assessing the accessibility of thiol groups in the protein interior Protein surface hydrophobicity in the various meals (measured by means of a hydrophobic fluorophore) related to the different nature of the most represented protein classes, as determined by SDS-PAGE Predominance of albumins before the “milky” phase resulted in the highest surface hydrophobicity The same trend was evident for thiol accessibility, with a sharp drop in this parameter at the beginning of the “milky” phase Western blotting experiments carried out with anti-gliadin antibodies showed that gliadin development begins suddenly at the onset of the “milky” phase Altogether these results suggest that the use of immature grain for the production of gluten-free food or functional food requires particular attention as for the choice of the most appropriate time for grain harvesting 7.2 BINDING OF GLUTEN PEPTIDES TO THE COELIAC DISEASE-ASSOCIATED HLA-DQ2 MOLECULE BY COMPUTATIONAL METHODS S Costantini1§*, G Colonna1, M Rossi2, A M Facchiano1,2 CRISCEB – Research Center of Computational and Biotechnological Sciences, Second University of Naples, via Costantinopoli 16 – 80138 Naples, Italy Institute of Food Science and Technology, CNR, via Roma 52 A/C – 83100 Avellino, Italy § PhD fellowship supported by E.U * susan.costantini@unina2.it Coeliac disease is an enteropathy, affecting mainly the proximal small intestine, and can be defined as an intolerance in genetically susceptible individuals to certain gluten peptides found in the cereals wheat, barley and rye The large majority of patients express the HLA-DQ2 [DQ(α1*0501,β1*02)] and/or HLA-DQ8 [DQ(α1*03,β1*0302)] molecules The DQ2 and DQ8 molecules confer susceptibility to celiac disease by presenting gluten-peptides to T-cells in the small intestine Gluten-specific DQ2- and DQ8-restricted T cells have been found at the site of the lesion in the gut This finding suggests that the intestinal damage (in this disease) is caused by mucosal T cell recognition of gluten-derived peptides in the context of disease-associated DQ2 and DQ8 molecules The peptide-binding motifs of DQ2 and DQ8 show a preference for negative charges at anchor positions of the bound peptides The gluten proteins contain few negative charged residues and are rich of proline and glutamine Experimentally it has been found that lesion-derived T cells recognize deamidated gluten peptides and that this deamidation can be mediated in situ by the transglutaminase type enzyme1,2 In our work, we have predicted the three-dimensional structure of HLA-DQ2 molecule by homology modelling and of its complex with some gluten peptides by superimposition and energy minimization on the basis of the experimental structure of DQ8-insulin B9-23 complex (PDB code: 1JK8)3 Moreover, we have 72 evaluated the energies of interaction for each peptide/DQ2 complex We have studied the peptide binding motif for DQ2 and observed why this molecule has the preference for negatively charged residues in specific anchor positions Infact, when a single glutamine residue in these anchor positions is exchanged with glutamic acid, the peptide-DQ2 complex appear more stable This finding gives an explanation at molecular level of the experimental results reported in literature We are applying this strategy to evaluate the effects of other peptide modifications as well as the interaction of DQ2 with other peptides4,5 Sollid, L.M Molecular basis of coeliac disease Ann Rev Immunol 18, 53-81 (2000) Sollid, L.M Coeliac Disease: dissecting a complex inflammatory disorder Nat Rev Immunol 2(9), 647-55 (2002) Lee K.H et al Structure of a human insulin peptide-HLA-DQ8 complex and susceptibility to type diabetes Nature Immunol 2(6), 501-507 (2000) Arentz-Hansen H et al Celiac lesion T cells recognize epitopes that cluster in regions of gliadins rich in proline residues Gastroenterology, 123, 803-809 (2002) Vader W et al The gluten response in children with celiac disease is directed toward multiple gliadin and glutenin peptides Gastroenterology, 122, 1729-1737 (2002) 7.3 USE OF SELECTED LACTOBACILLI TO PRODUCE A SOURDOUGH BREAD MADE OF WHEAT AND NON TOXIC FLOURS: IN VITRO AND IN VIVO HUMAN GLUTEN TOLERANCE * R Di Cagno1, M De Angelis2, S Auricchio3, L Greco3, C Clarke4, M De Vincenzi5, F Landolfo3, G Parrilli3, F Minervini1, M Gobbetti1 Department of Plant Protection and Applied Microbiology, University of Bari, 70126 Bari, Italy 2Institute of Sciences of Food Production, CNR, 70100 Bari, Italy 3European Laboratory for Food Induced Disease (ELFID), Department of Gastroenterology, University of Naples Federico II, 80131 Naples, Italy 4Department of Food and Nutritional Sciences, University College Cork, Cork, Ireland 5Istituto Superiore di Sanità, Laboratorio di Metabolismo e Biochimica Patologica, I-00161 Rome, Italy e-mail of presenting author: gobbetti@agr.uniba.it Sourdough lactic acid bacteria were preliminarily screened for proteolytic activity by using a digest of albumin and globulin polypeptides as a substrate Based on their hydrolysis profile patterns, Lactobacillus alimentarius 15M, Lb brevis 14G, Lb sanfranciscensis 7A e Lb hilgardii 51B were selected During