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Wheat Structure, Biochemistry and Functionality

Edited by

J. David Schofield Department of Food Science and Technology, University of Reading, UK

RS·C ROYAl. SOCIETY OF CHEMISTRY

The Proceedings of a Conference organised by the Royal Society of Chemistry Food Chemistry Group, held on 10-12 April 1995, in Reading UK

Special Publication No. 212 ISBN 0-85404-777-8 A catalogue record for this book is available from the British Library © The Royal Society of Chemistry All rights reserved. Apart from any fair dealing for the purpose of research or private study, or criticism or review as permitted under the terms of the UK Copyright. Designs and Patents Act. 1988, this publication may not be reproduced. stored or transmitted. in any form or by any means, without the prior permission in writing of The Royal Society of Chemistry. or in the case of reprographic reproduction only in accordance with the terms of the licenses issued by the Copyright Licensing Agency in the UK, or in accordance with the terms of the licenses issued by the appropriate Reproduction Rights Organization outside the UK. Enquiries concerning reproduction outside the terms stated here should be sent to The Royal Society of Chemistry at the address printed on this page. Published by The Royal Society of Chemistry, Thomas Graham House, Science Park, Milton Road, Cambridge CB4 OWF, UK For further information see our web site at www.rsc.org Printed by MPG Books Ltd, Bodrnin, Cornwall, UK

Preface

In 1985, John Blanshard, Peter Frazier and Terry Galliard organised a highly successful international conference on behalf of the Royal Society of Chemistry's (RSC) Food Chemistry Group on the "Chemistry and Physics of Baking". The proceedings of that conference, which those organisers edited, were published under the same title by the RSC. A similar conference was not repeated in the UK until in 1995 the "Wheat Structure, Biochemistry and Functionality" conference was organised in Reading, albeit with a slightly different focus. During the intervening 10 years, substantial progress was made in our understanding of the structural, compositional and physicochemical factors that determine wheat's technological quality for flour milling, for the production of bread, biscuits, pasta and other products, and for other end uses. Significant gaps remain to be filled even now, but it was felt that, 10 years on from the "Chemistry and Physics of Baking" meeting, it would be valuable to bring together an international audience, both of established experts and of scientists new to the area, to review progress and hopefully to identify ways forward. Those then were the aims of the Reading conference, which was again organised by the RSC Food Chemistry Group, and, thus, of this book, which represents its proceedings. Progress in any scientific area is dependent on committed people with good ideas, but also, importantly, on the availability of effective experimental techniques and approaches, which can be used to tease out the information required. In his excellent and thought provoking introductory chapter, which represented the plenary lecture for the conference, Colin Wrigley, one of the undoubted leaders in this field for many years, imaginatively reviews the impact that older and more recent techniques and approaches have had in helping us to advance our understanding of structure-function relationships. In fact, this is a recurrent theme in the various sections of the book. The chapter also contains some timely reminders, not only of the great strides that have been made, but also of some of the pitfalls that await both the researcher carrying out the work and those who come along after and try to build on earlier 'discoveries' if a careful and critical approach is not adopted. The first section of the book deals with macroscopical and microscopical aspects of wheat grain structure. Here, application of newer techniques, such as image analysis for morphological characterisation, fracture mechanics approaches, and newer sample preparation techniques for electron microscopy, are helping to provide new insights into grain structure and relationships to technological properties. Undoubtedly, the greatest research activity over the past two decades has been in determining the structural, physicochemical and genetical characteristics of wheat proteins, in particular the gluten proteins, and in defining how such properties relate to the functionality of those proteins in bread making in particular, but in other applications also. The next three sections contain chapters that describe progress in understanding the structural features of the gluten proteins and relationships to functional properties, such as rheological characteristics, in defining relationships between genetical differences in polypeptide structure and composition and quality indicators, and in demonstrating how molecular biology and genetic engineering approaches can help to answer questions about structure-function relationships amongst the gluten proteins.

vi


But the gluten proteins of wheat, although extremely important, are not the only components of the wheat grain that have potentially important effects on the functionality of dough or batter systems. The chapters in the section of the book dealing with low molecular weight sulphydryl compounds examine how redox compounds, such as glutathione, may have significant effects on flour functionality, and they offer experimental approaches for tackling the complex question of the involvement of redox phenomena in flour and dough technology. Similarly, the polar lipid components of flour have potentially important roles, and there interaction with relatively recently discovered lipid binding proteins is of considerable technological importance. Recent progress in this area is dealt with in the next section, and the functionality of added emulsifiers, which to some extent simulate the actions of the natural polar lipids, is also considered. Rheology is often said to provide a link between understanding of structure at the molecular level and technological performance in the bakery. The next section contains several chapters that consider experimental approaches for characterising the rheological properties of dough systems under relevant conditions of shear rate and strain, and, in particular, how those properties relate to gas cell stability in bread doughs. The final section contains several contributions that deal with the importance of the non-starch polysaccharides, particularly the arabinoxylans, in flour and baked product systems, and, in particular, the effects of enzymes on those arabinoxylans that provide novel ways of improving baking performance. For a book such as this, which gathers together offered contributions from a conference, it is difficult to achieve complete and well-balanced coverage of the overall subject area. Likewise, styles of individual contributions may vary considerably. Nevertheless, it is hoped that this book provides an overview of the progress that has been made to date in some important areas and provides insight into some of the newer approaches that may be used in future to solve outstanding problems.

J. D. Schofield

Contents

Windows on Wheat Quality: Fresh Insights and Their Dependence on New Research Technologies C. W Wrigley and F. Bekes

Grain Structure and Quality Grain Size and Morphology: Implications for Quality A . D . Evers

19

The Shape of the Wheat Kernel and its Influence on Fracture J. F. V. Vincent, A. A. Khan and J.-H. Liu

25

Ultrastructure and Technological Properties of Wheat E. Quattrucci, L. A. Pasqui and J. Fornal

31

Microscopical Methods for the Study of Wheat (Triticum aeslivum) Caryopsis Development, from Anthesis to Maturity G. D. Lunn, P. Echlin, P. J. Frazier and N. W R. Daniels Effects of Variable Environment on Wheat (Triticum aestivum) Caryopsis Protein Body Morphology and Protein Matrix Development During Grain Filling and Dehydration G. D. Lunn, P. Echlin, P. J. Frazier and N. W R. Daniels

37

44

Wheat Protein Structure and Functionality The Structures of Wheat Proteins A. S. Tatham

53

Disulfide Bonds of a- and y-TypeGliadins H. Wieser and S. Muller

63

Purification and Characterisation of lBx and IBy HighM,Glutenin Subunits from Durum Wheat Cultivar Lira F. Buonocore, C. Caporale and D. Lafiandra

70

Further Analysis of the Carbohydrates Associated With HighM, Subunits of Wheat Glutenin K. A. Tilley and J. D. Schofield

74

Presence ofGlycosylated Polypeptides inGliadin andGlutenin Fractions M Lauriere, I Bouchez, C. Doyen and G. Branlard

79

viii

Wheat Structure. Biochemistry and Functionality

Identification of Dimers Formed by the Low Molecular Weight Glutenin Subunits Belonging to the D Group S. Masci, T. A. Egorov, D. D. Kasarda, E. Porceddu and D. Lafiandra

85

Composition and Structure of Gluten Proteins A. Graveland, M H. Henderson, M Paques and P. A. Zandbelt

90

Time-Temperature Superposition for Networks Formed by Gluten Subfractions A. Tsiami, A. Bot, W. G. M Agteroj, A. Graveland and T. Henderson

99

The Role of Gluten in the Heat-Induced Changes that Occur in Dough Rheology During Baking A. Nakonecznyj, S. J. Ingman and J. D. Schofield

106

Biochemical Characterisation of Wheat Flour Proteins Using Gel Chromatography and SDS-PAGE E. L. Sliwinski, T. van Vliet and P. Kolster

112

Wheat Protein Composition and Quality Relationships Structural Differences in Allelic Glutenin Subunits of High and Low Mr and Their 117 Relationships with Flour Technological Properties D. Lafiandra, S. Masci, R. D 'Ovidio, T. Turchetta, B. Margiotta and F. MacRitchie Capillary Electrophoresis: A State-of-the-Art Technique for Wheat Protein Characterization J. A. Bietz, G. L. Lookhart, S. R. Bean and K. H. Sutton

128

Electrophoretic and Chromatographic Characterization of Glu-AI Encoded HighMr Glutenin Subunits B. Margiotta, M Urbano, T. Turchetta and G. Colaprico

134

HMW and LMW Subunits of Glutenin of Triticum tauschii, the D Genome Donor to Hexaploid Wheat M C. Gianibelli, R. B. Gupta and F. MacRitchie

139

Relationships Between Biochemical Parameters and Quality Characteristics of Durum Wheats M C. Gianibelli, M Ruiz, J. M Carillo and F. MacRitchie

146

Effects of the lBLlIRS Translocation on Gluten Properties and Agronomic Traits in Durum Wheat G. Boggini, P. Tusa, S. Di Silvestro and N. E. Pogna

153

Durum Wheat for Bread Making: Relationships Between Protein Molecular Properties and Technological Parameters M Carcea, N. Guerrieri and L. A. Pasqui

160

Contribution of the Hordeum chilense Genome to the Endosperm Protein Composition of Tritordeum J. C. Sillero, J. B. Alvarez and L. M Martin

167

Gliadin Components and Glutenin Subunits in Wheat Breeding A. 1. Abugalieva

173

Contents

ix

Gliadin and High Molecular Weight (HMW) Glutenin Subunits in the Collection of Polish and Foreign Winter Wheat Cultivars and Their Relation to Sedimentation V��

lW

Pathogenesis-Related Proteins in Wheat

184

Investigation of Hypersensitivity to Wheat Gliadin from Gluten-Free Dietary Products UsingDot-Blot Assay

189

The Brewing Value and Baking Qu�ity of Polish Winter Wheat Cultivars

192

J. Waga and J. Winiarski

C. Caruso, G. Chilosi, C. Caporale, F. Vacca, L. Bertini, P. Magro, E. Poerio And V. Buonocore

l. M Stankovic, /. Dj. Miletic and V. D. Miletic

J. Winiarski and J. Waga

Wheat Protein Molecular Biology and Genetic Engineering

Wheat Protein Molecular Biology and Genetic Engineering: Implications for Quality Improvement

199

The Use of Biotechnology to Understand Wheat Function�ity

206

P. R. Shewry, A. S. Tatham, J. Greenfield, N. G. Halford, S. Thompson, D. H. L. Bishop, F. Barro, P. Barcelo and P. Lazzeri

A. E. Blechl and O. D. Anderson

Construction ofDx5 Genes Modified in the Repetitive Domain and Their Expression in Escherichia coli 211 R. D 'Ovidio, 0. D. Anderson, S. Masci, J. Skerritt and E. Porceddu

coli for Biophysical Studies J. J. A. Greenfield, L. Tamas, N. G. Halford, D. Hickman, S. B. Ross-Murphy, S. Ingman, A. S. Tatham and P. R. Shewry

Expression of Barley and Wheat Prolamins in E.

215

Low M. Sulphydryl Compounds in Wheat Flour and Their Functional Importance

Measurement and Reactivity of Glutathione in Wheat Flour and Dough Systems

221

Determination of Low Molecular Weight Thiols in Wheat Flours and Doughs

235

J. D. Schofield and X Chen

B. Hahn, R. Sarwin and W. Grosch

Nature and Functionality of Wheat Lipids, Lipid Binding Proteins and Added Emulsifiers

Wheat Lipids and Lipid-Binding Proteins: Structure and Function

245

Starch Lipids, Starch Granule Structure and Properties

261

D. Marion and D. C. Clark

W. R. Morrison


x

Monoclonal Antibodies Against Wheat Glycolipids: New Tools to Investigate Mechanisms of Gas Retention in Bread Dough

271

Aspects on the Functionality ofDATEM in Breadmaking

279

Chang:�s of Wheat Flour Components Induced by Bread Improver

286

Z Gan and J. D. Schofield

N. C. Carr and P. J. Frazier

M Soral-Smietana, M Rozad and A. Cielem�cka

Rheological Properties and Functionality of Wheat Flour Doughs

Experimental and Conceptual Problems in the Rheological Characterization of Wheat Flour Doughs

295

Physical Factors Determining Gas Cell Stability in a Dough During Bread Making

309

Strain Hardening and Dough Gas Cell Wall Failure in Biaxial Extension

316

Stress Relaxation of Wheat Flour Doughs Following Bubble Inflation or Lubricated Squeezing Flow and Its Relation to Wheat Flour Quality

323

Gluten Microstructure and Changes in Hard Biscuit Doughs as Determined by Light Microscopy and Rheology

332

E. B. Bagley, F. R. Dintzis and S. Chakrabarti

T. van Vliet

B. J. Dobraszczyk

J. C. Bartolucci and B. Launay

A. Jurgens, T. V. P. Maarschalkerweerd, J. F. C. van Maanen and W. J. Rottier

Non-Starch Polysaccharides and Enzymic Improvement of Bread Quality

The Effects ofXylanases in Baking and Characterization of Their Modes of Action

343

Peroxidases in Breadmaking

350

A Method for Testing the Strengthening Effect of Oxidative Enzymes in Dough

361

Arabinoxylan in Wheat Flour Milling Fractions

368

Wheat Dough Properties Affected by Additives

371

Subject Index

377

T. S. Jakobsen and J. Qi Si

M van Oort, H. Hennink, P. Schenkels and C. Laane

P. Bak, I. L. Nielsen, H. Thogersen and C. H. Poulsen

R. Andersson and P. Aman

E. Torok

Acknowledgements

It is a pleasure to acknowledge the financial support given to this conference by the following companies. Their donations provided grants to enable a number of delegates, especially those from former Eastern bloc countries, to attend the conference: Allied Bakeries Ltd Dalgety PLC Kellogg Company of Great Britain Ltd Northern Foods PLC PBI Cambridge Ltd United Biscuits (UK) Ltd Weetabix Ltd

WINDOWS ON WHEAT QUALITY: FRESH INSIGHTS AND THEIR DEPENDENCE ON NEW RESEARCH TECHNOLOGIES

C. W. Wrigley and F. Bekes CSIRO Division of Plant Industry Grain Quality Research Laboratory North Ryde (Sydney) NSW 2113 Australia

1 INTRODUCTION Research advances in the elucidation of wheat quality have involved the opening of a series of windows to gain new insights into our understanding of composition-function relationships with respect to the wheat grain, dough and baked products. The opening of these windows has often involved the application of a new technique, or perhaps a new approach has been used in asking an old question. For example, one hundred years of advances in methods of protein composition/function analysis (and the opening of many new windows) have changed our view of gluten-p'rotein composition. As a result, there is a great contrast between Osborne's modee of only two protein components (gliadin and glutenin) and the current view of gluten as a complex of many polypeptides interacting via covalent and non covalent bonds to constitute a vast macro-molecular matrix. Many windows have been used to provide these new insights, such as dough-testing methods, gel electrophoresis in various forms (one- and two-dimensional) and chromatographic methods (most recently size-exclusion and reversed-phase HPLC). New insights are promised with the introduction of further techniques, including capillary electrophoresis, flow field-flow fractionation, immunoassay and a range of gene technologies. 2 METAPHORS: WINDOWS, HOUSEHOLDS AND COMMUNITIES 2.1

Looking in at Windows

Have you gone for a walk on a hot summer evening past houses with the windows wide open? Your don't mean to pry, but you can see in at the windows - someone watching the news on television, someone else writing at a desk, a family at the evening meal. Through another window you see someone reading, a student doing homework, a group playing cards. A glance in each window of the house will give a little more information about the household - how many people in the family, their interests (what television sessions are watched, what books are read), a look into the kitchen and dining-room windows will tell us their eating habits. All these glimpses through windows should enable one to build up an integrated profile on the family'S characteristics. Do this for many households and one should be able, in turn, to obtain a profile of the community (Fig. 1), though it would be important to do so for a representative population of households. This information-gathering activity might involve more than peaking in at windows around a house. Other "windows" available include less obvious opportunities, such as the phone (what is said to whom), the family'S balance sheet from the bank, mail, credit card

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accounts, and the bills that come in. In a similar manner, our investigations of flour composition and function must involve a range of approaches ("windows") - microscopy, protein extraction and fractionation, composition analysis, functionality testing - each in tum on a diverse population of samples, trying to make consistent sense of the various pictures seen through those various windows. However, with both of these scenarios, there is the likely problem that we will obtain a fragmented view depending on what windows we look through and when. The conclusions will also depend on how many households or samples we examine. 2.2

Possible Misconceptions

For example, if we look through the bedroom window and see the daughter of the family doing her French homework, we might conclude (incorrectly) that this a French speaking family. If our observation of the family meal happens to be on Christmas day (windows open for summer in Australia), we would conclude (probably incorrectly) that this is an extravagant family of gluttons. In observing a casual game of cards, we may draw conclusions about the group being a gang of gamblers. Our glimpse of someone watching the news on television may lead to the conclusion that he is vitally interested in current affairs, unless we look closely enough to see that he is asleep. The reliability of our conclusions is thus limited by the resolution of our methods of observation and by their frequency. Observations made must be made at many windows on many occasions to obtain a representative and reliable impression of the family. For the same reasons, the compilation of a representative picture of a community requires many observations at the windows of many houses, with intelligent integration of all these glimpses, bearing in mind the limitations of drawing conclusions from the view available at any window.

2.3

Making Good Use of Windows on Wheat

There has been a century of "modem" observation of wheat structure, biochemistry and functionality, the focus of this book. Progressively, new windows have been opened through which we have been able to make fresh observations. So often, we have obtained isolated glimpses that have, in tum, seemed to yield inconsistent and fragmented information. For example, conflicts and misconceptions have arisen when conclusions have been based on statistical observations of limited national sets of cuItivars. Attempts to reconcile the disparate information due to genotype versus environment provide recurring examples of these dilemmas. It is the function of a book such as this to help us to broaden our views on the range of windows available to us and to realise the importance of extending the population of windows through which we seek information.

3

TECHNIQUES

Can we correctly integrate all this information, bearing in mind the limited view provided by each window? What are some of these windows on wheat composition and function?

3.1 Microscopy

2

"What is unique about wheat gluten?" was the question posed by Eckert et al. at the latest International Workshop on Gluten Proteins. It is a recurring question. These authors addressed it by microscopy, observing the differences in the behaviour of flour particles from various grains upon wetting. Rye, barley and com contained insoluble protein that formed "network-like structures filling the space between the starch granules", but wheat flour alone provided a dough that had the "elasticity and aggregation behaviour" needed to hold growing gas cells during fermentation and oven rise. In a companion pape�, they reiterated the long-standing hypothesis that these gluten-specific characteristics are due to


4

the unique combination of gliadin and glutenin, "gliadins existing as monomers imparting viscosity to dough whereas the glutenin fraction is responsible for dough strength and elasticity". These observations reinforce the dramatic video sequences and the micrographs published by Bernardin and Kasarda4 showing how eagerly gluten fibrils form when wheat flour particles are wetted. 3.2

Protein Fractionation

3. 2. 1 Fractional Extraction. Centuries ago, the discovery that gluten could be washed from dough opened up a window on the nature of proteins themselves, the word "protein" being more recently devised than "gluten"s. The distinction between gliadin and glutenin as the two major components of gluten! provided an initial means of attempting to relate composition to function, but this window proved to be particularly blurred. Several research groups around the world (American, Australian, English, French) late last century used the Osborne procedure of fractional extraction to characterise various wheat-flour samples, obtaining results varying from 22% to 80% for the proportion of gliadins. The Australian Guthrie6 embraced the method enthusiastically, applying it to a range of flour samples, initially claiming success in reporting6 a positive relationship between glutenin content and dough strength (defined as water absorption) for tabulated results, which look less convincing when tho/ are treated graphically (Fig. 2) and statistically. With further experimentation, Guthrie finally concluded that this relationship "is not as simple as I at first thought; nor is the separation and accurate determination of the two proteins quite satisfactory. This method has, therefore, been abandoned in this laboratory". Though this

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5

method of protein fractionation may today be regarded as "bucket chemistry", Osborne's method and nomenclature have stood the test of a century of criticism, although many authors have variously redefined the terms gliadin and glutenin.

3.2.2 Gel Electrophoresis. A succession of gel electrophoretic methods opened up a vista of seemingly endless degrees of heterogeneity for Osborne's two fractions from gluten. Electrophoretic methods, first in starch gels, then in polyacrylamide, and later combined two-dimensionally with isoelectric focusing, have revealed gliadin to be a complex mixture of monomeric polypeptides, coded by genes on the short arms of group 1 and 6 chromosomes8. Gel electrophoresis in the presence of sodium dodecyl sulfate (SDS)9 opened an important window on the mysteries of the glutenin complexlO, particularly after the incorporation of methods to exclude non-glutenin protein from the patternll. Although the necessary rupture of disulfide bonds also destroys some of the function-composition information, the resulting classification of high M,9,1O,12 and low M,ll polypeptides of glutenin has led to important relationships, permitting the prediction of genetic potential for dough properties. 3.2. 3 High Performance Liquid Chromatography (HPLC). Whilst not replacing gel electrophoresis, reversed-phase HPLC has provided a valuable alternative means of defining the compositions for gliadin and for low and high M, glutenin polypeptides 12-14 and thus of predictin��enetic potential for dough-forming properties. Change the column type to size exclusion 7 and a molecular-size profile is provided, promising more reliable prediction of dough properties for the combined effects of genotype and environmental factors, especially if the methodology can be made to accentuate the larger-sized aggregates of gluten proteinsls-17. -

3.3

Emerging Methods

3.3. 1 Size Distribution Analysis. Indications from size-exclusion HPLC of the importance of very large glutenin aggregates have stimulated attempts to extend the analysis of size distribution into the millions of molecular weight. Two such "emerging" methods are multilayer SDS-gel electrophoresisl8,19 and flow field-flow fractionation . (FFF)20 21. The former involves conventional SDS electrophoresis in a series of layers of gel, increasing in polyacrylamide concentration in steps - 4, 6, 8, 10, and 12%T. The extent of staining in the respective gel layers provides quantitative indications of gluten protein content in size classes well over 100,000 in size. Difficulty in obtaining suitable standard proteins in the very large size ranges has so far precluded satisfactory calibration of the method. Nevertheless, our surveys of size distribution for various sets of wheat samples reinforce the likelihood that it is the very large aggregates of glutenin that are most effective in providing dough-strength properties (defined, for example, as resistance to extension in the Brabender Extensograph). FFF, on the other hand, is theoretically an absolute method, permitting size-distribution measurements up into particle-size ranges20. Figure 3 illustrates some of the potential of this method to distinguish between the various sizes of gluten proteins21. 3.3.2 Capillary Electrophoresis (CE). This method appears currently to be revolutionising protein and polypeptide analysis in general. According to Breliminary reports, it promises to offer advantages for wheat-protein analysis, for gliadins -24 and for glutenin polypeptides23. Figure 4 shows how CE profiles can be obtained in less than ten minutes to provide rapid varietal identification based on gliadin composition with considerable discrimination, three of the varieties shown in Figure 4 being indistinguishable by conventional acidic-gel electrophoresis. 3.3.3 Immunoassay. The specificity of antibodies in their ability to target defined amino-acid sequences (epitopes) offers the possibility of simplified mass screening of flour

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samples for appropriate quality-related structures in the gluten proteins, taking advantage of methodology devised for medical diagnostics. Some examples of this approach have involved the raising of antibodies directed towards specially synthesised peptides, representing specific gluten proteins2s. Alternatively, combinations of extraction conditions, antibodies and delivery systems have been sought that would provide correlations to specific quality attributes, particularly to screen for dough strength26

(primarily based on Extensograph height, Rmax). Detection, in turn, of the epitopes identified by the strength-relevant antibodies27 reinforces evidence28 that the �-spiral conformations of the high Mr glutenin polypeptides are important for dough properties.

3.3.4 Genetic Probing Methods. Results such as these, indicating amino-acid sequences, are vital steps towards the obvious progression from the identification of functional groups in proteins to the isolation of corresponding quality-related genes. Intermediate in this process is the use of this information to probe at the gene level for nucleotide sequences relevant to quality. For example, restriction fragment length polymorphism (RFLP) procedures and probes have been described for identification of Glu-l alleles using leafDNA!O,29. In addition, the wide range of gene-grobing techniques are also valuable tools for discriminating identification of cereal varieties o. 3.3.5 Expressing and Modifying Genes. The isolation and characterisation of genes is, in turn, only a means to ultimate goals of modifYing the genes in a beneficial way and using them to improve grain quality through transformation3!. Intermediate goals involve discovering more about the aspects of protein composition that affect functional properties, and possibly finding that these properties are already available in natural germplasm stocks.


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The molecular-biology approach involves expressing the respective gene (native or modified) in a heterologous system, such that the functionality of the expressed polypeptide can be tested, and proceeding to show that the gene can be introduced into the target plant, with the desired functional properties being contributed in the grain. Progress to this end has been demonstrated by the expression, in tobacco, of modified glutenin genes lacking a cysteine residue32. In this case, the degree of aggregation of the modified polypeptides could be tested, but not the functional properties in dough. It has thus been important to develop a system for doing so in order for the contributions to dough properties to be evaluated for minute amounts of purified or expressed polypeptides. 3.4

Micro Dough Testing

The development of very small-scale tests for functional properties has permitted the direct evaluation of very small samples of purified or expressed Erotein samples, which was not previously possible. Use of the direct-drive Mixograph for this purpose has generally confirmed hypotheses about the relative contributions to dough properties of specific gliadins and glutenin polypeptides34. All gliadin polypeptides tested contributed a

8


weakening effect to dough, measured as a considerable shortening of time to peak mixing resistance (as shown in F ' but also as decreased peak resistance and faster resistance breakdown after the peak '. For the gliadins, the results were essentially the same whether or not a partial oxidation-reduction cycle were used35; it was therefore not applied routinely for gliadins. Simple addition of glutenin subunits (high or low Mr types) also produced a weakening effect, similar to that caused by the gliadin addition or incorporation. The strength-conferring properties of the glutenin polypeptides could only be demonstrated if they were incorporated into the disulfide-crosslinked gluten matrix by the partial reduction and re-oxidation of disulfide bonds35 Figure 6 shows that purified (native) polypeptides of glutenin behaved similarly in dough to the corresponding polypeptides expressed in a bacterial system34a. Furthermore, their individual contributions to dough strength (shown as an increase in the mixing time of the base flour of 1 80 sec) were generally proportional to their size. There was a statistically significant difference between the increases in mixing time due to the incorporation of subunits 2 and This may be due to the higher proportion of cysteine residues in subunit than in subunit 2, rather than the size differencelO Figure 6 also shows the contributions of the low M, subunits (from three sources) after they had been incorporated into the dough matrix by partial reduction and re-oxidation of disulfide bonds35. Their shorter length presumably accounts for their proportionately lower contribution to dough strength.

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figure 5. Schematic representation of the domain structure of a-gliadin, y-gliadin and a LMW subunit ofglutenin. * Indicates a cysteine residue. The y-gliadins typically contain about 280 residues, the repetitive domain accounting for about one half of the sequence41•42, the repeat is related to that of the co-gliadins


60

(consensus PQQPFPQQ) minus one glutamine residue, PQQPFPQ. These sequences form {Hum and poly-L-proline II-like structures43• The C-terminal domain is predominantly a-helicaI43• Preliminary SAXS studies indicate a prolate ellipsoid with an axial ratio of anout 1 : 1 . 5 , while STM images of y-gliadins deposited onto a graphite surface indicated proteins with dimensions of about 10 x 3nm, with a slight broadening at one end44• The shape could result from the combination of a rod-shaped repetitive domain and a more compactly folded non-repetitive domain. Images of the protein deposited from a higher concentration produced a monolayer, with the long axis of the molecules lying perpendicular to the graphite surface44, forming an ordered array. Little information regarding the LMW subunits of glutenin is known despite being one of the major prolamin classes. They are divided into three groups B, C and Irs. B types subunits form a discrete group with two sub-classes called LMWs and LMWm on the basis of their N-terminal sequences46• The C-types are related to the a-type and y type gliadins, with additional cysteine residues for disulphide bond formation46• The D types appear to be related to the w-gliadins with the addition of cysteine residues47• A limited number of complete sequences are known and these are only for the minor forms of the LMW subunits. The proteins are difficult to purify in large amounts for physico chemical work, cd spectra of mixtures of LMW subunits would indicate a similar secondary structure content to the monomeric alf3-gliadins and y-gliadins48•

5 CONCLUSIONS This paper has concentrated on the structure of the prolamins of wheat, this work being stimulated by an interest in the unique technological properties of wheat flours and doughs, particularly in relation to breadmaking. It is difficult to explain the basis of gluten viscoelasticity based on our present knowledge of protein structure and interactions. Gluten is a complex system and so far we have only started to study and understand protein-protein and protein-water interactions, a whole range of other interactions involving, for example, starch and lipids, are probably also important in determining physical properties. There is still a considerable amount to understand regarding some of the unique protein structures found within the cereal prolamins. The application of a number of different techniques, including rheology, should enable a better understanding as to the structure-function properties of the prolamins.

References 1. 2. 3.

4. 5. 6.

7. 8. 9.

J.B. Beccari , Inst. Acad. Comm. Bologna , 1745, 2, 122. T.B. Osborne, 'The Vegetable Proteins' , Longmans, Green and Co. , London, 1924. L. Krejci and T. Svedberg, J. Am. Chem. Soc. , 1935, 57, 946. O. Lamm and A. Poulsen, Biochem. J. , 1936, 30, 528. P.P. Entriken, J. Am. Chem. Soc. , 1941 , 63, 2127. P.1. Payne, Ann. Rev. Plant Physiol. , 1987, 38, 141 . P.R. Shewry, N.G. Halford and A.S. Tatham, J. Cereal Sci. , 1992, 15, 105. A.S. Tatham, P.R. Shewry and B.J. Miflin, FEBS Lett. , 1984, 177, 205. A.S. Tatham, A.F. Drake and P.R. Shewry, J. Cereal Sci. , 1990, 1 1 , 189.

Wheat Protein Structure and Functionality

61

10. J.M. Field, A.S. Tatham and P.R. Shewry, Biochem. J. 1987, 247, 215. 1 1 . N. Matsushima, G. Danno, N. Sasaki and Y. Izumi , Biochem. Biophys. Res. Commun. 1992, 186, 1057. 12. M.J. Miles, H.J. Carr, T. McMaster, K.J. I' Anson, P.S. Belton, V.J. Morris, J.M. Field, P.R. Shewry and A.S. Tatham, Proc. Natl. Acad. Sci. (USA) , 1 991 , 88 , 68. 13. P.Y. Chou and G.D. Fasman, Ann. Rev. Biochem. , 1978, 47, 251 . 14. D.W. Urry, J. Prot. Chem. , 1988, 7, 1 . 1 5 . P.S. Belton, I.J. Colquhoun, J.M. Field, A . Grant, P.R. Shewry and A.S. Tatham, J. Cereal Sci. , 1994, 19, 1 15. 16. N . Matsushima, C.E. Creutz and R.H. Kretsinger, Proteins, 1990, 7, 125. 17. D . D . Kasarda, G. King and T.F. Kumosinski, ACS Symposium Series 576

'Molecular Modeling: From Virtual Tools to Real Problems' , American Chemical Society 1994, Chapter 13, p. 209. 18. M. Pezolet, S. Bonenfant, S. Dousseau and Y. Popineau, FEBS Lett. , 1992, 299, 247. 19. P.S. Belton, I.J. Colquhoun, J.M. Fild, A. Grant, P.R. Shewry, A.S. Tatham and N . K. Wellner, Int. J. Bioi. Macromol. , 1995, 17, 74. 20. J.T. Weeks, O.D. Anderson and A.E. Blechl, Plant Physiol. , 1993, 102, 1 077. 2 1 . F. Bekes, P. W. Gras, R. Gupta, D.R. Hickman, D.R. and A.S. Tatham, J. Cereal Sci. , 1994, 19, 3. 22. F. Bekes and P.W. Gras, 'Gluten Proteins 1993' Association of Cereal Research, Germany, 1993, 170. 23. P. Kohler, H.D. Belitz and H. Wieser, Z. Lebensm. Unters Forsch , 199 1 , 192, 24.

234. P. Kohler, H.D. Belitz and H. Wieser, Z. Lebensm. Unters Forsch , 1993, 196,

239. 25 . H.P. Tao, A.E. Adalsteins and D.D. Kasarda, Biochim. Biophys. Acta , 1992, 1 159, 13. 26. A.S. Tatham and P.R. Shewry, J. Cereal Sci. , 1995, In press. 27. J.C. Pernollet and J. Mosse, Int. J. Peptide Prot. Res. , 1983, 22, 1 3 1 . 28. A.S. Tatham, A.F. Drake and P.R. Shewry, Biochem. J. , 1985, 226, 557. 29. A.S. Tatham and P.R. Shewry, J. Cereal Sci. , 1985, 3, 103. 30. A.S. Tatham, A.F. Drake and P.R. Shewry, Biochem. J. , 1989, 259, 471 . 3 1 . J.M. Field, A.S. Tatham , A.M. Baker and P.R. Shewry, FEBS Lett. , 1986 , 200, 32. 32. Y. Popineau and F. Pineau, Lebens. Wis'sen. Technol. , 1988, 2 1 , 1 13 . 3 3 . K.J. I' Anson, V.J. Morris, P.R. Shewry and A.S. Tatham, Biochem. J. , 1992, 287, 183. 34. P.R. Shewry, M.J. Miles and A.S. Tatham, Prog. Biophys. Mol. Bioi. , 1 994, 6 1 , 37. 35. J.M. Purcell, D.D. Kasarda and C.S.C. Wu, J. Cereal Sci. , 1988, 7, 2 1 . 36. N . K . Wellner, P.S. Belton and A.S. Tatham. Submitted. 37. E.W. Cole, D.D. Kasarda and D. Lafiandra, Biochim. Biophys. Acta , 1984, 787, 44. 38. D.D. Kasarda, J.E. Bernardin and R.S. Thomas , Science , 1967, 155 , 203. 39. J.E. Bernardin, D.D. Kasarda and D.K. Mecham, J. Bioi. Chem. , 1967, 242 , 445. 40. A.S. Tatham, M.N. Marsh, H. Wieser and P.R. Shewry, Biochem. J. , 1990, 270,

62


313. 41 . J.A. Rafalski , K. Scheets, M. Metzler, D.M. Peterson, C . Hedgecoth and D . SoU, EMBO J. , 1984, 3, 1409. 42. T.W. Okita, V. Cheesbrough and C.D. Reeves, J. BioI. Chern. , 1985, 260, 8203 . 43 . A.S. Tatham, P. Masson and Y. Popineau, J. Cereal Sci. 1990, 1 1 , 1 . 44 . N.H. Thomson, M.J. Miles, A.S. Tatham and P.R. Shewry, Ultrarnicro. , 1992, 42-44, 1 204. 45. P.I. Payne and K.G. Corfield, Planta , 1979, 145, 83. 46. E.J.-L. Lew, D.D. Kuzmicky and D.D. Kasarda, Cereal Chern. , 1 992, 69, 508. 47. S . M . Masci, E. Porceddu, G. Colaprico and D. Lafiandra, J. Cereal Sci. , 1 991 , 14, 35. 48. A.S. Tatham , J.M. Field, S.J. Smith and P.R. Shewry, J. Cereal Sci. , 1987, 5 , 203.

DISULFIDE BONDS OF a- AND V-TYPE GLIADINS

H. Wieser and S. MOller Deutsche Forschungsanstalt fur Lebensmittelchemie and Kurt-Hess-Institut ffir Mehl- und EiweiBforschung LichtenbergstraBe 4, D-85748 Garching, Germany

1 INTRODUCTION Gliadin, the alcohol soluble protein fraction of wheat flour and gluten, consists of numerous monomeric proteins. They can be classified into three different types (a-type, v-type, w type) according to the primary structure. Whereas W-type gliadins are mainly free of cysteine, most of the a- and v-type gliadins contain six and eight cysteine residues, respec tively. Two cysteine residues are located in domain

V, the others in domain III (designation

1 of domains according to Kasarda et al. ). Within these domains, a- and v-type gliadins show a high degree of sequence homology. Because of the absence of free sulfhydryl groups, it can be assumed that all cysteine residues are linked by intramolecular disulfide bonds. So far, no direct experimental proof for the locations of disulfide bonds has been available; therefore, two a-type gliadins and one v-type gliadin isolated from gluten have been investigated. 2 EXPERIMENTAL Gliadin was extracted from gluten of the wheat variety Rektor with 70 % (vIv) aqueous 2 ethanol adjusted to pH 5.5 with acetic acid and then separated by preparative RP-HPLC on C 16 h.

18

3 silica gel . Single gliadins were digested with th�rmolysin at 37°C and pH 6. 0 for

The

digests were

analysed

for

cysteine peptides

by

differential

RP-HPLC

2 (chromatography prior to and after reduction of disulfide bonds) on C l a silica gel . The cystine peptides detected were isolated, reduced and alkylated with 4-vinylpyridine. The resulting cysteine peptide derivatives were analysed for their amino acid sequences using a pulsed liquid protein sequencer. 3 RESULTS AND DISCUSSION Gliadin obtained from gluten of the wheat cultivar Rektor was separated by preparative RP HPLC into 15 a-type (peaks 1-15) and 8 V-type (peaks 16-23) components (Figure 1). Peak

64


3 (a3-gliadin), peak S ( as-gliadin) and peak 17 (V17-gliadin), which appeared pure on the basis of rechromatography on C silica gel, were selected for the analysis of disulfide bonds. s The proteins were digested with thermolysin at pH 6. 0 to minimise sulfhydryl/disulfide 2 exchange reactions, and the digests were analysed by differential RP-HPLC . Two cystine peptides (a3

23/26) were detected in the digest of a3-gliadin, three cystine peptides Th

(aSTh9/ 1 1 / 12) in the digest of as-gliadin and three cystine peptides (V17 9/ 1 1 / 17) in the Th digest of V 17-gliadin. After preparation and purification by RP-HPLC, these eight peptides were reduced and alkylated with 4 -vinylpyridine. A further HPLC separation demonstrated that the cystine peptides consisted of two or three fragments (cysteine peptides), which then were isolated and analysed for amino acid sequences. The sequences and linkage types are shown in Table 1 . Peptide a3

23 was composed Th

of three fragments connected by two disulfide bonds. The order of attachment to the 26 adjacent cysteine residues of the fragment a3 23-2 was not determined. Peptide a3 Th Th consisted of two fragments linked by one disulfide bond. The sequences determined were 4 identical with corresponding sequences of the a-type clone A1235 (cv. Cheyenne) . Thus, 4 I 120 ) and C2 1 are linked to the adjacent residues C 150 /e 5 1 and cysteine residues 120 (C 4 2 9 residue C 163 is linked to C .

16 17

21 22

5

1.5

7

4

1.0

8

20

13 9

11 18

6

3 0.5

23 •• • • • •• • • • • • • • •

20

Figure 1 Preparative RP-HPLC ofgliadin (cv. Rektor)

••

40

60

min

65


The cystine peptides aSTh 1 1 and aSTh 1 2 derived from as-gliadin have structures homologous to the peptides of a3 -gliadin. They were identical to the corresponding sequen ces of a-type clone A212 (cv. Cheyennel As a consequence, residues C 123 and C252 are linked to the residues c 1 53 /c 154, and residue c 1 66 is linked to residue C260. An additio

nal cystine peptide (aSTh9) detected in the digest of as-gliadin was homologous to peptide

aSTh 1 1 . The only difference was found at position 4 of the fragment 9-1 (E instead of Q). Glutamic acid has not been described at this position in the literature until now. It is possible that a glutamine residue at this position was deamidated during the preparation procedure, or that peak S of the gliadin separation contained two different proteins modified at least at this position. Table 1 Amino acid sequences and linkage types ofcystine peptides from a- and V-type gliadins

Cystine peptides

a3 Th 2 3

a 3 Th 2 6 --

- -

-----

aS Th 9

aS Th l l

aS Th 1 2 --------V 1 7 Th 9

V 1 7 Th l l

V 1 7 Th 1 7

Fragments

23-1

Sequences

23-3 23-2

26-1

I PCXD I

-

---

ttQQb

L

-

-----

12 - 2 --------

LPAMCN

tt

L QQb -----------------------LQC

I

9-2

VYVPPECS

11-1

11-3 11-2

LNPCKN I

ttQEb

LQQCKP

I

IWPQSDCQ

a See Fig. 2. b The order of attachment to CC was not determined.

Ce ,

cy

Cf 1Cf2

--------CW CZ

LQTLPTMCN

VMQQQ

CZ --------CW

CZ

L I PCRD I

Cf lCf 2

CW

VYIPPYCT 12 - 3

cy

CZ

I

- - -

17 - 2

-

SRCQA

12 - 1

Ce ,

CW

I

11-2

17 - 1

I

VYI PPYCT

11-1

9-1

I

VYI PPYCSTT --------- -------SRCEA

9-2

-

LPAMCN

FQI PEQSXCQA

26-2 ----------9-1

--

Linkage typea

I

Ce ,

cy

Cf l Cf 2 Cd Ce

66


a

- type

W C

I

I II I

I

:m

I

C TIl

"

cc-cf1 cf 2

y.

Z

I

I

cy

y - t ype I

I

:nr

"

I NI

y.

I

Cc - C f1 C f2 ---- C y Figure 2 Schematic disulfide bond structure of 0'- and V-type gliadins (designation of domains according to Kasarda et al. 1 and of cysteine residues according to Kohler et al. 6)

The thermolytic digest of V17-gliadin contained three cystine peptides. One of them (V17Th 1 1) was composed of three fragments linked by two disulfide bonds, each of the others (V17Th9, V17Th 17) consisted of two fragments and had one disulfide bond. Five out

of seven cysteine peptides (9-1, 9-2, 1 1-3, 17-1, 17-2) were identical to sequences present in V-type clone pTag143 6 (cv. Chinese Spring)5, the remaining peptides (1 1-1, 1 1 -2) were modified in one or two positions.

The results obtained until now indicate that the homologous cysteine residues of 0'- and V-type gliadins are connected by the same intramolecular linkage types (Table 1, Figure 2). Common are three disulfide bonds, which reflect one loop within domain III (cysteine residues CC/Cft or Cf2) and two loops between domain III and V (ef1 or Cf2/cY and CW /CZ). V-Type gliadins contain an additional loop within domain III (CdICe). It can be assumed that the intramolecular disulfide bonds of 0'- and V-type gliadins are not formed randomly but strongly directed. The two-dimensional models shown in Figures 3 and 4 give an indication of the structural features and relationship of 0'- and V-type gliadins within the domains III, IV and V. Remarkably, the six (Q1-type) and eight (V-type) cysteine residues are concentrated to a relatively small area. Two small rings and a big ring are formed by the three disulfide bonds present in the Q1-type. In the case of the v-type, the first ring of the 0' type is divided into two smaller ones by an additional disulfide bond. Comparing the different 0'- and V-type gliadins described in the literature, the sizes of the small rings are generally constant; the big ring, however, is variable in size.


67

Figure 3 Partial two-dimensional structure of OI.-type gliadin (domains III-V of clone A 123s4)

Corresponding linkage types have also been found in glutenin-bound V-gliadins (CcICfl or Cf2, CdICe, Cfl or Cf2IcY, CWICZ) and LMW subunits of glutenin (CCICfl or Cf2, CdICe, d1 or Cf2Icy)6,7. For reasons of homology, it has been proposed that these bonds

are also intramolecular. Compared with monomeric V-type gliadins, glutenin-bound V gliadins have an additional cysteine residue Cb * within domain I, which is linked to aggregated LMW subunits. For the aggregative nature of LMW subunits, the cysteine

68


200

Figure 4 Partial two-dimensional strncture of V-type gliadin (domains III- V of clone

p Tag14365)

* residues eb of domain I and eX of domain IV appear to be responsible. They are absent in

monomeric

Ci.-

and V-type gliadins. In contrast to intramolecular disulfide bonds, different

linkage types have been found for the intermolecular disulfide bonds of LMW subunits; * thus, eX is linked with eb of LMW subunits or glutenin-bound v-gliadins or with cY of y type HMW subunits7.


69

References 1.