wheat sourdough fermentation, under conventional technological conditions, all the four species showed a great hydrolysis of albumins, globulins and gliadins These sourdough lactobacilli showed a pattern of specialised peptidases which dealt adequately with Pro-rich peptides, including the 33-mer peptide Hydrolysis of this epitope was complete after 24 h of treatment with cells and cytoplasm extracts (CE) A sourdough made of a mixture of wheat and non toxic oat, millet and buckwheat flours was started with lactobacilli After 24 h of fermentation, wheat gliadins were almost totally hydrolysed as well as low molecular mass alcohol-soluble polypeptides Proteins were extracted from this sourdough and used to produce a peptic-tryptic (PT) digest for in vitro agglutination tests on K 562 (S) subclone cells of human myelagenous leukaemia origin The minimal agglutinating activity was ca 250 times higher than that of a dough chemically acidified or started with baker’s yeast Two types of bread, containing ca g of gluten, were started with baker’s yeast or with lactobacilli and CE An in vivo challenge was carried out on Celiac Sprue (CS) patients based on intestinal permeability tests CS patients showed a marked alteration of the intestinal permeability after ingestion of the bread started with baker’s yeast When the same CS patients ingested the same dose of gluten with breads started with lactobacilli and CE, they showed values of rhamnose and lactulose adsorption which did not significantly differ from those determined in optimal conditions of permeability level This work shows that a bread biotechnology which include selected lactobacilli, non toxic flours and long time fermentation is a probable way to eliminate or decrease the level of human gluten intolerance 7.4 EVIDENCE OF PANICUM MILIACEUM AS A SAFE FOOD FOR COELIAC PATIENTS 8th “Gluten Workshop”, Viterbo 8-10 settembre 2003 73 F Fusari1, A Petrini1, M De Vincenzi2, N Pogna3 CERMIS – Centro Ricerche e Sperimentazione per il Miglioramento Vegetale “N Strampelli”, Via Abbadia di Fiastra 3, Tolentino (MC), I-62029, E-mail: cermis@tin.it Laboratorio di Metabolismo e Biochimica Patologica, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome, I-00161 Istituto Sperimentale per la Cerealicoltura, Via Cassia 176, Rome, I-00191 In this work toxicological and nutritional analyses provided evidence of the validity of Panicum miliaceum, a cereal species phylogenetically close to maize, as a safe food for coeliac people A collaboration with some public and private institutions, from both Italy and other countries, has allowed to recover 23 genotypes of Panicum miliaceum, which have been grown in the field and studied for their main agronomic characteristics Peptic-tryptic (PT) digests of ethanol soluble proteins from Panicum miliaceum have been submitted to agglutination tests on human myelogenous leukemia K562(S) cells, the agglutinating activity of the PT digests being strongly associated with toxicity for the coeliac intestine The analysis has evidenced absence of agglutination up to 2300 mg/l of PT digests as compared with 100% agglutination caused by 70 mg/l of PT digests from the positive check sample Triticum aestivum cultivar S Pastore, suggesting the absence of toxicity of Panicum miliaceum for coeliac patients Moreover, the nutritional analyses showed the absence of any inhibition effect on the in vitro growth of CaCo-2 cells by the alcohol-soluble proteins of the Panicum miliaceum genotypes analysed here, even at protein concentration as high as 10 g/l 7.5 DETECTION OF GLIADIN IN FOOD, BASED ON A NEW MONOCLONAL ANTIBODY U Immer, S Lindeke, R-Biopharm AG Patients with gluten intolerance enteropathy, celiac disease and related allergic reactions have to avoid contact with prolamins from wheat, rye and barley throughout their whole life In Germany one in every 1000 people contract celiac disease The highest rate of celiac disease in Europe we find in Western Ireland with one person in every 300 people The assay methods to determine gliadin have to become more sensitive then in the last 10 years to support the industry guaranteing the new limit of 10 ppm gliadin which is in discussion now On the basis of a new monoclonal antibody R-BIOPHARM has developed a family of very sensitive gliadin kits Two of the assays are based on the ELISA technique The RIDASCREEN ® Gliadin test (3x 30 minutes) has included standards (5 – 80 ng/ml) and shows a detection limit of 1.5 ppm gliadin The second one represent the RIDASCREEN ® FAST Gliadin ELISA (3x 10 minutes) including standards (10 – 80ng/ml) and reaching a detection limit of ppm gliadin The standards are adapted to the new European Gliadin Standard, a standard, purified from 40 European wheat varieties on behalf of the Prolamin Working Group (PWG) Our third product, RIDA ® QUICK Gliadin represents an immunochromatographic assay, based on a lateral flow technique, simple and easy to perform, which get a result within five minutes This test is especially suitable for quick checking of incomming raw materials Moreover this dipstick can be used for searching of contamination of lab equipment (swabbing method) and to inspect the hygiene of the production process The RIDASCREEN ® Gliadin assay successful participated on an international ring trial with 20 participants under the supervision of the PWG All samples were extracted under implementation of the cocktail extraction method, recommended by Mendez (1), especially for heat treated samples Four negative samples were found clearly negative Three spiked maize samples (heated) and three spiked rice samples ( non heated) as well as positive samples from the market (content of gliadin in the range of 12 – 15 ppm) were found with a mean recovery of 94.7% All of the three assays are controlled during development and each production process by using gliadin spiked bread samples (heated, extracted with cocktail) We yield a mean recovery of 98.1% Gliadin kit) and 97.7% (FAST Gliadin kit) All of the spiked samples in the range of 33 – 139 ppm gliadin are positive with the QUICK Gliadin kit The RIDASCREEN Gliadin kit has included the recommended cocktail solution of Mendez We will data comparing the traditional extraction (60% ethanol) with the cocktail extraction method (1) Lopez, E., E Reyes, M Hernandez, M Llorente, E Mendez and H Wieser: A quantitative gluten „Cocktail extraction“ procedure for heat – processed foods Proceedings of the 15th meeting of Working Group on Prolamin Analysis and Toxicity Nov 2000, Meran, page 129 74 7.6 EFFECTS OF RYE AND BARLEY IN CELIAC DISEASE L Sabbatella, S Vetrano, M Di Tola, C Casale, M.C Anania, A Picarelli Department of Clinical Sciences University of Rome “La Sapienza”, Viale del Policlinico 155 - 00161 Rome, Italy (l.sabbatella@email.it) INTRODUCTION Antiendomysial antibodies (EMA) are highly specific for celiac disease (CD) They are detected not only in sera, but also in culture supernatants of the intestinal mucosa specimens of untreated CD patients, as well as in culture media of treated CD patients upon in vitro gliadin-challenge The organ culture method could be an useful tool to test the immunological effects of cereals on cultured intestinal mucosa from treated CD patients In fact we have previously used this system to show that oats in not capable to induce immunological activation typical of CD This result has also been confirmed by other in vivo studies In order to better define the role of rye and barley in CD, we have tested whether these cereals are capable to induce EMA production in treated CD patients METHODS Intestinal samples from 11 treated CD patients were cultured in the presence and in the absence of peptic-triptych (PT) digest of gliadin (1 g/l), ordein (2 g/l) and secalin (2 g/l) Intestinal specimens from 15 patients with gastroenterological diseases other than CD were cultured with and without gliadin and other cereals EMA were sought in these culture supernatants by means of indirect immunofluorescence analysis RESULTS EMA were present in supernatants from treated CD patients only when they were cultured with PT-gliadin No EMA were detected in any of the specimens from treated CD patients cultured with PTordein and PT-secalin CONCLUSIONS Our findings seem to suggest that in vitro challenge with PT-ordein and PT-secalin is unable to induce an immunological response typical of CD, evidenced by EMA production in treated CD patients and, therefore they could suggest the safety of rye and barley consumption in the gluten-free diet of celiac disease patients 7.7 NUTRITIONAL COMPONENTS OF MILL STREAM FRACTIONS L.D Simmons, K.H.Sutton, S.P.Rao New Zealand Institute for Crop & Food Research, Private Bag 4704, Christchurch, New Zealand Mail to SimmonsL@crop.cri.nz The commercial flour milling of wheat grains results in a considerable number of fractions or millstreams Composition of the final product depends on the proportion of different millstream fractions used Previous work has shown that there is significant segregation of molecular level components during the milling process This work investigates the changes in distribution of nutritional components such as phytic acid, dietary fibre and antioxidant compounds that occur as a result of milling of a number of wheat varieties These changes could allow the manipulation of flour composition to yield products with improved nutritional performance or find further uses for mill steams that are currently under-utilised The relationship between the distribution of these complex compounds and simpler compounds such as proteins is also investigated with a view to using simpler forms of component analysis to provide estimates of nutritional performance and to determine what, if any, interactions exist between them 7.8 FERMENTED WHEAT GERM EXTRACT AS A MEDICAL NUTRIMENT FOR CANCER PATIENTS R Tömösközi-Farkas, M Hidvégi* Central Food Research Institute, Budapest, 1022, Herman Ottó út 15, Hungary, r.farkas@cfri.hu 8th “Gluten Workshop”, Viterbo 8-10 settembre 2003 75 *Biromedicina first Hungarian Corporation for Cancer Research and Oncology, Budapest Madách I út Hungary The possible cytotoxic effect of methoxy-substituted benzoquinones, especially 2,6-dimethoxy-p-benzoquinone and 2-methoxy-p-benzoquinone has been proposed by Szent-Györgyi Using the theory of