D.D. Kasarda, T.W. Okita, J.E. Bernardin, P.A. Baecker, C.C. Nimmo, E.J.-L. Lew, M.D. Dietler and F.C. Greene, Proe. Natl. Aead. Sci. USA, 1984, 81, 4712.

2.

P. Kohler, H.-D. Belitz and H. Wieser, Z. Lebensm. Unters. Forseh. , 1991, 192, 234.

3.

H. Wieser, W. Seilmeier and H.-D. Belitz, ]. Cereal Sci. , 1994, 19, 149.

4.

T.W. Okita, V. Cheesbrough and C.D. Reeves, J. BioL Chem. , 1985, 260, 8203.

5.

D. Bartels, J. Altosaar, N.P. Harberd, R.F. Barker and R.D. Thompson, Theor. AppL

Genet. ,

1986, 72, 845.

6.

P. Kohler, H.-D. Belitz and H. Wieser, Z. Lebensm. Unters. Forseh. , 1993, 196, 239.

7.

B. Keck, P. Kohler and H. Wieser, Z. Lebensm. Unters. Forseh. , 1995, 200, 432. Acknowledgement

This work was supported by the FEI (Forschungskreis der Ernahrungsindustrie e.V., Bonn), the AlF and the Ministry of Economics, Project No.: 8684.

PURIFICATION AND CHARACTERISATION OF l Bx AND l By HIGH M r GLUTENIN SUBUNITS FROM DURUM WHEAT CULTIVAR LIRA

F. Buonocore, C. Caporale and D. Lafiandra Department of Agrobiology and Agrochernistry, University of Tuscia, Via S. Camillo de Lellis, 0 1 1 00 Viterbo, Italy

1 INTRODUCTION The glutenin fraction is essential for the viscoelastic properties of wheat flour doughs. This fraction is usually divided into two types of subunits depending on their electrophoretic mobilities in SDS-PAGE under fully reduced conditions. The subunits with the slowest mobilities are referred to as high M glutenin subunits and the group with r faster mobilities as the low M glutenin subunits. The high M glutenin subunits are one of the most widely studied group of wheat prolamins, mainfy because of their role in determining breadmaking quality of wheatl,2. They are encoded at the Glu-I loci on the long arm of group 1 chromosomes (lA, IB e l D). Molecular analyses have indicated that each locus consists of two tightly linked genes encoding a low M y-type subunit and a high M x-type subunit3.

In particular, two all�les were reported at the Glu�Bl locus possessing only the x-type subunit, namely subunits 7 and 20. More recently, based on reversed phase high performance liquid chromatographic analyses, it has been shown that subunit 20 is accompained by another subunit, termed 2Oy, whose chromatographic behaviour is typical of the y-type subunits4. Moreover, same chromatographic data allowed to hypothesize that subunit 20 possesses only two cysteine residues compared to the other x-type

subunits that usually contains four cysteine residues (five in I DxS only). This last hypothesis was confirmed by Morel and Bonicel5 by electrophoretic data. We report here the purification and characterization of the high M glutenin subunits present in the durum wheat cultivar Lira to confirm the presence of a y-type subunit accompanying subunit 20 and to determine number and position of cysteine residues in subunit 20.

2 EXPERIMENTAL 2. 1 Purification of subunits 20 and 20y High M glutenin subunits 20 and 20y were purified from the durum wheat cultivar Lira. They �ere extracted from flour with a procedure based on Marchylo et al. 6 and purified by RP-HPLC using the System Gold (Beckman) apparatus, with a model 1 26 solvent delivery system and a model 166 detector. Proteins were separated on a Vydac Cs


71

semi-preparative column at 50 °c with a 30 minutes linear gradient of 28-35% (v/v) aqueous acetonitrile containing 0.05% TFA at a flow rate of2.5 mVmin. Amino acid analyses were performed with the Pico-Tag Work Station and the N terminal amino acid sequencing with a pulsed-liquid amino acid sequencer (model 477A, Appli� Biosystems). 2.2 Determination of cysteine peptides in subunit 20

The purified subunit 20 was reduced and alkylated in a single step according to Kelso et al. 7. The reduction was performed adding tributylphosphine and the alkylation using the fluorogenic agent 7-fluoro-4-sulfamoyl-2, I ,3-benzoxadiazole (ABD-F)8; both reagent were added in lar e excess over the supposed number of -SH groups. The reaction was performed at 50 C for 10 min. The sample was promptly injected into the RP-HPLC system above mentioned modified by the addition of a spectrofluorescence detector with a xenon source (Shimadzu Fluorescence HPLC-Monitor RF-530) connected in series with the UV absorption detector. Excitation wavelenght was at 385 nm; emission was monitored at 5 1 5 nm. Elution conditions were the same as those used for the purification step. The peak corresponding to subunit 20 was collected and freeze-dried. The protein, alkylated with ABD-F, was then digested with trypsin in a ratio (w/w) 1 :25 at 37 °c for 6 hours. Peptides were fractionated by RP-HPLC, at room temperature, with a 200 min linear gradient of 5-35% aqueous acetonitrile containing 0.05% (v/v) trifluoroacetic acid at a flow rate of 1 . 5 mVmin, detecting both UV absorption and the fluorescence. Peaks showing fluorescence were collected and sequenced.

§

3 RESULTS 3 . 1 Amino acid compositions and N-terminal sequences of subunits 20 and 20y The amino acid composition of subunit 20 is slightly different from that presented by Tatham et al.9 for the same subunit purified from the durum wheat cultivar Bidi 17. The higher level of histidine, tyrosine and methionine and the lower level of proline and phenilalanine make the composition of subunit 20 from cultivar Lira more similar to that of subunit IBx7 present in bread wheat. The amino acid composition of subunit 20y from Lira is similar to that of y-type subunits 8 and 16 from durum wheat and consistent with that of the other y-type subunits. The N-terminal amino acid sequence of subunit 20 (34 residues) is identical with that of subunit 20 from Bidi 1 79 (Fig. 1 ), except that glutamic acid was found at positions 13 and 18 of subunit 20 from Bidi 1 7, while glutamine was recovered in subunit 20 from Lira, and that glutamine substitutes arginine in subunit 20 from Lira at position 3 1 . The former substitutions might result from experimental errors, the latter could be the result of a point mutation.

72

Wheat Structure. Biochemistry and Functionality 1

10

20

2 0a E-G-E-A- S -G-Q-L-Q-C-E -R-Q-L-R-K-R-Q-L-E-A-Y-Q-Q30

V-V-D-Q-Q-L-Q-D-V-S b 1

20

10

20

E- G-E-A- S -G-Q-L-Q-C-E-R-E - L-R-K-R-E-L-E-A-Y-Q-Q30

V-V-D-Q-Q-L-R- D-V- S Figure 1 . Comparison of N-terminaJ. amino acid sequences of subunits 20 from cuJtivar Lira f) andfrom cuJtivar Bid; J 7 f). Differences are bolded The N-tenninal amino acid sequence of subunit 20y (35 residues) is identical with that of the y-type subunit 89 (Fig. 2), except that glutamate was recovered at positions 1 1 , 1 3, 16 and 20 of subunit 8 and glutamine in subunit 2Oy, and more generally with that of the other y-type subunits. 1

10

20

2 0y E -G-E-A- S -R-Q- L-Q-C-Q-R-Q-L- Q-Q- S - S - L-Q-A-C -R-Q30

V-V- D-Q-Q- L-A-G-R-L- P B

1

10

20

E - G-E-A-S -R-Q-L-Q-C-E -R-E -L-Q-E - S - S -L-E-A-C -R-Q30

V-V- D-Q-Q-L-A-G-R-L-X Figure 2. Comparison of N-terminaJ amino acid sequences of subunits 20y and 8. Differences are bolded

3 . 2 Detennination of cysteine residues in subunit 20 Tatham et al.9 hypothesized that subunit 20 lacks two cysteine residues in the N terminal region in contrast with the other Bx-type subunits, which could be the result ot

two independent Cys to Tyr substitutions due to two point mutations. To detennine the number and the position of cysteine residues of subunit 20, the purified subunit was alkylated with ABD-F, which reacts with thiol groups and specifically labels the cysteine residues present in the proteins. Among the different peptides obtained after the digestion with trypsin of the alkylated subunit, only two showed fluorescence. These peptides were collected and sequenced. Peptides 1 belongs to the C-tenninal region of subunit 20; its amino acid sequence (Fig. 3 ) is, in fact, identical to the same region of


73

subunit IBx7 and very similar to that of the other x-type subunits. Peptides 2 (Fig.3) matches the N-ternrinal region of subunit 20. A- Q-Q-L-A-A-Q-L-P-A-M-C-R

peptide 1

�-A-Q-Q-L-A-A-Q-L-P-A-M-C-�- L-E-G-S -D-A-L-S- T-R-Qa E-G-E-A- S-G-Q-L-Q-C-E-R E-G-E-A- S - G-Q-L-Q-C-E- �-Qb

peptide 2

a Figure 3 . Amino acid sequences of fluorescent peptides of sgbunit 20. C-terminal sequence of subunit 7from nucleotide sequence of cloned gene. N-terminal sequence of subunit 20. The underlined amino acids are the cleavage sites of trypsin; the cysteine residues are bolded 4 CONCLUSIONS

Present results confinn that an expressed y-type subunit is associated with subunit 20. Moreover, it is definitively proved that subunit 20 possesses only two cysteines residues, one in the N-ternrinal region and the other in the C-tenninal domain. Owing to its structural characteristics, subunit 20 provides a valuable model to determine the influence of number and position of cysteine residues in the pattern of disulphide bond formation of the glutenin polymers.

References 1 . Payne, P. I., Corfield, K.G. and Blackman, lA, Theor. Appl. Genet. 1 979, 55, 1 53 . 2 . Payne, P. I., Corfield, K.G., Holt, L.M. and Blackman, lA, J. Sci. Food Agric. 1 98 1, 32, 5 1 . 3 . Harberd, N.P., Bartels, D . and Thompson, R .D., Biochem. Genet. 1 986, 24, 579. 4. Margiotta, B., Colaprico, G., D'Ovidio, R. and Lafiandra, D., J. Cereal Sci. 1993, 17, 22 1 . 5 . Morel, M.H. and Bonicel, l,."Wheat Kernel Protein: Molecular and Functional Aspects", Universita della Tuscia, C.N.R., Viterbo, p. 1 83, 1994. 6. Marchylo, B.A., Kruger, IE. and Hatcher, D.W., J. Cereal Sci. 1 989, 9, 1 1 3 . 7 . Kelso, G.l, Kirley, T.L. and Harmony, lAK, "Techniques in Protein Chemistry", Vol. II (J.J. Villafranca, ed.), Academic Press, New York, 1 99 1 . 8. Masci, S., Lafiandra, D., Porceddu, E., Lew, EJ.-L., Tao, H.P. and Kasarda, D.D., Cereal Chem. 1 993, 70, 58 1 . 9 . Tatham, AS., Field, 1M., Keen, IN., Jackson, PJ. and Shewry P.R., J. Cereal Sci. , 1 99 1 , 14, 1 1 1 .

FURTHER ANALYSIS OF THE CARBOHYDRATES ASSOCIATED WITH HIGH Mr SUBUNITS OF WHEAT GLUTENIN

K.

A. Tilley and 1. D. Schofield

Montana State University, Department of Plant, Soil and Environmental Sciences, Bozeman, MT 597 1 7, USA and The University of Reading, Department of Food Science and Technology, P.O. Box 226, Whiteknights, Reading RG6 6AP, UK.

INTRODUCTION The glutenin proteins of wheat have been studied intensively due to their direct involvement in the variation that occurs in the bread making potential amongst different cultivars. Many proteins are co- or post-translationally modified, and those modifications play vital roles in the structures of those proteins. Only recently have such modifications been detected within the structure of the glutenin proteins with the discovery that the high Mr glutenin subunits are glycosylated I . The sugars found associated with those subunits were glucose (Glc), mannose (Man) and N-acetylglucosamine (GlcNAc). It was assumed that, because of the presence of GlcNAc, the sugars were linked to the polypeptide backbone via a linkage to asparagine (N-linked). Such a mode of linkage is inconsistent with the sequences that have been deduced from the sequences of cDNA corresponding to the central domains of the high Mr glutenin proteins, however2. The sequences reported for the high Mr subunits do not include the necessary site for N-linked glycosylation, Asn-Xaa-SerlThr; in fact, asparagine residues are absent from the central repetitive domain. Recently, evidence was obtained indicating that the sugars associated with the high Mr glutenin subunits are bound via O-glycosidic linkages to serine and/or threonine (O-linkages) rather than through N-linkages3 Man, which was previously shown to be associated with high Mr subunits I , has been shown to be linked O-glycosidically (i.e. the carbohydrate moieties were covalently attached to the amino acids serine and/or threonine) to the backbone of high Mr. It remains to be seen whether mannose is linked to the proteins as single residues or possibly disaccharide or trisaccharide structures, which may include Man and/or G\CNAc. These data are consistent with the published sequences, and they help to rationalize the hypothesis that the high Mr glutenin subunits are glycoproteins with the published sequence data. The predicted glycosylation site is the repeat sequence Tyr-Tyr-Pro-Thr-Ser, which occurs several times throughout the central repetitive domain of high Mr subunits4.


75

2 METHODS AND RESULTS 2.1

Extraction and Lectin Analyses

Total glutenin extracts of the cultivar Chinese Spring were prepared from approximately 3 or 4 kernels, which had been freshly hand-ground. Selective extraction and fractionation by sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS PAGE) were performed on the flour as described previously) . Lectin binding analyses, which were performed according to the procedure issued with the Glycan Differentiation kit (Boehringer Mannheim)', indicated positive reactions with Galanthus nivalis agglutinin (GNA) and with Datura stramonium agglutinin (DSA) as described previously),3. GNA is capable of binding both to N-linked terminal Man residues and to single O-linked Man residues6. DSA binds to terminal GaIB 1 �GlcNAC residues in heterosaccharide moieties linked N-g1ycosidically to asparagine or to single GlcNAc residues bound O-glycosidically to serine and/or threonine8. Galactose is not present in high M, glutenin subunits). Therefore, the lectin binding analyses, together with the amino acid sequence data reported previously, which indicated no N-linkage sites were present, suggested that the Man and GlcNAc were present as single sugar residues (or perhaps di- or trisaccharide moieties) linked O-glycosidically to serine and/or threonine residues. 2.2

Deglycosylation Using Enzymic and Chemical Methods

To investigate further the type of linkage by which the sugars were attached to the protein backbone, electroblots of SDS-PAGE fractionated high M, glutenin subunits were subjected to several specific deglycosylation procedures. When the blots were incubated with the enzyme N-Glycosidase-F (Oxford GlycoSystems) and the presence or absence of sugars was evaluated subsequently using the Glycan Detection system (Boehringer Mannheim), not all, if any, of the carbohydrate was removed from the proteins by this enzyme. Since N-Glycosidase-F cleaves N-glycosidic linkages specifically8, the results indicated that at least not all, if any, of the sugars associated with the high M, subunits were glycosidically linked to asparagine residues. The 13-elimination reaction specifically removes only carbohydrate moieties that are linked O-g1ycosidically. Electroblots of SDS-PAGE fractionated glutenin polypeptides were subjected to the 13-elimination reaction (2M NaBHJO. l M NaOH at 45°C for 1 6 h). Carbohydrate was detected using the Glycan Detection system. No carbohydrate was detected after the blots had been exposed to 13�elimination reaction conditions. A control blot was incubated with 2M NaBHJO. l M NaOH and stained for protein with Coomassie Brilliant Blue. The control indicated that protein remained on the blot after the incubation process. These results indicated, therefore, that the sugars associated with high M, glutenin subunits were likely to be linked O-glycosidically to serine and/or threonine residues. The enzyme 13-N-acetylhexosaminidase is capable of cleaving single GlcNAc residues from glycoproteins if those residues are linked O-glycosidically to serine and/or threonine but not N-g1ycosidically to asparagine9,1O. When electroblots of SDS-PAGE fractionated glutenin subunits were incubated with 13-N-acetylhexosaminidase (Oxford GlycoSystems) carbohydrate could not be detected using the Glycan Detection system indicating that all the carbohydrate had been removed from the glutenin polypeptides by the enzyme. A control blot, which was incubated with the enzyme buffer alone, gave a typical positive


76

reaction for carbohydrate with the Glycan Detection system. �-N-acetylhexosaminidase may be capable of removing single O-glycosidically linked Man residues as well as O-glycosidically linked GlcNAc residues although this has not been documented in the literature. The work with the deglycosylation methods, while intriguing, did not provide unequivocal evidence that the carbohydrates were involved in O-glycosidic linkages to the protein backbone. 2.3

Detection of O-linkages via �-Elimination and Alditol Acetate Derivatization

In order to provide more definitive evidence that the sugars associated with the glutenin polypeptides were linked to Ser/Thr residues, the following analyses were performed. Sugars were removed by �-elimination from electrophoretically purified glutenin polypeptides and their a1ditol acetate derivatives were analyzed at the Complex Carbohydrate Research Center, University of Georgia, by gas-chromatography-mass spectroscopy. The a1ditol forms of the sugars would be detected if they had previously participated in O-glycosidic linkages to the glutenin polypeptide backbone. For example, if Man had been O-Iinked directly to the polypeptide backbone, then mannitol would be detected, and if GIcNAc had been O-glycosidically linked to the polypeptide backbone, then N-acetylglucosaminitol would be detected. Recent results indicate that mannitol was detected providing direct evidence that Man was O-linked to the glutenin polypeptides. These results indicate that the glutenin polypeptides are true glycoproteins and provide evidence for the nature of the glycosylation type.

3

DISCUSSION

The exact sites of glycosylation are being investigated. However, we propose that these sites are likely to be the repeated Tyr-Tyr-Pro-Thr-Ser amino acid sequences, which occur several times throughout the central repetitive domains of the high M, subunits2. This site has also been predicted as a potential �-turn site in these proteins 10. These results fit extremely well with data obtained for other proteins, which are O-glycosylated. For example, typical O-glycosylated proteins contain high amounts of proline, serine and threoninel2 and have �-turns in their structure. Glutenin polypeptides contain high levels of Pro, Ser and Thr and contain large numbers of �-turns in their repetitive domains1o. Pro, Ser and Thr are common in reverse turns, which comprise four amino acid residues. O-glycosylation tends to occur in regions of the protein that exist as reverse (�) turns, but not necessarily coincident with the turnsJ3. Also O-glycosylation seems to be significantly enhanced if Pro occupies the - 1 or +3 position in the sequence13 In the case of glutenin, it seems likely that Thr may be glycosylated in the sequence Tyr-Tyr-Pro-Thr-Ser since Pro is in the - 1 position relative to Thr. The location of the sugar residues remains to be determined, however. Since �-turns are often located at the surface of proteins, these results are consistent with a post-translational model of O-glycosylation. If O-glycosylation takes place in the Golgi apparatus, as most data indicate, the protein has already been folded at this stage so accessibility to the glycosylation site would be a determining factor in determining the O-glycosylation site.


77

Another significant feature of many O-glycosylated proteins is that they commonly contain phosphorylated tyrosine residues in their structures10, 14. We have recently detected phosphorylated tyrosine in high Mr glutenin subunits4 . Glutenin subunits appear to be very highly phosphorylated as evidenced by the intensity of the binding reaction with an anti phosphotyrosine monoclonal antibody. It would seem likely that it is the Tyr residues involved in the repeat Tyr-Tyr-Pro-Thr-Ser that are phosphorylated, although that remains to be determined. For glycoproteins, in which GlcNAc and/or Man are linked O-glycosidically directly to the polypeptide moieties, the carbohydrates are typically present as single residues along the backbone of the protein rather than as branching structures containing other sugar residues or they may exist as di- or trisaccharides, in which the entire structure is made up of only Man or GlcNAc or a combination of the two 10, I4- 16 . The findings presented here are consistent, therefore, with the known amino acid sequences of high Mr subunits of glutenin and with the structures of other glycoproteins, in which Man and GlcNAc occur as single residues linked O-glycosidically to the hydroxyl groups in the sides chains of serine and/or threonine residues. The presence of O-glycosylation is a significant discovery as much of the debate regarding the glycosylation of glutenin subunits centres around the fact that the known glycosylation site for typical N-glycosidically linked carbohydrates (Asn-Xaa-SerlThr; where Xaa is any amino acid except Pro) has not been found in the sequences of high M. glutenin subunits published to date2. It is clear now that the sugars are linked to the polypeptide backbone in a rather unexpected way. Linkage of sugars through serine or threonine (O-glycosidic linkage) is not unusual. However, much less is known about this type of glycosylation than is known about glycosylation involving linkage to asparagine (N-glycosidic linkage), and analyses of these glycoproteins typically prove to be more difficult due to the fact that O-glycosidically linked sugars are often present in substoichiometric amounts.

References 1.

2. 3. 4.

5. 6. 7.

K. A. Tilley, G. L. Lookhart, R. C. Hoseney, and T. P. Mawhinney, Cereal Chem., 1 993 , 70, 602. N. G. Halford, J. Forde, O. D. Anderson, F. C. Greene and P. R. Shewry, Theor. Appl. Genet., 1 987, 75, 1 1 7. K. A. Tilley and J. D. Schofield, In 'Wheat Kernel Proteins - Molecular and Functional Aspects' . Universita Degli Studi Della Tuscia, Viterbo, Italy, 1 994, p. 2 1 3 . K . A . Tilley and J . D. Schofield, J. Cereal Sci., I n press. A. Haselbeck and W. Hosel, In 'Protein Glycosylation: Cellular, Biotechnological, and Analytical Aspects' Gesellschaft fur Biotechnologishe Forschung mbH, 1991, p. 1 7 1 . N. Shibuya, I. 1 . Goldstein, E. J. M . Van Damme, and W . J . Peumans, J. Bioi. Chem., 1 988, 263, 72 8 . J. F. Crowley, I. J. Goldstein, J. Amarp, and J. Lonngen, Arch. Biochem. Biophys. , 1 984, 231, 524.

S. Alexander and 1. H. Edler, Methods in Enzymol., 1 982, 179, 505. 9. N. G. Hanover, C. K. Cohen, M. C. Willingham, and M. K. Park, J. Bioi. Chem., 1 987, 8.

262, 9887.

10. S. P. Jackson and R. Tijian, Cell, 1 988, 55, 125.

78


A. Tatham, A. Drake, .and P. Shewry, J. Cereal Sci. , 1 990, 11, 1 89. B . C. O'Connell, F. K. Hagen and L. A. Tabak, J. Bioi. Chem., 1 992, 267, 250 1 0. 1 3 . I. B. H. Wilson, Y. Gavel, and G. von Heijne, Biochem J. , 1 99 1 , 275, 529. 14. E. P. Roquemore, A. Dell, H. R Morris, M. Panica, A. 1. Reason, L. -A. Savoy, G. 1. Wistow, 1. S. Zigler, B. 1. Earless and G. W. Hart, J. BioI. Chem. , 1 992, 267, 5 5 5 . 1 4 . C. RTorres and G. W. Hart, J. Bioi. Chem., 1 984, 259, 3 308. 1 5 . R S. Haltiwanger, W. G. Kelly, E. P. Roquemore, M. A. Blomberg, L. Y. Dong, L. Kreppel, T. Y. Chou and G. W. Hart, Biochem. Soc. Trans., 1 992, 20, 264. 1 6 . H. Nishimura, S. Kawabata, W. Kisiel, S. Hase, T. Ikenaka, T. Takao, Y. Shimonishi and S. Iwanaga, J. Bioi. Chem., 1 989, 264, 20320. 11. 12.

PRESENCE OF GLYCOSYLATED GLUTENIN FRACTIONS

POLYPEPTIDES

IN

GLIADIN

AND

M. Lauriere, I. Bouchez. , C. Doyen and G. Branlard* Laboratoire de Chimie Biologique. Centre INRA de Grignon, F-78850 Thiverval Grignon, France. *Station d' Amelioration des Plantes, INRA, Domaine de Crouelle, F-63039 Clermont Ferrand cedex 02, France.

1 SUMMARY

Glycans covalently bound to proteins, were investigated on gliadin or glutenin fractions, using specific chemical derivatization of glycans, anti-carbohydrate antibodies and lectins. Glycosylated proteins were evidenced in both gliadin and glutenin fractions. All high molecular weight glutenin subunits produced hydrazide conjugates after periodate oxidation; a reaction specific of bound carbohydrates. On the contrary, only some gliadins or low molecular weight glutenin subunits developed the same response or reacted with anti-carbohydrate antibodies. None of them specifically reacted with the tested lectins. Glycosylated polypeptides differed from one variety to an other and seemed to be present in low amount among wheat storage proteins. 2 INTRODUCTION The presence of carbohydrates along with proteins in gluten is known for a long timel. Most of these carbohydrates originate from starch or from the cell wall. They can be eliminated more or less easily from gluten. On the contrary, some carbohydrates are difficult, or impossible to separate from proteins, even after thorough purification, which raises the question of their covalent binding to proteins. When the amount of these sugars is compared to that of proteins, the calculated number of carbohydrate units is generally lower to the number of polypeptidic chains2,3. These observations lead to the assumption that wheat storage proteins are not glycosylated. This contrasts with legume storage proteins that also are synthesized on the secretory pathway, where most of the glycosylations occur, and which are often glycosylated4• The possibility that some isoforms of gliadins or glutenins are glycosylated cannot be excluded. The only way to detect them, is to evidence carbohydrates directly on electrophoretic separations of the protein subunits. The periodic acid Schiff or thymol sulfuric reagents always give negative results5,6. Lectins as concanavalin A, wheat germ agglutinin7, or Galanthus nivalis agglutininS, have already been described to react with wheat storage proteins but reactio�s are weak and difficult to interpret. Recently Tilley el al. S (and these proceedmgs) reported detection of carbohydrates bound to high molecular weight glutenin subunits (HMW-GS) using derivatization of

Abbreviations used: HMW-GS, high molecular weight glutenin subunit(s); LMW-GS, Low molecular weight glutenin subunit(s); PVDF, polyvinylidene difluoride; SDS PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.


80

the glycan moiety after periodate oxidation according to Haselbeck and Hosel9 . The present work explores with the same method and by using specific anti-carbohydrate antibodies and lectins, the possibility of the presence of glycosylated wheat storage proteins. 3 METHODS Unless stated, reagents were of analytical grade and from Prolabo (Fr.). or from Sigma Chemical Co. (Saint Louis MO). Immobilon P a polyvinylidene difluoride (PVDF) membrane was from Millipore Corp. (Bedford, MA). Triticum aestivum cv. Capelle, Capitole, Chinese Spring and Courtot, were field grown.

3.1 Preparation of Gliadin and Glutenin Fractions Wheat grains without their embryo and their pericarp were ground in a ball mill. The flour (40 mg) was extracted without delipidation in Eppendorf tubes, with 0.5 ml of solvent under continuous gentle agitation for 30 min. Gliadins were extracted first with 65 % (w/v) ethanol at 20 °e, then glutenins with 35 % (w/v) I-propanol, 0. 1 M acetic acid, 5 % (v/v) 2-mercaptoethanol at the same temperature. Proteins were recovered from the centrifugated extracts, by overnight precipitation with 2 volumes of 7.5 % (w/v) NaCI at 4 °C, and centrifugation . Centrifugations were at 1 3 ,000 g for 5 min .

3.2 Electrophoretic and Blotting Techniques SDS-PAGE was according to LaemmlilO• In most experiments ready made 420 % gradient gels (Novex, San Diego, CA), were used. Semi-dry electroblotting on PVDF membranes of separated proteins, was according to Laurierell to ensure complete transfer of omega-gliadins and HMW-GS. Total proteins were stained on gels with Coomassie Blue according to Neuhoff et al. 12 and on blots with amido black lOB, according to Gershoni and Palade1 3

3.3 Detection of Carbohydrates on Blots Total glycoproteins were evidenced after periodate oxidation of proteins either in solution before the electrophoresis or after blotting, using the method of Haselbeck and Hosel9 . Complex N-glycans with xylose, which appear in the Golgi apparatus during protein targeting, were evidenced using anti-carbohydrate antibodies and anti-rabbit IgG conjugated to alkaline phosphatase (Biosys S. A . , Fr.), as already described by Laurierel4. Other glycan structures were searched using lectins, conjugated to digoxigenin from the Glycan Differentiation Kit (Boehringer Mannheim, Ger.) or conjugated to alkaline phosphatase from E. Y. Laboratories inc. (San Mateo, CA.). Each lectin was assayed according to the recommendations of the manufacturers. Adapted controls were included in each experiment, to test the specificity of the reactions.

Figure 1 Detection of glycoproteins in storage protein fractions of cultivated bread wheats. Analyses ofprolamin (P) and glutenin (G) fractions of wheat cultivars: Capelle, Capitole, Chinese Spring and Courtot, separated with gradient SDS-PAGE; (a), polyacrylamide gel stained with Coomassie blue; (b), blot processed according to Raselbeck and RlJsel9 for detection of total glycoproteins; (c) immunoblot with anti complex N-glycans containing xylose antibodies.

81


Capelle CapitoIe C.Spring Courtot p

Mr (kOa)

G

P

G

G

P

G

P

1 16.3 97.4 66.3

a

HMW GS

-

-

GLIADINS

55.4

+

36.5 31 .0

LMW-GS

-

21.5 1 4.4

_

P

Mr

(kOa)

1 1 6.3 97.4 66.3

b

55.4

G

P

G

P

G

P

G

_

-

HMW-GS

-

GLiADINS

_

+

36.5 31.0

LMW-GS

_

21 .5 14.4

P

Mr

(kOa)

1 1 6.3 97.4

C

_

-

G

P

G

P

G

P

G

HMW-GS

66.3

-

55.4

-

GLIADINS

31 .0

-

LMW-GS

21 .5

-

+

36.5

1 4.4


82

4 RESULTS AND DISCUSSION

4.1 Assay of Gliadin and Glutenin Fractions Using Lectins Gliadin and glutenin preparations of hexaploid wheats were separated by SDS PAGE and blotted on PYDF membranes. Blots were overlayed with the following lectins: Galanthus nivalis agglutinin, which reacts with mannose present in high mannose N-glycans; wheat germ agglutinin, which reacts with the core mannose6( 1-4)N-acetylglucosamine6(1-4)N-acetylglucosamine characteristic of N glycans; Datura stramonium agglutinin, which binds specifically to galactose13(1-4)N acetylglucosamine in complex and hybrid N-glycans or N-acetylglucosamine in 0glycans; Dolichos bijlorus agglutinin specific of terminal non-reducing N acetylgalactosamine of both N- and O-glycans; Jacalin and peanut agglutinin, primarily specific of O-glycans terminated with 13-D-galactose or in the case of the peanut lectin, with the disaccharide galactose 13(1-3) N-acetylgalactosamine. No reaction was observed between these lectins and the proteins present in the extracts of the four wheat varieties tested (data not shown). These results differ from those of Tilley et al·s who observed reactions with Galanthus nivalis agglutinin and Donovan and Baldo7 who observed reactions with several lectins, among which only wheat germ agglutinin reacted more specifically.

4.2 Detection of Glycans Covalently Bound to Proteins Among Gliadins and

Glutenins

Gliadin and glutenin preparations, oxidized with �riodate and derivatized with digoxigenin-hydrazide, according to Haselbeck and Hose19, were separated using SDS PAGE and blotted onto PYDF membranes. Digoxigenin labelled proteins were detected on blots using anti-digoxigenin antibodies conjugated to alkaline phosphatase. Numerous protein bands were evidenced in both preparations (Figure 1 b) . Their molecular weights were similar to those of prolamins and glutenins. Except for HMW GS, their distribution upon SDS-PAGE did not match to the total protein pattern evidenced by Coomassie Blue staining (Figure l a), suggesting that they were minor components. These components differed from one variety to an other. They were genotype dependent. Some of them were common to several genotypes. It is noteworthy that for each variety studied, the bands evidenced in the prolamin fraction appeared also in the glutenin fraction, but to a lesser extent. The similarity of their pattern suggested that they corresponded to the same components, with unequal distribution between prolamin and glutenin fractions. These components were found also in gliadin or LMW-GS fractions further purified by chromatographic techniques (data not shown). This makes unlikely that they were unrelated contaminants. Work is in progress to determine to which storage protein sub-group(s) they belong.

4.2 Detection of Glycans Covalently Bound to HMW-GS HMW-GS were separated using linear SDS-PAGE, blotted and either stained with amido black or oxidized with periodate and processed according to Haselbeck and Hosel9. The figure 2 shows that all the HMW-GS of the four varieties, stained by amido black, could be oxidized and derivatized with digoxigenin hydrazide conjugate. This reaction is generally considered as characteristic of bound carbohydrates; it shows that HMW-GS behave as glycoproteins. The absence of N-glycosylation sites in the published sequences, suggests a probable O-glycosylation of these glutenins.

83


Mr (kDa)

1 2 3 4 1

2 3 4

1 1 6.3 97.4

66.3 a

b

Fi&ure 2 Direct detection on blots of glycans linked to HMW-GS of cultivated bread wheats: (a), blot processed according to Haselbeck and HiJseI9,' (b), blot stained with amido bklck; 1-4, wheat cultivars: (1) Chinese Spring, (2) Capitole, (3) Capelk, (4) Courtot.

4.3 Assay of gliadin and glutenin fractions using anti-xylose containin&-glycan antibodies Gliadin and glutenin fractions were blotted from gradient SOS-PAGE as previously, and overlayed with antibodies reacting with complex N-glycans modified with xylose14 that are characteristic of proteins targeted through the Golgi apparatus. Numerous glycoproteins were evidenced in the glutenin fraction of each variety (Figure 2). Their molecular weights were in the range of those of HMW-GS and LMW-GS. Much less glycoproteins was detected among prolamins. Some of them seemed to be specific of this fraction. They migrate preferentially in the alpha-, beta- and gamma gliadin region. Glycoproteins reacting with the antibodies are normally part of the total glycoproteins evidenced by digoxigenin hydrazide derivatization. More bands were observed in the glutenin fraction than by digoxigenin hydrazide derivatization, without evident matching. This suggested that antibody detection was even more sensitive than digoxigenin hydrazide derivatization, and that glycoproteins bearing complex N-gI)'cans with xylose are in very few amounts. This low level questions about the identity of these glycoproteins: are they isoforms of glutenin or gliadin polypeptides, with glycosylation sites in their sequences, or contaminating membrane proteins that often bear these types of epitopes? To answer to these questions, would bring new information abOut the diversity of proteins that participate to the formation of gluten. 5 CONCLUSION There are proteins in gliadin and glutenin fractions that show specific reactions of glycoproteins. Among them, the HMW-GS were clearly identified. Their lack of N glycosylation site leads to hypothesize an O-glycosylation of these proteins. The other proteins evidenced behaved as prolamins or glutenins. They were present in low level in the varieties studied. Some were conjugated to N-glycans with xylose, which means that they were processed in the Golgi apparatus. They were numerous in the glutenin fractions. More work is needed to precise to which protein sub-group they belong.


84

References 1 . C . W. Wrigley and J. A. Bietz, 'Wheat: chemistry and technology ' , Y. Pomeranz ed. , Am. Assoc. Cereal Chern . : St Paul, MN 3rd ed. , 1988, Vol. 1 , Chapter 5, p. 159. 2. J. E. Bernardin, R. M. Saunders and D. D. Kasarda, Cereal Chern. , 1976, 53, 612. 3. T. Terce-Laforgue, L. Charbonnier and J. Mosse, Biochirn. Biophys. Acta, 1980, 625 , 1 1 8. 4. M. J. Chrispeel s, Ann. Rev. Plant Physiol. Plant Mol. Bioi. , 199 1 , 42, 2 1 . 5 . G . Danno, K . Kanazawa and M . Natake, Agric. Bioi. Chern. , 1978, 42, 1 1 . 6. A. Graveland, P. Bosveld, W. J. Lichtendonk, H. E. Moonen and A. Scheepstra, l. Sci. Food Agric. , 1982, 33, 1 1 17. 7. G. R. Donovan and B. A. Baldo, l. Cereal Sci. , 1987, 6, 33. 8. K. A. Tilley, G. L. Lookhart, R. C. Hoseney and T. P. Mawhinney, Cereal Chern. , 1993, 70, 602. 9. A. Haselbeck and W. Hosel, Glycoconjugate 1. , 1990, 7, 63. 10. U. K. Laemmli, Nature, 1970, 227, 680. 1 1 . M. Lauriere, Anal. Biochern. , 1993, 212, 206. 12. V. Neuhoff, N. Arold, D. Taube and W. Ehrhardt, Electrophoresis, 1988, 9, 255. 13. J. M. Gershoni and G. E. Palade, Anal. Biochern. , 1982, 124, 396. 14. M. Lauriere, C. Lauriere, M. J. Chrispeel s, K. D. Johnson and A. Sturm, Plant Physiol. , 1989, 90, 1 1 82.

IDENTIFICATION OF DIMERS FORMED BY THE LOW MOLECULAR WEIGHT GLUTENIN SUBUNITS BELONGING TO THE D GROUP Masci S. l , Egorov T.A· l ,2 , Kasarda D.D. 3 , Porceddu E. l and Lafiandra D. l I Dipartimento di Agrobiologia ed Agrochimica, Universita della Tuscia, Via S.Camillo de Lellis, 01 100 Viterbo, Italy 2Group of Analytical Proteins and Peptide Chemistry,

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, UI. Vavilova 32, 1 1794 Moscow, B-334, Russia 3 U.S. Department of Agriculture, Agriculture Reserach Service, Western Regional Research Center, 800 Buchanan St. , Albany CA 94710

1 INTRODUCTION An increased amount of glutenin subunits is generally associated with improved quality characteristics because these subunits contribute positively to the amount and size of glutenin polymers. However, the presence of gliadin-type sequences in glutenin polymers of both breadl and durum wheat2 and the uneven number of cysteines found in some y- and a.-gliadin type clones3 , 4, 5 , makes it reasonable to suggest that these particular subunits may act as glutenin chain terminatorsl by having only one cysteine residue available for forming intermolecular disulphide bonds, and, in this way, having a negative influence on quality. For the same reason, D low molecular weight glutenin subunits, which have ro-gliadin type sequences6 , and likely possess only one cysteine residue7, may also contribute negatively to gluten quality. Because intermolecular disulphide bonds in glutenin polymers play a key role in quality and are poorly characterized, to some extent because of the great size of these aggregates, dimers of D subunits represent a simpler system for their study. Here we report a procedure for the identification and purification of dimers of D low molecular weight glutenin subunits.

2 MATERIALS AND METHODS Proteins were extracted from 50 g of flour of the bread wheat cv. Chinese Spring with 250 m1 of 70% ethanol with gentle stirring fot 2 hrs at room temperature, so as to have a starting fraction richer in the smallest polymers. No reducing agent was used. To confirm the presence of naturally formed dimers of D subunits, ImM iodoacetic acid (IAA) was once added in the extraction solution (70% ethanol), in order to avoid possible disulphide interchanges during stirring . Because the results were comparable, IAA was omitted in the successive experiments. After centrifugation at 10.000 RPM for 10 min, the supernatant was freeze-dried. Approximatively 3 g of protein were obtained (about 60% extraction). 300 mg of these proteins was solubilized in 60 m1 of a solution containing 4M Urea, and an ampholyte mixture (Pharmacia) that was 0.67%


86

ampholyte with pH range 2.5-4, 0.67 % ampholyte with pH range 4-6, and 0.67% ampholyte with pH range 5-7. This mixture was subjected to free flow isoelectrofocusing (Rotofor, Bio-Rad) for 4 hrs at 12 W power constant. Because D subunits are among the most acidic storage proteins, the dimers they form should be present in the most acidic fractions. 4 �l (5-30 �g) of each of the 20 Rotofor fractions was analyzed with a Mini SDS PAGE under both reducing and non reducing conditions. Fractions containing bands in the molecular weight range of D subunit dimers (about 100 Kd) were checked on two dimensional SDS-PAGE (unreduced vs. reduced) after extensive dialysis against O. I M acetic acid and freeze-drying. These fractions were also used to purify D subunits dimers by size exclusion (SE)-FPLC by using the column Protein Pak Glass 300SW (8.0 x 300 mm) (Waters). The eluent was 50% acetonitrile containing 0 . 1 % trifluoroacetic acid, and the flow rate was 0.45 mVmin.

3 RESULTS The ftrst Rotofor separation gave a ftnal pH range from 3.6 to 7 (Figure 1). The ftrst and the last fractions were not taken into consideration because they are in contact with the electrodes. C8 1

2'

3

..

5

6

7

6

9

10

CS 11 12 13 14 15 16 17 18 19

20

I

I pH lI....;ent 2.7 3.0 3.1 4.1 4.3 4.5 4.7 4.9 5.1 5,3

U U

1.0 6.2 6.4

""

.8

7.0 7.1

U

Figure 1 SDS-PAGE separation of the first Rotofor fractions of the 70% ethanol soluble proteins from Chinese Spring (eS) flour. In the upper panel, fractions have been reduced with added 1 % DIT. The first traclc in each gel represents the electrophoretic paltem ofthe total 70% ethanol soluble proteins of Chinese Spring as a reference. The pH value of each fraction is reponed at the bottom.


)

CS1

87

2 3 4 5 1 7 • • 10

CS11 12 13 14 11 11 17 11 1. 20

pH grIIdIent 3.3 ... 1 U .... 4.7 4.1 4.' 1.0 1.1 U 1.3

5.4 5.5 5.1 1.7 1.1

I.'

e.2 e.1 1.1 •.,

Figure 2 SDS-PAGE pattern 0/fractions obtained lJy re-submitting fractions 5-11 to Rot% r separation.

Unreduced

Figure 3 Two-dimensional (unreduced vs. reduced) SDS-PAGE separation o/proteins present in fractions 8-15 0/ the second Rot% r. The only proteins present below the diagonal have a mobility corresponding to D type 0/ low molecular weight glutenin subunits.


88

Because fractions 5- 1 1 were richer in D subunits, while low and high molecular weight glutenin subunits were present in a lesser proportion (see the reduced fractions), they were combined and re-separated by means of the Rotofor system, without adding further ampholytes. This procedure produced a narrower pH range (4.5-6.8), allowing a better separation. Figure 2 shows the SDS-PAGE pattern of this latter Rotofor separation. Fractions 8- 15 containined dimers of D subunits and ro-gliadins, as shown by two-dimensional SDS-PAGE (Figure 3) Separation of D subunits dimers from ro-gliadins was performed by SE-FPLC on the freeze-dried fractions 8-15. A typical chromatogram is shown in Figure 4 along with the SDS-PAGE pattern of the collected SE fractions.

0 ..J Q. Ll-t

W fn

1 �

(;:)

M e, "-

or, '-'J

,

z

Figure 4: In the upper panel, the size exclusion chromatographic separation of the same proteins used for the gel in figure 3, shows the separation of dimers from monomers. Peak 1, in fact, co"esporuis to dimers of D subunits, while peak 2 cOn/ains w-gJiadins (gel shown in the lower panel).

89


4 CONCLUSIONS The presence of dimers formed by the D subunits of low molecular weight glutenin in wheat endosperm was demonstrated by the presence of bands in the unreduced Rotofor acidic fractions, corresponding to a molecular weight of about 100 Kd.

These bands, after reduction, were identical in their electrophoretic migration to D subunits. Based on the observation that three D subunits are present in the bread wheat cultivar Chinese Spring, one coded by the B genome and two by the D genome8, we

expect six different possible dimers, corresponding to all the possible combinations of the

three bands.

The procedure here reported allowed to distinguish at least 4 bands in

the unreduced fraction. D subunits of low molecular weight glutenins are present only in those bread wheat cultivars with Chinese Spring-type ID-encoded oo-gliadins9 .

It bas been hypothesized

that they have a negative correlation with quality because they likely act as chain terminators of the glutenin polymers6, 9, 10 . Varieties possessing good bread-making

this The presence may favour the

properties have larger amounts of the more insoluble glutenin proteins, and insoluble

material consists of aggregates of higher

molecular weightll,12.

of chain terminator proteins such as the gliadin-like glutenin subunits,

decrease this ratio. The ability of D subunits to form the presence of dimers of D subunits might therefore contribute to the poor quality of the Chinese Spring-type bread wheat

formation of oligomers that

intermolecular disulfide bonds as indicated by

cultivars. Moreover, study of the disulfide bonds formation in D subunit dimers might contribute to the understanding of gluten

structure .