SzentGyörgyi, and by the reason of our previous studies we increased the concentration of 2-MBQ and 2,6-DMBQ in wheat germ by yeast (Saccharomyces cerevisiae) fermentation An orally applicable and water-soluble granulate has been developed by chemical and biotechnological transformation of wheat germ According to acute and subacute toxicological investigations with laboratory rats and mice, Avemar was not toxic, and showed no mutagenic effects In several series of animal experiments, the preparation, when administered alone, strongly inhibited the metastasis formation also of the most aggressive neoplastic tumors In tumor-bearing animals, parallel administration of Avemar and cytostatic drugs resulted in complete inhibition of the formation of metastases Avemar treatment reduced the side effects of chemotherapy, and significantly improved the regeneration of the hematopoetic system in mice receiving irradiation and cytostatic therapy In animals with artificially destroyed immune system Avemar treatment resulted in a complete reconstruction of normal immune response In a carcinogenesis study Avemar reduced the development of colon cancer by 70 per cent According to early clinical experiences, supportive Avemar treatment resulted in abrupt improvement of quality of life in also last stage cancer patients The preparation also increased performance status and it has been suggested that it may significantly delay disease progression In oral cancers Avemar treatment also significantly reduced the number of metastases plus that of relapses Session 8: Enzymatic modifications and non gluten components 8.1 UTILIZATION OF RAPID VISCO ANALYZER FOR ASSESSING THE EFFECT OF DIFFERENT LEVELS OF TRANSGLUTAMINASE ON GLUTEN QUALITY A Basman1, H Köksel1, P.K.W Ng2 Department of Food Engineering, Hacettepe University, 06532 Beytepe, Ankara, Turkey Department of Food Science & Human Nutrition, Michigan State University, East Lansing, MI, 48824-1224 Transglutaminase (TG: protein-glutamine g-glutamyl transferase, EC 2.3.2.13) is an enzyme capable of catalyzing the formation of nondisulfide covalent crosslinks between peptide-bound glutaminyl residues and e-amino groups of lysine residues in proteins Application of this crosslinking to wheat gluten proteins would be of particular interest because of their high glutamine content The formation of protein polymers as the result of TG action can modify the rheological properties of gluten The objective of the present study was to assess the effect of increasing levels of added transglutaminase on gluten quality of two wheat cultivars by using a Rapid Visco Analyzer (RVA) In the study, flours of wheat cultivars Roane (soft red winter wheat) and Sharpshooter (hard red spring wheat), and a bacterial TG (Ajinomoto, Teaneck, NJ) were used The RVA (Newport Scientific Warriewood, NSW, Australia) with data analysis software (Thermocline, Warriewood, NSW, Australia) was used to evaluate gluten quality by the wheat gluten acid method Control and TGsupplemented flour suspensions prepared in 1.0 M lactic acid were incubated in a water bath (45°C) for 30 and then tested by RVA in a 10 test The profile included initial equilibrium at 25°C for min, heating to 50°C during min, and holding at this temperature for The viscosity values at 3.0 and 10.0 were determined from the RVA curves The breakdown viscosity was calculated as the difference between viscosities at 3.0 and 10.0 The results indicated that RVA shows great promise as a quick method for evaluation of enzyme effect on gluten quality 8.2 ANTIOXIDANTS, FREE RADICALS, STORAGE PROTEINS, PUROINDOLINES AND PROTEOLYTIC 76 ACTIVITIES IN BREAD WHEAT SEEDS DURING ACCELERATED AGING L Calucci1, A Capocchi2, L Galleschi2, S Ghiringhelli2, C Pinzino1, F Saviozzi2, M Zandomeneghi3 Istituto per i Processi Chimico-Fisici, CNR, Area della Ricerca, V G Moruzzi 1, I-56124 Pisa Dipartimento di Scienze Botaniche, Università di Pisa, via L Ghini 5, I-56126 Pisa Dipartimento di Chimica e Chimica Industriale, Università di Pisa, via Risorgimento 35, I-56126 Pisa, e-mail: rino@ipcf.cnr.it Accelerated aging was performed by incubation of bread wheat (Triticum aestivum cv Centauro) seeds at 40 °C and 100 % relative humidity for 3, 4, and 10 days.1 The effects of the treatment on seed germinability and on several biochemical characteristics of flour (carotenoids, free radical and protein contents, and proteolytic activities) and gluten (free radical content and flexibility) were evaluated In particular, organic free radicals content in flour and gluten was measured by Electron Paramagnetic Resonance (EPR) spectroscopy and carotenoids in flour were determined by both front-face absorption spectroscopy2 and RPHPLC Storage proteins and puroindolines in flour were analyzed by SDS-PAGE, whereas proteolytic activities in flour were detected and quantitatively determined by using exogenous substrates, such as Hb, CBZPhe-Ala and azocasein, and suitable inhibitors, that is PMSF, pepstatin A and E-64.3 Gluten flexibility was investigated by means of the spin labeling EPR technique.4 Accelerated aging resulted in a decreased seed germinability, the germination being completely inhibited after 10 days A progressive reduction of the carotenoid content in flour was found, accompanied by an increase of the free radical content in both flour and gluten A progressive deterioration of the gluten polymeric matrix was also observed by spin labeling EPR spectroscopy and SDS-PAGE analyses of storage proteins, which can be associated to the marked increase of three proteolytic enzymes of dry wheat seeds, that is carboxypeptidase, aspartic and serine proteinases, found during accelerated aging On the other hand, puroindolines revealed to be quite resistant to the aging treatment (Footnotes) J C Delouche, C C Baskin, Seed Sci Technol., 1, 427 (1973) M Zandomeneghi, C Festa, L Carbonaro, L Galleschi, A Lenzi, L Calucci, J Agric Food Chem., 48, 2216 (2000) A Bottari, A Capocchi, D Fontanini, L Galleschi, Phytochemistry, 43, 39 (1996); A Capocchi, M Cinollo, L Galleschi, F Saviozzi, L Calucci, C Pinzino, M Zandomeneghi, J Agric Food Chem., 48, 6271 (2000) Spin Labeling: Theory and Applications, L J Berliner Ed., Academic Press, New York, 1976; Biological Magnetic Resonance, L J Berliner and L J Reuben Eds., Plenum Press, New York, 1989 8.3 USE OF 13C AND 1H SOLID STATE NMR TECHNIQUES TO PROBE THE CHANGES INDUCED IN FLOUR BY ACCELERATED AGING OF BREAD WHEAT SEEDS L Calucci1, C Forte1, L Galleschi2, M Geppi3, S Ghiringhelli2, G Mollica3 Istituto per i Processi Chimico-Fisici, CNR, Area della Ricerca, via G Moruzzi 1, I-56124 Pisa Dipartimento di Scienze Botaniche, Università di Pisa, via L Ghini 5, I-56126 Pisa Dipartimento di Chimica e Chimica Industriale, Università di Pisa, via Risorgimento 35, I-56126 Pisa e-mail: c.forte@ipcf.cnr.it Different 13C and 1H Solid State Nuclear Magnetic Resonance techniques were employed to study the changes induced in structural and dynamic properties of flour by accelerated aging (storage at 40 °C and 100 % relative humidity) of bread wheat (Triticum aestivum cv Centauro) seeds for periods ranging from to 10 days Structural information was obtained through 13C Single Pulse Excitation, Cross Polarization, Non Quaternary Suppression and other selective experiments performed under Magic Angle Spinning (MAS) and Dipolar Decoupling conditions, as well as 1H-fast MAS spectra and 2D-WISE (WIdeline SEparation) experiments1 Proton FID analysis and wideline measurements of proton spin-lattice relaxation times in both the laboratory (T1) and rotating (T1ρ) frames were performed over a broad range of temperatures in order to get insight into the changes induced by the aging process of seeds in the molecular dynamics of flour components To this aim, we used two recently developed procedures: (a) the use of the Power Weighted Rate Average2 which allows the proton relaxation to be completely interpreted in terms of dynamics, eliminating the spin diffusion effects; (b) the T2/T1ρ correlation experiment3 which yields the degree of correlation between the different exponential components of the T1ρ decay and the different FID components Static 8th “Gluten Workshop”, Viterbo 8-10 settembre 2003 77 and MAS 1H spectra at different spinning rates were also recorded in order to investigate the nature of the magnetic interactions responsible for spectral line broadening The experimental results highlighted a clear correlation between the dynamic properties of flour at a molecular level and the effects of seed aging N Zumbulyadis, Phys Rev B, 33, 6495 (1986); K Schmidt-Rohr, J Clauss, H W Spiess, Macromolecules, 25, 3273 (1992) M Geppi, R K Harris, A M Kenwright, B J Say, Solid State NMR, 12, 15 (1998) M Geppi, A M Kenwright, B J Say, Solid State NMR, 15, 195 (2000) (Footnotes) 8.4 A LIPID TRANSFER PROTEIN FROM FARRO (TRITICUM DICOCCUM SCHRANK) AND COMMON WHEAT A Capocchi*, D Fontanini*, L Galleschi*, L Lombardi*, F Saviozzi*, M Zandomeneghi†, L Carbonaro† *Dipartimento di Scienze Botaniche, Università di Pisa, via L Ghini 5, I-56126-Pisa, Italy (lgalles@dsb.unipi.it); † Dipartimento di Chimica e Chimica Industriale, Università di Pisa, via Risorgimento 35, I-56126, Italy Lipid transfer proteins (LTPs) are ubiquitous in plants Their biological role is still unknown (1), although they have been initially considered ideal candidates in membrane biogenesis and in regulating intracellular lipid exchanges Among LTPs, LTP constitutes a multigenic family of secreted plant lipid binding proteins, constitutively present in some plant tissues or induced in response to several stresses (1) Since LTPs have been also localized in the cell wall, two other possible roles have been suggested, i.e their participation in the synthesis of hydrophobic polymers, like cutin and suberin (2), and their involvement in plant defence mechanisms against pathogenic fungi (3, 4) Recently LTP from seeds has been recognized to be a new potential panallergen present in foods of plant origin (5) The Italian word “farro” designates a group of cereals characterized by their hulled habit; in fact, after threshing, their grains still appear covered by the spikelet glumes The increasing popularity of hulled wheats as environmentally-friendly cereal crops for the production of niche products is prompting research