5 REFERENCES 1 . E.J.L.Lew, D.D. Kuzmicky and D.D. Kasarda, Cereal Chem. , 1992, 69, 508 2. Masci, E.J.L. Lew, D. Lafiandra E. Porceddu and D.D. Kasarda Cereal Chem. , 1995 72, 100 3. Okita, V. Cbeesbrough and C.D. Reeves, J. Bioi. Chem. , 1985, 260, 8203 4. Scheets and C. Hedgoth, Plant Sci. , 1988, 57, 141 5. D'Ovidio, M. Simeone, S. Masci, E. Porceddu and D.D. Kasarda Cereal Chem. , 1995, in press ,

,

,

Lafiandra, E. Porceddu, E.J.L. Lew, H.P. Tao and D.D. Kasarda Cereal Chem. , 1993, 70, 581 7. Masci, F. Buonocore, D.D. Kasarda, E. Porceddu and D. Lafiandra "Wheat Kernel Protein: Molecular and Functional Aspects", Universita della Tuscia,

6.

Masci, D.

,

,

C .N.R. , Viterbo, p. 2(J7 8. Jackson, L.M. Holt and P.I. Payne, Theor. Appl. Genet. , 1983, 66, 29 9. Masci, E. Porceddu and D. Lafiandra, Biochem. Genet. , 1991, 29, 403 10. Masci, E. Porceddu, G. Colaprico and D. Lafiandra, J. Cereal Sci. , 1 99 1 , 14, 35 1 1 . R.A. Orth and W. Bushuk, Cereal Chem. , 1972 , 49 , 268 12. R.B. Gupta, K. Khan and F. MacRitchie, J. Cereal Sci. , 1993, 18, 23.

COMPOSITION AND STRUCTURE OF GLUTEN PROTEINS

A. Graveland, M.H. Henderson, M. Paques, PA Zandbelt Unilever Research Laboratory V1aardingen P.O. Box 1 1 4 3 1 30 AC Vlaardingen The Netherlands

1 INTRODUCTION Wheat flour has many diverse end-uses, whether it is for human food, animal feed or industrial use. In companies, such as Unilever, in which the role of flour based and flour improving products is increasing rapidly, technological innovation is essential to safeguard long term growth. The basis for innovation is on the one hand, a better understanding of the product systems and on the other, sound knowledge of and response to market trends. This report relates to wheat flour as the raw material for bakery products and especially for bread. The complexity of this material will be examined and an attempt made to show how, using sophisticated techniques, the functional properties of flour can be determined, and how, on basis of that information, the correct choice of raw material can be made. Wheat flour is the preferred raw material for the production of yeast leavened bread because of its gas retention capacity. Doughs from other cereals lack the ability to retain gas because of the absence of gluten proteins. The main question is 'why does gluten have this property and why does it vary amongst wheat cultivars?' Both gluten quantity and quality are important. Reconstitution studies have shown that the gluten proteins are mainly responsible for quality differences amongst different varieties. The structural and physical characteristics of the glutenin fraction have been a particular focus for research l . To gain a better understanding of the structure-functionality relationships of gluten proteins, we have attempted to characterise the glutenin aggregates/polymers by establishing a new fractionation system, particularly for glutenin polymers. The different glutenin fractions were analysed by SDS-PAGE, gel filtration chromatography and reversed phase-high performance liquid chromatography (RP-HPLC). The structures of some glutenin fractions were also studied by transmission electron microscopy (TEM).

2 EXPERIMENTAL AND RESULTS 2.1

Isolation of Gluten Protein Fractions and Their Subunit Compositions

A fractionation procedure has been developed for the gluten proteins, during which no denaturation of the proteins occurs, in order to obtain information about the composition and structure of glutenin polymers.

91


First of all, the albumin and globulin fractions were extracted from flour using O. I M NaCI. After centrifugation, starch and dilute acetic acid were added to the wet residue and the mixture mixed into a dough. The pH of this artificial dough must be 3 .0. The mixing-step at pH 3.0 is necessary to disrupt all intermolecular interactions, such as non-covalent entanglements, between the glutenin polymers and to ensure their complete disaggregation. The mixed artificial dough was then suspended in dilute acetic acid at pH 3 . 5 . After centrifugation at 1 000 x g a precipitate of starch and acetic acid-insoluble glutenin (Res. 1) was obtained. Re-centrifugation of the supernatant at 20,000 x g resulted in a gel-layer (Res.2) at the bottom of the centrifuge tube. Both fractions (Res. 1 and Res.2) are designated here as 'gel-protein' because they form a gel in dilute acetic acid as well as in 1 .5% (w/v) SOS. Four glutenin fractions (Res.3, ResA, Res.5 and Res.6 6) were precipitated by adding sequential amounts of NaCI to the supernatant. Gliadin and aggregates, comprising low M, glutenin subunits, remained in the supernatant (Res.7). The subunit compositions ofthe fractions were analysed by SOS-P AGE under reducing conditions (Figure 1). Res.2 to 5 were glutenin polymers, which contained high M, glutenin subunits. Res.2 had a relatively high proportion of x-type high M, subunits and Res. 5 a relatively high proportion of y-type subunits. The proportion of medium M, subunits increased from Res.2 to Res.5. ResA and 5 contained small amounts of gliadin, but most of the gliadin occurred in Res.7. Res. 1 and 2, as well as Res.3 and 4, contained high-, medium- and low M, glutenin subunits. Although the four fractions had different subunit compositions, they were designated collectively as 'glutenin I'

Figure 1 SDS-PA GE patterns ofgluteninfractions I, II,

III and of the gliadinfraction


92

Res.5, containing a relatively high proportion of y-type high Mr subunits, was separable into two main fractions on a Sepharose CL-4B column, one containing high Mr subunits, especially y-type high Mr subunits, and medium Mr subunits, and the other containing only low Mr subunits (Figure 1). The glutenin in the first fraction was designated 'glutenin II' and that in the second 'glutenin III'. Res.6 6 and 7 comprised only glutenin III and gliadin, which were separable on a Sepharose CL-4B column. The ratios of high Mr subunits to medium Mr subunits plus low Mr subunits in the above mentioned glutenin fractions were determined by dissolving the fractions in 1 . 5% (w/v) SDS/5% (v/v) 2-mercaptoethanol and adding ethanol to a final concentration of 70% (v/v) to precipitate the high Mr subunits selectively; the medium- and low Mr subunits remained in solution. Protein in the fractions was determined as Kjeldahl nitrogen . . The glutenin I sub-fractions contained the highest proportions of high Mr subunits and glutenin II the lowest; glutenin III contained no high- or medium Mr subunits (Table I). The molecular weight characteristics of glutenin III (Res.6) were determined using 2D electrophoresis. The results demonstrated that this fraction comprised a restricted range of polymers differing in polymer size (results not shown). The subunits of glutenin III were identical to glutenin I low Mr subunits on the basis of their reactivities with monoclonal antibodies specific for low Mr subunits of glutenin I. These experiments showed that glutenin polymers could be differentiated into three distinct fractions: • Glutenin I: relatively high proportions of high Mr subunits, especially x-type (gel protein), insoluble in SDS • Glutenin II: relatively low proportions of low Mr subunits (mainly y-type), more medium Mr subunits and low Mr subunits • Glutenin III: only low Mr subunits and no medium Mr subunits or high Mr subunits

Table 1 Subunit Compositions of Glutenin Polymer Fractionsfrom Hereward Flour

Fraction

Molecular Structure

Subunit Composition High Mr (%)

Medium Mr (%)

Low Mr (%)

50 45 40

2

R2

R4

39

3 4

47 52 60 60

network network n.d. linear chain

Glutenin II R5

20

5

75

linear chain

Glutenin III R6 R7

--

1 00 1 00

spherical globules spherical globules

Glutenin I RI

R3

-

3

-

--

93

Wheat Protein Structure and Functionality 2.2

Structure of Glutenin Polymers

It is well-established that, the gluten proteins, and especially the glutenin polymers, determine the rheological properties of a dough. In 1 985 we proposed a model for the structure of glutenin polymers2. In this model of glutenin polymers the high M, glutenin subunits are linked together via head-to-tail interchain disulphide bonds to form a linear backbone. The low M, glutenin subunits form clusters, which are linked via disulphide bonds to the linear backbone. There is now considerable evidence to show that the composition and the relative proportions of the high M, glutenin subunits account for a substantial proportion of the variation in the breadmaking performance of wheat. It is plausible, therefore, to assume that the length of the glutenin polymer backbone may be determined by the type, composition and proportions of the high M, glutenin subunits),4. To obtain further insight into the structure of the different groups of glutenin polymers we carried out experiments using transmission electron microscopy (TEM). The different glutenin polymer fractions described above were suspended in a dilute acetic acid and examined by TEM using a negative staining technique (Figures 2, 3 and 4). Glutenin I fractions Res. I and Res.2 (gel protein), which had relatively high proportions of high M, glutenin subunits (see above), comprised a network of elongated strands with spherical globules alongside (Figure 2). These fractions therefore appeared to consist of a huge network or aggregate of polymers, in which the strands (backbone) consisted of high M, subunits and the spherical globules (clusters) low M, subunits. The main differences in the structure between the gel proteins from different flours were in the compactness and ;ize of the aggregates, and in the distribution of the globules along the strands.

Figure 2

rEM ofglutenin I polymers/aggregates or 'gel-protein ' (network structure)

.

94

Figure 3


TEM ofglutenin /-B (concatenous structure)

The Res.3 and 4 fractions of glutenin I had more linear structures (Figure 3). Those glutenin I polymers had the appearance of concatenations. It may be hypothesised that those glutenin I polymers are the precursors of the gel-protein polymer types. The glutenin I polymers may, therefore, be categorised into two different types: fraction A (Res. l + Res.2), polymers having a network structure (glutenin I-A) and a fraction B (Res.3 + Res.4), polymers having a linear chain structure (glutenin I-B). It is envisaged that, in a flour particle, glutenins I-A and I-B are likely to be interconnected very strongly with each other, forming large compact and folded aggregates. During dough mixing these aggregates become hydrated, disaggregated, unfolded and stretched. When the glutenins I-A and I-B in a dough are fully unfolded and stretched there will be an optimal intermolecular interaction between the glutenin polymers. It is further envisaged that, during the mixing of a dough, glutenin I-A, having a network structure, may be broken down into linear fragments (glutelljn I-B) and that, during subsequent resting of the dough, reassembly of glutenin 1-B takes place resulting in the re-formation of glutenin I-A. It could also be demonstrated by TEM, that the aggregates of glutenin I-A and B could have different conformations. Depending on the concentration of the glutenin I in a dilute acetic acid solution and depending on the pH or the presence of small amounts of salt, the large aggregates could be converted from a network structure into large compact spheres. The acetic acid soluble glutenin II, which contained a higher proportion of low M, glutenin subunits than the acetic acid insoluble glutenin I and a higher proportion of y-type high Mr glutenin subunits, had a linear structure of spherical globules, which were linked together (Figure 4). It is plausible to envisage that the spherical globules comprise low M, and medium M, subunits (which were shown to be present in glutenin II by SDS-PAGE: Figure 1 ) linked together by y-type high M,-subunits. The glutenin III fraction, which contained only low M, subunits, appeared in the TEM as discrete spherical globules that differed in size. The average diameter of those globules was about 20 nm, corresponding to a molecular weight in the order of 500,000. It is reasonable to hypothesise that the aggregates of glutenin I, as well as the acetic acid soluble glutenin II, are present in protein bodies in flour in the form of spheres and that, in a dough, they are unfolded and stretched as a consequence of mixing. It is also


Figure 4

95

TFM ofglutenin If (concatenous structure)

plausible that those different conformations of the gel-proteins are present in dough. Conformational changes in the gel proteins would undoubtedly have a big effect on dough properties. When the gel proteins in a dough are fully stretched, thus having a network structure, there would be an optimal intermolecular interaction between the glutenin aggregates. Directly after mixing of a dough, the gel-proteins are likely to be fully stretched. This would explain the relatively high maximum resistance of a developed dough to extension. During dough resting, the network structure of the gel proteins would return to a spherical structure. The gel-proteins with a spherical structure would have fewer intermolecular interactions, resulting in a dough with lower extensibility. It seems likely that the glutenin II polymers would be present in fuIly stretched linear form in a fuIly developed dough. 2.3

Variation in the Proportions of Glutenin Polymer Types Among Wheat Flours

The relative proportions of the different glutenin polymer types depended strongly on the type of flour (Table 2) The strong flour, Fresco, had a relatively high proportion of glutenin I (gel-protein), a lower proportion of glutenin III and even less gliadin, whereas the weak flour, Ritmo, had a relatively low proportion of glutenin I and higher proportions of glutenin III and gliadin. The medium strong flour, Hereward, was intermediate between those extremes in terms of the relative proportions of the different glutenin polymer types. 2.4

Effect of Different Glutenin Polymer Types on the Rheological Properties of Dough

Dough made from the strong flour (Fresco) had a much higher resistance to extension than a dough from a weak flour (Ritmo). Addition of glutenin I (1% on flour basis) to a dough of Ritmo flour had a large effect on the resistance of the dough. The addition of glutenin II had a far smaller effect, and addition of glutenin III did not increase resistance. In other words the proportion of glutenin I in any particular flour appeared to be the main determinant of the rheological properties of doughs made from that flour.

96


Table 2 Glutenin Polymer and Gliadin Compositions of Flours of Different Quality as a Proportion (%) of Total Gluten Protein

Fraction

Fresco

Hereward

Ritmo

R4

25 14 6 18

17 2 7 21

12 7 5 31

Glutenin II R5

7

6

4

Glutenin III R6 R7

3 2

3 2

4 2

Gliadin

25

32

35

Glutenin I RI R2

R3

2.5

Changes in the DilTerent Glutenin Polymer Types During Dough Mixing and Resting

Another essential factor determining the viscoelasticity of a dough is the size of the glutenin polymers. During dough mixing, disaggregation of the large aggregates, as well as breakdown (depolymerisation) of the fully-stretched glutenin I-A polymers to glutenin I-B polymers takes place. During resting, reassembly (polymerisation) of the glutenin I-A polymers from glutenin I-B polymers occurs. These processes also affect the changes in the rheological properties of a dough during mixing and resting. Table III shows the changes in the composition of gluten proteins as a consequence of mixing. The breakdown and reassembly of the glutenin polymers are due to the reductive cleavage of disulphide bonds and the reformation of disulphide bonds through re-oxidation of SH-groups, respectivell,6. These processes can be followed by monitoring the solubility of the glutenin polymers in SDS. The large acetic acid insoluble glutenin I-A fragments (gel proteins), which are present in the flour, are also insoluble in SDS. During mixing those polymers or aggregates become soluble and during resting they again become insoluble in SDSM The rate of breakdown and the degree of reassembly are important parameters in relation to gluten quality. Different flours have their own individual and characteristic breakdown! reassembly curves. In a dough made from a weak flour, there is rapid breakdown of glutenin I and hardly any reassembly, whereas in a dough made from a strong flour, the breakdown of the glutenin polymers is slow. Thus, the rate of breakdown and reassembly of the glutenin aggregates of a particular flour is an important parameter for characterising the quality of the gluten and also that of the flour. The degrees of breakdown and reassembly, respectively, during processing of a dough, determine the ultimate size of the glutenin I-A polymers and also the rheological properties of a dough. To obtain more information about the breakdown of glutenin I-A polymers,


97

the glutenin fragments from a overmixed dough were isolated and fractionated using the acetic acid dispersion and salt precipitation procedure described above. Overmixing resulted in the acetic acid-insoluble gel-protein becoming soluble in acetic acid. All the solubilised glutenin resulting from overmixing was precipitated in the first step of the salt fractionation procedure. The other fractions remain unchanged. Examination of the fractions by TEM confirmed that the glutenin I-A polymers were broken down to smaller glutenin I-B fragments and that the glutenin II and III polymers were unchanged. During dough mixing, therefore, only the acetic acid-insoluble glutenin I-A or gel protein is broken down into smaller linear glutenin I-B fragments. Using the same fractionation and analysis procedures, it was also shown that, during dough resting, those glutenin I-B fragments could be converted back into acetic acid-insoluble glutenin I-A polymers. It is likely that the structure of this reassembled glutenin I-A network will differ considerably from that of the original glutenin I-A network. In addition to structural changes in the acetic acid-insoluble glutenin I-A polymers during mixing and resting, the ratio and the size of the different glutenin polymer types plays an essential role in the intermolecular interactions between those glutenin polymers. The larger the glutenin I-A polymers are after reassembly during dough resting, the stronger the intermolecular interactions will be. Since the gas-retention capacity of a dough is determined mainly by the gluten network, it is evident that this is determined, on the one hand, by the concentration of the different glutenin polymer types and, on the other, by the conformation and the size of the glutenin I-A polymers after breakdown and reassembly in a fully developed dough. In general, wheat flours suitable for bread-making contain large glutenin I-A polymers, which are broken down rapidly, but also in those types of flour reassembly of the glutenin I-A polymers takes place rapidly. If breakdown proceeds too rapidly, it may easily lead to overmixing, and the formation of glutenin I-B fragments that are too small. This results in a very slack dough, which is difficult to handle, but with a reasonable gas-holding capacity. If the breakdown is too

Table 3 Glutenin Polymer and Gliadin Compositions oj Flour and Dough (Hereward) as a Proportion oj Total Gluten Protein Fraction

Flour

Dough

R3 R4

17 12 7 21

3 6 16 32

Glutenin II R5

6

7

Glutenin III R6 R7

3 2

4 2

Gliadin

32

33

Glutenin I R1 R2

98


slow or if there is no breakdown at all, this will lead to discontinuities in the gluten network and, in turn, to poor gas-holding capacity. 3 DISCUSSION

The baking performance of a wheat flour is primarily related to flour protein content and composition and dough mixing. Amongst other factors, adequate gluten quantity and quality are essential for the quality of bakery products. A generally accepted model for gluten in a fully developed dough is that of a framework of large glutenin polymers. The monomolecular gliadins are located between, where they act as plasticisers and weaken the intermolecular interaction between the glutenin polymers. Relationships between various quality parameters of gluten and end-use quality have been analysed here in attempts to understand the basis of the functional quality of wheat flour. A fractionation procedure, involving acetic acid extraction and selective salt precipitation, was used to produce a number of glutenin polymer fractions, which differed in subunit composition and size. The results of transmission electron microscopy experiments showed that the acetic acid-insoluble glutenin polymers, which contained high proportions of high M, subunits, had a network structure, whereas the acetic acid-soluble glutenin polymer fraction, which had a relatively high proportion of low M, subunits, has a concatenous structure. Addition of gliadin to a dough decreases the number of intermolecular interactions between the glutenin polymers, which results in a more viscous or extensible dough. Addition of glutenin, in contrast, increases the number of intermolecular interactions and makes the dough more elastic and less extensible. The effects of glutenin addition on dough properties were shown here to depend strongly on the type of glutenin polymer. Glutenin I aggregates had much more effect on the dough properties than glutenin III, the so-called low M, glutenin subunit clusters. Important structural changes occurred during dough mixing in the acetic acid-insoluble glutenin I-A polymer fraction such that those polymers were depolymerised to acetic acid-soluble glutenin I-B polymers. The other glutenin polymer types present in the original flour were unaffected. It is concluded that the quality of gluten, which in turn determines the viscoelastic properties of dough, is determined by (a) the proportions of the different glutenin polymer types, (b) the sizes of the different types of glutenin polymer, and (c) structural changes in the glutenin I (gel-protein) polymer type during processing.

4 REFERENCES 1 . P.R. Shewry, N.G. Halford and A.S Tatham, 1. Cereal SCi., 1 992, 15, \ 05 . 2. A . Graveland, P . Bosveld, W J . Lichtendonk, J.P. Marseille, J.H.E. Moonen and AJ. Scheepstra, 1. Cereal Sci. , 1985, 3, 1 . 3 . P.K.w. Ng, C. Xu and W . Bushuk, W . Cereal Chem. , 199 1 , 68, 3 2 1 . 4. L . Gao, P.K.w. Ng and W . Bushuk, W . Cereal Chem. , 1 992, 69, 452. 5. A. Graveland, Getreide, Mehl Brot, 1988, 42, 3 5 . 6. A. Graveland, P. Bosveld, W J . Lichtendonk and J.H.E. Moonen, Biochem. Biophys. Res. Commun. , 1980, 93, 1 1 89.

TIME-TEMPERATURE SUPERPOSITION FOR NETWORKS FORMED BY GLUTEN SUBFRACTIONS

Amalia Tsiami, Arjen Bot, Wim G.M. Agterof, Aris Graveland and Thijs Henderson Unilever Research Laboratorium Vlaardingen Olivier van Noortlaan 120 NL-3 133 AT Vlaardingen The Netherlands

1 INTRODUCTION

The rheological properties of a dough are of paramount importance for its performance during baking. For this reason, many investigators have studied the rheology of this material using complete dough systems. However, due to the complex nature of dough it is very difficult to obtain results which allow unambiguous interpretation in terms of microscopic or mesoscopic phenomena taking place in the dough. Another approach is the separation of the dough system into its components. This route has been followed by many investigators as well. One of the conclusions from their work is that the main component of dough, starch, does not gelatinize before the baking stage. After gelatinization, starch dominates the rheological behaviour of the baked product. Before gelatinization however, starch granules act as inert filler particles. The component that determines the rheology of the dough most before baking is the most abundant protein fraction, the so-called gluten. Gluten forms a class of proteins which customary is subdivided in glutenins and gliadins according to differences in solubility. In this paper, we will present our first results that relate the molecular weight of glutenin fractions to their rheological behaviour over a wide range of frequencies and temperatures. It will be shown that different fractions have different functionalities. Our approach is similar to that by Comec et al. , who studied the relation between molecular weight and rheological properties at room temperature. ! Such work is a first step to achieve a more quantitative relation between the physical properties of the gluten and the rheology of a dough. 1 . 1 Time-Temperature Superposition

A thermoreversible gel is a dynamic structure, that may behave differently depending on the time scale under study : a gel behaves as a viscous fluid on very long time scales and as a strong gel on short time scales. This is illustrated in Figure 1 , which shows the frequency dependence of the shear modulus G for a typical polymer system. The general interpretation of this dynamic rheological spectrum is that the modulus consists of two contributions: 2 (i) Glassy modes (or Rouse modes) at high frequencies. These are coordinated movements of several backbone atoms and bond rotations in a single polymer, and can be described by a power-law dependence on the frequency w: G' cx G" cx w(3·dr)12 , where df is

100


the fractal dimension of the polymer. 3

(ii) Network modes at low frequencies. There , a cross-over from viscous behaviour at low frequencies to elastic behaviour at higher frequencies can be observed. For thermorheologically simple materials, these modes are a function of the product of the frequency and a temperature dependent shift factor aT ' Different expressions for this shift factor have been proposed , the simplest of which is the Boltzman factor e-U/RT • Here U is the activation energy of the associations between network chains,

R is the gas constant and

T is the absolute temperature. In a dynamic rheological experiment only a limited frequency window is accessible experimentally . However, it is possible to shift this frequency window over the full spectrum shown in Figure 1 by performing measurements at different temperatures. This procedure is called time-temperature superposition and will be applied in the present experiments . 2 Experiments

on

synthetic

polymers showed that an increase

1

of the molecular weight of the

o

polymer results in a widening of the range of frequencies for which

-1

the polymer acts as a ge1.4,5 This is

-2

illustrated in Figure 1 . The rheolo

-3

gical behaviour of gluten may be

-4

pictured

as

behaviour weight

of

the

sum

narrow

subfractions,

of

.�

-5

the

molecular where

G"

-6

the

high molecular weight fractions

G'

L-��__��__��__��

-8 -7 - 6 -5 - 4 - 3 -2 - 1

0

2

will show a wider plateau than the low molecular weight fractions . In this paper we will proof this for fractions

of glutenin.

Since

no

phase transitions should occur to apply

time-temperature

super

Figure 1 Dynamic rheological spectrum for a polymer system. Solid lines, high molecular weight; dotted lines, low molecular weight (after Groot and Agterof3) .

position, we studied the effect of temperature on glutenin. This will give a better understanding of the thermal behaviour of wheat protein. Schofield and coworkers suggested that denaturation and setting of the protein ( - 50 °C) contributes significantly to the rheological properties of the dough, and the resulting final product.6•7 Changes between 75-90 °C are due to weakening of non-covalent bonds, with a reduction in G ' values.8 Above 90 °C there i s a n increase o f cross-linking o r polymerization of the protein.

2 MATERIAL AND METHODS Glutenin fractions were isolated from wheat flour (Hereward and Soissons) by washing out the water soluble proteins, dissolution of the remaining sample in a solution of acetic acid (pH 3 . 9) , and precipitation by sequential addition of NaCl.9 To standardize the ionic strength of different fractions, all samples were brought to a NaCI concentration of 0 . 03 molll afterwards . Precipitated protein was collected and stored frozen at -20 °C . Rheological tests were carried out using a Carrimed-500 CSL with cone and plate geometry

(diameter 60

thermocouple within

mm,

angle 2°) .

The

temperature

± 0 . 1 °C of the required temperature.

was

controlled

with a

For the superposition

experiments , the sample (Hereward) was equilibrated for 15 min at each new temperature ,

101


which was found sufficient because the modulus was stable for at least 1 h afterwards. A fixed peak strain of 0.03 was applied during a frequency sweep over the range 0 .01-30 Hz. Preliminary data showed that the linear range for stress-strain is up to a strain of 0.05 for glutenin subfractions. The particle size distribution was measured using a Malvern system 4700c sub-micron particle analyzer with a 128 channel 7032CE Multi 8 7032 CE Autocorrelator, a Spectraphysics laser model 127 operating at A =632. 8 nm and an output power of 25 mW, and a spectrometer goniometer with computer control. As a detector a Malvern PCS5 photomultiplier was used.


3 . 1 Size of Glutenin Subfractions Dynamic Light Scattering ,....., (DLS) demonstrated that the � 40 ,------, present fractionation method RS = separates the glutenins according to .g molecular size (see Figure 2) : *E polymers in class R3 have sizes 20 ell :a higher than 4 ILm; in class R4 sizes are in the range 0. 2-4 ILm; in fractions R5 and R6, which were o L-��L_�� collected with higher salt concen 101 102 103 tration, even lower molecular sizes were observed. Gel electrophoresis Particle size (nm) after reduction with mercapto ethanol showed that R3 contains higher amounts of high molecular Figure 2 Particle size distribution of glutenins weight subunits, while R4 contains according to light scattering data. more low molecular weight subunits. 9 60 To relate the relative ...... = abundance of these fractions to the 50 'd I\) strength of different flours, we ... :os carried out a fractionation for a b'o 40 strong (Soissons) and a weak -;; 30 '0 (Hereward) flour. The major ... 20 difference (Figure 3) was found � � between the occurrence of fractions '-' 10 R2 + R3 and R4. The gliadin � 0 content was not investigated. The R2/R3 R4 R5 R6 R7 strong flour had higher amount of high molecular weight glutenin and the weak flour a higher Figure 3 Concentration of each fraction to weak concentration of R4. Therefore, we (open column) and strong (hatched column) flour. will only describe the two most important fractions in the present

]

102


paper,

R3 and R4.

400 ,-------� 3.2 Effect of Heating on Glutenin To

apply

time-temperature

superposition it is show

that no

important to

phase

transitions

,-.. ..

�

C,

(,

is used. In Figure 4, a temperature

1

moduli for a glutenin fraction (R4, 12%

w/w)

is

presented .

At

a

G'

•

o

o •

. o

• o

•

• o

o

L--__�__��__�__�_____'

o

20

60

40

80

1 00

Temperature (OC)

temperature of 1 °C the glutenin has

..

•

10

occur in the temperature range that scanning of the storage and loss

•

0

100

> G" and with increasing

temperature

G'

and

G"

show the

4 The effect of heating of glutenin system (R4, 1 2 %). (Modulus has been extrapolated at zero polymer. However, at - 50 °C, G' peak strain, frequency 1 Hz), (e ) G ', (0) G ". is suddenly higher than G". This

expected behaviour for a melting

Figure

confirms that changes occur in the glutenin at higher temperatures. Some investigators found that the temperature at which structural changes of protein start is much higher, and that changes in the rheological properties at lower temperatures ( - 60 °C) were due to starch gelatinization.1O In the present experiment, however, starch gelatinization can be excluded as the origin of the changes because the starch content of the glutenin solution is only 0. 1 % . In addition, the temperature at which the changes occur is much lower than the gelatinization temperature of starch. Differential Scanning Calorimetric (DSC) studies did not show any thermal changes due to starch gelatinization or glutenin denaturation. Irreversible changes in the rheology of gluten at - 50 °C have been observed by other groups as well . 6•7, 1 1-13 To apply time-temperature superposition successfully, one should therefore distinguish two cases : (i) samples which have not been exposed to temperatures higher than 30 °C (called non-heated samples), and (ii) samples which have been heated at 70 °C for 1 5 min and measured at the temperatures indi-cated

(called

heat-treated

10'

samples) .

3 . 3 Non-Heated Glutenin Frac

tions R3 and In

103

R4

Figure

5,

a

102

dynamic

101

rheological spectrum obtained by

.-------�

,-_.. ..... ... .._ .... ... . . . . . .. . ..... • 40 °C which results in an increase of the rheologicaUy effective linkages. -

In our experiments we found an activation energy of network associations of 14-22 kllmol


lOS

for both glutenin fractions. This is considerably lower than the values obtained by Matsumoto, who found 1 25 kJ/mol from stress relaxation-time experiments. 14 It has been observed that the network of the heat-treated gluten R4 is weaker than the network of the R3 . Fraction R3 will give the elastic properties to the dough during baking that can support the lamellar films against the CO and water pressure, which will prevent 2 the collapse of the bread foam due to the breaking of the bubbles. This could be an explanation why a strong flour (rich in R3) does not collapse during the baking process. These results indicate the viability of the approach to relate the rheological properties of glutenin fractions to their molecular weight. However, further work on mixtures will be needed to be able to predict quantitatively the rheological behaviour of a dough from its gluten composition. Acknowledgement: We would like to thank L.L. Hoekstra for technical assistance and R . D . Groot for fruitful discussions. This work was supported financially by the E.E.C. Human Capital and Mobility Program.

References 1. 2. 3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 13. 14.

M . Cornec, Y . Popineau, I . Lefebvre, J. Cereal Sci. , 1994, 19, 1 3 1 . R.G. Larson, ' Constitutive Equations for Polymer Melts and Solutions' , Butterworths, London, 1988. R.D. Groot, W.G.M. Agterof, Macromolecules, 1995, 28 (in press). S. Onogi, T. Masuda, K. Kitagawa, Macromolecules, 1970, 3, 109. T. Masuda, K. Kitagawa, T . Inoue, S . Onogi, Macromolecules, 1970, 3, 1 16. I . D . Schofield, R.C. Bottomley, M.F. Timms, M.R. Booth, J. Cereal Sci. , 1983, I, 241 . I . D . Schofield, R.C. Bottomley, G.A. LeGrys, M.F. Timms, M.R. Booth, ' Gluten Proteins ' , eds. A. Graveland, I.H.E. Moonen, TNO, Wageningen, 1984, p. 8 1 . H . Levine, L . Slade, ' Dough Rheology and Baked Product Texture' , eds. H . Faridi, I.M. Faubion, Van Nostrand Reinhold, New York, 1990, p. 157. A. Graveland, M.H. Henderson, M. Paques, P.A. Zandbelt, Proceedings 'Wheat Kernel Proteins, Molecular and Functional Aspects' , Viterbo, 1994, p. 55. A . C . Eliasson, K. Larsson, ' Cereals in Breadmaking ' , Marcel Dekker, New York, 1993, chapter 7 , p. 325 . R. Bale, H.G. Muller, J. Food Technol. , 1970, 5, 295 . G. Attenburrow, D.I. Barnes, A.P. Davies, S.1. Ingman, J. Cereal Sci. , 1990, 12, 1. A.H. Bloksma, Cereal Foods World, 1990, 35, 237 . S. Matsumoto, ' Food Texture and Rheology ' , ed. P. Sherman, Academic Press, London, 1979, p. 291 .

THE ROLE OF GLUTEN IN THE HEAT-INDUCED CHANGES THAT OCCUR IN DOUGH RHEOLOGY DURING BAKING

A. Nakonecznyj *, S. 1. Ingman* and 1. D. Schofieldt *Unilever Research Colworth Laboratory, Colworth House, Shambrook, Bedfordshire MK44 I LQ, UK and tThe University of Reading, Department of Food Science and Technology, PO Box 226, Whiteknights, Reading RG6 6AP, UK.

I INTRODUCTION Wheat flour dough is a viscoelastic material ' . During baking, chemical and physical interactions occur at the molecular level to cause changes in a dough's viscoelastic nature thus influencing final baked product structure. The temperature-induced interactions have been attributed to starch gelatinisation2,3 , protein crosslinking4,5 and water redistribution between starch and protein fractions4,6. Although these processes may occur concurrently during heating, the quantitative contribution each of them makes to the rheological properties of a dough is not known. The aim of this work was to determine how starch free gluten crosslinks during heating in order to assess how that process contributes to the rheological properties of dough. A method of preparing dough samples with varying protein/starch (PIS) ratios was adopted, which retains the main dough structure, causing minimal processing and damage. Previous experiments have been cited7.' 2 describing methods for reconstituting doughs by mixing commercially or laboratory prepared wheat protein and wheat starch. Such reconstituted doughs may not be truly comparable to original, native doughs, and may not achieve a homogenous gluten network. Dough and gluten rheological characteristics were evaluated as a function of temperature, using dynamic mechanical testing at small deformation. Gluten-protein crosslinking on heating was also monitored by SDS extractability methodsI 3 , and correlated with rheological data. 2 MATERIALS AND METHODS 2.1

Dough Sample Preparation

Dough from single variety, Mercia wheat flour (62% water absorption) was mixed in a Brabender Do-Corder to optimum development as measured by peak torque. Test samples of varying PIS ratios were prepared by progressively washing out starch from the dough (Fig. I ). Samples were taken at various stages of washing out and were frozen and freeze dried. Freeze-dried samples were milled to 'flour' using a Retsch mill (250 mm screen) and re-hydrated to 'doughs' on the Brabender Do-Corder by mixing until peak torHue. Protein content (N x 5 .7) was determined using the Foss-Heraeus Macro N technique ' 4 . 2.2

Rheological Measurements

Measurements of rheological changes during heat setting were performed on doughsl glutens of varying PIS ratio by heating from 25-JOO°C in a Rheometries RDA II rheometer. Measurements were made using parallel plate geometry with a gap setting of

107


•

RETAIN MAIN DOUGH STRUCTURE, ElJT INCREASE PIS RATIO

•

MINIMAL PROCESSING AND DAMAGE

ROUR +

WATER

•

WASHING OUT STARCH

DOUGH

I

l' +

STARCH WASHING

Figure 1

1

FRACTION

1

I

I

1 FRACTION +

STARCH WASHING

2

I

1, I FRACTION 3 I

2

+

STARCH WASHING

3

I FRACTION n I +

STARCH WASHING n

Schematic diagram ofthe methodfor preparing samples ofdifferent PIS ratio

2-3 mm. The strain was 0.2% and the frequency I Hz. After loading onto the rheometer, samples were allowed to relax for 30 min prior to making the measurements. A heating rate of 1 .5°C/min was used plus a 'soak' time of 30 s. Temperature is controlled by the use of a convection oven in the Rheometrics rheometer. Thus, a Moisture Loss Reduction (MLR) chamber was built to reduce drying out of samples at high temperatures (Fig. 2).

Oven

Dough Sam ple

011

o < .. "

Oven

Oscillation

Figure 2

Schematic representation ofthe Moisture Loss Reduction chamber (MLR)

108


2.3

SDS Extractability of Protein

Mercia dough samples were placed between two metal plates separated by a rubber gasket, with a thin thermocouple inserted into the dough. The samples were rested for min and then uniformly heated to a range of temperatures between - 1 00°C. Once the sample had reached the desired temperature, as measured by the thermocouple, it was removed from the plates and quenched into liquid N2. Samples were freeze-dried and milled. Milled samples g) were extracted with SDS solution w/v; mL) for h with occasional stirring. The mixture was centrifuged min at rpm), and the UV absorbance of the supernatant was measured at 280 nm, against a SDS blank 1 3. The extractability of the dough protein was determined from a calibration curve constructed from unheated Mercia dough sample.

30

40

(2%, 20 30,000

(20

3 3.1

(0. 1

24

RESULTS AND DISCUSSION

Rheological Measurements on Dough Heat Setting

3�

On heating Mercia dough has shown an increase in G' above 50°C (Fig. indicating the occurrence of a major physicochemical process(es), as previously reported . At higher PIS ratios, this rapid G' increase - 80°C) was considerably reduced, implying that the G' change is predominantly due to starch (gelatinisation). Although, the G' increase from 50°C was not as prominent for gluten-like samples (PIS ratios > 1 ) in comparison to dough, G' was observed to increase quite sharply at temperatures near and above 70°C (Fig.

(50

3 ).

200,000 100,000

�

., ::;) oJ ::;) 0 0 :E (J >=

� w

50,000

10,000

----- ------

.

_

-·

�

.

·-

--

--. .· . '

'* • • * . . . . .. . . . . . • 20

OOUGH 1

___

3.2

---------- - -

.

2,000

Figure 3

--

0 , · " . - - o-c'* - - - - - - - - -'- ·

® .�" .. ·l- :�- . . !Q} * � * '* .

5,000

1 ,000

- - .- - -

--------------

20,000

··t·

� DOUGH 2

____

..

DOUGH 3

0

50

. ..

-

. •

TEMPERATURE ('e)

DO� "

U

DOUGH 5

.*-

•

•

.

50

DOUGH 6

•

roo DOUGH 7

--

DOUGH 8

120

•

Temperature induced changes in G ' ofdoughs/glutens varying in PIS ratio

Theoretical Prediction of Gluten Heat Setting

In order to predict the contribution of gluten on dough heat setting properties G' values


109

for PIS doughs, at selected temperature intervals, were plotted against protein content (Fig, 4), Data between 40 and 60°C nicely fitted a power regression curve in the form of:-

b y = ax while data = 70°C conveniently fitted exponential curves in the form of:

y = aix y = G' modulus values; x = protein content; a, b = constants for selected isotherms,

where:

100,000

Ii

�

•

30,000 �Cl, - - - - -ti - - - - - - - - - - - - - �-�:

10,000

2 U �

. . .. >',*:

_

3,000

!w

1 ,000 300

20

0 4O'C

Figure 4

5O'C

-0 ..

40

TOTAl % PROTEIN (calc, at 0%

8O'C G

70'C

---

*- - -

60 mOisture) SO'C

.. .

. . . -

80 9O'C

----

100

100'C

Regression analysis ofG ' versus dough/gluten protein content

A regression analysis based on the data was carried out to predict a theoretical heat setting curve for pure gluten (Fig, 5), The results of this analysis confirmed that the G' increase above 70°C was due to gluten, 3.3

Protein Crosslinking as Determined by Extractability in SDS Solution

Extractability in SDS, which was used as a measure of temperature-induced protein cross linking in doughlgluten 1 3 , began to fall at about 70°C (Fig, 6), Above 70°C less protein was extracted, indicating that crosslinking had probably taken place, These observations complement those on rheological changes in G' on heating, 4 CONCLUSION

By progressively lowering the starch content of a dough by means of the washing procedure adopted here, it was possible to produce doughs of varying protein/starch (PIS)

1 10


1,000,000

!

100,000

§:

Ul ::::> ...J ::::> C

�

0 >=

10,000

5w

I

._---- ----.--

1,000 20

40

60

TEMPERATURE

('c)

80

100

120

Predicted theoretical effect ofheat on pure gluten

Figure 5

100 <J> 0 <J> a;

�

�

�

w z

�'" Q, :I:

80

g

40

z 0 ;:: '" 0 Q,

20

0 0

is �

� Q,

•

60

a

Figure 6

20

40

60

TEMPERATURE

80 ('c]

100

120

Changes in the SDS extractability of dough/gluten protein after heating to du.Terent ten1peratures

ratios, while retaining a dough-like structure with a continuous gluten network, These samples were then used to predict gluten heat setting rheology, Using a regression analysis based on the rheological data of the doughs of increasing PIS ratio, the


111

rheological changes during heat setting o f pure gluten were determined. The main heat setting rheological changes were shown to commence at -70°C and to continue increasing to temperatures beyond 95°C. References 1. 2. 3. 4. 5.

6.

7. 8. 9. 1 0. 1 1. 12. 13. 14.

R . K. Schofield. and G. W. Scott Blair, Proc. Roy. Soc. (London), 1 932, 138, 707. A. H. Bloksma and W. Nieman, J. Texture Studies, 1 975, 6, 343. P. C. Dreese, 1. M. Faubion and R. C. Hoseney, Cereal Chern. , 1 988, 65, 348. G. A. LeGrys, M. R. Booth, and S. M. Al-Bagdadi, In 'Cereals, a Renewable Resource', Eds L. Munck and Y. Pomeranz, American Association of Cereal Chemists, St. Paul, Minnesota, 1 980, p. 243. R. Bale and H. G. Muller, J. Food Technol. , 1 970, 5, 295. A.-C. Eliasson, J. Cereal Sci. , 1 983, 1 , 1 99. G. E. Hibberd, Rheol. Acta, 1 970, 9, 50 1 . J. R. Smith, T. L. Smith and N. W. Tschoegl, Rheol. Acta, 1 970, 9, 239. A. S. Szczesniak, 1. Loh and W. R. Mannell, J. Rheol. , 1 983, 27, 537. L. L. Navickis, R. A. Anderson, E. B. Bagley and B. K. Jasberg, J. Texture Studies, 1 982, 13, 249.

A. Abdelrahman and R. Spies, In ' Fundamentals of Dough Rheology', Ed. H. Faridi and J. M. Faubion, American Association of Cereal Chemists, St. Paul, Minnesota, 1 986, p. 87. S. Cavella, L. Piazza and P. Masi, Ita!' J. Food Sci. , 1 990, 4, 235. 1 . D . Schofield, R . C. Bottomley, M . F. Timms and M . R . Booth, J. Cereal Sci. , 1 983, 1, 24 1 . D. Smith, Analytical Proc. , 1 99 1 , 28, 320.

1.

BIOCHEMICAL CHARACTERISATION OF WHEAT FLOUR PROTEINS USING GEL CHROMATOGRAPHY AND SDS-PAGE

E.L. Sliwinski1.2, T. van Vliee & P. Kolster' , ATO-DLO, P.O.Box 1 7, 6700 AA Wageningen, the Netherlands 2 WAU, Dairying and Food Physics Group, P.O.Box 8 1 29, 6700 EV Wageningen, the Netherlands

INTRODUCTION The study of wheat gluten proteins is hampered by the poor solubility of these proteins. To overcome this problem, often sonication in combination with appropriate buffers is used, although it is known that with this technique covalent bonds in proteins could be broken. A method has been optimized to extract total protein from wheat flour without the use of sonication or reducing agents. Results of a study will be presented in which the dissolved gluten proteins are separated by gel chromatography and the composition of fractions is determined (HMWILMW glutenin subunit-ratio, presence of disulfide bonds). 2

MATERIALS AND METHODS

A sample of the wheat variety Soisson was obtained from Meneba BV. Soisson is a wheat variety with good breadmaking potential and strong dough properties (table 1 ) .

Table 1 Some properties of Soisson flour protein content (% dry matter) damaged starch (% dry matter) ash content (% dry matter)

l OA 7.6 0.46

Ext: H (BU' s) Ext: L (BU's) Ext: A (BU's)

590 1 56 1 25

Proteins were extracted from flour and from freeze-dried dough in a 2% SDS/50 mM TrislHCI, pH 8.0 buffer (Bottomley et aI, 1982). The flour was carefully suspended in this buffer and left gently shaking for 24 hrs. After centrifugation the dissolved protein was studied by gel chromatography using a Superose-6 column, SDS-PAGE and laser-scanning densitometry.

1 13

Wheat Protein Structure and Functionality 3

RESULTS

From the defatted flour 92% of the total nitrogen was extracted. 94% of total nitrogen was extracted from defatted flour after a mixing and freeze-drying procedure.