in new nutritious and attractive foods (6) However, knowledge on these cereals is still lacking being particularly poor on the allergen and anti-nutrient factors that these plants contain The main goal of this study is to isolate and characterize LTP from T dicoccon Schrank in comparison with that isolated from common wheat LTP was purified from bran of T dicoccon Schrank according the modified procedure of Charvolin et al (7) Farro bran was extracted in water and fractionated with ammonium sulphate After centrifugation the precipitate was dialyzed and then loaded on a cation-exchange resin eluted with an ammonium acetate linear gradient LTP was detected by SDS-PAGE and immunoblotting with an antibody for barley LTP The fractions containing LTP were precipitated by ammonium sulphate and, after centrifugation, the precipitate was resuspended and dialyzed overnight The solution was applied onto a Sephadex G50 column and the recovered fractions were purified by preparative and then by analytical C18 reversed phase HPLC using a water/acetonitrile gradient in the presence of 0.05% trifluoroacetic acid, at 50 °C This purification procedure was also used to isolate and purify LTP from Triticum aestivum cv Centauro (common wheat) bran and bran supplied by an industrial mill Our results have shown that a strong heterogeneity exists among LTPs isolated from “commercial” bran and those purified from common wheat In particular, RP-HPLC of commercial bran extracts separated many LTP isoforms while a simpler chromatographic pattern was given by Centauro and farro bran extracts References Kader 1996 Annu Rev Plant Physiol Plant Mol Biol 47, 627-654 Douliez et al 2000 J Cereal Sci 32, 1-20 Buhot et al 2001 FEBS Lett 509, 27-30 Guiderdoni et al 2002 Plant Mol Biol 49, 683-699 Pastorello et al 2002 Allergy 57, 106-110 Cubadda R and Marconi E (2002) In: Pseudocereals and less common cereals Belton P and Taylor J, eds Springer-Verlag, Heidelberg, Germany, pp 153-175 Charvolin et al 1999 Eur J Biochem 264, 562-568 78 8.5 BUG DAMAGE TO WHEAT: SPECIFICITY OF ACTION OF THE WHEAT BUG PROTEINASE D Every1, K H Sutton1, P R Shewry2, A.S Tatham2, T Coolbear3 NZ Institute for Crop & Food Research Limited, Private Bag 4704, Christchurch, New Zealand 2Rothamden Research, Harpenden, Hertfordshire AL5 2JQ, UK 3Fonterra Research Centre, Private Bag 11029, Palmerston North, New Zealand D Every E-mail: everyd@crop.cri.nz The New Zealand wheat bug (Nysius huttoni) injects a salivary proteinase into immature wheat kernels while feeding This proteinase remains in flour made from bug-damaged wheat, and in dough digests gluten to produce slack, sticky dough and poor quality bread A variety of techniques were used to test the following proteins as substrate for the enzyme: glutenin, gliadin, high molecular weight glutenin subunits (HMW-GS), low molecular weight glutenin subunits, wheat albumins and globulins, D-hordein from barley, HMW-secalin from rye, haemoglobin, BSA, cytochrome c, cytochrome c oxidase, papain, elastin, collagen, gelatine, keratin (hide powder), fibrin, azo-casein, α-casein, β-casein and k-casein Only the HMW-GS, HMW-secalin, D-hordein and β-casein were substantially hydrolysed All these proteins are glutamine rich Sequence analysis of the peptide products of enzyme reaction for both HMW-GS and β-casein revealed that glutamine occupied the P1 position relative to the scissile bond at all cleavage sites A variety of fluorogenic substrates made from synthetic peptides with glutamine in the P1 position relative to the fluorogenic group were tested with the enzyme, but none reacted 8.6 RELATIONSHIP BETWEEN SEQUENCE POLYMORPHISM OF GSP-1 AND PUROINDOLINES IN TRITICUM AESTIVUM AND AEGILOPS TAUSCHII A Massa, C F Morris USDA ARS Western Wheat Quality Laboratory, E-202 Food Sci & Human Nutrition East, Washington State Univ., Pullman, WA 99164-6394; e-mail: morrisc@wsu.edu GSP-1 (Grain Softness Protein-1) was coined by Rahman and colleagues and has evolved to define a set of homoeologous genes which are closely related to the indolines–most notably, puroindoline a and b; and closely co-located in chromosome 5DS Indolines appear in most taxa of the Triticeae and Aveneae tribes They are members of the same cysteine-rich protein family as the CM proteins and non-specific Lipid Transfer Proteins Our primary interest in GSP-1 stems from the unique properties of this family of proteins, and their utility in phylogenetic studies Hexaploid wheat (Triticum aestivum L.) was formed some 7,000-10,000 years ago through one or more rare hybridization events between a wild AABB tetraploid species and a DD genome-bearing Aegilops tauschii (Coss.) Consequently, modern wheat is limited by this «catastrophic» bottleneck of evolution, and the specific genetic composition of those few individual Ae tauschii plants of long ago Surveys of hundreds of hexaploid wheat varieties has indicated limited diversity in the puroindoline genes; of the seven variants defined to date, are single-nucleotide polymorphisms; all seven result in hard-texture kernel phenotype Surveys of Ae tauschii accessions have indicated a much greater degree of polymorphism for the two puroindoline genes compared to that found in wheat Surveys of GSP-1 gene sequences have not been conducted in Ae tauschii Consequently, we undertook the present study which involved 50 accessions of Ae tauschii drawn from the collection of the Wheat Genetics Resource Center, Kansas State University, and provided by Prof Bikram Gill Genome-specific primers were produced based on GSP-1 sequences provided by S Cloutier, Winnipeg, EST databases, and Turner et al (1999) Gsp-A1, Gsp-B1, and Gsp-D1 gene sequences were obtained for Chinese Spring and each was assigned the ‘a’ allele designation Complete Gsp-D1 gene sequence was also obtained for the 50 Ae tauschii accessions A total of polymorphic nucleotide positions were found for Ae tauschii GSP-1 gene sequences which translated to a total of amino acid differences These sequences were assigned alleles ‘b’ through ‘h’ Since none of these GSP-1 genes matched exactly that found in Chinese Spring, we concluded that none of these Ae tauschii accessions embody the specific ancestral D-genome donor of wheat These results will be combined with those for the puroindoline a and b genes and will be reported elsewhere 8th “Gluten Workshop”, Viterbo 8-10 settembre 2003 79 8.7 EFFECTS OF SOME PLANT EXTRACTS ON THE PROPERTIES OF BUG (EURYGASTER SPP.) DAMAGED WHEAT B Olanca, D Sivri Hacettepe University, Faculty of Engineering, Food Engineering Department, Beytepe Campus, 06532, Ankara / TURKEY e-mail: olanca@hacettepe.edu.tr Wheat bugs, the general term for certain Heteropterous insects, i.e Eurygaster spp., Aelia spp and Nysius huttoni, causes major quality losses in several wheat-growing countries including Turkey Wheat grain infested by the insects contains protease enzymes that hydrolyze gluten during breadmaking Dough prepared from bug damaged wheat flour has an unusual consistency and produces poor quality bread The aim of the present study was to investigate the possibility of using a plant extract to decrease the negative effects of bug proteases on the breadmaking quality of bug-damaged wheats Four bread wheat cultivars (hard red wheat, medium-hard red wheat, soft white wheat and medium-soft white wheat cultivars) damaged by wheat bugs were selected for this study The level of bug-damage was almost the same in all samples as revealed by modified Zeleny sedimentation test The crude plant extract was prepared and added to the bug-damaged wheat samples at various levels The effects of the crude extracts on the activities of bug protease in the flours were investigated by acid polyacrylamide gel (A-PAGE) and sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis Rheological and baking properties of the bug damaged wheat flours added crude extract were also tested 8.8 EVIDENCE OF A GENE CODING FOR GRAIN SOFTNESS PROTEIN (GSP-1A) ON THE LONG ARM OF CHROMOSOME 5D IN BREAD WHEAT L Gazza, A Niglio, F Nocente, N.E Pogna Experimental Institute for Cereal Science-Section of Applied Genetics-via Cassia,176 00191, Rome, Italy pognanorberto@mclink.it The 2S protein family of wheat endosperm includes three main components, i.e puroindoline a (pin a), puroindoline b (pin b) and grain softness proteins (GSPs) Minor variations in cDNA sequences have been found in genes coding for GSP-1a, GSP-1b and GSP-1c proteins, which show about 40% amino acid sequence homology with puroindolines Each of the short arms of group chromosomes of bread wheat (Triticum aestivum L.) was found to contain a GSP-1-encoding gene, that on chromosome 5DS being closely linked to the Pina-D1 and Pinb-D1 loci coding for pin a and pin b, respectively GSP-1 genes have never been detected in tetraploid durum wheat (Triticum turgidum spp durum) In this work, durum wheat genotypes, in which the Pina-D1 and Pinb-D1 loci from bread wheat had been introduced through allosyndetic recombination, were crossed with the commercial durum wheat cv Colosseo and the resulting F2 progeny analysed for their GSP-1 compositions The segregation data suggested the presence of two independent genes coding for GSP-1 on chromosome 5D PCR amplification of DNA from the ditelosomic 5DL line of cv Chinese Spring gave an amplification product identical to the published GSP-1a sequence, confirming that chromosome 5D contains two GSP-1a genes, one located on short arm close to Pina-D1 and Pinb-D1, the other on the long arm 8.9 INTERCULTIVAR VARIATION OF THE RHEOLOGICAL AND BAKING PROPERTIES OF BUG (EURYGASTER SPP.) DAMAGED WHEATS D Sivri*, B Olanca*, A Atlı** , H Köksel* *Hacettepe University, Faculty of Engineering, Food Engineering Department, Beytepe Campus, 06532, Ankara / TURKEY e-mail: sivri@hacettepe.edu.tr ** Harran University, Faculty of Agriculture, Department Food Technology, 63040, anlıurfa / TURKEY 80 Pre-harvest damage to wheat caused by some Heteropterous insects is called suni bug damage The damage is characterized with high protease activity in bug-damaged flours and has been associated with the genera of Eurygaster and Aelia in Turkey These insects are also responsible for the same problem in European and Middle Eastern countries In this study, intercultivar differences in terms of susceptibility of gluten