AU's

25 20 15 10 5 0 -5 20

25

30

35

40

45

50

55

time in min

Figure 1 A typical elution pattern of extracted Soisson flour protein M

2

3

4

5

6

7

8

9

10

11

12

M


1 14

Figure 2 SDS-PAGE-pattern of fractions obtained by gel chromatography of Soisson protein. Upper photo: unreduced samples; lower photo: reduced samples. Numbers refer to fractions. Using SDS-PAGE under unreduced conditions large protein polymers are shown that cannot enter the gel or result in a smear (figure 2., upper foto). The protein composition after reduction with B-mercapto-ethanol of these fractions is shown on the gel on the lower photo. Using this gel the HMWILMW-ratio of the glutenin subunits of the separate fractions obtained by gel chromatography is determined by laser-scanning densitometry. Table 2 Ratio between HMW and LMW glutenin subunits

fraction HMWILMW-ratio

0.36

2

3

4

5

0.34

0.26

0.20

0.22

It can be concluded that the higher the molecular weight of the polymeric proteins the higher the HMWILMW-ratio.

4

CONCLUSION

These results show that for a successful interpretation of elution-patterns the use of SDS-PAGE in combination with laser-scanning densitometry is very useful. With this method the ratio between groups of gluten proteins of flour and gluten of various varieties with similar protein content and a large range in dough properties will be studied. Bottomley et aI, 1. Sci. Food Agr., 1 982, 33: 48 1 -49 1 .

Wheat Protein Composition and Quality Relationships

STRUCTURAL DIFFERENCES IN ALLELIC GLUTENIN SUBUNITS OF HIGH LOW Mr

AND AND THEIR RELATIONSHIPS \-\lITH FLOUR TECHNOLOGICAL

PROPERTIES

D. Lafiandra l , S Masci l , R. D'Ovidio l , T. Turchettal, B. Margiotta2 and F. MacRitchie3 IDepartment of Agrobiology and Agrochemistry, University of Tuscia, 0 1 1 00 Viterbo, 2Germplasm Institute, C.N.R., Bari, Italy 3 C . S.I.R.O. Division of Plant Industry,

Italy

North Ryde, Australia

1 INTRODUCTION It is widely accepted that glutenins, which are polymeric proteins whose subunits are held together by disulfide bonds, are the major determinant of dough strength and elasticity l,2; when disulfide bonds are broken by reducing agents and the resulting mixture separated on SDS-PAGE, two groups are found which have been termed high- and low molecular weight glutenin subunits, being encoded by genes at the complex loci on the long and short arms of the homoeologous group 3, respectively3,4.

1 chromosomes and designated Glu-1 and Glu-

Correlation studies have stressed the relative importance of certain subunits compared

to others, but the mechanisms by which certain allelic subunits confer superior dough properties is not fully understood and is matter of intensive investigations. Qualitative effects may be related to differences in the amount of subunits produced by' the different alleles or result from differences in their structure which can affect their ability to form polymers with other high or low Mr subunits.

Major structural features, of both high and low Mr glutenin subunits, which are

supposed to play a role in determining allelic differences are number and position of cysteine residues and the presence of a repetitive domain; these aspects will be reviewed in this presentation.

2

STRUCTURAL CHARACTERISTICS OF HIGH Mr GLUTENIN SUBUNITS

Bread wheat cultivars possess . from three to five high Mr glutenin subunits, as

determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-

PAGE): one or none encoded at the Glu-A l complex locus, one or two at the

GJu-Bl

locus and two at the Glu-Dl locus. Molecular analyses have indicated that each complex locus contains two tightly linked genes, one encoding a higher Mr subunit designated

as x

type and the other encoding a lower Mr y-type subunit. Absence of subunits in some cases

has been proved to be due to gene silencing. DNA sequencing of genes encoding high Mr

glutenin subunits has revealed structural features of these proteins, in fact the presence of three distinct structural domains has been reported in both x- and y-type subunits: a central repetitive domain flanked by non repetitive N- and C-terminal domains5-7


118

1

2

3

4

5

15

7

8

8

10

Figure 1 S1JS-PAGE separation of high Mr glutenin subunits on 10% concentration gel (upper) and 10% concentration gel containing 4M urea (lower).

x- and y-type subunits differ in number and type of repeat motifs. Both contain in fact hexa- and nona-peptides, but only x-types possess tripeptides. Analyses by polymerase chain reaction

(peR)

of novel subunits of unusually high

Gill-A 1

Mr

present at the

Glu-DI

and

loci have demonstrated that the repetitive central domain is responsible for observed size variation8 .9 This supports a previous suggestion that the repetitive block structure of the central domain provides the basis for a more rapid evolution and divergence by duplication and or deletion of whole blocks, or several blocks of residues, by unequal crossing over, whereas unrepetitive domains evolved by a combination of single amino acid substitutions and small insertion and/or deletions ! ! . According to Tatham et aL ! 2 these repetitive sequences appear to form a loose spiral supersecondary structure which is based on repeated f3-turns, and this spiral nay be

intrinsically elastic and responsible for gluten elasticity.

1 19


Table 1 Characteristics ofhigh Mr glutenin subunits§ SuhlUlit

Cultivar

Dx5

Cheyenne Hope Yamhill Cheyenne Cheyenne

Axl

Dx2 Ax2*

Bx7

Molecular Weight

Nwnber of residues

N-terminal

Domain

Repetitive

Domain

C-terminal

Domain

Cysteine residues

88128

827

89

696

42

5

87680

809

86

681

42

4

87022

817

88

687

42

4

86309

794

86

666

42

4

82865

770

81

647

42

4

Bx 1 7

L86-69

80750

734

81

61 1

42

4

By9

Cheyenne Ch. Spring Cheyenne Cheyenne

735 1 8

684

104

538

42

7

68650

639

1 04

493

42

7

67476

627

1 04

481

42

7

63027

581

1 04

435

42

6

Dyl2

DyIO Ay (silent)

§Modified from Shewry et aJ7; data for subunit

1 7 are from Reddy and AppelslO

SDS-PAGE separations, have been extensively used to characterize different high

Mr 1 ) and to assess their relationship with flour breadmaking properties. DNA sequencing of high Mr glutenin subunit genes have made it possible to deduce correct molecular weights of corresponding subunits (Table 1 ). Such data have indicated that molecular weight of high Mr glutenin subunits, as determined

glutenin subunit alleles (Fig.

by their relative mobility in SDS-PAGE, are overestimated; additionally discrepancies between the migration of certain subunits and their molecular weight have also been

observed. For instance the migrations of allelic pairs I Dx2I 1 DxS and I Dy I O/1DyI2 have been shown to be anomalous13 In fact subunit I DxS has higher mobility than the smaller allelic subunit I Dx2; similarly subunit l Dy l O has lower mobility than the larger subunit I Dy I 2. Other discrepancies have appeared with the complete sequences published for genes corresponding to subunit 1 and 2* encoded at the Glu-A l locus. Mobilities on SDS PAGE of these two components are in fact slower than subunit S encoded at the Glu-Dl locus (Fig. 1 ), though molecular weight of this latter is larger than subunits 1 and 2*' Goldsborough et aP4 suggested that anomalous relative mobility of subunits 10 and 1 2 i s due to conformational differences between the proteins, because the anomalous behaviour is destroyed by the addition of a strong denaturant, such as 4M urea, to the SDS-containing gels. Even though the relative mobility of pairs SI2 and 1 0/ 1 2 is correct when 4M urea is added to SDS-PAGE, anomalies have been observed for other subunits like subunits I and 2 1 5 Moreover under the same conditions By-type subunits migrate faster than Dy subunits though the Mr of the former is larger than the latter (Fig. I ). This

suggests that full denaturation for all high Mr subunit is not accomplished in 4M urea, and

that conformational differences between allelic subunits still exist. In accordance with this Field et al. 16 reported that subunit 20 was incompletely denatured in 6M guanidinium chloride, though denaturation appeared complete when a stronger chaotropic agent, such as guanidinium thiocyanate was used. Conformational changes can be observed in detail using transverse gradient gel electrophoresis (TGGE) in which a gradient of urea, from 0 to 8M, perpendicular to the direction of migration, is formed 1 7 (Fig. 2). Observation of these separations shows differences between x- and y-type subunits, and helps in clarifying some of the anomalies observed in one dimensional gels with a constant 4M urea concentration.

1 20


o

U R E A•

8M P 1 20

Figure 2 Transverse gradient gel electrophoresis of high Mr glutenin subunits present in

two different bread wheat cultivars

Unfolding patterns, as seen on TGGE, are different for x- and y-type subunits. It appears that x-type subunits have a slow and continuous change of mobility across the urea gradient whereas y-type have a sudden change as urea concentration increases. The mid point of the unfolding transition is different for Dy and By subunits and this accounts for anomalous mobilities observed between these homeoallelic subunits in 4M urea. The unfolding pattern reveals that as urea concentration increases, the mobilities of subunits 1 0 and 1 2 are strongly affected becoming slower compared to larger subunits 7+8 and 1 7+ 1 8 present at the Glu-Bl locus. Further increase i n urea concentration results in these latter alleles being more affected in order of mobility in accordance with molecular weight of the subunits. A peculiar behaviour is seen for subunits 1 and 2*. Both reverse their migration compared to subunits 4 and 5 as urea concentration increases, but whereas the order is in agreement with molecular weights for the pair 5 and 2* this is not the case for pair 4 and I , the latter being larger than the former. From these observations it i s difficult to firmly assess what are the factors for different conformational changes' among different allelic subunits. Several features of high Mr glutenin subunits can affect conformation and their differential behaviour under denaturing conditions and in presence of SDS . As mentioned

FIG

121


earlier, a striking difference between x- and y-type subunits is represented by the presence of a tripeptide motif which is present only in the former type of subunits but not in the latter; also the number of repeats is different between allelic subunits. This variation can affect and stabilize certain conformations compared to others, resulting in differential behaviour of certain alleles toward strong denaturating agents. The other important feature of high Mr glutenin subunits is the number and distribution of cysteine residues as they play major role in the formation of glutenin polymers. Three and five cysteine residues are present in the N-terminal region of x- and y-type subunits respectively, whereas one only is present in the C-terminal part of the molecule. In addition an extra cysteine residue is present in the repetitive domain toward the C-terminal region in y-type subunits, except Ay-type subunits, and near the N-terminal region in the Dx type subunit 5 . Recent studies have demonstrated that variation i n the number of cysteine residues of high Mr glutenin subunits can occur and is detectable by RP-HPLC separations. In fact comparative analyses of reduced and reduced and alkylated subunits, using 4-vinylpyridine as alkylating agent, revealed a differential effect of the alkylation on proteins encoded at different loci and on x- or y-type subunits, according to their different number of cysteine residues1S, Such analyses on subunits encode.d at the Glu-Bl locus, allowed Margiotta et aI· 18 to postulate that subunit 20 would have a lower number of cysteine residues, on the basis of its chromatographic behaviour compared to subunits 7 and 1 7 which have been shown to possess four cysteine residue (Table 2). This was supported by results of Tatham et al. 19 who reported, on the basis of N-terminal studies, that two cysteine residues present in the N-terminal region of subunit 20 had been replaced by two tyrosine residues and postulated that subunit 20 possess only two cysteine residues. The same chromatographic studies also gave strong support to the existence of a By-type subunit associated with subunit 20 which was termed 20y. The number of cysteine residues in subunit 20 was recently confirmed and their relative positions assessed20,21 The presence of one cysteine residue in the C- and the N-terminal regions was established (Fig. 3).

Table 2 Retention times (min) oj reduced and reduced and alkylated high Mr glutenin subunits encoded at the Glu-B I locus. Time difference was calculated (a) and this difference was expressed as % oj the retention time ojreduced subunits (b)

SublUlit

Reduced

Reduced Alkylated

Time difference (8)

% (b)

Cysteine number

7

67.9

35.9

32.0

47.1

4

17

68.9

36.5

32.4

47.0

4

13

61.1

30.9

30.2

49.4

50.0

34.7

1 5.3

30.6

50. 1

42.4

7.7

1 5.4

6

20

n.d. n.d. 2

1 22


Dx5

�

1

.... fWpetltlYe 1Iom.1n �� I--�-----------------------------� � �

7

7m

1

�7 �� Bx20 �-----�----'�__ __ __ __ __ __ __ __ __

1

m

Figure 3 Representations of high Mr glutenin subunits Dx5, Bx7 and Bx20. Linked bars represent intramolecular disulfide bridges

3

EFFECT

ON

QUALITY

CHARACTERISTICS

OF

STRUCTURAL

DIFFERENCES IN HlGH Mr GLUTENIN SUBUNITS

It appears always more evident that cysteine residues play a major role in affecting dough viscoelastic properties. At the

Glu-D1

locus the two allelic pairs

5+ 1 0

and

2+ 1 2

have been associated with good and poor technological properties respectively. The absence of recombination between the two pairs has prevented any conclusion about the relative importance of the x- and the y-type subunits. Comparison of the DNA sequences of subunit

10

and

12

led Flavell et al 22 to suggest that a more regular pattern of J3-turns in

the central repetitive domain of subunit

as a consequence of a higher proportion of

10,

consensus-type repeats, was responsible for providing better elastic properties to this subunit with a consequent effect on dough elasticity. On the other hand Greene et aI . 1 3 in considering the difference between the pairs

2/5

on the one hand and

pointed out that the additional cysteine residue present in subunit

2

5

1 01 1 2

on the other,

compared with subunit

would affect the dough system more profoundly since it would promote a differential

cross-linking, thus endowing dough with increased strength. Very recently Gupta and MacRitchie2 3, using several biotypes and a set of recombinant inbred lines, showed that superiority of the allelic pair locus and of

1 7+ 1 8

over

20

at the

Glu-B1

5+ 1 0

over the

2+ 1 2

at the

Glu-D1

loci was essentially due to the significantly

greater size distribution of polymeric proteins, associated with

5+ I 0

and

1 7+ 1 8,

as

indicated by the proportion of % of unextractable polymeric protein in the total polymeric protein and confirmed that dough strength is primarily controlled by the proportion of the larger sized or unextractable polymers. These authors reported that there was no difference in the quantities of the pairs

5+ 1 0

vs

2+1 2

and

1 7+ 1 8

vs

20

(now known to be made of

two subunits) in the total reduced polymeric proteins, suggesting that contrastating effects of these subunits are caused by a factor other than quantitative differences between them. The observed differences in polymerizing capacity of the pair

2+ 1 2

5+ I 0

compared to the pair

has to be ascribed to differences in their structure; e.g., the extra cysteine residue of

subunit

5 vs 2 as suggested by Greene et

al. 13

A similar explanation can be offered to explain the results obtained comparing pairs

1 7+ 1 8 vs 20.

As subunits

18

and

20y

should be structurally very similar, like all the y-type

subunits, the observed qualitative differences, between the pair

1 7+ 1 8

versus the pair


1 23

20+2Oy, are most likely the result of structural differences between subunits 1 7 and 20. The most important difference between the two pairs of subunits is the absence of two cysteine residues in subunit 20 compared to subunit 1 7+ 1 8, in particular, the former lacks the second and the third cysteine residue in the N-terminal region. This, as also speculated for subunits 2 and 5, might have a profound effect on glutenin polymer formation and on their size distribution. Recently, Kohler et al 24 found that in x-type subunit 7 the first and the second cysteine residues are involved in an intra-molecular disulphide bond and, hence, should not affect glutenin polymer formation; moreover, the third and the fourth cysteine residues, present respectively in the N- and C-terminal regions, are very likely involved to form inter molecular disulphide linkages. It is very likely that the two cysteine residues of subunit 20 are involved in intermolecular disulfide linkages, the only difference with subunit 7 being the absence of an intra-molecular disulphide bond and the position of the cysteine residue in the N-terminal region possibly involved in an inter molecular disulphide bridge. If this is the case, the differences in the amount of insoluble glutenin polymers observed by Gupta and MacRitchie23 remain puzzling, unless the particular position and environment of the cysteine residue in the N-terminal domain of subunit 20 affects the formation of glutenin polymers and their size distribution. Less clear is the influence that the central repetitive domain and the structure that it can adopt might have on dough properties. Andrews and Skerritt25, based on antibody studies, postulated that certain amino acid sequences contributed more effectively to dough strength. The sequences they identified are those that would promote the formation of /3-tum secondary structure, with a greater proportion of these epitope sequences associated with greater dough strength. As suggested by Kasarda 1 /3-spiral regions might interact with one another, perhaps through side by side alignment of the spirals. In this respect subunits possessing a larger repetitive domain, such as 2 .2, 2.2*, 1 2 l > or 2 . 1 * (Fig. I), might have a different influence on dough properties compared to allelic subunits with a smaller repetitive domain. Investigation on the contribution of single high Mr glutenin subunits to the functional properties of a dough using the 2g-Mixograph, in which purified high Mr glutenin subunits have been incorporated into the dough, have shown a significant positive correlation between subunit molecular weight and mixing time26. Preliminar studies incorporating subunits 2.2, 2.2*, 1 2 1 have shown that they have positive intluence on dough mixing properties compared to smaller allelic subunits 2 or 12.

4 LOW Mr GLUTENIN SUBUNITS AND MUTATED GLIADIN COMPONENTS Differently from high Mr subunits, low Mr subunits, due to their complexity and heterogeneity, have been less characterized, though their effect on flour technological properties and results of their characterization are being produced in an attempt to claryfY their role in glutenin structure. Low Mr glutenin subunits are usually subdivided into B, C and D subunits based on their mobilities in SDS-PAGE and their isoelectric points27•28 Most subunits are included in the B group which are the most basic of the major storage proteins and have lower mobilities than <X.-, /3- and y-gliadins; the C group has a wide range of isoelectric points and mobilities in SDS-PAGE similar to that of <X.-, /3- and y-gliadins.

1 24


The D group includes the most acidic subunits and possesses the lowest mobilities among the low Mr subunits. DNA sequences reported for low Mr subunits have shown that they are closely related to y-gliadins as they have similar structure and identical number of cysteine residues (eight). Disposition of cysteine residues are, however, different; in tact in y-gliadins cysteine residues are all located in the large C-terminal domain, whereas in low Mr subunits one cysteine is present in the short N-terminal region29 N-terminal sequence studies have indicated the existence of two main types of low molecular weight glutenin subunits30,3 1 . The sequence of the most abundant type begins with serine (LMW-s) whereas the complete sequence is available only for the least abundant type whose N-terminal sequence starts with methionine (LMW-m). Both type of sequences were found in the B-group of low molecular weight glutenin subunits, whereas N-terminal sequences of the C-group corresponded mainly to y- and a-type gliadins. Strong similarities exist between the LMW-s and LMW-m type of sequences, the major difference between them is the absence of the cysteine residue at position 5 in the LMW-s type compared to the LMW-m, though it has been speculated that additional cysteine might be in a different position32. In recent years it has appeared always more and more clearly the presence, in the glutenin complex, of subunits with N-terminal sequences corresponding to those of monomeric gliadins32. Masci et al.33 found that of the two biotypes present in the bread wheat cultivar Newton, differing in their I D encoded ro-gliadins, gliadins which resembled the electrophoretic patterns of Chinese Spring (CS) and Cheyenne (CNN), only the first one possessed two l D-coded D-type low Mr glutenin subunits. Purification and determination of N-terminal amino acid sequences of these two subunits revealed homologies with I D coded ro-gliadins present in Chinese Spring. Moreover, when these subunits were treated with a fluorogenic reagent (ABD-F), which specifically alkylate sulfhydryl groups, both subunits were fluorescent contrary to what is observed for ro-gliadins34 This led Masci et al.34 to postulate that D subunits were very likely formed as a consequence of a mutation of an ro-gliadin gene (or genes), such that one or more cysteine codons was produced. Similarly to D subunits, that are clearly related to ro-gliadins from which they differ by the presence of cysteine and that makes them part of the glutenin fraction, a- and y-gliadins also may behave like glutenin subunits if the number and/or the position of the cysteine residues allow the possibility to form intermolecular disulphide bonds. a- and y-gliadin type sequences have in fact been found in the residue of both bread and durum wheat32,35 In particular, the y-gliadin type sequences found present a cysteine in position 26, not present in the true y-gliadins. This extra cysteine has also been reported in the nucleotide sequence of y-gliadin clones by Scheets and Hedgcoth36 and recently by D'Ovidio et a1 37 and it is likely to be involved in intermolecular disulphide bonds because the other eight cysteines, known to be involved in intramolecular disulphide bonds, are normally present. The possibility that these kinds of y-gliadin may be part of the glutenin fraction has also been shown by Kohler et al. 24 who found a disulphide bond between two peptides with sequences belonging to low-molecular weight glutenin subunits and y-gliadin respectively. However, the cysteine residue present in the y-gliadin peptide did not correspond to the one present in position 26, but to position 8 1 , in which a phenylalanine is usually reported.

1 25


S STRUCTURAL DIFFERENCES OF LOW Mr GLUTENIN SUBUNITS AND THEIR EFFECT ON QUALITATIVE PROPERTIES Correlative studies have contributed to establish relationships between different low Mr allelic type and flour technological properties. As far as qualitative differences between LMW-s and LMW-m type are concerned, no data is available but again the role of cysteine residues and their position is very likely to be of extreme importance in their contribution to glutenin polymer formation as already stressed for the high Mr glutenin subunits. Lew et aJ32, comparing the structural differences between low Mr subunits and y-gliadins, have hypothesized that the cysteine residues forming intermolecular disulfide bonds are positioned in the N- and C-termini for the LMW-m-type sequences. Because LMW-s-type do not have any cysteine residue at the N-terminus, at least in the first fifty arninoacid residues, is not clear what makes them chain extenders, how they likely act because of their predominance in the best quality wheat. According to them, the presence of cysteine residues at the N- and C-termini might restrict interactions of the repeating sequence region located in the N-terminal half of the molecule, whereas their presence at only the C terminus might leave this region available to interact with equivalent region of other molecules. A possible relation with quality of the two different types of low Mr subunits comes from results of Masci et al. 35 who have found that between the two biotypes of the durum wheat cultivar Lira, one possessing the y-gliadin 42 and the associated LMW- I , and the other possessing the y-gliadin 4S and LMW-2, the latter possessed a higher amount of LMW-s type compared to the former. It has been hypothesized that mutated gliadins having an odd number of cysteine residues may act as chain terminator and tend to limit the molecular weight distribution of glutenin polymers, with consequent negative effects on quality characteristics such as dough strength1 . Though this has yet to be experimentally proved, some hints of the negative effects of mutated gliadins comes from the work of Masci et al. 32. These authors reported that the two biotypes, present in the bread wheat cultivar Newton, one possessing and the other not possessing D subunits of low Mr differ also in quality characteristics, as assessed by the SDS sedimentation test. Because the biotype without D subunits presented higher values of SDS-sedimentation volume, these authors suggested, for these subunits, a similar role to that proposed for certain (l- and y-type glutenin subunits as the effect on quality is concerned. These subunits present in fact an extra cysteine residue compared to the ancestral (l- and y-type gliadins due to a mutation of a serine codon that makes them able to link intermolecularly and to act as chain terminators. Table 3 SE-HPLC separation ojproteins extractedfrom the two biotypes, present in the

bread wheat cultivar Newton, differingjor the absence (CNN-type) or presence (CS-type) oj the D subunits

Peak I (%) Newton CNN Newton CS

51.1 5 1 .3

Peak 2 (%) 4 1 .7 39.6

Peak 3 (%) 7.2 9.2

UPP (%) 53.0 50.0

126


Separation of proteins present in the two biotypes, on SE-HPLC, according to Gupta et a) 38, indicated no difference in the amount of total polymeric glutenin (% Peak I ) between the two biotypes, whereas a slightly larger amount of unextractable proteins (% UPP) was associated with the CNN-biotype compared to the CS-biotype.

6 CONCLUSIONS Biochemical and molecular studies are contributing to the elucidation of structural differences among allelic subunits of high and low Mr glutenin subunits in order to establish the role these differences have in affecting flour functional properties. Whereas there is increasing confirmation of the importance of the number and position of cysteine residues in both types of glutenin subunits, the role of length and structure adopted by the central repetitive domain needs further investigation. To this end, novel genes are being produced and drastic changes in the structure of corresponding polypeptides generated. For instance high Mr glutenin subunits varying in the length of the repetitive region have been constructed. Further incorporation of these modified genes into wheat, with established transformation procedures, will offer an additional approach to explore structure functionality relationships.

References D.O. Kasarda, 'Wheat is Unique', Am. Assoc. Cereal Chem., St Paul, MN, 1 989, p. 277. F. MacRitchie, Adv. Food Nutr. Res., 1 992, 36, 1 . P.I. Payne, Ann. Rev. Plant Physiol. , 1 987, 38, 1 4 1 . R B . Gupta and K.W. Shepherd, Proc. 3rd Int. Workshop Gluten Proteins, (R Lasztity and F. Bekes, eds.), 1 987, p. 1 3 . 5. P.I. Payne and G.J. Lawrence, Cereal Res. Commun. , 1 983, 1 1 , 29. 6. N P. Harberd, D. Bartels and R D. Thompson, Biochem. Genet. , 1 986, 24, 579. 7. P.R. Shewry, N.G. Halford and A S . Tatham, J. Cereal Sci., 1 992, 1 5, 1 05 . 8 . R. D'Ovidio, E. Porceddu and D. Lafiandra, Theor. Appl. Genet., 1 994, 88, 1 75 . 9. M. Tahir, A Pavoni, G.F. Tucci, T. Turchetta and D. Lafiandra, Theor. Appl. Genet. , In press. 1 0. P. Reddy and R. Appels, Theor. Appl. Genet. 1 993, 85, 6 1 6. 1 1 . P.R Shewry, N.G. Halford and A S . Tatham, Oxford Surveys of Plant Molecular and Cell Biology, 1 989, 6, 1 63 . 1 2 . A S . Tatham, P . R Shewry and B.1. Miflin, FEBS Letts., 1 984, 177, 205 . 1 3 . Greene, F.e., Anderson, 0.0., Yip, R.E., Halford, N.G., Malpica-Romero, J-M. and Shewry, P.R. Proc. 7th Int. Wheat Genet. Symp. IPSR, Cambridge, 1 988, p. 73 5 . 1 4. A.P. Goldsbrough, N.J. Bulleid, R B . Freedman and R B . Flavell, Biochem. J., 1 989, 263, 837. 1 5 . D. Lafiandra, R D'Ovidio, E. Porceddu, B. Margiotta and G. Colaprico, J. Cereal Sci., 1 993, 18, 1 97. 1 6. 1M. Field, A S . Tatham and P.R Shewry, Biochem. J , 1 987, 247, 2 1 5 . 1 7. D.P. Goldenberg and T.E. Creighton, Anal. Biochem., 1 984, 138, 1 .

1. 2. 3. 4.

.


127

1 8 . B. Margiotta, G. Colaprico, R D'Ovidio and D. Lafiandra, J. Cereal Sci. , 1 993, 1 7, 22 1 . 1 9. A S . Tatham, lM. Field, IN. Keen, PJ. Jackson, and P.R Shewry, J. Cereal Sci. , 1 99 1 , 1 4, I l l . 20. M.H. Morel and l Bonicel, Proc. Wheat Kernel Proteins, 1 994, p. 1 83 . 2 1 . F . Buonocore, C. Caporale and D . Lafiandra, J. Cereal Sci. , 1 995, I n press. 22. RB. Flavell, AP. Goldsbrough, L.S. Robert, D. Schnick and R.D. Thompson, Bio/Technology, 1 989, 7, 128 1 . 23 . RB. Gupta and F . MacRitchie, J. Cereal Sci. , 1 994, 1 9, 1 9. 24. P. Kohler, H.-D. Belitz and H. Weiser, Z. Liebenm. Unters. Forsch., 1 993, 1 96, 239. 25. lL. Andrews and lH. Skerritt, J. Cereal Sci. , 1 994, 19, 2 1 9. 26. F. Bekes, O.D. Anderson, PW. Gras, RB. Gupta, A Tam, C.w. Wrigley and R AppeJs, 'Improvement of Cereal Quality by Genetic Engineering', (RJ. Henry and lA Ronalds, eds.), 1 994, p. 97. 27. p.r. Payne and KG. Corfield, Planta, 1 979, 1 45, 83. 28. Al Jackson, L.M. Holt and P.I. Payne, Genet. Res. Camb., 1 985, 46, 1 1 . 29. P.R Shewry, MJ. Miles and A S. Tatham, Prog. Biophys. Molec. Bioi. , 1 994, 61, 37. 30. D.D. Kasarda, H.P. Tao, P.K. Evans, AE. Adalstein and S W. Yuen, J. Exp. Bot. , 1 988, 39, 899. 3 1 . H.P. Tao and D.D. Kasarda, J. Exp. Bot. , 1 989, 40, 899. 32. E.lL. Lew, D.D. Kuzmicky and D.D. Kasarda, Cereal Chem. , 1 992, 69, 508. 33. S. Masci, E. Porceddu, G. Colaprico and D. Lafiandra, J. Cereal Sci. , 1 99 1 , 1 4, 35. 34. S . Masci, D. Lafiandra, E. Porceddu, EJ.-L. Lew, H. Peggy Tao and D.D. Kasarda, Cereal Chem., 1 993, 70, 58 1 . 35. S . Masci EJ.-L. Lew, D. Lafiandra, E. Porceddu and D.D. Kasarda, Cereal Chem., 1 995, 72, 1 00. 36. K Scheets and C. Hedgcoth, Plant Sci. 1 988, 67, 1 4 1 . 37. R D'Ovidio, M . Simeone, S . Masci, E. Porceddu and D.D. Kasarda, Cereal Chem. , 1 995, In press. 38. RB. Gupta, K Khan and F. MacRitchie, J. Cereal SCi. , 1 993, 1 8, 23.

CAPILLARY ELECTROPHORESIS: A STATE-OF-THE-ART TECHNIQUE FOR WHEAT PROTEIN CHARACTERIZATION

1. A. Bietz', G. L. Lookhartb, S. R. Beanc and K. H. Suttond •

b C

d

Food Physical Chemistry, USDA-ARS, National Center for Agricultural Utilization Research, 1 8 1 5 North University, Peoria, IL 6 1 604 U.S.A. USDA-ARS, U.S. Grain Marketing Research Laboratory, 1 5 1 5 College Ave., Manhattan, KS 66502 U.S.A. Dept. of Grain Science and Industry, Kansas State University, Manhattan, KS 66506 U.S.A. New Zealand Inst. for Crop and Food Research, Ltd., Private Bag 4704, Christchurch, New Zealand

1 INTRODUCTION It has long been recognized that wheat is critically important to humanity, and that many of its important properties, such as its role in breadmaking, are closely associated with its gluten proteins. Knowledge of gluten's composition is thus useful in evaluating wheat quality, and for identifying genotypes during breeding and marketing. Gluten's unique functional properties also permit many nonfood applications. Gluten is extremely heterogeneous and complex, however, demanding powerful analytical methodsl. Many methods of chromatography and electrophoresis can be used to analyze gluten. Until recently, however, electrophoresis techniques have not advanced as rapidly as those of chromatography [especially high-performance liquid chromatography (HPLC)2]. Most electrophoresis procedures have been manual, labor-intensive, and slow, and results are difficult to quantify. In recent years, however, methods of capillary electrophoresis (CE) have evolved that significantly enhance resolution and reproducibility of electrophoresis, while reducing analysis time and permitting accurate quantitation. Commercial CE instruments are now available, and CE has been shown applicable to proteins3• CE is, in principle, a simple technique. A tube connects two buffer reservoirs, to which high voltage is applied. A means is provided to introduce samples, and the capillary is cooled to dissipate heat and maintain constant temperature. Proteins may be detected on-column, based on UV absorbance. A computer provides system control and data acquisition. The chemistry behind CE separations is a little more complex. Protein mobilities depend both on net charge, reflecting amino acid compositions and buffer pH, and on electroendosmosis, which moves buffer and solutes toward the cathode. Net mobility is thus the sum of these forces. Mobility and selectivity vary with buffer pH, and even electrically neutral molecules can be separated. Many further modifications are possible. The capillary surface can be modified to change its adsorptive characteristics. Detergents, denaturants, or organic solvents can be added to the buffer. Sieving media can be introduced into the capillary. Column dimensions, temperature, or voltage can be varied. Thus, although CE is a simple method in principle, many variables affect separations, making myriad types of separations possible. For these reasons, we have begun to develop and apply CE methods for separation of wheat gluten proteins. Progress to date is here reviewed and summarized.

2 CE OF GLIADIN Wheat proteins were first fractionated more than 35 years ago by a process analogous to CE, moving boundary electrophoresis4• In this method, proteins migrate in an open tube under the influence of an applied electric field. The success of this method strongly suggested that CE should be applicable to gluten proteins. Indeed, the first CE separations of gliadins extracted

129


10

Figure 1 CE in pH 9.0 0.06M sodium borate buffer, containing 20% ACN and 1% SDS, of Centurk wheat proteins extracted with 30% ethanol. From Biett.

15

20 Minutes

25

30

35

Figure 2 CE in pH 2.5 phosphate buffer, containing polymeric additive, of 30% ethanol-soluble Centurk wheat proteins. From Bietz6•

with 30% ethanol, done in an alkaline borate buffer containing sodium dodecyl sulfate (SDS) and acetonitrile, had resolution roughly comparable to that of RP-HPLC (Figure 15•7). Separations were rapid, automatic, easily quantified, and complemented other methods. Wheat varieties were readily differentiated by their CE patterns. Reproducibility was a serious problem with this buffer, however - migration times gradually increased from run to run. Protein adsorption or insolubilization can change current, column dimension, or electroosmotic flow, causing migration times to vary. This can be a real problem for gluten proteins because of their interactive and aggregative tendencies, and their low solubility in aqueous buffers. This reproducibility problem, although controllable by extensive between-run washes, prompted testing of alternate CE buffer systems. An acidic phosphate buffer containing a cellulose derivative separated ethanol-soluble wheat proteins better than did the first buffer system (Figure 2). With this buffer, later-eluting proteins are primarily gliadins, while proteins eluting early « 1 0 min) are albumins and globulins, which migrate rapidly due to high charge densities at low pH. Run-to-run reproducibility with this system was excellent, both for migration times and injection volumes. Comparison of analyses by CE and reversed-phase high-performance liquid chromatography (RP-HPLC) for the same protein extracts showed that both methods could differentiate most wheat cultivars - even ones closely related - but that resolution of CE was slightly higher. This was confirmed by CE of gliadin peaks from an RP-HPLC separation: CE frequently resolved multiple components from a single isolated peak. Thus, the real advantage of CE is that it complements RP-HPLC, providing another high-resolution method of protein fractionation. Werner et al.8 developed still another system for gliadin fractionation. They used an acidic aluminum lactate buffer and a coated capillary that prevented proteins from adsorbing to the silica. Good separations resulted, which easily differentiated wheat varieties. Obviously many possible types of gliadin CE separations exist; we may still not yet know the best methods.

3 IMPROVEMENTS IN CE METHODS Recent studies of Lookhart and Bean9 significantly improved CE separations of gliadins under acidic conditions. By optimizing temperature, voltage, injection time, and extraction conditions, and by reducing capillary length and diameter, excellent and reproducible gliadin separations were achieved in an acid phosphate buffer in about ten minutes, less than half the time previously required (Figure 3). In another recent studylO, Lookhart and Bean modified capillary cleaning protocols and added 20% acetonitrile or detergents (such as lauryl

1 30

Wheat Structure, Biochemistry and Functionality 10 "'" IDcaplllry l

y

B

-_ a -

TIme, min

Figure 3 CE (pH 2.5 phosphate buffer) of 30% ethanol-soluble TAM 1 07 proteins on 50 11m and 2 0 11m capillaries. From Lookhart and Bean9•

2

4

I \ ,

. .

Minutes

8

10

Figure 4 CE (pH 2.5 phosphate buffer) of Shawnee gliadins. Migration times for each protein type were identified by CE of individual peaks from a RP-HPLC separation of gliadin. From Lookhart and Bean12•

sulfobetain) to the buffer. These further improved resolution and reproducibility of many CE separations in an acid phosphate buffer system. To be widely used and accepted, CE results must be reproducible between laboratories. Bietz and Lookhart recently tested this by comparing the same samples with similar instruments and procedures in two laboratoriesll. While results between laboratories generally agreed well, elution times sometimes shifted significantly when different lots of the same commercial buffer were used. Constant buffer pH and ionic strength are critical for satisfactory reproducibility. Clearly, individual lots of commercial buffers may vary in pH or ionic strength; homemade buffers, prepared entirely by gravimetric and volumetric methods, are probably preferable. We are now beginning to understand the basis of CE separations better, and can begin to relate CE data to results of other methods. Lookhart and Beanl2 isolated gliadin peaks by RP-HPLC, and then characterized them by gel electrophoresis and by CE. Multiple components were often resolved by CE from single HPLC peaks, again showing the complementary nature of CE and RP-HPLC. In addition, this study showed the order in which ethanol-soluble wheat proteins elute upon acid CE (Figure 4): albumins and globulins elute first, followed by (X - and p -gliadins, y -gliadins, and finally by w -gliadins. This order of mobility is the same as in acid gel electrophoresis, showing that protein charge is the primary determinant of mobility in acid CEo

4 APPLICATIONS OF CE As noted above, one major reason for analysis of gluten proteins - especially gliadins - is to differentiate varieties. Such analyses may be especially valuable in breeding programs, and during marketing for identifying and selecting wheats having desirable functional characteristics. Each of the above-described CE procedures readily discriminates among wheat cultivars, even ones very closely related, based on their differing gliadin "fingerprints." A typical example, showing CE patterns of gliadins of several hard red spring wheat varieties using the rapid acid phosphate analysis system of Lookhart and Bean9, is shown in Figure 5 . Many qualitative and quantitative differences exist among the .patterns. In a related application, Bietz and Schmalzried7 used CE to differentiate U.S. hard red spring and hard red winter wheat classes. Examination of CE results for many wheats revealed apparent differences between peak distributions for gliadins from wheats of these two classes.

131

Wheat Protein Composition and Quality Relationships 50

40

}

30 20 '0

11"". "n

Figure 5 CE (pH 2.5 phosphate buffer) of gliadins from hard red spring wheat varieties. From Lookhart and Bean9•

·' 0 '---,.120-�-!: ' 5-�-'c 30:--� 25-�--:' 0 -�--'Minutes

Figure 6 Averaged acidic CE separations of gliadins from 16 hard red winter and 16 hard red spring wheat varieties. Differences in peak distributions indicate characteristic class-related differences. From Bietz and Schmalzrietf.

This became much more apparent when the averaged data sets were compared, however (Figure 6). Most hard red winter wheats contain much more late-migrating proteins - probably y- or w -gliadins - than do hard red spring wheats. Many other applications for CE are already emerging. For example, the ability to quantify CE data accurately permits analysis of effects of environment on wheat protein synthesis. Lookh art has found that amounts of early-eluting albumins and globulins in acid phosphate CE patterns are greater in wheats grown under hot, dry conditions. Such quantitative differences may help explain, and be predictive of, the lower baking quality of such wheats. Lookhart has also identified specific late-eluting peaks in electrophoregrams of gliadins from wheat varieties containing rye translocations that reliably indicate y -secalins, coded by rye genes in translocated chromosome segments. CE also gives useful separations of other wheat protein classes. As shown in Figure 2, rapid, high-resolution separations of albumins and globulins can be achieved simultaneously with analyses of gliadins in an ethanol extract. CE is also useful for evaluating the nature and composition of proteins sequentially extracted from wheat. For example, Shomer et alt3 used CE to compare proteins sequentially extracted with 80% ethanol and O. I M acetic acid from Israeli and U.S. spring and winter wheat varieties. Other yet-unpublished studies, both at our Peoria and Manhattan laboratories, have achieved good CE separations of reduced glutenin subunits by acid phosphate CEo These examples show that excellent CE separations are now possible for each major wheat polypeptide type.

5

CE OF HIGH MOLECULAR WEIGHT (HMW) GLUTENIN SUBUNITS

Analysis of wheat glutenin, especially its unique HMW subunits, is of special interest because of the relationship of the composition of HMW glutenin subunits to breadmaking quality14.1S. Werner et alB first used CE to separate glutenin's HMW, quality-associated subunits using the Applied Biosystems ProSort system. This method provides a sieving matrix that separates protein-SDS complexes primarily based on size, as in SDS-polyacrylamide gel electrophoresis (PAGE). Excellent resolution of HMW glutenin subunits occurs, permitting their identification and relation to quality characteristics. Werner16 recently provided an excellent example of how this method of glutenin HMW subunit analysis can be used. Total storage proteins were extracted from wheat, and analyzed at different dilutions of ProSort sieving matrix, causing migration rates to vary. Ferguson plots of the resulting data revealed molecular weights of HMW glutenin subunits that closely

132


0.00. ,----------

0.015

Olano

0.010

OJ''''

0.005

E c ...

O.oo:J

" .

�

0.000

0.010

0.005

0.000

�.-�.-�.-�.-�.-�,-�.-�,

I

10

12

Karamu

��-L�-L��L-�

ElcdroehtioII Ti_(min)

Figure 7 Size-based CE separation, by modified ProSort method, of HMW glutenin subunits of the wheat varieties Karamu and Tritea.

10

Electroelution Time (min)

12

14

Figure 8 Size-based CE separation, by modified ProSort method, of HMW glutenin subunits of the wheat varieties Dtane and Karamu.

matched those from DNA sequence analyses. Results suggested that the anomalously high molecular weights typically found for HMW glutenin subunits upon SDS-PAGE may result from decreased binding of SDS, possibly due to subunit glycosylation 17. Sutton recently modified the ProSort method to examine HMW glutenin subunits of New Zealand wheats. Use of a 95:5 mixture of ProSort buffer and methanol, combined with enrichment of the HMW subunits by a precipitation step, gives improved separations. An example, showing a standard mixture of eight HMW subunits from the varieties Karamu and Tritea, is shown in Figure 7. Excellent resolution of all HMW subunits results, with no overlap from earlier-eluting low MW subunits. Sutton previously used RP-HPLC to characterize HMW glutenin subunits of New Zealand varieties18• Otane, of high baking quality, and Karamu, of poor baking quality, were studied in detail. Both varieties had HMW subunits 2+1 2 and 7+8 (according to standard SDS-PAGE nomenclature), but Otane also contained subunit 2*. RP-HPLC also showed that the subunits designated "8" in these two varieties are different, and that Otane has more subunit 7, which may contribute to its better performance. Sutton recently compared these results with those of CE using the modified ProSort method. CE, however, like SDS-PAGE, did not differentiate subunit 8 in these two varieties (Figure 8). Quantitative results from CE agreed closely with those of RP-HPLC, however. Thus, SDS-PAGE, RP-HPLC, and CE each provide different analyses of HMW glutenin subunits. This comparison provides another example of the complementary nature of CE and RP-HPLC, and reminds us that all methods are and will remain highly useful. Sutton also used CE to characterize nonstandard HMW glutenin subunits in European varieties provided by Dr. Domenico Lafiandra. SDS-PAGE had shown the presence of HMW Glu- 1 D subunits designated 2.2* in the line MG3 1 5 and 2.2 in MG7249. Upon CE using the modified procedure, these subunits migrated much slower than did "normal" HMW glutenin subunits: 2.2* had an apparent MW of ca. 3 1 0 kD, and 2.2 eluted at a position corresponding to ca. 330 kD. The MWs and elution order of these subunits were reversed between SDS-PAGE and CE, however, possibly due to differences between sieving characteristics of the two methods. This study again emphasizes the complementary nature of CE to other separation methods.

6 CONCLUSIONS Capillary electrophoresis, though recently introduced, has already been shown valuable for fractionation and characterization of wheat proteins, and for identifying wheats and predicting


133

their functional properties. CE methods are rapid, versatile, sensitive, and offer high resolution. Most important, however, CE is the first electrophoresis procedure to be automated, it is the first electrophoresis procedure for which accurate quantitation is readily achieved, and it provides separations complementary to those of other protein fractionation methods. CE does, of course, have some disadvantages, and has not yet been optimized for all applications - but these problems are being overcome as our understanding of CE methodology grows. Clearly, CE has become a valuable addition to other methods of wheat protein analysis.

References 1 . C. W. Wrigley and J. A. Bietz, "Wheat Chemistry and Technology," Y. Pomeranz, ed., Amer. Assoc. Cereal Chemists, St. Paul, MN, 1 988, Vol. J, Chapter 5, p. l 59 . 2. F. R. Huebner and 1. A. Bietz, "High-Performance Liquid Chromatography of Cereal and Legume Proteins," Amer. Assoc. of Cereal Chemists, St. Paul, MN, 1 994, p.97. 3. 1. P. Landers, R. P. Oda, T. C. Spelsberg, 1. A. Nolan and K. 1. Ulfelder, BioTechniques, 1 993, 14, 98. 4. R. W. Jones, N. W. Taylor and F. R. Senti, Arch. Biochern. Biophys., 1 959, 84, 363. 5. J. A. Bietz and E. Schmalzried, Cereal Foods World, 1 992, 37, 555. 6. J. A. Bietz, "Gluten Proteins 1993", Association of Cereal Research, Detmold, Germany, 1 993, p.404. 7. J. A. Bietz and E. Schmalzried, Food Science and Technology, 1 995, 28, 1 74. 8. W. E. Werner, J. E. Wiktorowicz and D. D. Kasarda, Cereal Chern., 1 994, 71, 397. 9. G. Lookhart and S. Bean, Cereal Chern., 1 995, 72, 42. 1 0. G. L. Lookhart and S. R. Bean, Cereal Chern., submitted. 1 1 . J. A. Bietz and G. L. Lookhart, Cereal Foods World, 1994, 39, 603. 1 2. G. Lookhart and S. Bean, Cereal Chern., in press. 1 3 . I. Shomer, G. L. Lookhart, R. Vasiliver and S. Bean, 1. Cereal Sci. , in press. 14. J. A. Bietz and J. S. Wall, Cereal Chern. , 1 972, 49, 4 1 6. 1 5 . P. I. Payne, Ann. Rev. Plant Physiol. , 1 987, 38, 1 4 1 . 1 6. W . E . Werner, Cereal Chern. , 1995, 72, 248. 17. K. A. Tilley, G. L. Lookhart, R. C. Hoseney and T. P. Mawhinney, Cereal Chern., 1 993, 70, 602. 1 8 . K. H. Sutton, 1. Cereal Sci. , 1 99 1 , 14, 25.

ELECTROPHORETIC AND CHROMATOGRAPHIC CHARACTERIZATION OF GLU-A l ENCODED HIGH Mr GLUTENIN SUBUNITS

B. Margiotta\ M. Urbano·, T. Turchetta2, G. Colaprico· • Gerrnplasm Institute, C.N.R., Via Amendola 1 65/A, 70 1 26 Bari, Italy 2 University of Tuscia, Department of Agrobiology and Agrochemistry, 0 1 1 00 Viterbo, Italy

1 INTRODUCTION Variation at the Glu-l loci has been studied extensively in recent years primarily because of the effects produced by corresponding encoded high U glutenin subunits on flour technological properties. At the Glu-A l locus in particular, three alleles have been described by Payne and Lawrence designated as a, b and c and respectively known as 1 , 2 * and null, the latter corresponding to a silent gene, which does not code for a detectable protein on SDS-PAGE. New allelic variants at the same locus have been identified subsequently by SDS-PAGE analysis of various Triticum species2-4. The characterization of new allelic variants detected at the Glu-A l locus in hexaploid and tetraploid wheat by a combination of electrophoretic and chromatographic techniques, is reported in this communication.

2 MATERIALS AND METHODS Several durum and bread wheat cultivars and lines maintained at the Germplasm Institute were used. SDS-PAGE analysis was carried out on 1 0% polyacrylamide gels according to Payne et aI., and 1 0% urea SDS-PAGE were prepared following the procedure reported by Lafiandra et a1. 6 . Two-dimensional electrophoretic separations, were carried out according to Holt et aC, combining isoelectric focusing (IEF) or non-equilibrium pH gradient electrophoresis (NEPHGE) in the first dimension with SDS-PAGE in the second. Transverse gradient urea gels were performed following the procedure reported by Goldenberg and Creighton8 with some modifications. High Mr glutenin subunits prepared as described by Marchylo et al. 9 were also analysed by RP-HPLC.

3 RESULTS 3. 1 One-Dimensional SDS-PAGE

SDS-PAGE separation of different high Mr glutenin subunits encoded at the Glu-A l locus is shown in Figure 1 . The mobility of the novel subunits (Figure la and Ib, lanes 3 ,

135


1

2

3

4

5

B

2

3

4

5

6

P1

Z.l·

I)

b)

Figure 1 . One-dimensional SDS-PAGE (10%) separation of high M, glutenin subunits from hexaploid and tetraploid genotypes a) and in the presence of urea 4M b). 1, PK15684 (2. 1 *, 7+8, 2**+ 10�; 2, Drago (1, 6+8); 3, Fenix biotype b (1 ', Bx+ 15); 4, Duramba (2 *, 13+ 16); 5, MG 826 (2 *" Bx*+By); 6, MG 2984 (2 *2, 20). 5, 6) are compared with those of subunits I (lane 2), 2 * (lane 4) and 2. 1 * (lane I ) described recently by Tahir e t al. \0. The durum wheat cultivar Fenix has subunit l ' described by Branlard et ae, which had a mobility slightly different from subunit 1 . Two subunits with greater mobility than subunit 2 * , indicated as 2 * \ and 2 * 2, were found in some T. durum lines. Separation of the same subunits in 4M urea gels, is shown in Figure lb. The presence of urea in the gels had different effects on the mobilities of different subunits. In particular the differences among 2 * , 2 * \ and 2 *2 were enhanced. 3.2 RP-HPLC Analyses

The results of RP-HPLC separations of reduced and reduced/alkylated subunits from each genotypes carrying the different IA allelic subunits are reported in Table I . The order of elution of reduced and reducedlalkylated subunits was: 2. 1 * , I , 2 * , I ', 2 * \, and2 * 2.These analyses indicate that allelic variants can be separated into two groups: the first, including 2. 1 * , I , 2 * and l ' having longer retention times in both the reduced and reduced and alkylated forms and the second with subunits 2 * . and 2 * 2 having lower retention times.


136

Table I. Comparison oj the retention times oj reduced and reducedlalkylated subunits and the retention time differences.

Glu-AI

Subunit x

Reduced (min)

Reduced and alkylated

60.9

J��2 48.6

J�l�

60. 8

46.4

14.4

2*

60.0

45.7

1 4. 3

I'

59.2

45.6

1 3 .6

32.3

24.9

7.4

_ _ _______________________________

2. 1 *

_ _

Time Difference

��

��

________________

��

___________

__________

_____.

12.3

___________

1}

______.

3.3 Two-Dimensional Separations of High Mr Glutenin Subunit Variants Encoded on Chromosome la

Two dimensional electrophoretic separations (IEF x SDS-PAGE and NEPHGE x SDS-PAGE) of new allelic variants were also performed (Figure 2). The new subunits had different isoelectric points from allelic subunits 2. 1 * , 2 * and 1 . In particular, subunits 2 * . and 2 *2 were more basic than 2 * and 1 , and subunit 2 * 2 was detected only by using NEPHGE x SDS-PAGE. 3.4 Transverse Gradient Gel Electrophoresis (TGGE)

The conformational behaviour of different high Mr glutenin subunits was examined in transverse gradient urea gels. All lA subunits analysed showed a similar electrophoretic transverse gradient profile with a midpoint transition at approximately 6M urea. Interestingly, the migration of subunit 2. 1 * at 4M urea concentration was greater than that of the smaller subunit 2 * *. This may be ascribed to incomplete unfolding of subunit 2. 1 * (Figure 3a) at urea concentrations between 4M and 8M. The patterns for subunits 2 * . and 2 * 2 (Figure 3c) reflect the same differences in Mr with respect to subunit 2 * (Figure 3b), 1 and I ' (Figure 3b and d). The equilibrium midpoint of the transition for these subunits was at approximately 6M urea and was the same for all subunits.

4 DISCUSSION The combination of different chromatographic and electrophoretic techniques provided additional information on gluten subunits encoded at the Glu-l loci. Further heterogeneity was revealed with alleles differing in size, surface hydrophobicity and charge. The importance of these differences with regard to their effects on gluten structure and flour functionality remains to be established.

137


Ilf

•

..

I)

fr

-

r

-

I ..

...

h

L

Figure 2. Two-dimensional electrophoresis (IEF x SDS-PAGE) oj high M, glutenin subunits present injIour mixtures: PK 15684 + MG 7249, a); MG 826 + Cheyenne, b); Fenix biotype a + Fenix biotype b c). The high Mr glutenin subunits present in all the cultivars and lines are indicated

REFERENCES I.

P. 1 . Payne and GJ. Lawrence, Cereal Research Comm, 1 983, 11, 29. J.G. Waynes, P. 1 . Payne, Theor Appl. Genet., 1 987, 74,7 1 . G. Branlard, J.C. Autran, P.Monneveux, Theor.Appl. Genet., 1 99 1 , 78, 3 5 3 . B. Margiotta, G . Colaprico, R . D'Ovidio, D. Lafiandra, J Cereal Sci. , 1 993 , 17, 22 1 . P. 1 . Payne, L.M. Holt, C.N. Law, Theor. Appl. Genet. , 1 98 1 , 60, 229. D. Lafiandra, R. D'Ovidio, E. Porceddu, B Margiotta, G. Colaprico, J. Cereal Sci. , 1 993, 18, 1 97. 7. L.M. Holt, R. Austin, P. l . Payne, Theor. Appl. Genet. , 198 1 , 60, 237. 8. D.P. Goldenberg and T.E. Creighton, Anal. Biochem., 1 984, 138, I . 9. B A Marchylo, I.E. Kruger, D.w. Hatcher, 1. Cereal Sci., 1 989, 9, 1 1 3 . 10.M. . Tahir, A . Pavoni, G.F. Tucci, T. Turchetta, B. Margiotta, i n "Wheat Kernel Proteins", CNRlUniversity of Tuscia, 1 994, p.253. 2. 3. 4. 5. 6.

1 38


I

i lE A (II

... I

I

i lE A 'II

.. I p

II

cl

Figure 3 . Transverse gradient urea gels (O-8M urea): Pk-15684 a); Solitaire b); MG 2984 + MG 826 c); Fenix biotype a+ Fenix biotype b d).

BMW AND LMW SUBUNITS OF GLUTENIN OF Triticum tauschii, THE D GENOME DONOR TO HEXAPLOID WHEAT.

M. C.Gianibelli, 1,3, R.B. Gupta, 2 and F. MacRitchie 1 1.CSIRO Division ofPlant Industry, Grain Quality Research Laboratory, North Ryde, NSW, 2 1 13, Australia. 2.CSIRO, Division ofPlant Industry, GPO Box 1 600,Canberra, ACT 260 1 ,Australia. 3.Permanent Address: Facultad de Cs. Agrarias y Ftales, Cat. Cerealicultura, UNLP, P.O.Box 3 1 , 1900, La Plata, Argentina.

1 INTRODUCTION The wild grass Triticum tauschii (Aegilops squarrosa 2n=2x=14, DD) is considered to be the D genome donor to Triticum aestivum 1,2 being a potentially rich source of variation for agronomically important traits 3 . The endosperm storage proteins of hexaploid wheat are important components because of their influence on the baking quality characteristics of the flour. This protein consists of two major fractions: gliadins and glutenins. Gliadins are monomeric proteins whilst glutenins are polymeric aggregates of High-Molecular-Weight (BMW) or A subunits and Low Molecular-Weight (LMW) or B and C subunits held together by disulphide bonds. Allelic variations in both glutenin subunits are important in controlling dough properties. Changes of up to 80% in dough resistance to extension and up to 25% variability in extensibility can be accounted for by LMW and BMW glutenin subunits together 4 . Studies on the composition of BMW glutenin subunits and gliadins (endosperm proteins) have been reported S,6 on 60 and 79 lines respectively. However, no research has been conducted on the composition of LMW glutenin subunits in T. tauschii. This could be due to the fact that these proteins ,when separated by SDS-PAGE, overlap with monomeric proteins making it difficult to distinguish them. The recent development of the technique of one-step one-dimensional SDS-P AGE of reduced and alkylated proteins 7 permits a clear resolution in the LMW area (B and C subunits). The present paper describes the variation in BMW and LMW glutenin subunits in a collection of Triticum tauschii. ,

2 MATERIALS AND METHODS A collection of 1 7 1 Triticum tauschii accessions belonging to the botanical varieties typica (97), meyeri (24), strangulata (20) and intermediate (30) with origins in Turkey, Iran, Afghanistan, Pakistan, Azerbaijan and Turkmenia, were studied. Chinese Spring and Hartog were included as controls for BMW glutenin subunits for the alelles 2+12 and 5+10 of Triticum aestivum and one accession of Triticum macha for the allele 2. 1+13. Controls for LMW subunits were: Gabo (lBL- IRS), Chinese SpringiGabo (IAL-IRS; lBL-IRS), Halberd (IBL- IRS), Hartog (IAL-IRS; IBL-IRS) and Norin-61A. BMW and LMW glutenin subunits were analysed by one-step SDS-PAGE 7 . This electrophoretic method separates the glutenin subunits A (HMW), B and C (LMW), without overlapping of albumins, globulins and gliadins. , As reported previously S 6 , the BMW glutenin subunits have been designated by the superscript " both for the Glu-D'1 locus and for the first reported Glu-IY3 locus. Superscript I has been used to distinguish gene symbols of the D genome of T. tauschii from its homologous gene loci in the D genome of hexaploid wheat.


140

3 RESULTS

AND

DISCUSSION

3.1 High Molecular Weight glutenin subunits

A wide variation in HMW glutenin subunit composition was found between different accessions of Triticum tauschii in the collection under study. Forty different alleles were observed , resulting from the combination of different x and y-type subunits. The frequencies of these alleles are presented in Table 1 . Table 1 : Frequencies ofA, B and C glutenin subunit patterns of Triticum tauschii Band Pattern A

Frequency

Band Pattern B

Frequency

Band Pattern C

Frequency

2 + 10

26

1

36

1. 5 + 10.1

24

2

23

2

16

2 + 10.1

23

3

15

3

12

1. 5 + 10

12

4

12

4

12

2 + 12.2

9

5

9

5

9

3 + 10

8

6

8

6

9

2 + 12

7

7

7

7

8

5 + 10

6

8

7

8

8

1.5 + 10.2

9

7

9

7

5 + 12

5 5

10

6

10

7

3 + 10.1

4

11

6

11

4

12

5

12

4

2.1 + 10.2

3

1

17

1.5 + 12.2

3

13

5

13

4

2 + 12.3

2

14

4

14

4

2

15

3

15

1 + 12

2

16

2

16

1 + 10.1

2

17

2

17

1 + 10

3

3

3

2.1 + 10

2

18

2

18

3

1.5 + 12

2

19

2

19

3

3 + 12

2

20

2

20

2

4 + 12

2

21

21

2

4 + 12.2

2

22

22

2

null - l0

23

23

2

2.1 + 12.2

24

24

2

25

25

2

1.5 + 1 1

26

26

2

1.5 + 10.3

27

27

2

2 + 11

28

28

2

2 + 12.4

29

29

2

2 + 12.1

30

2

2 - null

31

1

3 + 10.2

32

1

2.1 + 12.3

3 + 12.2

33

4 + 10

34

4 + 10.2

35

5 + 11

36

5 + 10.1

37

5* - null

39

5 + 12.2

5* + 1 2

38 40 41 42 43


14 1

The larger number of alleles found (40) ,compared with the results of Lagudah and Halloran ' and William et al. 6 , could be due to the larger screening performed, the presence of five new bands and more recombination between x and y-type subunits. In contrast with the huge polymorphism found in the Glu-lY1 in T. tauschii, the Glu-Dl locus in T. aestivum encoded a lesser number of allelic variants 8,9. Although the alleles 2+ 12 and 5+ 10 are the most common in bread wheat cultivars, their frequency in the T. tauschii accessions is much lower. Only 1 3 accessions presented these alleles and most of them belonged to spp. strangulata var. strangulata. Eight subunits of the slow mobility x-type were identified and have been numbered I, 2.1, 1.5, 2, 3, 4, 5, 5 * in order of ascending mobility',6. From them 2. 1 and 1.5, have been previously observed in T. tauschii ',6 . Subunit 1 , with lower mobility than 2 . 1 , is first reported in this study in T. tauschii and its mobility is slightly less than subunit 1 of hexaploid wheat coded by the Glu-Al locus. The other subunit that has not been reported previously is 5*. This subunit has hi&her mobility in SDS PAGE than subunit 5 in T.tauschii and bread wheat. Recent studies in a bread wheat variety (Fiorello) show a 5 * subunit with higher mobility than 5 ; when we compared our subunit 5 * with that from Fiorello , it appears to have similar mobility. In agreement with Lagudah and Halloran and William et al ',6 the subunit 2.2 , the highest molecular weight glutenin subunit of bread wheat, was not observed in this collection , adding more evidence to the hypothesis of Payne et al. 11 that subunit 2.2 is the result of a rare unequal crossing-over with another gene which coded for a HMW glutenin subunit. Among the y-type, ten (10) subunits were observed, which were named 1 0.3, 10.2, 10. 1, 10, 1 1, 1 2, 12. 1, 12.2, 12.3 and 12.4 in accordance with their relative mobilities. The subunits 10.3, 10.2 and 10. 1 could correspond to those named 1 0.3, 1 0.2 and 10. 1 by Lagudah and Halloran '. In our study, 10.1 has only slightly higher mobility than the subunit 10. The small difference in molecular weight observed in our SDS-PAGE could be a result of minor mutations and/or deletions that affect protein charge and/or size. The subunit 10.3 is the y-type found in T.tauschii with higher molecular weight and it has slightly less mobility than subunit 8, coded by the Glu-B 1 locus in hexaploid wheat. Subunit 12.2 has the same mobility as subunit t2 ,,6. Both papers reported this subunit as associated with another subunit, called t l , which has similar relative mobility to subunit 10. Along with the x-type (2. 1 , 1 . 5, 2 and 3), they form a complex of three subunits of high molecular weight 6. In our study, no accessions showing three HMW glutenin subunits were found although 17 accessions with the subunit 12.2 (t2) were encountered. The subunit with 12** (t2) was not accompanied by a corresponding subunit similar to t 1 . Recently, Mackie 12 found that the subunit t l did not precipitate with 60% propan-1-01 as the other HMW subunits 13 and was also not soluble in NaCI, therefore not belonging to albumins or globulins. However this protein was present in the unreduced ethanol extract of storage proteins. This indicates that the t 1 subunit is not a HMW glutenin subunit in spite of its high molecular weight, due to the absence of intermolecular disulfide bonds and should be considered a monomeric protein 12.The protein extraction method that was used in this study first eliminates all the monomeric proteins , i.e. those that do not possess disulfide bonds in their structure. This is the reason why it is unexpected for t 1 protein to be present in our gels. Subunits 12. 1 and 12.3 showed lower and higher mobility respectively when compared with 1 2.2 .However, the other new y-type found, subunit 12.4, is very different to the other subunits of this group with a molecular weight markedly less than those reported previously for HMW glutenin subunits. This large difference in molecular weight may be explained by a loss of nucleotides corresponding to the repetitive domain of the coding genes. This is in agreement with the findings of D'Ovidio et aI. 14 that the differences in size of the allelic subunits detected at the Glu-D1 locus in hexaploid wheat is due to the variation in the central repetetive domain.

142


Figure 1 HMWglutenin subunit (A) patterns in Triticum tauschii lines. The controls (from

left side) are

T.

macha (lst lane), Fiorello (9th lane) and Chinese Spring (J2th lane).

Interestingly, there was one accession that was null for x-type and similarly two were null for y-type. The lack of expression of Glu-D1] has not been previously reported. However, Payne et al. IS and Margiotta et al. 9 have found null forms in Nepalese bread wheat landraces. This lack of subunits was explained as a deletion of the gene 9 or as a silencing of the encoded gene for x-type subunits of the hexaploid wheat 14.

3.2 Low Molecular Weight glutenin subunits

Genes coding for LMW glutenin subunits are located on the short arms of group 1 chromosomes IA, IB and lD (Glu-3 loci). The D genome of T. tauschii is genomically identical to that of the D genome of hexaploid wheat 16. Lagudah and Halloran 17 have located the G/u}] and GIi-'] on the long and short arms respectively of the ID chromosome. Because of the linkage between GIi-] and Glu-3 in hexaploid wheats, it may be assumed that the Glu-1J locus is located , as the homologous Glu-3 locus, on the short arm of the ID chromosome in T tauschii. . This group of proteins has been subdivided i n B and C glutenin subunits, i n accordance with their relative mobility in SDS, the B group being the one with higher molecular weight and therefore, lower mobility.


143

This group of proteins has been subdivided in B and C glutenin subunits, in accordance with their relative mobility in SDS-PAGE, the B group being the one with higher molecular weight and therefore, lower mobility. 3.2. J B Subunits. An extensive variation in the number and relative mobility of these subunits was observed in the material under analysis. Twenty nine different patterns were identified, while in T. aestivum, genes belonging to the Glu-D3 locus coded only for five 18. different patterns The number of bands varied from 1 to 4 ; a wider variation in mobility than those corresponding to Glu-D3 in hexaploid wheat was observed. (Some of them appear in Fig. 2) In some accessions , strongly stained bands could be observed , which may be the result of two or more bands with similar mobility and therefore similar molecular weight. Consequently, more genes are involved in those accessions . Other subunits with the same mobility and different staining could be also the result of differences in gene expression. A very sharp difference in the frequency of the patterns has been observed in the collection (Table 1 ) . Around 43 % of all the lines belonged to three major groups ( pattern # 1 : 2 1 .05%; pattern # 2 : 1 3 .45% and pattern # 3 : 8.77%) 3. 2. 2 C Subunit. Forty three different patterns were observed (some of them appear in Figure 2) for C subunits, higher than that observed in the B zone. This compares with the results reported in bread wheats 18 where only three different pairs of bands were recognised for the Glu-D3 locus in the faster mobility area. A greater number of genes are involved in the Glu-113 gene cluster. Components of LMW in most of the accessions were located in all regions of LMW patterns as occurs with the bands controlled by the D genome in polyploid wheat. Faster and slower bands were also detected in the C zone. A number of subunits ranging from 2 to 6 with a relatively wide difference in mobility was observed for the C subunits. Bands with different staining intensity were found, as previously described for B subunits. Despite the large variability observed in LMW subunits, the number of band combinations found in LMW glutenin subunits of T. tauschii is much lower than the expected number of such combinations on the basis of random association (29 B-subunits x 43 C-subunits = 1 247 combinations) indicating that genes coding for these bands are closely linked. The polymorphism of proteins controlled by gene clusters, as is the case of the proteins of wheat endosperm, could be explained by the following : 1 ) the number of active genes present in the gene cluster; 2) the number of alleles of each active gene; 3) the combination of the different alleles of active genes resulting in different band patterns. 4) recombination which occurs at low frequency might further increase the number of combinations. It is not possible to consider each of these patterns as allelic variants because hybridologic studies have not yet been done. Any of the cases mentioned before could happen given the 1 06 different patterns for LMW subunits ( B and C bands together ) found in the collection. A maximum number of9 subunits for LMW was present in T. tauschii (three B subunits and six C subunits). However, only a maximum of 5 subunits was found in the Glu-D3 b allele of hexaploid wheat. In the latter case, the lesser number of bands could be the result of the dyploidisation process which can cause gene inactivation and gene dosage compensation due to differential gene expression, both of which have been involved in the evolutionary process of polyploid wheats . Galili et af. 19 and Ciaffi et af. 20 have suggested this as the cause of gene inactivation for HMW glutenin subunits coded by the A genome. The high polymorphism found for LMW glutenin subunits (Glu-1f3 loci) should be useful genetic markers for genotype identification. More importantly, allelic differences in LMW gutenin subunits have been shown to be significantly related to flour qualities in bread 2 , 2 and durum wheat 23,24 . New LMW glutenin subunits could improve technological quality for bread flour. Recently, Cox et a1. 25 have reported a successful method for transferring genes from T. tauschii directly to hexaploid wheat.

144


Figure 2: LMW glutenin subunit (B and C) patterns in Triticum tauschii lines. The controls from left are Chinese Spring (1st lane), Halberd (2nd lane), Gabo ( 1 3th lane), Gabo l AL. I RS/I BL. I RS (14th lane) and Hartog l AL. IRS/lBL. I RS ( 1 5th lane). References

1 . H. Kihara, Agric. Hort. 1 944, 19, 889. 2. E. S. McFadden and E. R. Sears, J. Hered., 1 946, 37, 8 1 . 3 . R . AppeJs and E . S . Lagudah, J. Plant Physiol., 1 990, 17, 253 . 4. R. B . Gupta and F. MacRitchie, J. Cereal Sci., 1 994, 19, 19. 5. E. S. Lagudah and G. M. Halloran, Theor. Appl. Genet., 1 988, 75, 592. 6. M. D. H. M. William, R. 1. Pena, A. Mujeeb-Kazi, Theor. Appl. Genet., 1 993, 87, 257. 7. R. B. Gupta and F. MacRitchie, J. Cereal Sci., 1 99 1 , 14, 105. 8. P. R. Shewry, N. G. Halford and S. Tatham, J. Cereal Sci., 1 992, 15, lOS. 9. B . Margiotta, G. Colaprico, R. D'Ovidio and D. Lafiandra, J. Cereal Sci., 1 993, 17, 22 1 .


145

10. D. Lafiandra, R. D 'Ovidio, E. Porceddu, B. Margiotta and G. Colaprico, 1. Cereal Sci., 1 993, 18, 197. 1 1 . P. I. Payne, L. M. Holt and G. 1. Lawrence, 1. Cereal Sci., 1983, 1, 3 . 1 2 . A . Mackie, PhD Thesis, University of Sydney, 1994. 1 3 . B. A. Marchylo, 1. E. Kruger, D. W. 1. Hatcher, 1. Cereal Sci., 1989, 9, 1 13. 14. R. D'Ovidio, E. Porceddu and D. Lafiandra, Theor. Appl. Genet., 1994, 88, 1 75. 15. P. I. Payne, L. M. Holt, E. A. Jackson, C. N. Law, Philos. Trans. R. Soc. London Ser. B., 1984, 304, 359. 16. G. Kimber and Y. H. Zhao, Can. 1. Genet. Cytol., 1983, 25, 5 8 1 . 1 7 . E. S. Lagudah and G . M . Halloran, Theor. Appl. Genet., 1989, 77, 8 5 1 . 1 8. R . B.Gupta and K . W. Shepherd, Theor. Appl. Genet., 1990, 80, 65. 1 9. G. Galili, T. Felsenburg, A. A. Levy, Y. Altschuler, M. Feldman, Proc. 7th. Int. Wheat Genet. Symp., Cambridge UK., 1988, p. 8 1 . 20. M . Ciaffi, D. Lafiandra, E . Porceddu and S . Benedettelli, Theor. Appl. Genet., 1993, 86, 474. 2 1 . R. B. Gupta , K. W. Shepherd and F. MacRitchie, 1. Cereal Sci., 1 989, 10, 169. 22. R. B. Gupta, 1. G Paul, G. B. Cornish, G. A. Palmer, F. Bekes and A. 1. Rathjen, 1. Cereal Sci., 1994, 19, 9. 23 . 1. C. Autran, B. Laignelet and M. H. Morel, Biochemie, 1987, 69, 699. 24. M. Ruiz and 1. M. Carrillo, Plant Breeding, 1995, 114, 40. 25. T. S. Cox , R. G. Sears, R. K. Bequette and T. 1. Martin, Crop Sci., 1995, 35, 9 1 3 .

RELATIONSHIPS BETWEEN BIOCHEMICAL PARAMETERS AND CHARACTERISTICS OF DURUM WHEATS.

M . C . Gianibelli

1

"

M . Ruiz 2, l M . Carrillo 2 and F. MacRitchie

QUALITY

I

I

CSIRO Division of Plant Industry, Grain Quality Research Laboratory, North Ryde, NSW, 2 1 1 3 , Australia. 2 Department of Genetics, E T S . I . Agronomos, Universidad Politecnica de Madrid, 28040 Madrid, Spain. 3 Permanent address: Facultad de Cs. Agrarias y Ftales, Cat. Cerealicultura, UNLP, P OBox 3 1 , 1 900, La Plata, Argentina .

I INTRODUCTION It has been shown that gluten composition is the major factor that determines differences 14 in quality parameters, such as firmness and elasticity in durum wheat flour doughs Gliadins and glutenins are the main groups of gluten proteins. Gliadins have single polypeptide chains, whereas glutenins form polymers of protein subunits linked by interchain disulphide bonds. Glutenins are subdivided into high (HMW) and low (LMW) molecular weight subunits. The association between HMW subunits and pasta quality characteristics are controversial . However a strong correlation has been observed between LMW subunits and quality parameters, such as the SDS sedimentation test and mixograph parameters 5 Differences in the quantity of LMW subunits were associated with variations in quality parameters between two French cultivars of durum wheat 6 The proportion of polymeric protein in total protein ( mainly HMW and LMW glutenin subunits) and the relative size distribution of polymeric protein have been strong correlated with dough strength in bread wheat 7 . The aim of this study was to investigate the effect of allelic variation for LMW glutenin subunits and gliadins in progenies from a cross where both parents had the same HMW glutenin subunits and also to analyse the relationship between biochemical and quality parameters in durum wheat .

2 MATERIALS AND METHODS A durum wheat cultivar with poor gluten quality, Oscar, was crossed with a strong gluten quality cultivar, Mexicali. F2 derived F4 grains were analysed electrophoretically for glutenin (LMW and HMW) and gliadin composition. Gliadins and glutenins were extracted according to the method outlined by Gupta and Shepherd 8. Gliadins were fractionated by acid (pH 3 . 1 ) polyacrylamide-gel electrophoresis (A-PAGE) 9 Grains from each F3 line were tempered at 1 6% moisture and milled using a Uda; cyclone milL Gluten strength was estimated by the modified SDS Sedimentation Test with some minor modifications 3 Mixing properties were assessed by the l O-g mixograph based upon the folIowing parameters: mixograph dough development time (MDDT), maximum height (peak mixing resistance, PR) and the difference between height at PR and after three minutes (resistance breakdown, RBD).

147


Protein content (%P) was estimated by near infrared reflectance (NIR) with a Technicon Infralyzer 400. Vitreousness content (%V) was detennined in 200 kernels by the visual percentage of grains not showing yellowberry. A SE-HPLC technique was used to assess the molecular size distribution of proteins in SDS extracts following sonication and the amounts of different classes of proteins (polymeric, gliadins, albumins/globulins) as well as relative size distribution of polymeric proteins (Unexttactable Polymeric Protein, %UPP) were performed according to Gupta et a/I I . Chromatogram region Peak 1 was subdivided into two adjacent peaks p. and p • . corresponding to pure polymeric proteins and a mixture of smaller polymeric proteins and ID-gliadins, respectively · · . Peak 2 corresponded to gliadins and Peak 3 to albumins and globulins. The ratio of p. to p • . was also calculated.

3 RESULTS Differences in glutenins and gliadins from parents and some F2 grains are shown in Figures 1 , 2 and Table 1 .

8-LMW

C-LMW

2

3

6

7

8

9

10

11

Figure 1 Two-step one-dimensional SDS PAGE of glutenin subunits of the F2 progeny from the cross Oscar x Mexicali. Oscar (1) ; Mexicali (11)

1 48


2

3

4

5

II

7

8

9

10

Figure 2 A-PAGE fractionation of gliadins of the F2 progeny from the cross Oscar x Mexicali. Oscar (10) ; Mexicali (1)


149

Table 1 Allelic differences between parents for gluten proteins and location of the genes

controlling them Protein class

Locus

HMW glutenin subunits

Glu-AI Glu-B I Glu-A3 Glu-B3 GIi-A I G1i-BI GIi-A2 GIi-A3

LMW glutenin subunits Gliadins

Oscar

Mexicali

null

null

7+8 LMW 4 LMW 6 + I l

y-5 1 y-42 a.-2

7+8 LMW 4 LMW I + 14

y-5 1 y-45 a.-I

00-29.5 1 2. 1 3

respectively. LMW and HMW glutenin Gliadin bands and gliadin blocks arc numbered according to subunits are numbered according to 14, 1 5 respectively. LMW glutenin subunits, encoded at Glu-B3, 6+1 1 and 1+14 are also termed LMW-I and LMW2 glutenin subunits patterns, respectively.

Mixograms of both parents are shown in Figure

OSCAR

3

MEXICALI

Figure 3 Mixograms of cultivars Oscar and Mexicali. MDDT: mixograph dough development time. PR: peak mixing resistance and RBD:resistance breakdown. Differences between parents in monomeric (gliadins, albumins and globulins) and total polymeric (mainly g1utenins) proteins are shown in Fig. 4 as well as the different relative size distribution of polymeric proteins (Fig. 5) based on extractability. Correlation coefficients between quality and biochemical parameters obtained by SE-HPLC in F4 1ines are presented in table 2. No significant correlations were found for protein content (%P) and vitreousness (%V) with the other variables analysed. Quality characteristics, evaluated through the SDS sedimentation test (SDSS), mixograph dough development time (MDDT) and peak mixing resistance (PR), were highly correlated with each other. They were also positively correlated with P I, P ill' and unextractable polymeric protein (%UPP) at highly significant levels (>0.00 1 ) and negatively with P I ', P2 and P3, the last one showing significance only at the 5% level. (Table 2) When principal component analysis was performed, variables SDSS, MDDT, PR, PI, P ill' and %UPP appeared positively related with the first main axis while P I ' and P2 were


1 50

related in a negative way. These variables were shown to be independent of r-breakdown (RBD), %V (first principal axis) and %P (third principal axis). Fig 6 represents F4 lines and parents in the first factorial layer ( variance of 70%). Lines with subunits LMW 1+14 (LMW-2 pattern) had the highest values for SDSS, MDDT, PR, PI. PI/I' and %UPP while lines with subunits LMW 6+ 1 1 (LMW-1 pattern) showed the lowest values.

Table 2 Simple correlation coefficients between qUillity characteristics and SE-HPLC parameters. '!IoV

%P

%V

1 .00

lOSS

MDDT

PR

RBD

1'1(%)

P1T4)

P2(%)

1'3(%)

0.08

1.00

0.00

-0.05

0.00

-0.09

0.83 -

-0.12

0.11

0.70 -

0.80 �

1 .00

-0.28

-0.09

-0.07

0.03

0.19

PI(%)

0.03

0.00

0.89 -

0.84 -

0.72 - -0.14

Pl'(%)

0.05

0.10

-0.85 - -0.78 - -0.67 -

0.19

-0.91 -

1 .00

P2(%)

-0.07

-0.05

-0.81 - -0.78 - -0.66 -

0.04

-0.94 '"

0.74 -

1.00

P3(%)

-0.09

-0.12

-0,45 '

-0.42 '

-0.37 '

0.24

-0.54 "

0.39 '

0.40 '

P11P1'

0.02

-0.07

0.90 -

0.83 -

0.69 - -0.19

0.97 - -0.97 - -0.86 - -0.48 "

UPP(%)

-0.02

-0.03

0.92 -

0.83 -

0.75 - -0.06

0.90 - -0.84 - ·0.80 - -0.67 -

'!loP

SDSS

MOOT

PR

RSO

1 .00

P11P1'

UPP(%)

1 .00

1.00 1 .00

1 .00

1.00

0.89 -

1.00

%V: % of vitreousness; %P: protein content; SDSS: SDS sedimentation test; MDDT: mixograph dough development time; PR: peak mixing resistance; RBD: resistance breakdown. P1(%), P 1 '(%), P2(%), P3(%): percentage of the SE-HPLC measurements of peaks I , 1 ' , 2, 3 respectively. UPP (%): % of unextractable polymeric protein. .. , .... , .... refer to significance at probability levels of 0.05, 0.01 and 0.001 respectively. ]50 ••0

A

]00.00

2S0.GO

t

200.00

1.50.00

10G.00

SO.GO

_

0.00

l S ll . 0 0

lOO.OO

'lso.GO

B

tfr

P2

�L-

t 200.00 �o.oo

100.00

so.oo

o . O�

Jj' P,

_. _ _

I !

P2 '�

LL. _ l,f'3_ �

Figure 4:Size Exclusion HPLC patterns of total protein. Oscar (A) and Mexicali (B).

151


..

. .

A

- ...

.. ..

... ..

.. ..

... . ..

... ..

.....

.... 4----__2 I....

'" ...

.

-...

B

..i--.-L--L I

1....

.

-...

.

AA

' ", ..

I

a. ...

BB

.. ..

.. ..

, H."

.. ...

Figure 5 Size Exclusion HPLC patterns of: (A)-(AA) Extractable and unextractable protein from cultivar Oscar showing extractable (a) and unextractable (aa) polymeric protein and (B)-(BB) Extractable and unextractable protein from cultivar Mexicali showing extractable (b) and unextractable (bb) polymeric protein. J..

ABO

J..

"

P3 J..

J..

J..

J..

"

OSCAR

P2

J..

X

J..

.u.

J..

"

J..

P

UO-XCALI "

•

" PR

""

"

MOOT

"

SO�S p.,.

" "

'l.V

"

"

Figure 6 Plot of the 29 Fz derived F4 lines and the parentals in the first principal factorial axis defined by the parameters SDSS (SDS sedimentation test), MDDT, PR, RBD (Mixogram measurements), %P (Protein content), %V (Vitreousness), PI , PI" , Pz , P3 , P 1II" , and %UPP ( SE-HPLC measurements). .. LMW 1 pattern , • LMW 2 pattern.


152 4 CONCLUSIONS

SDS Sedimentation Test and the mixograph parameters , MDDT and PR are highly and positively influenced by the proportion of polymeric proteins (mainly glutenins) in the total protein content and the quantity of unextractable polymeric protein. The lines with the LMW l + 1 4 subunits (LMW-2 pattern) showed better quality, as estimated by SDSS, MDDT and PR, than the lines with the LMW6+ 1 1 subunits (LMW- l pattern). These differences are attributed to a greater proportion of polymeric proteins and a better polymerizing behaviour. The protein content (%P), vitreousness (%V) and r-breakdown (RBD) were not associated with dough strength parameters; therefore in a breeding program for durum wheat quality they should be independently estimated. The biochemical parameters obtained through Size Exclusion - HPLC have proved to be good indicators of dough strength and therefore a useful tool for breeding selection in early generations. Aknowledgements The authors wish to express thanks to O.R. Larroque for helpful collaboration. M.C.G. acknowledges support by the Consejo Nacional de Investigaciones Cientificas y Tecnologicas (CONICET) and Comision de Investigaciones Cientificas de la Provincia de Buenos Aires (CIC) of Argentina.

References N . E . Pogna, D. Lafiandra, P. Feillet and J. C. Autran, l. Cereal Sci., 1 988, 7, 2 1 1 . P . Feillet, O. Ait-Nouh, K. Kobrehel and J. C. Autran, Cereal Chern., 1 989, 66, 26. J . M . Carrillo, J. F. Vazquez and J . Orellana, Plant Breeding, 1 990, 104, 3 25 . M . I . P . Kovacs, N . K . Howes, D . Leisle and J . H. Skerritt , J . Cereal Sci., 1 993 , 18, 43. 5. M . Ruiz and J . M . Carrillo, Plant Breeding, 1 995, 114, 40. 6. J. C. Autran, B. Laignelet and M. H. Morel, Biochirnie, 1 987, 69, 699. 7. R. B . Gupta and F. MacRitchie, l. Cereal Sci., 1 994, 19, 19. 8. R. B. Gupta and K. W . Shepherd, Theor. Appl. Genet. , 1 990, 80, 65 9. D. Lafiandra and D. D. Kasarda, Cereal Chern., 1 985, 62, 3 1 4. 10. J. Dick and J. S . Quick, Cereal Chern., 1 983, 60, 3 1 5. 1 1 . R . B . Gupta, K. Kahn and F . MacRitchie, J. Cereal Sci. , 1 993 , 18, 23. 1 2 . H. D. Sapirstein and W. Bushuk, Cereal Chern., 1 985, 62, 372. 13. N . E . Pogna, J. C. Autran, F . Mellini, D. Lafiandra and P. Feillet, J. Cereal Sci., 1990,11, 1 5 . 1 4 . M . Ruiz and J. M. Carrillo, Theor. Appl. Genet., 1 993 , 87, 353 1 5 . P. I. Payne and G . J. Lawrence. Cereal Res. Cornmun . . 1 983. 1 1 , 29.

l. 2. 3. 4.

EFFECTS OF THE l BU1RS TRANSLOCATION ON GLUTEN PROPERTIES AND AGRONOMIC TRAITS IN DlJRUM WHEAT

Boggini G. ''', Tusa P. ''' , Di Silvestro S." and Pogna N . E .... Experimental Institute of Cereal Research, .. Section of Catania, Via Varese 43, 95 1 23 Catania, ** Section of Applied Genetics, Via Cassia 1 76, 00 1 9 1 Roma, Italy

Summary Six durum wheat lines containing the I BLlI RS chromosome translocation were grown in replicated plots in Sicily during two years of testing and analysed for their agronomical and quality characteristics. Yield and seed quality, as determined by SDS sedimentation volume and Alveograph and Mixograph parameters, were found to be lower and worse in the translocated lines as compared with those of three control durum wheat cultivars. The sticky dough and weak gluten characteristics of the I BLlI RS lines were likely due to the presence of secalins encoded by the G/i-RI locus on chromosome I RS and to the absence of low Mr glutenin subunits encoded by the Glu-3 locus on chromosome I BS. The occurrence of high M r glutenin subunits 7+9, which were shown to

exert positive effects on gluten strength in bread wheat, did not improve the poor dough properties of the translocated lines.

I INTRODUCTION The I BLiI RS chromosome translocation has been used extensively in bread wheat improvement programmes because of the high yield potential and disease resistence against leaf rust (Puccinia recondita spp. tritici), stem rust (Puccinia graminis spp. tritici) and stipe rust (Puccinia striiformis spp. tritici), conferred by genes located on chromosome I RS of rye I +4. However, most of the I BLl I RS bread wheat cuItivars are characterized by poor breadmaking quality and sticky doughS.;-7 The I BLiI RS translocation has been recently introduced into tetraploids wheat lines by crossing the bread wheat cv. Veery and

the durum wheat cv. CandoS. As expected, this lines showed a race-specific resistance against rust due to the Lr 26, Sr 3 1 and Yr 9 genes on rye chromosome I RS. The results reported in this communication concern the implication of the I BLlI RS translocation in grain quality and yield potential of six durum wheat lines containing high Mr glutenin subunits 6+8 or 7+9, the latter pair of subunits being very rare in durum wheat germoplasm.

154


2 MATERIALS AND METHODS 2. 1 Plant material

Seeds of six I BLl I RS translocation durum wheat lines (Titicum turgidum spp. durum) were kindly provided by B. Friebe. Each line was multiplied in head-rows for six generations and screened for morphological uniformity. Total proteins and ethanol-soluble proteins from seeds of the last self-pollinated generation were analysed by SDS-PAGE and A-PAGE respectively, and seeds from head-rows with identical protein patterns were bulked and used in this study. The I BLII RS lines, along with the durum wheat cvs Simeto, Ofanto and Duilio were grown in randomized blocks with three replications at Libertinia (Catania, Sicily) in 1 993 and 1 994 under husbandry conditions, similar to those used for commercial production. 2.2 Electrophoretic and technological analyses

Gliadins were extracted from single seeds with 70 % (v/v) ethanol and fractionated by A-PAGE as described previously9 The reduced total proteins from individual seeds were extracted and fractionated by SDS-PAGE according to Pogna el al. 9 "Straight run" tlours were produced from 3 K g o f grain from the bulk of three replications using a Bona 4RB laboratory mill with sieves of 54 and 42 GG. Protein content (% d. wt. ) was determined using the Inframatic 8 1 00 apparatus. The SDS sedimentation test was performed according to Mc Donald 1 0

Mixograph curves were obtained as described previously 1 1 + 1 2 The Alveograph evaluation of the I BLII RS lines and of the control durum wheat cultivars were carried out according to the I SO method 5530/4 and to the modified procedure developed by Boggini \ 3 , respectively. Yield and technological data were submitted to analysis of variance (ANOVA).