proteins to proteolytic degradation by the bug (Eurygaster spp and/or Aelia spp.) proteases were investigated using sixteen bread-wheat cultivars grown in Turkey The bread-wheat cultivars were selected to represent different gluten characteristics and breadmaking qualities The wheat samples were subdivided according to their damage levels (as high or low damage level) on the basis of protease activity as revealed by modified Zeleny Sedimentation test The effects of bug damage on gliadin and 50% 1-propanol insoluble glutenin proteins were compared among cultivars by acid polyacrylamide gel (A-PAGE) and sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis Rheological and baking properties of the bug damaged the wheat flours were also tested and compared to observe intercultivar differences at the same protein content and two bug damage levels 8.10 RELATIONSHIPS BETWEEN TIMING OF EURYGASTER MAURA ATTACKS AND GLUTEN DEGRADATION IN TWO BREAD WHEAT CULTIVARS P.Vaccino 1, M.Corbellini 1, A.Curioni 2, G.Zoccatelli 3, M.Migliardi 4, L.Tavella Istituto Sperimentale per la Cerealicoltura, via Mulino 3, 26866 S.Angelo Lodigiano (LO) Italy vaccino@iscsal.it 2Dipartimento di Biotecnologie agrarie, Università di Padova (Italy) 3Dipartimento Scientifico e Tecnologico, Università di Verona (Italy) 4Di.Va.P.R.A Entomologia e Zoologia applicate all’Ambiente, University of Torino (Italy) Infestations of wheat bugs (Heteroptera Pentatomidae and Scutelleridae) during the grain filling period sporadically occur in different areas of Italy These insects feed on ears, piercing the developing grains and injecting their saliva rich of proteolytic enzymes in the endosperm Flours derived from damaged kernels originate sticky doughs and poor bread due to the detrimental effect of proteases on gluten structure Up to date, no sources of resistance are known, thus the only way of reducing the damage is the chemical bug control in the crop To implement an efficient environment friendly control strategy there is a need to pinpoint the relationships between the timing of the bug attack and gluten degradation A two years study was carried out by caging plants of two bread wheat cultivars, characterized by different seed texture and breadmaking quality, and introducing adults of Eurygaster maura in four periods corresponding to different grain filling stages Technological performance of flours from sound and affected kernels was analysed by means of a modified SDS sedimentation volume test Significant differences were found among treatments, the maximum proteolytic degradation occurring with insect attacks at the milk-ripe stage Biochemical investigations on gluten were performed by means of SDS gel electrophoresis and HPLC analyses of storage proteins Degradation of two components of the HMW glutenins was observed in damaged samples, associated with a breakdown of the HPLC peak representing high molecular weight aggregates The predominant role of water for the activation of the proteolytic enzymes during dough formation has been revealed 8.11 EFFECT OF FUSARIUM PROTEASES ON BREADMAKING PROPERTIES D Vázquez1, 2, S Gonnet1, 3, M Nin1, 4, O Bentancur1, Mesa Nacional de Trigo, URUGUAY INIA La Estanzuela CC 39173 Colonia CP 70000 URUGUAY.Uruguay dvazquez@inia.org.uy 3Laboratorio de Bioqmica Facultad de Agronomía, Universidad de la República Av.Garzón 780, Montevideo CP 12900 Uruguay Departamento de Producción Vegetal Facultad de Agronomía, Universidad de la República Paysandú CP 60000 Uruguay 5Departamento de Biometría, Estadística y Computación Facultad de Agronomía, Universidad de la República Paysandú CP 60000 Uruguay 6obent@fagro.edu.uy During the last few years, Fusarium sp caused important damage in wheat producing area of Uruguay, as well as in many other countries This fungus causes both lost in grain yield and detrimental industrial quality The production of high levels of several mycotoxins by Fusarium makes the contaminated cereal 8th “Gluten Workshop”, Viterbo 8-10 settembre 2003 81 unsuitable for human consumption or animal feeding At the same time, this fungus produces proteolytic enzymes, which may act on gluten proteins It was observed that some flours obtained from wheat damaged by Fusarium, even containing low levels of toxins, had poor breadmaking quality The objective of this research was to evaluate the effect of Fusarium proteases on gluten proteins and its impact on breadmaking properties Samples of wheat grains obtained from four different farms with very high Fusarium damage (10-17%) were cleaned carefully handpicking affected grains For each farm sample, “as is” grains were blended with clean grains in different ratios (0:100, 25:75, 50:50, 75:25 and 100:0) All blends were obtained by duplicate Kernel damage was evaluated for each sample Flours extracted from the 40 samples (4 farms, blends, reps) were analyzed for protein content, wet and dry gluten and alveograms Proteolytic activity caused by Fusarium was measured in flours from grain samples of two farms, using azocasein as substrate Hearth-type breads were obtained, with a 3-hours fermentation time method Fusarium damage did not influenced significantly (P