3 RESULTS AND DISCUSSION 3_1 Prolamin composition of 1 BL/I RS durum wheats

According to the gliadin nomenclature system of Kudryavtsev l4, the six I BLl I RS lines contained alleles c, (J and a at the gliadin loci Gli-A I (on chromosome I A), Gli-A2 (on chromosome 6A) and Gli-B2 (on chromosome 68), respectively. The lines inherited these alleles from the parental durum cv. Cando. Moreover, they showed six secalin components encoded by the Gli-R I ( Sec I ) locus on the short arm of chromosome I R ( Fig. I , arrows). SDS-PAGE fractionations of the reduced total proteins from five I BLi I RS lines showed high Mr glutenin subunits 6+8, which =

are encoded by the Glu-Bl locus on chromosome I BL ( Fig. 2 ). This subunit pair also occurred in the parental cv. Cando.

1

2

3

I. A -FAGt-· Figure of fractionation prolamins from (Jj I BLI R5; translocation line CY. 256 8 10, (2) FI from the cross CY. 256 8 10 x CY. Cappelli (durum wheat) and (3) CY. Cappelli. Secalins encoded by I U.S are arrowed.

155


1

2 3 4 5 6

7

One line (25617127), contained Glu-HI encoded high Mr glutenin subunits

8 9 10 11 12

from the parental bread wheat cv. Veery. As far as we know, this is the l BU I RS sole translocation durum wheat genotype containing subunit pair 7+9, which is quite frequent in bread As cultivars. wheat the all expected, translocated lines were found to lack in low M r

7+9

glutenin subunits encoded by the Glu-B3 locus on chromosome l BS.

3.2 Agronomical and quality characteristics

.

, SDS-PAG�

fractionation of the reduced total proteins from the I BI., I �" lines provided hy Dr. B. r.riehe. , The lines 256 8'5 (C( umn nO 1 1), 25687 (column no 10) and

Figure 2.

�

256 8, 10 (column n 8) showed high Mr glutenin subumts 6+8, while the line 256 7 2 7 (column nO 3) showed high Mr glutenin subunits 7+9. The lines 256; 7,26 (column n ° 5) and 256/728 (column nO 2), which still appeared heterogeneous in this photograph, ajier six self-pollinated

generations showed only the high Mr glutenin subunits

Table 1 :

Source of variation

6 "- 8.

ANOVA showed significant variation for yield, heading time, kernel weight and test weight amongst the six I BLl I RS lines and the three control cultivars analysed ( Table 1 ). The effect of year was significant for all the agronomical traits except seed weight, whereas the e ffect of genoty pe x year interaction was significant at P < 0.05 for heading time.

Mean squares for agronomic traits in six 1 BUt RS durum lines and three control cultivan grown in Sicily during two yean of testing. Degrees

of freedom

Genotype (G) Year (Y) GxY interaction

u = p S O. O I ;

1 8

R

Y ield

1 7.75

8.64

Plant

Heading

height

date **

**

0.99 n. s.

... = p S O.05;

2 1 4 46

5 iO.30

\ 6.60

**

**

*

1 37 R6

1 ,557.4 1

n. �. **

1 33 . 1 9 n. s. n. s.

=

1 000 kernel

Hectolitre

weight

weight

444 . R 3

**

1 8.08 n. s.

23.46 n. s.

not significant.

88 57

1 0 1 .83

**

**

8.70 n. �,


156

Table 2:

Mean values over two years of testing for agronomic traits in six IBUIRS durum lines and three control cultivars. Yield

Genotype

Heading time (days

(tfha)

Hectoliter weight

from april 1 sl)

I BVI RS lines

b

1 000-kernel weight

Plant height

(g)

(kg)

(cm)

28. 1 bc 28.5 be 28.0 be 25.4 b 33.9 d 29.0 c

73.6 de 74.2 d 7 1 .2 ef 74.8 cd 70.5 f 73 . 1 de

27.8 24.8 26.3 27. 1 28.3 25.2

6.5 a 6. 1 a 5.8 a

1 6.8 a 18.7 a 1 7.7 a

79.7 ab 77.2 be 82. 1 a

44.5 a 43.2 a 43.2 a

80.3 a 85.7 a 76.7 a

Mean values of I BVI RS lines

2.8

28.8

72.9

26.6

82.4

Mean values of control cvs

6.2

1 7. 7

79.7

43.6

80.9

Friebe 25617, Friebe 25617, Friebe 25617, Friebe 256/8, Friebe 256/8, Friebe 256/8,

26 27 28 5 7 11

3.2 2.2 2.6 3.3 3.1 2.5

c

be b be be

b b b b b b

87.3 a 89.7 a 8 1 .0 a 8 1 .7 a 79.7 a 75.3 a

Control evs Simeto Ofanto Duilio

Values followed by the same letters in columns are not significantly different at P to Duncan's multiple range test

=

0.05 according

The I BLlI RS lines were not adapted to Sicilian environment because of their late heading, which resulted in lower kernel weight, test weight and yields as compared with the control cultivars (Table 2). Seed quality characteristics of the I BLlI RS translocation group were compared with those of the control group by analysis of variance (Table 3 ). There were significant differences (P < 0.0 1 ) amongst the genotypes for most of the characteristics; the effects of year and genotype x year interaction were significant for some quality parameters. However, the variance component for genotypes accouqted for most of the variation and the effects of year and genotype x year interactiorl� were small in magnitude for most quality characteristics. Table 3:

Source of variation

Mean squares for quality traits in six I BUIRS lines and three control cultivars grown in Sicily during two years of testing.

Genotype (G) Year (Y) GxY interaction **

=

Protein SDS content sedimen tation volume

Degrees of freedom

p > O.OI

5.27 * * 226.97 0. 1 3 n.s. 1 7·3.36

8

2.25

8 *

=

**

26.05

p > 0.05

**

**

ALVEOGRAPH PIL

W

5,006.68 1 ,88 1 .46

**

*

=

Judgement

Mixing time

Peack height

1 . 1 6 D.S. 10.76 * * 1 26. 1 8 D.S. 229.72 * * 2 . 1 2 D.S. 2.00 n.s. 80.22 D.S. 40.50 *

**

n.S.

MIXOGRAPH

not significant


157

TABLE 4: Mean values over two years of testing for quality traits in six IBUIRS

durum lines and three control cultivars. Genotype

Protein content

SDS

sedimentation

(% d. wt.)

index

(ml)

MIXOGRAPH

ALVEOGRAPH W

(j x 1 0-4)

P/L

Judge-

Mixing

Peak

ment time (mm) height (mm)

1BV1RS lines

47.5 be 37.5 d 39.5 d 46.5 c 4 1 .5 cd 39.5 d

6.5 a 5.5 a 5.5 a

52.0 a 40.5 a 49.0 a

62.5 a 67.5 a 53.5 b

3.2

1 .3

35.8

42.0

2.2

5.8

47.2

6 1 .2

1 .5 1 .0 1.5 1 .5

d

3 . 1 ab 3.6 a 3.0 ab 2.9 ab 2.7 ab 3.8 a

42.0 b 41.5 b 43.5 a

166.3 a 12 1 .7 be 157.6 ab

3.2 ab 1 .4 b 1 .9 ab

27.5

55.9

42.3

148.5

28.0 28.5 24.8 29.0 28.8 26.3

Simeto Ofanto Duilio

13.2 d 1 3.6 cd 1 3.6 c

Mean value of the lBV1RS lines

1 5.2

Mean value of the control cvs

1 3.5

26 Friebe 256/7, 27 Friebe 256/7, 28 Friebe 256/8, 5 Friebe 256/8, 7 Friebe 256/8, I I

46.5 a 3 1 .5 a 33.5 a 30.5 a 34.5 a 38.0 a

58.6 de 70.4 de 35.7 de 80.5 cd 33.4 e 56.7 de

1 3.4 cd 1 5. 5 b 15.5 b 15.2 b 1 5.5 b 1 6.2 a

Friebe 256/7,

c

c

e c c

b

b b b LO b 1 .0 b

Control cvs

Values followed by the same letter in columns are not significantly different at P

to Duncan's multiple range test

=

0.05 according

The IBLlIRS group was characterized by values of SDS-sedimentation volume significantly lower than the control group (Table 4). Significant differences amongst the IBLllRS lines for this quality parameter were observed only in 1 994 (data not shown). All the lines were significantly different from the control cultivars in Alveograph W and mixograph peak height and judgement, whereas there was no significant difference between the two groups for the Alveograph PIL and mixograph mixing time. These results suggest that the durum lines containing the IBLllRS translocation produce weak doughs as observed in most bread wheat cultivars containing this translocation5+6. The high protein content of the IBLllRS lines as compared with that of the control cuItivars was likely due to their poor yield potential in the Sicilian environment. In this context it is noteworthy that the presence of the IBLll RS translocation in bread wheat cultivars has been found to be associated with high yield potential and reduced protein content7, 15+ 1 6. Doughs from the IBLl l RS lines showed a strong tendency to stick to the mixing bowl and hands, and could not be pulled out cleanly from the mixing bowl; in contrast the control cultivars showed a low degree of dough stickiness. Finally, dough quality characteristics of line 256/7/27, which contains high Mr glutenin subunits 7+9, were not significantly different from those of the remaining IBLll RS lines, which possess glutenin subunits 6+8 (Table 4).

158


4 CONCLUSION None of the I BL/I RS durum wheat lines here analysed had quality characteristics that would make them suitable for production of pasta or bread. Their poor quality characteristics are likely to be largely due to two factors: (i) the presence of secalins encoded by the Gli-Rl locus, on chromosome I RS and (ii) the absence of low Mr subunits encoded by the Glu-B3 locus on chromosome l BS. The negative effects of the l BLll RS chromosome on dough quality (weak gluten and dough stickiness) were not mitigated by the presence of the high quality glutenin subunits "7+9"17. These negative effects on gluten quality can be eliminated or, at least, minimized through three possible genetic approaches that would not affect the rust resistance potential of the I RS durum lines. The first approach is deletion of a small chromosome segment containing the GIi-Rl locus by irradiation of the translocated lines. This approach has been successfully applied to bread wheat 1 8 Alternatively, the phI c allele, which promotes homreologous pairing, can be introduced in a homozygous condition into the l BLl I RL lines, and allosyndetic recombinants possessing the Glu-B3 locus along with genes for rust resistance can be selected in the self-pollinated progeny of Glu-B3IGIi-Rl heterozygotes. Six secalin-free allosyndetic recombinants containing the Lr 26 gene have been recently selected in the 1 2 8 progeny of one 1 BLlIRS line related genetically to those here analysed (manuscript in preparation). The third approach consists in the incorporation of the l ALIlRS translocation from the bread wheat cv. Amigo into tetraploid wheat cultivars possessing LMW-2 type glutenin subunits encoded by chromosome I BS, in order to mitigate the deleterious effects on dough strength due to loss of this chromosome arm. The I ALII RS translocation is currently being transferred into elite durum genotypes in our lab.

References

1.

6.

P . BARTOS and I. BARES, Euphitica, 1 97 1 , 20: 425 F. J. ZELLER, 'Proceedings 4th International Wheat Genetic Symposium', Columbia, 1 973, MO, p. 209. S. RAJARAM, Ch. E. MAAN, G. ORTIZ-FERRARA and A. MUJEEB-KAZI, 'Proceedings 6th International Wheat Genetic Symposium', S. Sakamoto ed., Plant Germoplasm Institute, Faculty of Agriculture, Kyoto University, Kyoto, Japan, 1 983, p. 6 1 3 . F. J. ZELLER. and S. L. K. HSAM, S. L. K., 'Proceedings 6th International Wheat Genetic Symposium', S. Sakamoto ed., Plant Germoplasm Institute, Faculty of Agriculture, Kyoto University, Kyoto, Japan, 1 983, p. 1 6 1 . D. J. MARTIN and B. G. STEWART, Euphitica, 1 986, 35: 225 . R. J. PENA, A. AMAYA, S. RAJARAM and A. MUJEEB-KAZI, Jour. Cer. SCi. ,

7.

D. FENN, O. M. LUKOW, W. BUSHUK and R. M. DEPAUW,

2.

3.

4. 5.

.

1990, 12: 105.

Cer. Chern. , 1994,

70: 1 89.

8. 9.

1 0. 11. 12.

B. FRIEBE, F. J. ZELLER and R. KUNZMANN, Theor. Appl. Genet., 1 987, 74: 423 . N. E. POGNA, J. C. AUTRAN, F. MELLINI, D. LAFIANDRA and P. FEILLET, Jour. Cer. Sci. , 1 990,

1 1 : 15.

C. E. Me DONALD, Cereal Food World, 1 985, 30: 674. G. BOGGINI and G. NILSSON, Cereal Res. Cornrn. , 1 976, 4: 3. G. BOGGINI and N. E. POGNA, Agricoltura e Ricerca, 1 990, 1 1 4:

59.


13. 14. 15. 1 6. 17. 1 8.

159

G . BOGGINI, Molini d'Italia, 1 990. 4: 1 5 1 . A. M. KUDRYAVTSEV, Russian J o/Genet. , 1 994, 30: 69. A . S . DHALIWAL, D . J . MARES and D . R . MARSHALL, Cer. Chern. , 1 987, 64: 72. R. L. VILLAREAL, S. RAJARAM, A. MUJEEB-KAZI and E. DAL TaRO, Plant Breeding, 1 99 1 , 106: 77. N . E. POGNA, F. MELLINI, A. BERETTA and A. DAL BELIN PERUFFO, J Genet. & Breed , 1 989, 43: 1 7. E. MILLET and M. FELDMAN, 'Proceedings VIII International Wheat Genetic Symposium', Beijing, China, 20+25 July 1 993. (Abstract).

DURUM WHEAT FOR BREAD MAKING: RELATIONSHIPS BETWEEN PROTEIN MOLECULAR PROPERTIES AND TECHNOLOGICAL PARAMETERS

M. Carcea, N. Guerrieri* and L. A. Pasqui National Institute of Nutrition, Via Ardeatina 546, 00178 Rome, Italy *Department of Agricultural and Food Molecular Sciences, University of Milano, Via Celoria 2, 20133 Milano, Italy

1 INTRODUCTION Durum wheat (Triticum durum Desf.) semolina is used almost exclusively for pasta making. However, around the Mediterraneum and especially in southern Italy, there has always been a traditional consumption of bread made with remilled semolina. 1 A number of publications recently reviewed by Boyacioglu and D'Appolonia2 testify to past and recent interest in the use of durum wheat for bread making, as main ingredient or as improver of the baking quality of soft wheat flour. In 1 985 Boggini in a study on durum wheat for breadmaking concluded that, amongst the Italian durum wheat cultivars, those with poor pasta making qualities showed the same negative behaviour during bread making.3 In 1 989 Boggini and Pogna associated bread making quality with gluten viscoelastic properties, protein content and protein composition.4 In particular most cultivars with 'Y-gliadin 42 had lower loaf volumes than cultivars with the allelic 'Y-gliadin 45. Moreover, the HMW glutenins subunit composition of the durum wheat cultivars examined appeared to affect bread making quality. Wheat flour is a multi-component raw material and obviously all of them interact to affect its baking quality. However, it is well known that proteins play a basic role in determining the rheological characteristics of a wheat dough and consequently bread quality. Therefore, within the frame of a study on durum wheat for breadmaking, we chose to focus our attention on possible relationships between protein molecular parameters and quality data, defined in a range of ways, in a set of Italian durum wheat cultivars of diverse origin, generally used for pasta making and possessing a range of gluten qualities. A common soft wheat flour was also used as a comparison. 2 MATERIALS AND METHODS 2 . 1 Samples

Remilled semolina was obtained by means of a Buhler experimental mill MLU-202 from the grains of 9 Italian durum wheat cultivars (named Creso, Duilio, Grazia, Appio, Messapia, Appulo, Capeiti, Plinio, Latino) tempered to 1 6% moisture. A blend in equal parts of grains of 2 Italian soft wheat cultivars, Mec and Centauro, was milled to flour and used as control. 2.2 Chemical and rheological tests

Moisture and protein (Nx5.7) of remilled semolina were determined according to AACC

161


approved methods 44- 1 5 A and 46- 1 1 A respectively.5 Falling number was determined according to the ICC standard method No. 107.6 The Manual Gluten Quality (MGQ score) was obtained according to Landi,7 whereas the Gluten Index and the dry gluten content were obtained according to Cubadda et al .. 8 The SDS test was done following Axford et al.,9 with 3% S.D.S .. Farinograph and alveograph tests were performed according to AACC approved methods 54-21 and 54-30 respectively.5 2.3

Baking test

A straight-dough method was used to prepare pan bread loaves with the following ingredients: flour l 000g, compressed yeast 4%, salt 1 .5% and variable water according to the Brabender Farinograph. Malt was also added in variable amounts to adjust the falling number of the remilled semolina to the optimum value for baking (about 250s). All ingredients were mixed to optimum dough development (6 min) in a spiral mixer 60 r/min). The dough was left to ferment in bulk for about 30 min in a proofing cabinet under controlled temperature (30°C) and relative humidity (about 80%). It was then punched and mechanically moulded to give 250g loaves. The loaves were proofed under the same conditions as before until the dough had risen to a fixed height. The loaves were baked in a revolving oven at 250°C for 1 5-20 min. Volume was measured 3 h after removal from oven by the rapeseed displacement method whereas crumb characteristics were scored according to Mohs's scale as reported by Dallmann.lO 2.4

Proteins fractionation and quantification

Durum wheat proteins were fractionated according to the following procedure: Remilled semolina

I

Tris-HCl 0.05 M, pH 8.5, 1M NaCl

I

Distilled water

I

70% ethanol

J,

>Globulins > Albumins >Gliadins

HMW + LMW Glutenins Soluble proteins were quantified by the Bradford dye-binding method as modified by Eynard et al. l I. Globulins, albumins, gliadins and glutenins were studied by SDS PAGE. 1 2 Gliadins were also analyzed by Acid-PAGE. 13 The HMW/LMW ratio was calculated from the video image of the glutenins SDS-PAGE (Software CREAM. Kern en-tee). 2.5 Statistical analyses Data were analyzed by ANOVA and Duncan's multiple range text. Simple and multiple correlations were elaborated with the programme Statistica for Windows (Release 4.5) of StatSoft. 3. RESULTS AND DISCUSSION The glutenins and gliadins subunit composition, protein content, dry gluten and some gluten quality parameters are reported in Tab. l .


162

Table 1 Gluten Composition, Proteins, Dry Gluten, Gluten Index, Manual Gluten QualifJ: (MGQ) and S,D,S. Test Value otDurum Wheat Cultivars* SampleZ

Gliadins

Y

Proteins Dry gluten Gluten M G Q N x 5, 7 (% d.m ) Index scorex HMW (% d.m.)

Glutenins

lMW

Control

1 2.9c

l O.5e

92a

8

S.D. S value (ml)

Creso

45

2

6+8

1 2.7d

l 1 . 1 cd

87b

8

50

Duilio

45

2

7+8

1 2 .7d

l 1 . 1 cd

78c

8

45

Grazia

45

2

20

1 2.0e

l 1 .2cd

77c

7

44

Appio

45

2

20

1 2.0e

1 1 . 3c

72d

7

38

Messapia

45

2

20

1 2.9c

1 2. 2b

62e

6

44

Appulo

45

2

20

13.1b

1 2 .9a

38f

5

33

Capeiti

45

2

20

1 1 . 8f

l 1 .2cd

39f

6

30

Plinio

45

2

7+8

1 2. 1 e

1O.9de

72d

7

45

Latino

42

7+8

1 3 .3a

1 1 .9b

3g

4

25

*

All the values, a part from the MGQ scores and SDS values, are the means of duplicate determinations. Means within columns with different letters are significantly different (p$ 0.05). Z Control was bread flour. All the other samples were remilled semolina. x Gluten Quality obtained by the Manual method (range: 1 -10). Boggini and Pogna4 found that the HMWglutenin subunit composition of durum wheat cvs in combination with gliadin 42145 could be used as an indicator of quality in bread making. Therefore, we analyzed our samples for their gliadins and glutenins subunit composition. All of the durum wheat cultivars studied, except the cultivar Latino, possessed the 'Y-gliadin band 45. Most of the cultivars had the HMW glutenin subunit 20, some had the subunit pair 7+8, whereas only the cultivar Creso had the subunit pair 6+8. The Gluten Index, the Manual Gluten Quality Score and the SDS value clearly indicated the existance of a range of gluten qualities within our group of samples; from Latino, lowest values, to Creso, highest values. Alveograph and farinograph param�ters of the samples under study together with bread volume and crumb texture are reported in Tab. 2. According to our experience on soft wheat flour, values of W above 1 7 0 indicate good baking quality provided that the P/L value is between 0.30 - 0.70. Below 1 10 the baking quality is generally inadequate. Following this classification, the control sample with a W of 198 and a P/L of 0.24 would fall into the good baking quality group whereas the only durum wheat cv, Creso, with a W above 1 7 0 ( l 80) would have too high a P/L ratio, indicating, as expected, higher tenacity versus elasticity. A high P/L ratio is, in fact, typical of durum wheat samples whose gluten is, as known, very tenacious and not very elastic. Farinograph absorption values for the durum samples were in general higher than the control as reported also in the literature.14 This behaviour could be due to a higher content of damaged starch in remilled semolina. Development time of durum samples was generally higher than the control as a consequence of the higher strength of durum gluten compared to soft wheat and differences were also observed in stability and mixing tolerance. The cultivars Appulo and Latino, which scored the lowest values of MGQ, gave also the lowest values of stability and development. The results of the baking test are also reported in Tab. 2. Significant differences were found in bread volume of durum wheat cultivars which were all but the cultivar Plinio

163


Table 2 Technolosical Characteristics otDurum Wheat Cultivars AlveographZ Sample *

W

G

P/L

Control

198a

30.2

0.24f

Bread

Farinograph

Absorp. Develop. Stability MTX (sec) (B. V. (sec) (%) 5 5 .2

90

Crumb

Volumez TextureY (cc)

5 10

30

567c

8

630b

7

656b

8

Creso

180b

2 1. 2

0.82c

6 1.8

150

255

60

Duilio

1 6 1d

19.0

1 .02a

6 1 .9

150

240

60

Grazia

167c

22.9

0.54e

59.3

165

225

60

657b

8

59.0

150

2 10

50

654b

7

59.5

150

225

70

65 l b

8

6 l2d

7

Appio

156d

19.3

0.93b

Messapia

1 l 8e

2 1. 8

0.54e

Appulo

1 l 7e

22. 1

0.56e

59.3

170

90

60

Capeiti

IOlf

2 l .3

0.55e

60. 5

170

105

70

675a

7

Plinio

l 56d

2 1 .0

0.70d

58.6

180

225

80

629c

8

16.9

O.77c

60. 1

l35

90

60

622cd

6

Latino *

67g

Control was bread flour. AIl the other samples were remilled semolina. Means of four or six (bread volume) determinations. Means within columns with different letters are significantly different (p $ 0.05). x Mixing Tolerance. y Mobs score, range 1 -8. Highest value given to a very close porosity with very small pores.

Z

higher than the control. Crumb structure of durum samples showed in general quite a close porosity. The different protein fractions of remilled semolina, i.e. globulins, albumins, gliadins, HMW and LMW glutenins were fractionated according to the procedure described in the Materials and Methods section and reported on a percentage basis (data are not shown). All the durum wheat cultivars compared to the control showed a sensibly lower content of glutenins, especially the HMW fraction. Moreover different proportions of the various protein fractions were noticeable in the durum cultivars examined. After quantification of technological and molecular parameters we went on to study possible correlations between them. Simple correlations between technological parameters are reported in Tab. 3. Table 3 Simple Correlations between Technological Parameters in Durum Wheat * Alveograph W

P/L

Farinograph

Gluten Index

S. D.S value

0.5770 0.6201

!!.Mll

0.4443

� 0. 3 8 14 0.5 1 1 8

0.02 1 4

0. 1407

Absorption Develop. Stability Mixing Tolerance

Bread Volume 0 .3009 0.5079

� Q..6.8!lli

Bread Voll Proteins

0.4265

*

0.0676 0.3404

Underlined correlation coefficients are significant at p$O.05.

Significant positive correlations were found between some farinograph parameters such as absorption and development and bread volume. Bread volume normalized for protein content was significantly correlated to development only. Boggini and Pogna4 also reported development time as being positively correlated to bread volume in durum

1 64


wheat. This parameter could therefore be considered a good predictor of baking quality in durum wheat. Simple correlations between technological and molecular parameters are instead reported in Table 4. Table 4 Simple Correlations between Molecular and Technological Parameters in Durum Wheat* Bread Volume! Volume Proteins

Farinograeh

Alveograeh W

PIL

Absorp. Develop. Stab.

0.6002 Q.8B1l

0.2472 0.0933

0.0467 0.4818 0.0505

Glutenins + 0.3095 0.3035 Gliadins

0. 1 1 20 0.3573

0.3785

Gliadinsl Glutenins

0.3861 0.3742

Gliadins

Proteins

Gluten S.D.S

Mixing Index Tolerance 0.2832

0.481 2 0.2067

Q.B.l82. 0.4866 Q.Bill

0. 1 1 10 0.4033

0.0696 0.0388

0.21 86 0.4423 0.0658 0.6140

0.3559 0. 1 2 1 5

0.4225 0.3922

0.0432 0.0014

0.2214 0.2982 0.0153

0.4963

0.1622 0.5059

LMW

0.2083 0.0833

0.3798 0.3240

0.2803 0.0849 0.4793

0. 1973

0.4194 0.5582

HMW

Q.1448 0.6129

0.3423 0.4092

Mill Q...8ill

Q.ll54 ll.lill

0.2678 0.4144

HMW+IMW 0.4338 0.4 1 80

0.0026 0.0932

0.3206 0.6146 0.2232 !illl12.

0.0829 0.6054

HMWIIMW !l.1125. 0.6068

0.41 87 0.4890

ll.12Ql QMQ1 !!.12!i1

.!l.1..ll1

0.3559 0.1221

Globulins

0.4326 0.4233

0.0358 0.4537

0.3977

Q.W1 0.4286 �

0.0067 0.5445

Albumins

0.4382 0.4266

0.2640 0.3461

0.0765 0.0363 0.1908 0.021 5

0.1409 0.3390

Albumins+ Globulins 0.3096 0.3035

0.1 1 20 0.3573

0.3785

Q.B.l82. 0.4866 Q.Bill

0.1 1 10 0.4033

Dry Gluten

Mill 0.0193

0.2337 0.3387

QJi651 0.2841

0. 1737 0.5922

*

0.0562 0. 1 966

Underlined correlation coefficients are significant at p:'>0.05.

Significant positive correlations were found between bread volume and the total amount of HMW glutenins and between bread volume and the ratio HMW/LMW glutenins. HMW glutenins seem therefore to play a key role in determining durum wheat baking qUality. Significant and high positive correlations were also found between some farinograph parameters such as development and mixing tolerance and the amount of glutenins+gliadins, the amount of HMW glutenins, the HMWILMW ratio, the amount of globulins and globulins+albumins. Furthermore, mixing tolerance was also positively correlated to the amount of HMW +LMW glutenins. Farinograph absorption was significantly correlated only to the total amount of HMW glutenins and to the HMWILMW ratio, whereas stability in addition to being correlated to the same molecular parameters in the same way, was significantly correlated also to dry gluten. The alveograph parameter W showed only a significant positive, but low correlation with dry gluten. The study of the linear correlations between our variables suggested the existence of more complex relationships between them so we expanded our study to the mutual relationships between three variables. We inserted our variables in three dimensional graphs and we examined a number of them reporting different combinations. However,

16 3t 0.325 0. 3 3: 0.275 J 0.25 0. 225

3

0. 375 0. 35 0. 325 � 0. 3 � 0.2 75 0. 25 � 0.225 :r 0. 2

�


166

the most meaningful appeared to be those where the y and x axes were respectively, bread volume and percentage of glutenins. Some tridimensional graphs where the z axes are HMWILMW glutenins, gliadins/glutenins, globulins+albumins are depicted in Figure 1 . Within the same figure, we considered also of interest to differentiate our samples according to their HMW glutenins subunit composition. Our group of samples was not homogeneous as far as gliadins were concerned because the cultivar Latino (L) had "( gliadin 42 which could already be considered an indicator of poor baking quality, as confirmed by its bread volume. By observing Figure 1 , we could say that our 7+8/"(45 samples (P and D) were both characterized by a high HMW/LMW ratio even if they differed for their percentage of glutenins. The highest the level of glutenins, the better was bread volume (D>P). Within the same group of samples a smaller gliadin/glutenin ratio seemed to be favourable to bread volume together with a low albumin+globulin percentage. This latter statement could be valid also for the cultivars of the 201"(45 group where Capeiti 8 (C 8), which gave the highest bread volume, was characterized by the lowest level of globulins+albumins and vice-versa the cultivar Appulo (A). The HMW 20 group seemed in general to be characterized by a low HMWILMW ratio whereas the gliadin/glutenin ratio varied a great deal. The two extremes in this group of samples as regard to bread volume, i. e. C 8 (highest bread volume) and A (lowest bread volume) had not too distant glutenins percentages and HMW/LMW ratios but they had markedly different gliadin/glutenin ratios and globulins+albumins contents. Unfortunately there was only one 6+8 sample (C). A low albumins + globulins percentage (monomeric proteins) and a high level of glutenins (polymeric proteins) seemed to be favourable to bread volume in all the samples studied. This observation is supported by indications in the literature about the destabilizing role of monomeric proteins during loaf expansion. Even if our findings should be confirmed and corroborated by the observation of a bigger population of durum wheat samples , we could nevertheless conclude that simple correlations can not be used to predict baking quality in durum wheat, but multiple correlations (3D and more) between molecular and technological parameters could be useful to understand the role of each component and to identify their best combinations.

Acknowledgements We acknowledge the help of Mr. E. Caproni and Mr. L. Bartoli. Research supported by National Research Council of Italy, Special Project RAISA, subproject 4, Paper No. 23 1 1 .

References 1. G.B. Quaglia, 'Durum Wheat Chemistry and Technology', G.Fabriani and C . Lintas, Am. Assoc. Cereal Chern., St. Paul, MN USA, p. 263. 2. M. H. Boyacioglu and BL D'Appolonia, Cereal Foods World. 1994, 39, 1 68. 3. G. Boggini, Tecnica Molitoria, 1 985, 6, 579. 4. G. Boggini and N.E. Pogna, 1. Cereal Sci., 1989, 9, 1 3 1 . 5 . A.A.C.C., 'Approved Methods o f the American Association of Cereal Chemists', St. Paoul , MN USA, 1983. 6. I.C.c . , 'Standard Methods of the I nternational Association for Cereal Science and Technology', Moritz Schafer, Detmold, Germany, 1987. 7 . A . Landi, 'The Future o f Cereals for Human Feeding and Development o f Biotechnological Reserch ( I n Italian), Chamber of Commerce, Foggia, Italy, 1988. 8. R. Cubadda, M. Carcea and L.A.Pasqui, Cereal Foods World, 1992, 37, 866. 9. D.W.E. Axford, E.E. McDermott and D.G. Redman, Milling Feed Fert. , 1978, 161, 1 8 . 1 0 H. Dallmann, 'Porentabelle. Fortschritte der Getreideforschung', Moritz Schilfer, Detmold, Germany, 1 969. 1 1 . L. Eynard, N. Guerrieri, P.Cerletti, Cereal Chern. , 1994, 71, 434. 12. U . K. Laemmli, Nature, 1 970, 227, 680. 1 3 . A. Dal Belin Peruffo, N.E. Pogna, C. Pallavicini , E. Pegoraro, F. Mellini, A. Bianchi, Sernenti Elette, 1984, 30, 1 . 14. M . H . Boyacioglu and B.L. D'Appolonia, Cereal Chern., 1994, 7 1 , 2 1 . ,

,

CONTRIBUTION OF THE Hordeum chilense GENOME TO THE ENDOSPERM PROTEIN COMPOSITION OF TRITORDEUM

J . C . Sillero, 1.B. Alvarez, and L.M. Martin. Departamento de Genetica Escuela Tecnica Superior de Ingenieros Agronomos y de Montes Universidad de Cordoba Apdo. 3048, E- 14080 Cordoba, Spain.

1 SUMMARY

Several studies have indicated that the breadmaking quality of tritordeum (Hordeum chilense-wheat amphiploid) are related with the presence of the H. chilense proteins into the endosperm of the amphiploid. The objective of this work has been to group these proteins by the Osborne's categories and stablish their degree of genetic variation. These protein fractions were obtained from eight lines of tritordeum (5 hexa- and 3 octoploid) and analyzed by SDS-PAGE. The results indicated that the H. chilense genome incorpo rate a variable number of protein components to each fraction. Likewise, although the globulin fraction did not presented any variation for the tested lines of tritordeum, a certain degree of variation could be observed in the other three fractions. 2 INTRODUCTION

Tritordeum (XTritordeum Ascherson et Graebner) is the amphiploid derived from the cross between a South American wild barley (Hordeum chilense Roem. et Schulz. ) and wheat. This amphiploid was obtained in the hexaploid form (H. chilense x Triticum turgidum conv durum Desf. em. M.K.) for the first time in 1 9791 • Before the production of hexaploid tritordeum, an octoploid form of low fertility had been obtained from the cross involving H. chilense and bread wheat (T. aestivum L. em. Theil, cv. 'Chinese Spring'2. From the beginning, the hexaploid form showed promising characteristics as a new 3 crop . Later studies confirmed these expectations4,s. The data suggested that tritordeum could be used as a protein source crop, because of its high grain protein content4• Nevertheless, recent studies have shown that the protein contents of tritordeum and wheat are not significantly different when their grain yields are similar6• Alvarez et al . 7, using several physicochemical tests, found that hexaploid tritordeum exhibited some potential for breadmaking. Similar results were also obtained for some lines of octoploid tritordeum derived from bread wheat cultivars with better agronomic characteristics than those of the parent wheat cultivar, cv. 'Chinese Spring'8,9. Likewise. the results suggested that careful selection of the H. chilense line for use in the crossing experimental was important with respect to the end-quality of the tritordeum producedJO• because of the endosperm storage proteins contributed by H. chilense8,l 1 .


168

The objective of the current work has been to study the nature of the H. chilense proteins expressed in the endosperm of tritordeum, as well as to analyze their capacity of variation. 3 EXPERIMENTAL 3 . 1 . Grain samples The following eight lines of primary tritordeum, five hexaploid ([H- l IT-22 l , [H1 2/T- 103 ] , [H-57/T-5S] , [H-6 1 /T-5S] , and [H-6 1 1T- 1 03]) and three octoploid ([H- l IT26] , [H- 1 2/T-65 ] , and [H-55/T-79]) were analyzed, along with their respective wheat parent (T). The genomic constitution of the hexaploid tritordeum l ines is AABBHcbHcb and the octoploid tritordeum lines is AABBDDHcbHcb, where Hch is the H. chilense genome. 3.2. Protein extraction The different fractions (albumins, globulins, gl iadins and glutenins) were extracted from 20 mg of wholemeal flour by sequential extraction according to Khan et al . 1 2• The glutenins were obtained from the last pellet. 3.3. SDS-PAGE procedure Reduced and alkylated proteins were electrophoresed in vertical SDS-PAGE slabs in a discontinuous Tris-HCI-SDS buffer system (pH = 6.S/S. S) at a polyacrylamide concentration of 12 % (w/v). Reduction and alkylation procedures of the proteins were performed as described by Graybosch and Morris 1 3 • Al iquots ( 7 . 5 JL I ) were transferred to the sample wells o f gel . Electrophoresis was performed at a constant current of 30 rnA for 3 . 5 h at lOOC. Samples were run with standard reference proteins: Phosphorylase B, 94 kD; Bovine seroalbumin, 67 kD; Ovoalbumin, 43 kD; Carbonic anhydrase, 30 kD. The molecular weights of protein components were determined by comparison with these proteins. Gels were fixed and stained according to Alvarez et al. 1 1 . 4 RESULTS AND DISCUSSION Figure 1 shows the albumin patterns from the tested l ines. As far as could be determined from one dimensional electrophoresis, the tritordeums presented some additional bands to those of their wheat parents, which have a sharp origin in the H. chilense line. So, the tritordeums derived from the line H- l and H - 1 2 presented one band, named as a, with a Mr of 54.6 kD, but this component was not observed in the other tritordeums. Likewise, these tritordeums and also [H-55/T-79] incorporate one second band (b, Mr= 3 2 . 2 kD) absent in the rest. On the contrary, a new band (c, Mr = 3 1 . S kD) appear in the rest of the lines, [H-57/T-58] , [H-6 1 1T-58] and [H-6 1 1T- I03] . These results suggest that the bands b and c could be allelic, but this hypothesis must be confirmed with new studies on these components . The differences observed between the tritordeum [H- l IT-26] and its wheat parent (Figure 1 , lanes 14 and 1 5 , respectively) in the zone marked with the asterisk, could be only consequence of an over-extraction in tritordeum . It is noteworthy that this tritor deum show the highest protein content face to its parene4• On the other hand , these differences can not have their origin in the H. chilense line because of the other line of


169

tritordeum derived from the l ine H� 1_ did not present any irregularity. 2

3

4

5

6

7

8

9

10

11

A

12

13

Figure 1 SDS-PAGE separation of albumins of wheat and tritordeum lines. Lanes are asfollows; 1, T-22; 2, [H-lIT-22]; 3, [H-J2IT-103]; 4, T-1 03; 5, [H-611T-103], 6, [H-

5 7IT-58]; 7, T-58; 8, [H-611T-58]; 9, T-65; 10, [H-J2IT-65]; 11, Mw,' 12, T- 79; 13, [H-55IT-79]; 14, [H-lIT-26]; 15, T-26. The molecular weight (MW) of the indicated reference proteins is as follow: A = 94 kD; B = 67; C = 43 kD; D = 30 kD. The aste risk indicate some differences between this line of tritordeum and its parent (see text) .

The globulins patterns of the tritordeums showed three bands in addition to those of their wheat parents (Figure 2). These bands, named as d (Mr = 73. 3 kD), e (Mr= �4. 3 kD) and r (Mr = 32.6 kD), were presented in all the tested lines of tritordeum. Any variation could be observed between the globulins from H. chilense, although a slightly variability was detected for the wheat globulins, principally in bread wheat (Figure 2 , lanes 10, 1 2 and 15). When the gliadin fractions were studied (Figure 3), four bands form H. chilense could be observed. The two first bands (marked as g and b) showed very close Mrs (56.5 kD and 56.0 kD, respectively). Both bands are present in the tritordeum derived from the lines H- l , H-12 and H-55 of H. chilense (Figure 3 , lanes 2 , 3 , 1 1 , 1 3 and 14). O n third band (called a s i, Mr= 54.5 kD) appeared i n the abovementioned tritordeums, and in the line [H-57rr-58] (lane 6). The fourth band (marked as j) was present in all the lines of tritordeum and shows a Mr of 50.5 kD. Likewise, in the wne indicated with a bracket, there is a group of bands present in all the tested lines; however, their high density has very difficult their analysis. It is noteworthy that the lane 14 showt",d newly higher intensity than it parent (lane 15), which suggest that the abovementioned differen ce in protein content could be responsible of it. In any case, the high number of components and problems for the analysis were found in the glutenins fractions. A total of seven additional bands could be observed in some lines of tritordeum, although the high overlapping of some components with those from wheat suggest that the number could be higher.


170 '2

3

4

6

5

7

8

9

10

11

12

13

14

15

Figure 2 SDS-PAGE separation of globulins of wheat and tritordeum lines. Lanes are follows; 1, T-22; 2, [H-1/T-22J; 3, [H-12IT-103J; 4, T-103; 5, [H-61/T-103J, 6, [H5 7IT-58J; 7, T-58; 8, [H-61/T-58J; 9, Mw,' 10, T-65; 11, [H-12IT-65J; 12, T- 79; 13, [H-55IT- 79J; 14, [H-1IT-26J; 15, T-26. The MW is similar to the indicated in Figure 1 . as

2

3

4

5

6

7

8

9

10

11

12

13

14

15

A

[

Figure 3 SDS-PAGE separation of gliadins of wheat and tritordeum lines. Lanes are as follows; 1, T-22; 2, [H-1/T-22J; 3, [H-12IT-103J; 4, T-103; 5, [H-61/T-103J, 6, [H57IT-58J; 7, T-58; 8, [H-61/T-58J; 9, MW; 10, T-65; 11, [H-12IT-65J; 12, T- 79; 13, [H-55IT- 79J; 14, [H-1/T-26J; 15, T-26. The MW is similar to the indicated in Figure 1. Th e bracket indicate one zone where several gliadins are presents.

171


In Figure 4, the bands marked as k (Mr = 107,7 kD) and I (Mr= 98. 8 kD) appear in all of the tested lines of tritordeum and to correspond with the bands in the high Mr zone indicated by Alvarez et alY. Data from other studies have suggested that both bands are linkage'o. The band I corresponds to the component 1 indicated by Payne et al Y , who used the line H- l of H. chiLense. Paradoxically, these authors did not detected the band k, which appear in a high number of lines of the H. chilense collection availa ble -100 accessions-. When both bands disappear, other two bands of low Mr appear in this zone (Gimenez-Alvear, pers. commun.). The third band (called m) shows a Mr of 56.0 kD and is present in the lines derived from H- J , H-12 and H-55; while the fourth band (named as n , Mr = 54.5 kD) is present in the all lines of tritordeum with exception of the derived tritordeum from H-6 1 . The three last bands (marked as 0, p and q) are present in all the lines of tritordeum and show a Mrs of 50.5 kD, 32.0 kD and 30.7 kD, respectively. 1

3

4

7

9

--.",---:""_ r.--

10

11

12.

13

14

15

Figure 4 SDS-PAGE separation of glutenins of wheat and tritordeum lines. Lanes are as follows; 1, T-22; 2, [H-lIT-22]; 3, [H-12IT-103]; 4, T-103; 5, [H-611T-103], 6, [H-

57IT-58]; 7, T-58; 8, [H-61/T-58]; 9, T-65; 10, [H-12IT-65]; 11, T- 79; 12, [H-55IT- 79]; 13, [H-l/T-26]; 14, T-26; 15, MW The MW is similar to the indicated in Figure 1 . .

Because o f the band o f 54.5 kD appear both gliadins (Figure 3 , band i ) and glute nins fractions (Figure 4, band n) , although with higher intensity in the last ones, it maybe a band of glutenins which had been partially extracted with 70° ethanol. For the same reason, the glutenins bands (Figure 4) with Mrs of 56.0 kD (band m) and 50.5 kD (band 0) could be bands of gl iadins (Figure 3 , bands h and j , respectively) which had not totally extracted with the respective solvent. On the other hand, the asterisks of Figure 4 indicate the found differences between the banding patterns of the line T-79 and its putative derived tritordeum [H-55rr-79] . These results suggest that this wheat line was not the pattern of this tritordeum, although it could be a sister line of it.


172

5 CONCLUSIONS The current work indicates that the H. chilense genome synthesized several components for each one of protein group of the endosperm of tritordeum . We have detected 3 components of albumin, 3 of globulins, 3 of gliadins and 5 of glutenins, together with an additional group of very close bands in the gliadins fraction. In general , a certain de gree of variation has been detected between the bands from H. chilense. The data confirmed that the majority of the H. chilense proteins are include in the group of storage proteins (gliadins and glutenins) . Likewise, these fractions present the highest variability in protein components, which could have a great importance for quality improvement in tritordeum by manipulation of such variability for storage proteins. Acknowledgments This work was supported by grant AGR91 -0844 of the Comision Interministerial de Ciencia y Tecnologfa (CICYT) of Spain. The first author thanks to MAPA for a predoctoral fellowship. Likewise, J.B.A. thanks the FPJ programme of the Spanish MEC for a postdoctoral fellowship. We want to thank to A. MartIn and J. Ballesteros from CSJC (Spain) for the seed materials.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 1 1. 12. 13. 14. 15.

References A . Martin and E. Sanchez-Monge Laguna, Euphytica, 1 982, 3 1 , 262. A . Martin and V. Chapman, Cereal Res. Commun. , 1 977, 5, 365 . A . Martin and J . 1 . Cubero, Cereal Res. Commun. , 1 981 , 9, 3 17 . J . 1 . Cubero, A . Martin,T. Millan, A . Gomez-Cabrera and A. de Haro, Crop Sci., 1 986, 26, 1 1 86. A . Martin, Rachis, 1 988, 7 , 1 2 . J. Ballesteros, Tesis Doctoral , Universidad de Cordoba, Spain, 1 993. (in Spanish). J.B. Alvarez, J. Ballesteros, J.A. Sillero. and L.M. Martin, Hereditas, 1 992, 1 16, 1 93 . J.B. Alvarez, I.M. Urbano and L.M. Martin, Cereal Chern. , 1 994, 71, 5 17. J.B. Alvarez and L.M. Martin, Cereal Res. Commun. , 1 994, 22, 49. J.B. Alvarez, Tesis Doctoral , Universidad de Cordoba, Spain, 1 993 . (in Spanish) . J.B. Alvarez, A . L. Canalejo, J. Ballesteros, W.J. Rogers and L.M. Martin, Plant Breeding, 1 993, 1 1 1 , 1 66. K . Khan, G. Tamminga and O. Lukow, Cereal Chern. , 1 989, 66, 39 1 . R.A. Graybosch and R. Morris, 1. Cereal Sci. , 1 990 , 1 1 , 20 1 . J . C . Sillero, Tesis de Licenciatura, Universidad de Cordoba, Spain, 1 994. (in Spa nish). P . I . Payne, L.M. Holt, S.M. Reader and T.E. Miller, Biochern. Genet., 1 987, 25, 53.

GLIADIN COMPONENTS AND GLUTENIN SUBUNITS IN WHEAT BREEDING

A. I. Abugalieva Kazakh Research Institute of Agriculture by V. R. Williams P. O. 483 133 Almalybak, Kazakhstan

1 INTRODUCTION Gluten proteins are the most important biochemical determinants of wheat grain quality. Because of their unique properties and biological specificities, they are effective markers of genes, genotypes and quality traits. It is important in breeding to define the methods that are available for identifYing important protein markers and to establish how the information they can provide can be used for quality improvement. In practice, such an approach has been directed towards answering a number of important questions: ( 1 ) the nature and the number of protein components that define a genotype and the value of this information in breeding, (2) how the protein composition and method for determining it can be used as a means of identifYing genomes, (3) how the information · on protein composition can be used in selecting appropriate genotypic/phenotypic traits that are of commercial value, and (4) facilitating achievement of the objectives of the breeding process using electrophoretic methods for monitoring protein composition, thus enabling lines with desirable gluten polypeptide compositions and hence quality characteristics to be selected.

2 IDENTIFICATION OF VARIETIES

AND

BIOTYPES

Wheat varieties are commercially important. The use of protein marker methods has enabled different varieties to be identified and genetical relationships to be established. The Kazakhstan breeding varieties are characterised by a substantial degree of heterogeneity in terms of the occurrence of biotypes, which has been revealed both in gliadin polypeptide and glutenin subunit composition (Table 1 ). The degree of heterogeneity varies from one variety to another It is related to the relative qualities and the biotypes present, which is in tum affected by the growing conditions. The predominant biotype found for any particular variety is usually the basis for that variety. Until recently, traditional breeding was limited to selection according to technological properties, growth characteristics, yield, morphology and disease resistance. Studies at the biochemical level, in particular by characterising the grain storage protein polymorphism and genetics have extended the boundaries and possibilities in breeding. In this regard it is


174

important to provide more accurate information on the protein compositions of wheats, which enables specific biotypes to be identified and selected for at the various stages of breeding. 2.1

Gliadin Polypeptides and Glutenin Subunits in the Identification of Genotypes

Experiments were carried out to study gliadin polypeptide and glutenin subunit heterogeneity among Kazakhstan varieties. Wheat storage proteins may be classified from 2 a functional perspective into two main groups: gliadin, which comprises monomeric proteins and glutenin, which comprises polymers in which the subunits are linked by inter chain disulphide bonds. Gliadins may be further sub-divided into sulphur-rich (S-rich) a-, p- and y-gliadin sub-fractions and a sulphur-poor (S-poor) co-gliadin sub-fraction. Using a polarographic method, we have found that the disulphide bonds contents increase in going from co- to a-gliadins. For some varieties and biotypes, e.g. the variety Bogamaya 56, the S-S-bonds contents of the co-gliadin area are greater than those in the y-area. This may explain the high degree of heterogeneity of the co-gliadin sub-fraction in this genotype, which has 8-1 1 components, the unusual cysteine contents, which are more like those of y gliadins, accounting for the more mobile co-components.

Table 1 Polymorphism Found in Kazakhstan Wheat Varieties Based on Their Gliadin

Component and Glutenin Subunit Compositions Number ofBiotypes Identified

Variety Bogamaya 56 Bezostaya 1 OPAKS- l Zemokormovaya 50 Lesostepka 75 Saratovskaya 29 Omskaya 9 Kazakhstanskaya 126 Kazakhstanskaya 1 5 Dneprovskaya 521 Kavkaz Alabasskaya Albidum 1 14 Marquis * data not available

gliadin 26 16 5 9 3 5 3 1 2 4 3 4 2 6

Proportion (%) of Most Common Biotype as Defined by Gliadin and Glutenin Polypeptide Composition

glutenin

gliadin

glutenin

7 4 2 10 1 3** 2* * 1 3 * * * * *

44-85 3 7-96 89-95 46-60 75-92 80-94 72-88 99- 100 95-98 20-75 65-70 80-89 90-95 66-75

5 1 -90 68-76 50-8 1 52 99-100 53-85 74-86 99- 1 00 60-89 * * * * *

* * data provided by K. M. Bulatoval

Wheat Protein Composition and Quality Relationsh!ps

175

As a result of comparative studies of the amino acid compositions of glutenin subunits, highly aggregated gliadins, low molecular glutenin and total fractions, the glutenin electrophoretic spectrum was differentiated into A, B and C zones, with clear identification of high M, subunits ranging from A8 to B263 . The S-rich and S-poor gliadins are referred to here as the gliadin extract and the high M, glutenin subunits as the glutenin extract. 2.2. Development of a Cataloguing System for Biotypes

The selection of biotypes was carried out from three types of material: cultivated varieties, accessions in competitive trial and hybrid combinations derived from two variants at the early and late stages of breeding. It was necessary to create a protein component cataloguing system that could be used in the wheat breeding process. Previously used ways of identifying varieties were not sufficiently discriminating for hybrid populations for a number of reasons: firstly, the number of gliadin components observed was greater than the number in the known spectrum4; secondly, the lack of differences in the relative electrophoretic mobility has limited the identification of allelic variants' , though this was rectified later6; thirdly, analysis based only on relative electrophoretic mobility of components7 had excluded the application from breeding of a large amount of information about cultivated wheat varieties. In our work, the system that we established for cataloguing the gliadin compositions of genotypes, including those of hybrid origin, enabled us to complete the spectrum with high precision. The system is essentially based on a combination of a description of gliadin composition according to the VIR (Russian Institute of Plant Production, St Petersburg) nomenclature, together with the relative electrophoretic mobilities of protein bands, which improves precision and objectivity. In this system, the 0.5 gliadin component is taken as having a mobility of 1 00; the mobilities of other gliadin components are then determined relative to the 0.-5 component, and this has enabled errors in the identification of components to be avoided. The choice of this reference component was as a result of its widespread occurrence in most of the cultivated wheat varieties. Where this reference band was absent, any other band could be used providing that it was present in both the standard variety and in the variety under investigation. 2.3

Use of Storage Protein Composition and Isozymes in Biotype Identification

The intervarietal polymorphism of gliadin components, glutenin subunits and isozyme systems was established. This information was used for selecting corresponding protein biotypes. By electrophoretic analysis of individual wheat variety grain samples, their heterogeneity was determined in relation to all the enzyme systems studied, i. e. peroxidase (PRX), alpha-amylase (AMY), acid phosphatase (ACPH) and glucose-6-phosphate dehydrogenase (GPDH). For each class of enzyme, two biotypes of each of the varieties studied were selected and thus the potential for discrimination was extended. For example, for the variety Bogarnaya 56 were observed 26 gliadin variants, five glutenin variants and two PRX variants. AMY, ACPH and GPDH appeared in various combinations, of which there were 48 in all. However, for breeding programmes for grain quality, the gliadin polypeptide and glutenin subunit compositions, as determined by electrophoresis, were more informative.


176

3 DIFFERENT BIOTYPES Further objectives in the grain quality investigations were to obtain lines representing biotypes based only on differences in the gliadin and glutenin compositions. Thus, 38 biotypes of the variety Bogamaya 56, 19 of Bezostaya I , 24 of Zemokormovaya 50 and two of OPAKS I were found. Lines were also characterised with respect to marker traits and on the basis of a combination of biochemical parameters, such as amino acid composition of the endosperm proteins, protein content, starch content, content of disulphide bonds, and the technological properties of the grain, flour, dough and bread. Annual screening of the biotypes showed significant variation in their quality traits, whether it was biological, technological or breadmaking quality. All the traits showed some variability but this varied depending on the genotype. Differences in quality in relation to technological properties were noted under different growing conditions, and the ranking of the different biotypes varied for different growing locations. Investigations of grain quality of biotypes of the variety Bogamaya 56 (the standard variety) over a number of years can be summarised as follows. The minimal rank corresponding to a high grain quality rating both under irrigation and under non irrigation conditions has been determined for each of 38 gliadin-glutenin biotypes9 This method of evaluation provides the prospect of detecting, and directing the selection of, genotypes that are of greater value and more constant with respect to grain quality under different growing conditions. It is extremely difficult to detect biotypes distinguishable as having all positive or all negative quality attributes simultaneously. Statistical analysis using multiple parameters can provide useful information on inherent grain quality evaluation, however. For example, some biotypes of the variety Zemokormovaya 50 variety are highly polar in relation to both technological and nutritional properties. Samples combining both these attributes were arranged more close together and the rest were dispersed across the spectrum of "technological traits --+ nutritional value".

4 GLUTEN PROTEINS AND GRAIN QUALITY Some gliadin components and subunits of glutenin occurred in all varieties but some occurred only in a certain proportion of the varieties and samples analysed. Furthermore, it was noted the levels of individual gliadin components and glutenin subunits could vary considerably depending on the growing conditions and how they affected different wheat cultivars. This occurred both for polypeptides that were common to all biotypes as well as for those which varied in occurrence between biotypes. Our studies have revealed relationships in terms of protein accumulation between components and groups of components. Most of those relationships were noted for polypeptides that varied from one biotype to another, such as glutenin subunits A8.5, A9, A 1 2, A l 3 , B21 and B27, and in some cases for polypeptides that were present in all biotypes, such as glutenin subunits A8 60 and A l l and y-gliadins y3 63 and y4 4.1

Variability in Gluten Protein Content

Both gliadin and glutenin levels varied significantly under different growing conditions; the coefficient of variation was 4- 1 4% for gliadin and 2-4 1 % for glutenin in the case of


177

spring sown wheats. For winter wheats there was greater variability in terms of gliadin content (coefficient of variation 1 0-25%) and glutenin content ( I S-43%). Intravarietal differences in the contents of individual polypeptides were also observed. The levels of glutenin subunits AS. 5 and A9 varied inversely as did those for subunits A 1 2 and A 1 5 . Factor analysis of the contents o f gliadin components and glutenin subunits showed that 32% of the common variability was due to gliadin, 50% to glutenin and l S% to interactions between the contents of gliadin components and glutenin subunits. Of considerable interest was the finding that four pairs of glutenin subunits accounted for 37% of total variation. Those subunit pairs were AS. 5 plus A9, A12 plus AI3, A I 4 plus A 1 5 and A 1 9 plus B24. Conditions during each growing year also had a considerable influence on the nature of the protein correlation according to the scheme: protein content -+ contents ofthe total albumin, globulin, gliadin and glutenin fractions ---+ contents of gliadin and glutenin subfractions -+ contents of individual gliadin components and glutenin subunits. The way in which the contents of gliadin components and glutenin subunits contents vary during the ripening phase is the result of genotype - environment interactions. This characterises the degree of stability within genotypes (USSR Patent 1 269292). 4.2

Gluten Protein Components and Grain Quality Prediction.

For the spring wheats, it was established that the contents of high Mr glutenin subunits A 1 2 (G/u IB; 7+S) and AI3 (G/u IB; 7+9) were the main determinants of dough physical properties, such as mixing time, elasticity and the Alveograph PIL ratio. The breeding programme for winter wheat involves a detailed study of gliadin-glutenin biotypes and the way they determine grain quality. Variation in the contents of glutenin subunits A9 (G/u ID; 3) was related to grain hardness for the variety OPAKS l . Variation in the contents of glutenin subunits A l l , AI3 and A 1 9 and y-gliadin components for the variety Bogarnaya 56 was related to gluten content and high Valorimeter values. Variation in the contents of glutenin subunit A19 was correlated with grain hardness (r=0.69). Variation in the contents of glutenin subunit B2 1 was correlated negatively with glutenin protein content and total gluten content (r=-0.73 and-0.7 1 , respectively), with Valorimeter values and with Alveograph PIL ratio (r=-0.72 and -0. 79, respectively) and was correlated positively with gliadin content and grain hardness (r=0.79 and O.SO, respectively). Variation in the contents of glutenin subunit B22 was correlated with both total protein and gluten contents (r=0.69 and 0.67, respectively) Thus, the results obtained with a large number of samples of cultivated Kazakhstan varieties, prospective accessions in trials, biotypes, isogenic lines and lines derived from the breeding programme showed that the contents of individual gluten fractions were related differently to factors, such as grain yield and flour, dough and bread technological parameters. Our investigation of the quantitative relationships among gluten proteins in these diverse materials has enabled us to show that there is no universal quality relationship between glutenin and gliadin electrophoresis patterns and parameters used as indicators of technological quality. This shows that, in addition to a simple qualitative approach to valuable genotypes based on the presence or absence of particular gluten protein components, the use of statistical modelling of quantitative information may also be of value in increasing the quality of agriculture commodities.


178

Table 2 Grain Quality Evaluation System and Interpretation in Breedingfor Quality Evaluation and Interpretation

Grain Quality Related Factors

Determination Method

Biological Level Genetical

Individual gliadin components

Protein markers

Individual glutenin subunits Groups of components Blocks of components Composition of individual gliadin components Composition of individual glutenin components Genes and chromosomes Phenotypical

statistical

Monosomic analysis

Total protein content

Spectrophotometry and

Contents of protein fractions

NIR analysis

Contents of specific gliadins Contents of specific glutenin subunits

Electrophoresis using densitometry and RP-HPLC

Disulphide bond contents

Polarography

Amino acid composition to indicate nutritional Huality

Amino acid analysis

Variability of genotypes

Abugalieva et aI. , 1 993 9

Stability of genotypes Technological Level Technological (empirical evaluation)

Gluten quantity

State standard-68

Gluten quality Dough elasticity (P)

Chopin Alveograph

Dough extensibility (L) Dough energy (W) Dough mixing time

Brabender Farinograph

Dough resistance Valorimeter evaluation Bread volume

State standard

Bread making value etc. Sedimentation value statistical

Principal component integral evaluation Quality rank Quality clusters

Abugalieva et aI. , 1 993 9


179

5 CONCLUSIONS ON USE OF PROTEIN MARKERS IN WHEAT BREEDING The work summarised here was aimed at optimising the wheat breeding process for improving grain quality through a study of grain storage protein composition both in qualitative and quantitative terms. The choice of which grain quality data to include is determined principally by the aims of the particular breeding programme. In turn, this governs which evaluation methods need to be used as selection tools in the breeding process (Table 2) . The traits of new lines, the methods used for their determination and the breeding parameters need to be characterised at each level of interpretation. References

1 . Y. V. Peruanski and AI.Abugalieva, J. Breeding and Seed Production, 1 985, 3, 23 2. P. R. Shewry, A S. Tatham, J. Forde, M. Kreis and B. J. Miflin, J. Cereal. Sci., 1 986, 4, 2, 97. 3. Y. V. Peruanski, A I. Abugalieva, K. M. Bulatova and L. M. Nechoroscheva, Soviet Agric. Sci., 1 986, 6, 6. 4. V. G. Konarev, I. P. Gavrilyuk and N. K. Gubareva, 'Genetical resources of wheat' Agropromizdat, Leningrad, 1 976, 1 13 . 5 . A A Sozinov, P. A Poperelya, Soviet Agric. Sci. , 197 1 , 2, 9. 6. A Sasek, J. Cerny and S. Sykorova, Sci. Agric. Bohemosl., 1 989, 21, 257. 7. W. Bushuk and R. R. Zillman, Can. J. Plant Sci. , 1 978, 58, 505. 8. A I. Abugalieva and S. I. Abugalieva, Sci. News Kazakhstan, 1 99 1 , 2, 52. 9. A I. Abugalieva, Y. V. Peruanski, K. M. Bulatova and V. V. Novochatin, Russian Agric. Sci. , 1 993, 4, 9.

GLIADIN AND HIGH MOLECULAR WEIGHT (HMW) GLUTENIN SUBUNITS IN THE COLLECTION OF POLISH AND FOREIGN WINTER WHEAT CUL TIV ARS AND THEIR RELATION TO SEDIMENTATION VALUE

J. Waga and J.Winiarski Plant Breeding and Acclimatization Institute 4 Zawila St. ,30-423 Krakow Poland

I INTRODUCTION In the last decades in Poland the wheat cultivation was mainly aimed at high grain yield from cultivars introduced to production. This resulted in the deterioration of technological parameters of the new cultivars. Due to recent economical changes, the quality criteria became stricter in all areas of production. It particularly concerns agricultural products, including the technological quality of wheat grain. Breeders want to adjust the standard of their products to the rising criteria and so they are interested in the development and practical use of new methods, which allow an effective acceleration of quality breeding process. One of the methods is the electrophoretic analysis of wheat storage proteins. The research conducted in Plant Breeding and Acclimatization Institute (PBAI) is aimed at determination of the relationship between the technological quality and set of storage protein blocks of cultivar, and also at showing the possibility of their application as markers of this feature in practical breeding.

2 METHOD Gliadin and glutenin proteins of 1 05 cultivars and strains of winter wheat coming from PBAI collection were investigated over the period of 3 years. Proteins were examined by electrophoresis, gliadins on starch gel and glutenins on poJiacrylamid gel SDS PAGE. The technological quality of the investigated material was assessed using the results of ZELENY test. 1 The examined forms were divided into 4 groups, according to the sedimentation value: A - very good cultivars, B - good cultivars, C - fair cultivars and D - poor cultivars.

3 RESULTS 3.1 The polymorphism of gliadin and glutenin proteins in the analysed groups of cultivars and strains

2 Based on the Poperella catalogue, 26 different gliadin protein blocks were identified in the examined materials. The chromosomes of the 1 st homeological group coded as follows: I A seven, 1 8 six, ID five different protein fractions.


181

The chromosomes of the 6th homeological group coded as follows: 6A three, 6B three, 6D two fractions of gliadin protein. When describing the fraction of glutenin proteins using the PAYNE catalogue3 3 blocks coded by chromosome l A, seven blocks coded by chromosome 1B and two blocks coded by chromosome ID were identified. 3.2 The relationship between identified storage protein fractions and the sedimentation value

The average sedimentation value of the groups of cultivars and strains, containing particular identified blocks of storage proteins were compared. The significance of the differences between the average values was assessed by FISCHER test. It appeared that only protein blocks coded by chromosome I B and lD strongly affected the sedimentation value during each year of the three year research period. This resulted in considerable differences between the forms examined as far as the sedimentation value is concerned. Gliadin blocks marked: Gld IB5 and Gld 105 as well as glutenin blocks marked Glu I B7+9 and 105+10 are connected to the high sedimentation value, while gliadin blocks Gld IB3 and Gld 101 as well as glutenin blocks Glu I B6+8 and Glu 102+12 are connected to the low sedimentation value of the investigated cultivars and strains. On the diagrams (Figure 1) the frequency of the occurrence of cultivars and strains containing g1iadins and g1utenins coded by chromosomes IB and ID is shown. The above mentioned blocks are indicated. 80 50

6 40 I:: Q) ::I C" Q)

cl::

-

30

20 10

0

....

10 18 18 18 18 18 18 1 2 3 4 IS 7

oth

er.

o

blocks

.n..r=..

188+ 187+ 187 187+ 1817 188 8 . • +1'

Gliadins

50

6 40

I::

g

cl::

>. t) I:: Q) ::I C" Q)

30

20

cl::

10 0

101

102

1015

1015

� blocks ...

Glutenins

80

C" Q)

-

1 -1

1 08

other blocks

50

0

102+12

1015+10

other.

Gliadins Glutenins Figure 1 Thefrequency ofthe occurence of cultivars and strains containing gliadins and

glutenins coded by chromosome IB and 1D


182

Table 1 Frequency of high quality g.motypes in the groups qfcui/ivaI's containing individual 'good ' protein blocks Protein block Number of cultivars Frequency ofhigh Number ofhigh quality culrivars quality cultivars Gld IB5 7 86 6 Gld IDS 20 85 17 Glu IB7+9 63 46 29 Glu I D5+ 1 0 28 44 64

Table 2 Frequency of high quality genotypes in the groups of cultivars containing one, two, three and{our 'good' protein blocks Frequency ofhigh Number of high Number ofprotein Number of cultivars blocks quality cultivars quality cui/ivaI's 1 8 25 32 2 73 15 11 3 87 15 13 1 00 4 4 4

3.3 Possibility of technological quality evaluation based on the electrophoregrams of gliadins and gluten ins

The percentage of high quality forms (class A + B), among the cultivars and strains containing favourable protein blocks was considered to be an indicator of efficency evaluation of the technological properties, based on electrophoretic picture of storage protein fractions. In the table 1 the percent of high quality forms in groups of cultivars and strains having single protein blocks considered advantageous was shown. Because of a much greater number of forms containing glutenins IB7+9 or ID5+ l 0 as compared to forms containing gliadins IB5 or IDS the obtained result of about 63 % can be regarded as fair. It means that the cultivar, in which the glutenin fractions IB7+9 or ID5+ l 0 were identified may be defined with the probability close to 63 % as high quality form. In the table 2 the percent of high quality forms in groups of cultivars and strains having one, two, three or four protein blocks considered adventageous was shown. As comparison shows, the lowest number of high guality cultivars was observed in the group of forms containing one adventageous protein variant. This number increases with the rise of the number of advantageous protein blocks reaching the highest value for cultivars combining all four such variants. The results presented show that increasing the number of protein fractions contributing to a high sedimentation value in one genotype raises its technological properties. It means that genes controling influence of gliadins and glutenins on technological properties are additive.

4 DISCUSSION The results shown prove the existence of the relation between the blocks of storage proteins and the sedimentation value. However, the technological quality is a complex

183


feature, affected by many genetical and environmental factors. That is reason, the evaluation of this feature, using exclusively the results of electrophoretic analysis of gliadins and glutenins shall be incomplete for it defines only one of the factors, the technological properties of wheat cultivars. This however does not change the fact, that stored proteins are a very important factor influencing this feature. From the genetic and practical breeding point of view, the electrophoretic analysis of storage proteins is a particulary interesting method, for its results depend exclusively on genotype but not on environmental factors. Another important and advantageous feature of this method is the possibility of quick and error free indication of genotypes having the desired protein blocks combinations. This is the reason why some Polish breeders in co-operation with the autors of this paper have attempted to practically apply the electrophoretic analysis of gliadins and glutenins in defining the following problems: - Identification and reproduction of homozygous hybrid genotypes based on the results of half seedling analysis of the F2 generation. - Selection of homogenic lines, among the heterogenic strains of generations F4 - F7. - Identification and selection of hybrid lines between Triticum aestivum and Triticum durum. - Analysis of inheritance of protein fraction and identification of progeny from cross combinations between Triticum aestivum and wild wheat species.

Summary During the last three years the set of

250

Polish and foreign winter wheat cultivars

investigated in Plant Breeding and Acclimatization Institute in Krakow has been analysed regarding to gliadin and HMW glutenin subunits using the method of polyacrylamid gel electrophoresis. The allelic variants of these proteins - so called blocks - were identified based on catalogues of Poperela (gliadin) and Payne (glutenin). Variancy analysis shows significant relation between gliadin and glutenin subunits coded by chromosomes l B and

I D. Additively influence of storage protein blocks on the sedimentation value was observed. Electrophoregrams of gliadin and HMW glutenin proteins can be utilized as one of the criterion of quality properties estimation in breeding of new wheat cultivars. Polish breeders from Plant Breeding and Acclimatization Institute have practical undertaken to test the electrophoretic analysis of storage proteins in selection of high quality genotypes.

References

1 . L. Zeleny, W. T. Greenway, G. M. Gurney, C . C . Fifield and K. L. Lebsock, Cereal Chem. , 1 960, 37, 673 . 2. A. A. Sozinow and F. A. Poperella, Ann. Techn. Agric., 1 980, 29, 229. 3. P. 1. Payne and G. 1. Lawrence, Cereal Res. Commun., 1 983, 11, 29.

PATHOGENESIS-RE L ATED PROTEINS IN WHEAT

C. Caruso, G. C h i l osi*, C. Caporale, E. Poerio and V. Buonocore

F.

Vacca,

L . Bert i n i , P. M a g ro* ,

D ipartimento di Agrobiologia e Agrochimica and *Dipartimento di Protezione delle Piante, U n iversita degli Studi della Tuscia,Viterbo, 0 1 1 00-ltaly

1 INTRODUCTION

In their natural habitat plants are challenged by several pathogen ic agents such as viruses, viroids, fungi and bacteria. 1 For their survival they have developed a complex variety of defense responses induced upon infection . The most frequently observed biochemical events are the rapid accumulation of antim icrobial compounds (phytoalexins, thionins, enzyme inh ibitors) , 2- 4 the reinforcement of the cell wall by deposition of callose and ligni n , acc u m ulation of hydroxyproline-rich glycoproteins 5 , 6 and enzymes involved i n phenyl propanoid and flavonoid metabolism,1-9 and production of a family of proteins collectively known as pathogenesis-related (PR) proteins.1 0-1 6 P R proteins were first described in tobacco plants infected with tobacco mosaic virus (TMV); 1 7, 1 8 since then, PR proteins have been found in a variety of infected plants such as tomato, potato, maize, parsley, etc.1 9 -22 and h ave been grouped into five classes. The function of PR proteins of group 1 has not yet been established while it has been shown that groups 2 and 3 have in vitro f3 - 1 ,3-glucanase and chitinase activities, respectively; PR5 proteins are structurally similar to the bifunctional trypsin/a amylase i n h ibitor from maize and to the sweet protein thaumatin. Up to date, only few members of the PR4 class h ave been described; in particular genes encoding PR4 proteins have been described in potato, tobacco, tomato a n d rubber tree (Hevea brasiliensis) , 23-26 whereas mature PR4 proteins have been characte rised from barley and wheat seeds. 2 7, 2 8 So far, most of the work has been done on infected plants (tubers, leaves) , while the expression of PR proteins in specific tissues of healthy plants has been less characterized; for example, it has been reported that PR proteins are

developmentally regulated in healthy tobacco plants during flowering29,30 o r d uring leaf senescence i n tomato.31 Moreover expression o f specifically induced PR proteins during germination in maize seeds in response to fungal infection has been reported.3 2 , 33 These findings suggest that the PR proteins could also play a role in plant defense against pathogens during different stages of deve lopment. Recently we isolated from wheat seeds two h ighly homologous PR4 proteins (wheatw i n 1 and wheatwin2) showing antifungal activity towards

185


phytopathogenic fungi of cereals. 2 8 ,34 Here we report that these PR4 proteins are specifically induced upon infection with Fusarium culmorum d u ring germi n atio n ; using antisera raised against wheatwin 1 the two PR4 proteins were detected 48 hours after infection . 2 MATERIALS AND M ETHODS 2.1

M aterials

Triticum a estivum , cultivar San Pastore, was kindly supplied from Istituto Sperimentale per la Cerealicoltura (S. Angelo Lodigiano, Italy). M acroconidia from sporodochia of Fusarium culmorum (Smith) Sacc.(isolate 485} were collected from 1 0 days cultures grown at 20 OC and suspended in sterile potato dextrose broth (PDB) . Reagents for sodium dodecylsulfate polyacrylamide gel electrophoresis (SOS-PAGE), including low molecular weight markers and reagents for immunoblotting, were from Bio-Rad (Italy). All other reagents were of the h ighest purity com mercially available. 2.2

Methods

2.2. 1 . Plant Materials. Wheat seeds were sterilised with hypochlorite 2% and dried i n a fume cupboard; dried sterilised seeds were placed o n sterile agar plate for 24 hours at 21

Oc to allow germinatio n . Embryos were then inoculated J.l1 (3 1 0S

with a conidial suspension of the fungus F. culmorum by adding 1 0

*

spores per m illiliter) to each embryo. Inocu lated and sterile control e mbryos were

allowed to continue germination at 20 Oc for the required period of time. 2.2.2. Protein extract and western blotting. Sterile and F. culmorum-infected seedlings were h arvested and ground to a powder in a pre-chilled mortar in liquid n itrogen . The extraction buffer, 50 m M sodium phosphate, pH 8.0 containing 0. 1 5 m M NaCI and 1 % PVP, was added to the powder ( 1 mllg fresh weight tissue). The buffer extracts were then centrifuged twice at 1 0,000 rpm for 20 min at 4 0C , and the clear supernatant was used for SOS-PAGE and i m munoblotting, following essentially the procedure of Lae m mli34 and Towbin35, respectively. Monoclonal antibodies were raised against wheatwin 1 (manuscript in preparation) and used in this work. Goat anti mouse horseradish peroxidase (SIGMA) was used as second antibody; both 4-chloro-1 -naphthol (Merk) and l uminol (Amersham) were used for detection of serological reactions.

3 RESULTS AND DISCUSSION We have isolated from the albumin fraction of wheat kernel two highly homologous proteins (wheatwin 1 and wheatwin2) belonging to the PR proteins of class 4 , which were found to be strong i nhibitors of fungal growth . 2 8• 3 6 The present study was undertaken to obtain a better insight into the capability of wheat seedlings to synthesize these particular PR4 proteins in response to fungal infectio n . In fact, protection of the germinating seeds is of vital importance for the survival of the species. Wheat seedlings germinated for 24 hours were inoculated with a suspension of spores of F. culmorum and allowed to continue germination for a total of 24. 48 and 72 hours; control experiments were carried out in the same conditions using sterile water instead of the fungal spores.

1 86


Analysis of protein extracts from inocu lated and control seedlings at different time after infection allowed us to analyse the ability of germinating seeds to express specific PR4 proteins. Figure 1 shows the protein extracts analysed by SOS-PAG E . Approximately 1 0 mg of proteins per g of seedlings were obtained. The difference in the protein patterns of infected and control seedlings were mainly quantitative, showing relatively high amount of some proteins in the high range of molecular weight. A monoclonal antibody raised against wheatwin1 was used to detect the presence of this protein as well as the presence of wheatwin2 in the protein extracts obtained from F. culmorum-infected and u ninfected wheat seedlings. The same SOS-PAGE gel was blotted onto n itrocellulose and incubated with the monoclonal antibody; the i m m unoblot analysis of protein extracts is shown in Figure 2. A PR4 protein accum ulates only in extracts from infected seedlings ; its presence is detectable starting at 48 hours after infection and its level remains similar in 72 hour infection . F. culmorum i s reported to b e one of the most widely spread pathogens of wheat; the fungus is a soil-inhabiting species that causes important damages in crops through foot and root rot. Moreover, F. culmorum is frequently isolated from wheat kernels, the inoculum source consequently being also seed-transmissed 37 . I nduction of plant defense proteins such as PR4 proteins in seed tissue of germinating seeds may play a remarkable role in reducing colonization of F. culmorum and can be considered as part of the complex mechanisms that seedlings may use to defend themselves against pathogen attack.

2

3

4

5

Figure 1 SOS-PAGE of protein extracted from uninfected (1,3) and infected (2, 4) wheat seedlings at 48 (1,2) and 72 (3, 4) hours after infection. Molecular markers are shown in lane 5.

1 87


1

2

3

4

5

Figure 2 Immunoblot analysis of protein extracts from uninfected (1,3) and infected (2,4) wheat seedlings at 48 (1,2) and 72 (3, 4) hours after infection. Purified wheatwin1 (5) was used as a control. R eferen ces

1 . K. Hahlbrock and D. Scheel, 'Innovative Approaches to Plant Disease Control', I. Chet ed. John Wiley, New York, 1 987, pp 229-254. 2. A.G. Darvill and P. Albersheim, Annu. Rev. Plant Physio/. , 1 984, 3 5, 243-275. 3. A.W. Williams and M. M. Teeter, Biochemistry, 1 984, 2 3, 6796-6802. 4. C.A. Ryan, Annu. Rev. Phytopathol. , 1 990, 2 8, 425-449. 5. A.M. Showalter, J .N . Bell, C .L. Cramer, J.A. Bailey, J.E. Varner and C .J . Lamb, Proc. NatJ. Acad. Sci. USA, 1 985, 8 2, 6551 -6555. 6. D. Mazau and M.T. Esquerre-Tugaye, Plant Physiol. , 1 986, 8 0, 540-546. 7. M .A. Lawton , A.A. Dixon, K. Hahlbrock and C.J. Lamb, Eur. J. Biochem. , 1 983, 1 2 9, 593-601 . 8. J. Friend, Ann. Proc. Phytochem. Soc., 1 985, 2 5, 367-392. 9. C .L. Cramer, J.N. Bell, T.B. Ryder, J .A. Bailey, W. Schuch, G .P. Bolwell' M .P. Robbins, A.A.Dixon and C.J. Lamb, .EMBO J., 1 985, 4 , 285-289. 1 0. D.J. Bowles, Annu. Rev. Biochem., 1 990, 59, 873-907. 1 1 . J.F. BoI, J.M. Linthorst and B.J.C. Cornelissen, Annu. Rev. Phytopathol. , 1 990, 2 8 , 1 1 3- 1 38. 1 2. T. Boller, 'Plant-Microbe interactions: Molecular and Genetic Perspectives', T. Kosuge and E .W. Nester, eds. Macmillan, New York, 1 987, Vol 2, pp 38541 3. 1 3. J.P. Carr and D.F. Klessing, 'Genetic Engineering: Principles and Methods', J .K. Satlow ed. Plenum Press, New York, 1 989, pp 65- 1 09. 1 4. A.A. Dixon and C.J. Lamb, Annu. Rev. Plant Physiol. Plant Mol. Bioi. , 1 990, 4 1 , 339-367. 1 5. H .J.M. Linthorst, Crit. Rev. Plant Sci., 1 991 , 1 0, 1 1 3-1 50. 1 6. LC. Van Loon , Plant Mol. Bioi. , 1 985, 4 , 1 1 1 - 1 1 6.

188


1 7. 1 8.

L.C. Van Loon and A. Van Kammen , Virology, 1 970, 4 0, 1 99-2 1 1 . S . Gianinazzi, C . Martin and J . C . Vallee, Comptes Rendus de I' Academie des Sciences, serie 0, 1 970, 2 7 0, 2383-2386. A. Camacho-Hen riquez and H-L. Sanger, Arch. Virol. , 1 984, 8 1 , 263-284. E. Kombrink, M. Schroder and K. Hahlbrock, Proc. Natl. Acad. Sci. USA ,

1 9. 20.

1 988, 8 5, 782-786. 21 . 22. 23.

W. Nasser, M. de Tapia, S. Kauffman n , S. Montasser-Kousari and G . Bu rkard, Plant Mol. BioI. , 1 988, 1 1 , 529-538. I . E . Somsich, E. Schmelzer, J. Bollmann and K. H albrock, Proc. Natl. Acad. Sci. USA, 1 986, 8 3, 2427-2430. A. Stanford, M. Bevan and D. Northcote, Mol. Gen. Genet., 1 989, 2 1 5, 200-

208. 24.

L. Friedrich , M. Moyer, E. Ward and J. Ryals, Mol. Gen. Genet. ,

1 99 1 , 2 3 0,

1 1 3- 1 1 9 . 25. 26. 27. 28. 29. 30. 31 .

H . J . M . Linthorst, N . Danhash, F.T. Brederode, J AL . Van Kan ,P.J . G . M . De Witt and J . F. Bol, Mol. Plant-Microbe Interactions, 1 991 , 4 , 585-592. W. Broekaert, H. Lee, A. Kush , N . H . Chua, N . H . , N. Raikhel, Proc. Natl. Acad. Sci. USA , 1 990, 8 7 , 7633-7637. B. Svensso n , I. Svendse n , P. Hojrup, P. Roepstorff, S. Ludvigsen and F . M . Pou lse n, Biochemistry, 1 992, 3 1 , 8767-8770. C. Caruso, C . Caporale, E. Poerio, A. Facchiano and V. Buonocore, J. Prot. Chern. , 1 993, 1 2, 379-386. A.D. Neale, J .A. Wahleithner, M. Lund, H .T. Bonnet, A. Kelly, D . A. Meeks Wagner, W.J . Peacoc and E.S. Den n is, Plant Cell, 1 990, 2 , 673-684. T. Lotan, N. Ori and A. Fluhr, Plant Cell, 1 989, 1 , 881 -887. P. Vera, J. Hernandez-Yago and V. Conejero, Plant Science, 1 988, 5 5, 223-

239. 32. 33. 34. 35.

J . M . Casacuberta, P. Puigdomenech and B. San Segundo, Plant Mol. BioI. , 1 991 , 1 6, 527-536. J . M . Casacuberta, J . M . Raventos, P. Puigdomenech and B. San Segundo, Mol. Gen. Genet. , 1 992, 2 3 4, 97- 1 04. U .K. Laemmli, Nature, 1 970, 2 2 7, 680-685. H. Towbin , T. Staehelin and J. Gordon, Proc.Natl. Acad. Sci. USA, 7 6, 4350-

4354. 36. 37.

C . Caruso, C . Caporale, G. Chilosi, F. Vacca, L . Bertini, P . Magro, and V. Buonocore, submitted to Planta H. Fehrmann 'European Handbook of Plant Deseases', I . M . Smith , D . H . Phillips, A.A. Elliot and S A Archer eds . Blackwell Pubblications, Oxford, London, Edinburg h , Boston, Palo Alto, 1 988, pp 287-289.

E. Poe rio J. Dunez, Scientific Melburne,

INVESTIGATION OF HYPERSENSITIVny TO WHEAT GLIADIN GLUTEN-FREE DIETARY PRODUCTS USING DOT-BLOT ASSAY

FROM

I. M. Stankovic·, I. Dj . Miletic· and V. D. Miletic" •

Faculty of Phannacy, Institute of Bromatology, Belgrade, Yugoslavia "Blood Transfusion Institute, Belgrade, Yugoslavia

1 . INTRODUCTION Coeliac disease is a pennanent intolerance to dietary gluten, resulting in small intestinal villous atrophy with consequent malabsorption and malnutrition. Gluten is the protein fraction of cereal grains that gives cohesiveness and elasticity to the dough. It is composed of prolamins and glutenins. Gliadin (wheat prolamins) are of family of grain kernel storage proteins with more than 40 closely related members. Prolamins are insoluble in water and soluble in ethanol. A gluten-free diet is life long treatment for patients with coeliac disease ( 1 ) and require strict avoidance of any food and other products containing prolamins from selected grains, e.g., wheat, barley, rye and oats. Maize and rice storage proteins are not generally included on this list. G liadins are found in foods, soups, sauces, beers and phannaceutical products, as well as in additives, colorings, emulsifiers, eXcipients, flavorings and preservatives that are derived from gluten containing grains. There is no consensus about the highest nontoxic level of gliadin in gluten-free food. In most countries the official limit for gluten-free dietary products is 0.3 g/100 g on a dry weight basis, but there are many different opinions; some researches suggested much smaller concentrations (2). There is no standardized methodology for gliadin detennination in dietary products and finally there are great individual differences in reactions on gliadin among coeliac patients (3,). The aim of our work was development of specific semiquantitative test for detection of gliadin in gluten-free dietary products using dot-blot assay.

2. MATER IALS AND METHODS

2.1 Materials Table 1 lists 7 gluten-free dietary products and positive control. The table gives commercial names of product. the company that manufactured it and the result of dot-blot assay detennined by the method described below.


190

2.2 Methods 2 .2 . 1 Extraction (�{ Gliadins from glutL'1'l-free jJroduct.s .

Extraction of

gliadins from gluten-free dietary products was performed both with 1 % SDS (sodium dodecyl sulphate) and

70% ethanol. Some researchers suggest that

SDS might be the best solvent for extraction of gliadins resulting in more

quantitative extractions and avoiding problems associated with the use of

ethanol in assays solvents for

15

(2). Samples (5 g/25 ml) were suspended in aforementioned 2200 g for 1 5

minutes at room temperature and centrifuged at

minutes. Supernatant was used for immunoassay.

2 .2 .2 Dot-blot assay . strips by applying

A dot-blot a..resent address: Sir William Dunn School of Pathology, South Parks Road, Oxford

OXl 3RE

3NERC Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OXl 3SR 4IACR-Rothamsted, Harpenden, Herts AI.5 2JQ

1 IN1RODUCTION Wheat gluten is a complex mixture of proteins, with over 50 individual components being revealed by two-dimensional electrophoresis. Although most, if not all, of these components contribute to its functional properties, their precise structures and roles have still not been established. There are several reasons for this. Firstly, although it is relatively easy to fractionate gluten proteins into groups (e.g. a-type gliadins, (a) gliadins and v-type gliadins), the purification of individual components of these groups can be extremely difficult. Secondly, they may have unusual structures, resulting from the presence of extensive repeated sequences, making them difficult to study using methods of analysis generally used for globular proteins. Thirdly, the functional properties of gluten proteins are expressed as part of the gluten network that forms in doughs. Attempts to reconstitute dough or gluten from their constituent parts have met with varied success, making it difficult to determine the functional properties of individual purified components. Molecular biology can help to provide solutions to all these problems. The sequences of cDNAs and genes provide complete amino acid sequences of the encoded proteins, which in turn facilitate structural studies. These DNAs can also be expressed in E.coU or other heterologous hosts to produce wild-type or modified proteins for structural studies, or for functional analyses using small scale systems such as the 2g mixograph.l Finally, the cDNAs and genes can be used for wheat transformation, in order to explore the functionality of the encoded proteins when expressed in the developing seed.

2

ISOLATION OF cDNAs AND GENES FOR GLUTEN PROTEINS

The isolation of cDNAs and genes, even for abundant proteins such as most gluten proteins, is still far from routine. In fact, the repetitive structures of wheat gluten proteins make this particularly problematical as recombination between the repeated nucleotide sequences present in the corresponding cDNAs and genes may lead to instability. As a result our knowledge of prolamin sequences is still incomplete, with no complete sequences of (a)-gliadins and limited knowledge of LMW subunits of glutenin.

200


Although we do have the complete sequences of (&)-gliadin homologues from barley (C hordeins)2.3 and rye «&)-secalins),4 the (&)-gliadins appear to be more diverse in structures and the isolation of corresponding cDNAs or genes remains an important target. Our lack of knowledge of the structures of LMW subunits is a major problem

in understanding their role in gluten. These proteins account for about 40% of gluten, and they are se arated by SDS-PAGE into three groups called B-type, C-type and D-type subunits.6. It is extremely difficult to purify single components for

�

detailed analysis. Nevertheless, Lew et aI.B have determined the N-terminal sequences of a number of individual components. This showed that the B-type components formed a discrete group, with two sub-groups called LMWs and LMWm on the basis of their N-terminal amino acids. The LMWm type were further divided into three subgroups on the basis of amino acid substitutions at position 5 (Table 1). It is unfortunate that all the available cloned cDNAs and genes correspond either to the

minor LMWmc5 type of LMW subunit or to variant types not identified by Lew et aI.B In contrast, we know little about the quantitatively major LMWs type. Lew et aI.B also showed that the C-type subunits were related to a-type and y type gliadins, and suggested that they correspond to mutant forms of these proteins which contain additional cysteine residues: cDNAs encoding such proteins have been isolated.9,IO The D-type LMW subunits also appear to be mutant forms of monomeric gliadins, being related most closely to (&)-type gliadinsY

3 THE

APPliCATION OF PROTEIN ENGINEERING TO ANALYSIS OF WHEAT GLUfEN PROTEIN STRUCIURE

THE

Protein engineering is the analysis of structure and function using protein expressed from a cloned cDNA or gene. The advantages include speed, high yields and ease of purification. However, the most important advantage is undoubtedly the ability to design and express proteins with specific mutations in order to determine the impact on structure and function. It has proved immensely powerful in studying globular metabolic proteins, such as enzymes, but has not so far had a major impact on analysis of food or structural proteins. The requirements for protein engineering are the availability of cDNAs or genes and of an expression system. The latter is usually based on microorganisms (e.g. E.coli, other bacteria, yeasts and filamentous fungi) or cultured cells (notably insect or mammalian cells), with expression vectors derived from plasmids or viruses. It is necessary not only to produce high levels of protein, but also that the protein should be correctly folded and, in some cases, post translationally processed and assembled in multi-subunit complexes. It is this latter requirement which has limited the application to some proteins. We have used protein engineering to study the structures and properties of wheat gluten proteins, including the formation of glutenin polymers, using yeast,12 baculovirus13 and E.colil4 expression systems.

3.1 Disulpbide Bond Formation

in

a

LMW

Subunit of Glutenin

Expression of a wheat LMW glutenin subunit gene in cultured insect cells gave yields of about 30-5Omg protein per litre of culture, accounting for about 25-30% of the protein extracted from the insect cellsP However, much of this protein appeared

201


Table 1 Availability of cloned cDNAs or genes for LMW subunits of wheat glutenin Protein Type! B-type LMW s

Clone

Gene or

(major type)

None

LMW mh5

(minor type)

None

mr5

(minor type)

None

meS

(minor type)

Variant types

C-type a-type V -type D-type

Reference

eDNA

cDNA LMWG-IDI Gene cDNA pTdUCDI

Bl1-33

Okita et aI,9

Colot et aI,15 Cassidy and Dvorak16

lP1211 pLMW21

Gene Gene

Pitts et aI,t7 I D'Ovidio et aI, B

A735

eDNA

Okita et aI,9

pWI020

cDNA

Scheets & Hedgcoth,10

None

IProtein groups as defined by Payne et aI.,6 Jackson et aI.,7 and Lew et aI.B to be incorrectly folded and the yield after in vitro refolding was less than 10% of the total. Nevertheless, this system was used to study the effects of substitution of specific cysteine residues with serines on the ability of the protein to form disulpbide-bonded polymers. Comparison of the numbers, distributions and sequence contexts of cysteine residues showed that six of the eight cysteines present in the LMW subunit encoded by the cloned gene are also present in monomeric y-gliadins and four of these also in monomeric a-gliadins. These six cysteines were therefore assumed to form three intra-chain disulpbide bonds, as shown in Figure lA The two additional cysteines present in the LMW subunit (SH in Figure lA) are assumed to be "unpaired" and available to form inter-chain disulpbide bonds. A series of mutants was therefore constructed, in wbich one or both of the "additional" cysteines were converted to serine and the expressed proteins purified and re-folded in vitro using a rapid dilution method. The results are shown in Figures 1 B-E. Refolding of the wild-type protein (Figure IB) gave a mixture of monomers and polymers (the latter being too large to enter the gel). Monomers were also observed when the two single mutants were re folded (Figures lC and D), with dimers also present but no polymers. The proportion


202

A Predicted Behaviour Wild type

II I

I

Repeals

SH

mutant 1

M

Serine

�

2 Cysteine

dimers

Serine

�

�

Serine

C

B

�

SH

Cysteine

�

double mutant

polymers

Serine

0

monomers

E

-

�imers fnonomers

Mr

a

b

a

b

a

b

a

b

Mr

Figure lA Schematic representation of the structures of the wild type LMW subunit and the three mutants, and their predicted behaviour. The proposed disulphide structure is based on comparisons with monomeric r-g1iadins and a-gliadins. Figures IB-E SDS-PAGE of reduced (tracks a) and reduced and refolded (tracks b) samples of the following expressed LMW subunit proteins: wild-type (A), mutant 1 (B), mutant 2 (C) and double mutant (D). Mr indicates molecular weight marker proteins (Mr 180,()()(), 116,()()(), 84,()()(), 58,()()(), 48,500, 36,500 and 26,600).


203

of dimers was higher with mutant 2 (Figure ID), which is consistent with the single "additional" cysteine close to the N-terminus being more readily accesis ble for inter chain disulphide bond formation. These dimers were absent when the double mutant was folded, which gave monomers with little or no polymer. At least two monomeric bands were observed with mutant 1 and the double mutant, which presumably resulted from the presence of forms with incorrect disulphide bonds. Although the results of this study were consistent with our hypothesis and with the patterns of disulphide bond formation revealed by direct analysis of peptides prepared from gluten,19 they were not conclusive. The levels of refolded proteins obtained were very low, and the proportions of monomers and polymers formed by the wild-type protein were found to be influenced by the protein concentration. In addition, it is necessary to make further mutants in which putative paired cysteines are also substituted in order to determine whether the generation of further "unpaired" cysteine residues also affects polymer formation. 3.2 Analysis of BMW Subunit Structure

The HMW subunits of glutenin consist of three distinct domains. These are unique N- and C-terminal domains of 81-104 residues and 42 residues, respectively, flanking a central repetitive domain v in length from about 630 to 830 residues. Analyses of whole HMW subunit proteins, 1 and of synthetic peptides corresponding to the repeat motifs in the central domain,22 have shown that the repetitive sequences form an unusual spiral structure based on fJ-reverse turns, but the details of this structure and its role in determining the functional properties of the HMW subunits have proved difficult to determine due to the complex multi-domain structure of the protein. We have therefore adopted a protein engineering approach, and expressed a peptide corresponding to residues 103 to 643 of HMW subunit IDxS in E.coli. This peptide, and a mutant form with cysteine residues present at the N- and C-termini, have been purified and are currently being studied to determine their conformations, ability to form polymers and rheological properties.

�

4 EXPRESSION OF GLUTEN PROTEINS IN 1RANSGENIC WHEAT Protein engineering is a powerful tool for studying the structures and biophysical properties of individual gluten proteins and it is even possible to determine the . functional properties of heterologously expressed proteins by incorporation into doughs using a small scale mixograph.1 However, such incorporation experiments are difficult to perform and it is not possible to ensure that the incorporated protein forms the same molecular interactions as it would if expressed in the developing wheat grain. The development of wheat transformation systems therefore provides an opportunity to determine the roles of individual proteins in wheat gluten structure and functionality. This will ultimately allow us to develop new varieties in which the composition and properties of the gluten proteins have been tailored for specific end use properties. We are using wheat transformation to determine the roles of HMW subunits in the biophysical and functional properties of wheat gluten. This exploits the availability of a series of near isogenic wheat lines, produced by Dr Greg Lawrence at CSIRO, Canberra.23 These lines include a triple null containing no HMW subunits,


204

a control with five HMW subunits (lAxl, lBx17 + lBy18, 1Dx5 + 1Dy10) and six lines containing one (lAxl), two (lBx17 + lBy18 or 1Dx5 + 1Dy10), three (lAxl, 1By17 + 1By18 or 1Ax1, 1Dx5 + 1Dy10) and four (lBx17 + 1By18, 1Dx5 + 1Dy10) subunits. These lines are currently being transformed by inserting genes encoding subunit 1Ax1,24 1Dx5lS or 1Dy10.26 The genes for subunits 1Dx5 and 1Dx10 are currently being used in separate transformation experiments, but further studies will include the introduction of both genes together, allowing the effects of transformation with subunits 1Dx5 and 1DylO alone and the allelic pair to be compared. Previous studies have indicated that the HMW subunits have both quantitative and qualitative effects on gluten quality.24,27 Comparisons of the amounts of HMW subunits synthesised in the transformants and the isogenic control lines, and the biophysical and functional properties of the glutens and doughs produced from the lines, will allow these effects to be dissected in detail. This will facilitate the use of genetic engineering to produce wheats with new and improved end-use properties. Acknowledgement IACR receives grant-aided support from the Biotechnological and Biological Sciences Research Council of the United Kingdom. References

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

F.Bekes, o. Anderson, P.W. Gras, RB. Gupta, A Tam, C.W. Wrigley, and R Appels, 'Improvement of Cereal Quality by Genetic Engineering', Eds RJ. Henry and JA Ronalds, Plenum Press, New York and London, 1994 p.97. J. Entwistle, S. Kundsen, M. Miiller,and V. Cameron-Mills, Plant MoL BioL 1991 17, 1217. O.V. Sayonova. Abstract of thesis, Moscow, 1992. G. Hull, N.G. Halford, M. Kreis, and P.R Shewry, Plant MoL BioL 1991 17, 1111. D.D. Kasarda, J.-C. Autran, EJ.-L Lew, C.C. Nimmo and P.R Shewry, Biochim. Biophys. Acta 1983 747, 138. P.I. Payne and K.G. Corfield, Planta 1979 145, 83. EA Jackson, LM. Holt and P.I. Payne, Theor. AppL Genet. 1983 66, 29. EJ.-L Lew, D.D. Kuzmicky, and D.D. Kasarda, Cereal Chem. 1992 69, 508. T.W. Okita, V. Cheesbrough and C.D. Reeves, J. BioL Chem. 1985 260, 8203. K. Scheets and C. Hedgcoth, Plant Sci. 1988 57, 141. S.M. Masci, E. Porceddu, G. Colaprico and D. Lafiandra, J. Cer. Sci. 1991 14, 35. K.A Pratt, PJ. Madgwick and P.R Shewry, J. Cer. Sci. 1991 14, 223. S. Thompson, D.H.L Bishop, P. Madgwick, A S. Tatham, and P.R Shewry, J. Agric. Food Chem. 1994 42, 426. L Tamas, J. Greenfield, N.G. Halford, AS. Tatham, and P.R Shewry, Protein Expression and Purification 1994 5, 357. V. Colot, D. Bartels, R Thompson and R Flavell, MoL Gen. Genet. 1989 216, 81. B.G. Cassidy and J. Dvorak, Theor. AppL Genet. 1991 81, 653. E.G. Pitts, J.A Rafalski and C. Hedgcoth, Nucleic Acids Res. 1988 16, 1 1376.


18. 19. 20. 21. 22. 23. 24. 25. 26.

27.

205

R D'Ovidio, AT. Oronzo and E. Porceddu. Plant MoL BioL 1992 18, 781. P. KOhler, H.-D. Belitz and H. Wieser, Z. Lebens. u-Forsch. 1991 192, 234. J.M. Field, AS. Tatham and P.R Sbewry, Biochem. J. 1987 247, 215. MJ. Miles, HJ. Carr, T. McMaster, KJ. rAnson, P.W. Belton, VJ. Morris, J.M. Field, P.R Sbewry and AS. Tatham, Proc. NatL Acad. Sci. USA 1991 88, 68. AS. Tatham AF. Drake and P.R Sbewry, J. Cer. Sci. 1990 11, 189. GJ. Lawrence, F. Macritchie and C.W. Wrigley, J. Cer. Sci. 1988 7, 109. N.G. Halford, 1.M. Field, H. Blair, P. Urwin, K. Moore, L Robert, R Thompson, RB. Flavell, AS. Tatham and P.R Sbewry, Theor. AppL Genet. 1992 83, 373. 0.0. Anderson, F.C. Greene, RE. Yip, N.G. Halford, P.R Sbewry and J.-M. Malpica-Romero, Nucleic Acids Res. 1989 17, 461. RB. Flavell, AP. Goldsbrougb, LS. Robert, D. Schnick and RD. Thompson, Bio/I'echnology 1989 7, 1281. P.R Sbewry, N.G. Halford and AS. Tatham, J. Cer. Sci. 1992 15, 105.

THE USE OF BIOTECHNOLOGY TO UNDERSTAND WHEAT FUNCTIONALITY

A. E. Blechl and 0.0. Anderson Agricultural Research Service U.S. Department of Agriculture Western Regional Research Center 800 Buchanan St. Albany, CA, USA 947 1 0

1 INTRODUCTION

The High Molecular Weight Glutenin Subunits (HMW -GS) are seed storage proteins that along with Low Molecular Weight Glutenin Subunits form disulfide-bonded high molecular weight polymers in the endosperm cells of wheat. There are two types of HMW-GS, designated x and y types, and each allelic pair is encoded by linked genes at the Glu-J loci on the long arms of the homeologous group 1 chromosomes . In any one cultivar, between 3 and 5 of the 6 genes are active in producing polypeptides. I Although HMW-GS constitute only 5-10% of the total flour protein, they have large effects on mixing and baking properties. The presence of specific allelic pairs, most notably those encoded by the 0 genome, has been correlated with bread-making quality parameters.v The total amount of HMW-GS is also important in determining the strength characteristics of bread doughs.2,4 Indeed, some allelic differences, for example those between IAx l or I Ax2* and their null alleles,S and those between I Bx7 and I Bx7*,6 have been attributed to quantitative differences in gene expression, The effects of having different numbers of HMW-GS in the same genetic background has been investigated by Lawrence et al. who derived wheat lines that express between 0 and 5 HMW-GS.7 Lines with fewer numbers of HMW-GS genes had lower levels of HMW GS polypeptides and exhibited decreased performance in mixing and baking tests.7,8,9 Using an independent set of near-isogenic lines from variety Sicco, Kolster et al. found that decreases in overall HMW-GS levels due to deletion of the D-genome-encoded subunits was compensated to some extent by increases in the other HMW-GS, but no compensation occurred when A- or B-genome-encoded subunits were missing.1o Galili et al. also found that there was no compensation by the D-genome-encoded HMW-GS when the Glu-Bl locus was deleted, but that an increase in B-encoded subunits in aneuploid substitution lines resulted in decreased synthesis of the other HMW-GS.II We were interested in investigating further the relationship between the quantity of HMW-GS and end-use properties, specifically whether increases in HMW-GS levels would lead to improvements in dough strength. The establishment of a reliable and efficient transformation protocol in our laboratoryl2 allows us to ask this question experimentally by introducing additional copies of HMW-GS genes into wheat. The first


results from these experiments

are

207

presented in this paper.


Among the cloned genes available to us for re-introduction into wheat were all six HMW-GS genes from cultivar Cheyenne. These same genes are already components of the genome of cultivar Bobwhite, the wheat variety used in our DNA transformation experiments because of its high efficiency of regeneration from tissue cultures. For our initial experiments, we wanted to be able to distinguish the products of the introduced gene from those of the endogenous genes. Shani et al. had previously demonstrated that hybrid HMW-GS, created by gene fusions and expression in the bacteria E. coli, migrated in SDS-PAGE at unique positions, often clearly distinguishable from those of the native HMW-GS.13 One such hybrid, made by joining the genes for the DylO and Dx5 subunits at a shared HindlII site just inside the repeat region, migrates at a position slightly ahead of that of the native Dx5 subunit. This result can be seen in Figure 1. The lane marked Y(H)X contains the extract from E. coli expressing the DylO:Dx5 hybrid glutenin subunit (marked by an arrow). This band is not seen in extract from bacteria that contain the empty expression plasmid, pet3A,14 without a HMW -GS coding sequence (lane pet3A). Lane BW contains seed extract from cultivar Bobwhite showing the HMW-GS Ax2*, Dx5, Bx7, By9 and DylO as well as a few minor bands in this region. To test whether the separation between the hybrid and native HMW-GS would be clear enough in seed extracts under these gel conditions, Bobwhite seed extract and extract from the bacteria expressing the hybrid HMW-GS gene were mixed and run in the lane marked "mix". The hybrid DylO:Dx5 GS is clearly distinguishable from the endogenous HMW-GS, migrating slightly ahead of the Dx5 subunit and slightly behind a minor band from the wheat seed extract. Based on these results, a DNA construction was made in which the 5' and 3' flanking sequences for bacterial expression were replaced by the original wheat sequences. The result, a hybrid gene between the native DylO and Dx5 genes with the fusion junction at the HindlII site, is diagramed in Figure 2. The promoter elements for expression in wheat endosperm are provided by the approximately 2800 bp of sequence 5' to the DylO coding region. It has been shown that 432 bp of DylO 5' flanking sequence is sufficient for full levels of expression of reporter genes in maize endosperm cell transient assayslS and that 433 bp of the allelic Dy12 gene is sufficient for endosperm-specific expression of reporter genes in transgenic tobacco.16.17 The coding region of the hybrid gene consists of the first 143 codons of DylO, including the signal peptide, and the last 720 codons of the Dx5 gene. The Dx5 gene also provides the translation and transcription termination sequences within approximately 2000 bp of 3' flanking DNA. This DNA was co-transformed into immature embryos of Bobwhite along with DNA containing a selectable marker gene encoding herbicide resistance, UbiBAR.12 The DNA's were introduced by the particle bombardment method as detailed in Weeks et al.12 Transformed tissues were selected based on their resistance to the selection agent, bialaphos, and plants were regenerated. The transformation efficiency was 1 independent fertile resistant wheat plant obtained for every 200 embryos bombarded. Immature seeds from these plants were removed for dissection between 3 and 4 weeks after anthesis. The embryos were plated on a precocious germination medium containing 3 mg/L

208


Figure 1 Expression of hybrid HMW-GS genes in transgenic wheat and E. coli Extracts from either transgenic wheat endosperm (left four lanes) and/or E. coli cells containing the pet3A expression plasmid (right three lanes) were subjected to SDS-PAGE in a 10% (0.05% bis) acrylamide gel. The gel was stained with Coomassie blue. Lane 1 and 2 contain protein extracts from Tj wheat seeds of 2 independent lines whose embryos germinated in the presence of 3 mg/L bialaphos. Lane BW contains protein extract from the parental nontransformed cultivar Bobwhite. The lane labelled Y(H)X contains protein extract from an E. coli culture transformed with the pet3A vector expressing the hybrid Dy1O:Dx5 glutenin subunit. The lane labelled "mix " is an artificial mixture of the contents of lanes BW and Y(H)X. The lane labelled pet3A contains extract from E. coli transformed with the empty (nonexpressing) pet3A vector plasmidu. The designations to the left identify endogenous HMW-GS of Bobwhite wheat endosperm. The numbers to the right indicate the sizes in kilodaltons and migration positions of molecular weight standards run in a different lane of the same gel. The arrows mark the location of the bands corresponding to the hybrid HMW-GS.

�--

1

Wheat 2

-----;=====+- E. coli ---'1 BW mix Y(H)X pet3A

Ax2*

'

Dx5

-

.......

B x7

-

By9, Dy1 0

- 94

- 67


209

bialaphos to confirm that the herbicide resistance was a stably inherited trait and thus that these were true transgenic wheat plants. The endosperms were used to prepare protein extracts which were then analyzed by SDS-PAGE. Two such extracts are shown in Lanes 1 and 2 of Figure 1 . The band corresponding to the hybrid HMW -GS is indicated by the arrow. This protein is clearly present at levels higher than those of the endogenous HMW-GS in these two independent transformed lines. Similar results were obtained in 1 2 other independent transformants: expression levels are at least as high as those of the endogenous genes. The basis of the relatively high level of expression of the hybrid HMW -GS is not known at this time. Analyses of later generations of seed progeny of these plants show that the high expression levels are inherited to at least the T3 generation (data not shown). Experiments are in progress to quantitate the accumulation levels of the hybrid HMW-GS relative to total protein and to the native HMW-GS. Figure 2 Diagram of the plasmid encoding the hybrid HMW-GS The plasmid was constructed by fusing the 5' flanking DNA and N-terminal coding region of the DyJO HMW-GS to the coding and 3 ' flanking regions of the Dx5 HMW-GS gene. The junction is a shared Hindlll site (arrow) just inside the codons for the repeated amino acid motif regions of the DylO and Dx5 genes (shown as circles corresponding to 20 codons). J The flanking sequences are denoted by thin lines and are not drawn to scale. The DyJO coding regions are shown in black and the Dx5 coding regions are shown in gray. The S letters mark the locations of cysteine residues in the encoded polypeptide. The wheat sequences were cloned into the EcoRl site of the vector pBluescript (Stratagene, La Jolia, CAY.

5'

-Ot--fp_ Dy1 0

Hitll

Dx5

()- 3 '

3 CONCLUDING REMARKS These experiments demonstrate that a gene encoding a HMW-GS subunit can be stably incorporated into the wheat genome by genetic transformation and that such a gene is expressed at levels at least as high a,o; those of the endogenous subunits. The fourteen independent wheat lines derived here will provide a near-isogenic series in which to examine the effects of this HMW-GS on the mixing and baking properties of doughs derived from their flours. Because the protein synthesized is an entirely new variant, any differences observed in functionality could be due to either qualitative or quantitative effects of the new HMW-GS. In order to determine whether there is a purely quantitative effect of HMW-GS levels on dough strength, we are currently introducing native Dx5 and DylO genes into wheat by transformation. The results of the experiments presented here predict that the relative levels of these proteins in transgenic endosperm will be increased.


210

4 ACKNOWLEDGMENTS We acknowledge the excellent technical assistance of Dafna Elrad and Eric Schlossberg. This work was supported by the Agricultural Research Service, U. S. Department of Agriculture CRIS No. 5325-21430-001-00D. References

1. P. R. Shewry, N. G. Halford and A. S. Tatham, J. Cer. Sci. , 1992, 15, 105. 2. P. Payne, M. A. Nightingale, A. F. Krattiger and L. M. Holt, J. Sci. Food Agric. , 1987, 40, 5 1 . 3 . O. M . Lukow, P. I Payne and R. Tkachuk, J. Sci. Food Agric. , 1989, 46, 45 1. 4. K. H. Sutton, J. Cer. Sci., 199 1 , 14, 25. 5. G. I. Lawrence, F. MacRitchie and C. W. Wrigley, J Cer. Sci., 1988, 7, 109. 6. N. G. Halford, I. M. Field, H. Blair, P. Urwin, K. Moore, L. Robert, R. Thompson, R. B. Flavell, A. S. Tatham and P. R. Shewry, Theor. Appl. Genet., 1992, 83, 373. 7. B. A. Marchylo, O. M. Lukow and I. E. Kruger, J Cer. Sci., 1992,15, 29. 8. L. Gao and W. Bushuk, Cereal Chern., 1993, 70, 475. 9. R. B. Gupta, Y. Popineau, I. Lefebvre, M. Cornec, G.I. Lawrence and F. MacRitchie, J Cer. Sci., 1995, 21, 103. 10. P. Kolster, C. F. Krechting, and W. M. 1. van Gelder, Theor. Appl. Genet. , 1993, 87, 209. 1 1. G. Gali1i, A. A. Levy and M. Feldman, Proc. Natl. Acad. Sci. USA, 1986, 83, 6524. 12. I. T. Weeks, O. D. Anderson and A. E. Bleehl, Plant Physiol., 1993, 102, 1077. 13. N. Shani, I. D. Steffen-Campbell, O. D. Anderson, F. C. Greene and G. Galili, Plant Physiol., 1992, 98, 433. 14. A. H. Rosenberg, B. N. Lade, D. Chui, S. Lin, 1. 1. Dunn and F.W. Studier, 1987, Gene, 56, 125 15. A. E. Bleehl, G. F. Lorens, F. C. Greene, B. E. Mackey and O. D. Anderson, Plant Sci. , 1995, 102, 69. 16. V. Colot, L. S. Robert, T. A. Kavanagh, M. W. Bevan and R. D. Thompson, EMBO J., 1987, 6, 3559. 17. L. S. Robert, R. D. Thompson and R. B Flavell, Plant Cell, 1989, I, 569.

CONSTRUCTION OF D16 GENES MODIFIED IN THE REPETITIVE DOMAIN AND THEIR EXPRESSION IN ESCHERICHIA COU

R. D'Ovidio1,2, O.D. Anderson2 , S. Masci1,2, J. Skerritt3 and E. Porceddu1 IDipartimento di Agrobiologia e Agrochimica, Universita della Tuscia, Via S. Camillo de Lellis, 01 100 Viterbo, Italy. 2US Department of Agriculture, A.R.S . , W.R.R.C . , 800 Buchanan Street, Albany, CA 947 10, USA. 3CSIRO Division of Plant Industry, GPO Box 1600 , Canberra ACT 2601 , Australia.

1 INTRODUCTION High molecular weight glutenins (HMW-GS) have been studied extensively both at biochemical and molecular level because of

their role in determining the viscoelastic

properties of wheat gluten, which in turn are responsible for the quality characteristics

and biochemical analyses indicated that bread making quality Glu-DI and Glu-AI locP·3. Detailed analysis at the Glu-DI locus has shown that, in general, the cultivars containing the allelic pair designated 1Dx5 + IDyl0 show superior bread making quality3.

of bread wheat. Genetic

is particularly

associated with variation at the

Nucleotide sequence analysis of HMW-GS genes made it possible to distinguish the primary

structure of these

proteins in three main regions: the non repetitive N-terminal

and C-terminal domains, and

the central repetitive domain. Sequence comparison of

genes encoding HMW-GS revealed the presence of small differences in aminoacid sequence and/or composition which have been indicated as responsible for determining differences

in the physical

properties

of gluten.

In particular,

two

structural

characteristics seem mainly involved in conferring such important role to these proteins: the number and position of cysteine residues,

and the

structure of the

repetitive domain. In order to verify the importance of the latter characteristic we have modified

the central repetitive domain of the 1Ox5 gene.

2. MATERIAL AND METHODS The modifications of

the repetitive domain contained in the Dx5 constructs were the expression in Escherichia coU was

carried out on the pET-3a-Glu-1Ox54 and

performed following the procedure reported by Studier and Moffatts.

SDS-PAGE was performed with a Mini-Protean II Cell (BioRad) according to the manufacture instructions. western blot analysis were carried out using monoclonal antibody which recognized specifically the HMW-GS6.


212 Table

I . Summary ofthe characteristics oftlze polypeptide expressed by each pET-3aDx5 plasmid constructs.

1

pET-3a-Dx5

Dx5-R853

696

853

1

Dx5-R576

1

1

Dx5-R44 1

1-

Repetitive domain length

576

441

+ 22 . 5

-17.2

-36.6

88,259

104,583

75,484

61 ,80 1

1 19,000

142,000

102,000

82,000

% of repetitive domain added or removed Calculated MW (daltons) Apparent MW on SDS-PAGE (daltons)

Relative surface hydrophobicities were measured on the basis of elution time on RP-HPLC. Proteins were eluted through a C8 column with a linear gradient from 28% to 35 % acetonitrile containing trifluoroacetic acid.

3. RESULTS AND DISCUSSION Three different constructs with variable lengths of the repetitive domain were prepared and expressed in Escherichia coli.

The Dx5 HMW glutenin variants,

designated Dx5-R853, Dx5-R576 and Dx5-R441 , produced subunits with repetitive domains which are 22. 5 % longer, and 1 7 . 2 % and 36.6% shorter expressed subunit, respectively (Table 1 ) .

than the naturally

On SDS-PAGE the modified Dx5 glutenin subunits show the anomalously slower

migration typical of this class of proteins (Figure I). The anomalous migration seems

the repetitive region. The length of the has a negative correlation with the surface hydrophobicity of the

to be positively correlated with the size of repetitive domain

molecule, measured by RP-HPLC.

These constructs might be very useful to establish the role of the repetitive domain in determining the viscoelastic properties of dough because specific insertion and deletions are present in the same background . The evaluation of the effects that these modifications

might cause

on

gluten

is currently being

verified

in vitro by

micromixographic experiments7, and could be tested in vivo by expressing the Dx5

213


constructs in transgenic wheat plant... In addition, the Dx5 constructs could be used in basic physical studies of the polypeptide structure.

M

1

2

3

4

5

KOa 200 1 16-

97.466 -

45-

Figure 1 SDS-PAGE analysis of wild-type and modified Dx5 HMW-GS extracted from E. coli harbouring the pET-3a-Glu-1Dx5 plasmid constructs. 1) pET-3a; 2) Dx5-R853; 3) pET-3a-Dx5; 4) Dx5-R576; 5) Dx5-R441. M) Molecular weight standard (KDa) is reported on the left site of the picture.

The possibility to generate Dx5 HMW-GS modified in the repetitive domain demonstrates the capability of creating new genetic variability in the laboratory for this class of proteins. This approach, besides eliminating the need to search for natural variability of HMW-GS, offers the opportunity to generate variation at specific region or sites. This possibility makes it possible to test the proposed hypotheses about the influence of some structural features of HMW-GS on gluten characteristicS8•10 and to achieve more informations about the molecular structure of single HMW-GS, and their modification after the formation of the backbone of the polymeric structure of wheat gluten. While there is in fact a well documented correlation between breadmaking quality and the presence of specific HMW -GS3 little is known about the precise organisation of HMW-GS in glutenin polymer.

4. REFERENCES 1. 2.

P.l. Payne, K.G. Corfield, I.A. Blackman, Theor. Appl. Genet. , 1979, 55, 153. P.1. Payne, K.G. Corfield, L.M. Holt, I.A. Blackman, J. Sci. Food. Agric. , 1981, 32, 51.


214 3. 4. 5. 6. 7. 8. 9. 10.

P . I . Payne, Ann Rev Plant Physioi, 1987, 38, 141 . N. Shani, J.D. Steffen-Campbell, 0.0. Anderson, F.e. Greene and G. Galili, Plant Physiol. , 1992, 98, 433 F.W. Studier and B.A. Moffatt, J. Mol. Evol. , 1986, 189, 1 1 3 . J.L. Andrew and J.H. Skerritt, J. Cereal Sci. , 1994, 19, 2 1 9 . F. Bekes and J.H. Gras, Cereal Chem. 1992, 69, 229 J.A.D. Ewart, J. Sci. Food Agric., 1968, 19, 617. A. Graveland, P. Bosveld, W.J. Lichtendonk, J.P. Marseille, J.H.E. Moonen and A. Scheepstra, J. Cereal Sci. 1985, 3, I . D.O. Kasarda, 'Wheat is Unique', Y. Pomeranz Ed., AACC, St. Paul, MN, p. 277.

EXPRESSION OF BARLEY AND WHEAT PROlAMINS IN BIOPHYSICAL STUDIES

E.COLI FOR

JJA Greenfield!, L Tamas2, N.G. Halford!, D. Hickman!, S.B. Ross-Murphyl, S. Ingman4, AS. Tatham1 and P.R. Shewryl

lIACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS18 9AF, UK. 2CSIRO, Division of Plant Industry, GPO Box 1600, Canberra, ACI' 2601, Australia. 3Division of Life Sciences, Biopolymers Group, King's College London, Campden Hill Road, Kensington, London W8 7AH, UK. 4Unilever Research, Colworth Laboratory, Colworth House, Sharnbrook, Bedford MK44 1LQ, UK.

1

INTRODUCTION

Wheat gluten is a complex mixture of proteins, which are classically divided into gliadins and glutenins. 1 These groups can be readily prepared by solvent extraction and gel filtration chromatography, and the gliadins fractionated into their component types (a, p, y, c.» by anion-exchange chromatography. However, each of these fractions contains a mixture of individual proteins and it is difficult, and in many cases has not yet proved possible, to purify single homogeneous components. The glutenins can be similarly divided into high molecular weight (HMW) and low molecular weight (LMW) subunits, and the latter into B, C and D types.2.3 Once again it is difficult to purify single proteins, especially of the LMW types. Since a number of cloned cDNAs and genes for gluten proteins are available, expression in heterologous systems provides an attractive opportunity to prepare large amounts of single homogeneous proteins for biophysical studies. In addition it is possible to make specific mutations, in order to explore the relationship between protein structure and function (or, in the case of food proteins, functionality). We are therefore using protein engineering to study the structure and functionality of wheat gluten proteins, focusing on the c.>-type gliadins4 and the HMW 5 subunits of glutenin. However, clones encoding c.>-gliadins are not currently available and it has therefore been necessary to study a homologous protein from barley, called C hordein.6 C hordeins have similar amino acid compositions, N-terminal amino acid sequences" and conformations' (as demonstrated by circular dichroism spectroscopy) to the c.>-gliadins encoded by chromosomes 1A and 1D of bread wheat.

2

PROTEIN ENGINEERING OF C HORDEIN

The barley genomic clone Ahor1-17 encodes a protein of 261 residues, including a 20 residue signal peptide.8 The mature protein has an Mr of 28,033 and consists of 233 residues of repeated sequences (based on the consensus peptide Pro.Gln.Gln.Pro.Phe.Pro.Gln.Gln) flanked by non-rep�titive sequences of 12 and 6 residues at the N- and C-termini, respectively. These repeated sequences appear to form a loose s iral structure based on a mixture of poly-L-proline II structure and p reverse turns. ,10 The C hordeins and c.>-gliadins also lack cysteine residues. This

�


216

means that they are monomeric and, in the case of the w-gliadins, appear to contribute to gluten viscosity rather then elasticity. The coding region of this clone was mutated to replace the signal sequence with an ATG (Met) initiation codon and expressed in E.coli using the pETId vector system.n,12 In addition, a mutant form was also constructed and expressed, with cysteines substituted for serine and

threonine at positions 7 and 236, respectively, of the encoded protein. The protein was readily extracted from the pelleted cells with 70% (vIv) aqueous ethanol and precipitated by the addition of 2 volumes of 1.5M NaCI and standing at 4 " C. The yield after 3 hours of induction was in excess of 30 mgll of culture. Aliquots of the preparation were also purified further by RP-HPLC for detailed physico-chemical analysis. SDS-PAGE of the wild-type protein showed a single band migrating slightly slower than an Mr 30,000 marker protein. In contrast, the mutant cysteine-containing

protein was not clearly resolved in the non-reduced state, showing only a ladder of faint bands towards the top of the gel. These were replaced by a single band of similar Mr to the wild-type protein under reducing conditions, indicating that the mutant protein formed disulphide-bonded polymers either in the E.coli cells or during extraction and purification. The wild-type and mutant proteins appeared to be correctly folded as determined by comparison of their surface hydrophobicities (by RP-HPLC) and their secondary structure contents (by circular dichroism spectroscopy in 70% (vIv) ethanol) with those of a mixture of C hordeins prepared directly from barley. The mutant and wild-type C hordeins are being subjected to rheological analysis. Samples of 20-30 mg dry weight were hydrated to various levels ranging from 50 to 70% water and tested using a controlled strain rheometer. Preliminary results indicate that the mutant (polymeric) protein is more elastic than the wild-type as determined by comparing the 1/ * slopes of frequency sweeps. More detailed studies are currently in progress.

3

EXPRESSION OF AN HMW SUBUNIT REPEAT PEPTIDE

The HMW subunits of glutenin consist of between 627 and 827 residues, with Mr of 67,495 to 88,137.5 They have three clear domains, with an extensive central domain consisting of repeated sequences flanked by shorter non-repetitive domains at the N terminus (81-104 residues) and C-terminus (42 residues in all subunits). Variation in the length of the repetitive domain is, therefore, largely responsible for differences between the Mr of the whole proteins. Four, five or seven cysteine residues are present in the HMW subunits of bread wheat, with three or five in the N-terminal domain, one in the C-terminal domain and, in some subunits only, single cysteines within the repeats. These cysteines appear to allow the HMW subunits to form high Mr polymers which contribute to the elasticity of gluten and dough. The repetitive domains are based on short peptide sequences, with hexapeptides (consensus Pro.Gly.Gln.Gly.Gln.Gln), nonapeptides (consensus Gly.Tyr.Tyr.Pro.Thr.Ser.Pro or Leu. Gin. Gin) and, in x-type subunits only, tripeptides (Gly.GIn.GIn). As in C hordein these sequences also appear to form a loose spiral structure, which is based only on p-reverse turns and called a p-spiral.1>1S The p spiral could contribute to gluten functionality, either by being intrinsically elastic13 or by forming strong hydrogen bonds with adjacent proteins, facilitated by the high

217


content of glutamine residues (about 40 mol %). However, the complex multidomain structure of the HMW subunits makes it difficult to carry out detailed studies of the structure and functional properties of the J3-spiral structure. In order to eliminate this problem we have subcloned a genomic fragment from the subunit IDx5 gene, encoding a peptide of Mr about 57,000 from the central repetitive domain of the protein (Figure 1). In addition, a mutant form has been constructed encoding a protein with cysteine residues added close to the N- and C-termini (Figure 1).

89

I

NH 2

SH S.r;� S�

103

M r 88,128

REPEATS

M r 56,894

106 G or e

Figure 1

785

I 643

827 I

eOOH

SH

640 Yor e

Schematic structure of HMW subunit lDx5 and of the Repetitive Fragment Expressed in E.coli

The wild-type and mutant proteins were expressed in E.coli using the pETI7b vector systemll and purified by extraction with 70% (v/v) ethanol followed by precipitation with ammonium sulphate. The wild-type protein has also been subjected to detailed spectroscopic analysis, using circular dichroism, fluorescence and Fourier-transform infra-red spectroscopy. This has confirmed the presence of a J3-turn-rich conformation, which was previously proposed on the basis of structure prediction, 13 and the analysis of short synthetic peptides based on the nonapeptide and hexapeptide repeat motifs.14 Future plans include more detailed spectroscopic analyses, and rheological comparisons of the wild-type and cross-linked forms. Further mutants will then be constructed in which the number and distribution of cysteine residues is altered to affect cross-linked formation, while the length and precise sequence of the repetitive domain are altered to affect the extent and properties (including hydrogen bonding capacity) of the J3-spiral structure. These studies will provide details of the precise structures of the HMW subunits and the molecular basis for their role in gluten.

Acknowledgement IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.


218

References 1.

D.O. Kasarda, J.E. Bernardin and C.C. Nimmo, 'Advances in Cereal Science Ed. Y. Pomeranz. American Association of Cereal and Technology' Chemists, St. Paul, Minnesota, 1976, 1, p.158.

2. 3. 4.

P.I. Payne and K.G. Corfield, Planta 1979, 145, 83. E.A Jackson, LM. Holt and P.I. Payne, Theor. AppL Genet.

5. 6.

1983, 66, 29.

D.O. Kasarda, J.-c. Autran, E.J.-L Lew, C.C. Nimmo, and P.R Shewry,

Biochim Biophys. Acta 1983, 747, 138.

P.R Shewry, N.G. Halford and AS. Tatham, 1 Cer. Sci. 1992, 15, 105. P.R Shewry, 'Barley: Chemistry and Technology' Eds. J. MacGregor, and R Bhatty. American Association of Cereal Chemists, St. Paul, Minnesota, U.SA

1993, p.131.

8.

AS. Tatham, P.R Shewry and P.S. Belton, 'Advances in Cereal Science and Technology'. Ed. Y. Pomeranz. American Association of Cereal Chemists, St. Paul, Minnesota, U.S.A 1990, 10, p.1. J. Entwistle, S. Knudsen, M. Milller and V. Cameron-Mills, PL MoL BioL 1991,

9. 10.

AS. Tatham, AF. Drake and P.R Shewry, Biochem 1 1989, 259, 471. K.J. I'Anson, VJ. Morris, P.R Shewry and AS. Tatham, Biochem 1. 1992, 287,

11.

AH. Rosenberg, B.N. Lade, D.-S. Chui, S.W. Lin, J.J. Dunn and F.W. Studier,

7.

12. 13. 14. 15.

17, 1217. 183.

Gene 56, 125. L Tamas, J. Greenfield, N.G. Halford, AS. Tatham and P.R Shewry, Protein Expression and Purification 1994, 5, 357. AS. Tatham, P.R Shewry and B.J. Miflin, FEBS Letts. 1984, 177, 205. AS. Tatham, AF. Drake and P..R Shewry, 1 Cer. Sci. 1990, 11, 189. MJ. Miles, HJ. Carr, T. McMaster, P.S. Belton, VJ. Morris, J.M. Field, P.R Shewry and AS. Tatham, Proc. Natn. Acad. Sci. U.S.A. 1991, 88, 68.

Low Mr Sulphydryl Compounds in Wheat Flour and Their Functional Importance

MEASUREMENT AND REACTIVITY OF GLUTATHIONE IN WHEAT FLOUR AND DOUGH SYSTEMS

1. D. Schofield and X. Chen The University of Reading Department of Food Science and Technology P. O. Box 226, Whiteknights Reading RG6 6AP United Kingdom

1 INTRODUCTION The importance to dough processing performance (processability) and baked product quality of the reduction-oxidation (redox) reactions that occur in wheat flour and dough ) has long been recognised . The (bio)chemistry of those reactions, on the other hand, is still poorly understood, and their technological manipUlation, including the use of exogenous oxidising bread improvers, such as potassium bromate, azodicarbonamide and ascorbic acid (after oxidation to dehydroascorbic acid), or reducing agents used in biscuit making, such as sodium metabisulphite, is based largely on empiricism. The lack of a satisfactory knowledge base concerning the nature of these reactions restricts the development of new, more effective and more consumer-acceptable technology for modulating them than that presently available. The withdrawal in EU countries of traditionally used oxidising improvers for use in bread making on toxicological grounds, especially of potassium bromate, leaves processors dependent on ascorbic acid as sole bread improver. Ascorbic acid performs less well than bromate, however, highlighting the need for alternative technologies. Consumer and media pressure may also make the use of other presently-allowed redox additives, whether toxicologically safe or not, problematical in the long-term. Moreover, although variation is known to occur in oxidant requirements for bread making from one flour to another, the basis of this, whether genetic, agronomic or processing related, is largely unknown. Such variation is also difficult to assess and measure efficiently so as to control raw material inputs, and hence processability and product quality. Definition of the reactions/reactants that are important could form the basis for developing new analytical methods that might provide users of grain and flour with the means of monitoring and controlling raw material quality more precisely. Similarly, defining the important reactions/reactants and the mechanism(s) by which variation in them is controlled, together with the provision of appropriate analytical methodology, could be of value to plant breeders in selecting for appropriate genetically controlled redox characteristics and developing new cultivars with improved end-use properties.

1.1

The Technological Importance of Glutathione

Particular attention has been focused on the role of the tripeptide glutathione (y-g1utamylcysteinylglycine), which occurs endogenously in flour in both free reduced 2s (GSH) and free oxidised GSSG) forms - , as well as in the form of protein-glutathione mixed disulphides (pSSG) ,6" . GSH is able to react with inter-polypeptide chain disulphide bonds (PSSP) in glutenin during dough mixing through sulphydryl-disulphide (SHlSS)

{


222

interchange reactions8, resulting in the formation of a PSSG and a free protein SH gouP (pSH). Glutenin may thus be depolymerised, resulting in a weakening of the dough3,9' I. If such reactions are indeed significant in dough systems, flours with high levels of GSH, or similar peptides, would presumably be desirable for biscuit making, specifically for semi sweet or hard sweet biscuits, which require weak dough structure with minimal elastic recoil after sheeting. Such a dough weakening is effected currently by adding fairly high levels of the SS reducing agent, sodium metabisulphite. On the other hand, low GSH levels would be desirable in bread flours since a strong elastic dough is required that can retain the gas produced during yeast fermentation as discrete small gas celIs. Consistent with this notion, it has been observed that dough rheological properties are inversely correlated with the GSH contents of flours milIed from wheat cultivars that differ in bread making qualityI2.ls. It has also been proposed that oxidising improvers used as in�redients in bread making .16, exert their effects by causing the oxidation of GSH to GSSG4,I thus preventing GSH from participating in reactions involving cleavage ofPSSP, which would otherwise weaken the dough and cause bread quality to diminish. In the case of ascorbic acid, this reaction is thought to be catalysed by the endogenous flour enzyme, glutathione dehydrogenase (also called dehydroascorbate reductase). The oxidising improver reaction can therefore be considered as competing with the disulphide bond cleavage reaction for the available GSH. Thus protein depolymerisation and dough weakening are prevented:

:::

P

�

GSH

GSH

OXidiSi Impro

S Interchange PSSG

:>1r

GSH

:��s

PSH

�

GSSG

In fact, GSSG is also able to react with flour PSH via SH/SS interchange reactions, resulting in the formation ofPSSG and GSW7: GSSG

PSH

SH/SS Interchange PSSG

GSH

This reaction does not result directly in the scission of flour PSSP. Addition of GSSG during dough mixing does result in dough weakening, however3,Io This can be explained on the basis that the released GSH can undergo further SH/SS interchange reactions with flour PSSP. However, the naturally occurring levels of GSSG in flour-water doughs are thought to be relatively low, and such low levels may not have any significant effect on dough rheologyu It could be, however, that the role of oxidising improvers is not simply to convert GSH to GSSG in a 'one-off' reaction, but that they must continue to oxidise GSH to GSSG until all available PSH groups are blocked and further release of GSH via reaction of GSSG with PSH is prevented.

2 MEASUREMENT OF GLUTATHIONE IN FLOUR AND DOUGH SYSTEMS 2.1

Establishment of Methodology for Measuring GSH, GSSG and PSSG

Notwithstanding the research summarised above, the technological significance of sulphydryl peptides, and in particular that of glutathione and its reactions, remains extremely unclear. A major problem has been uncertainty about the true levels of

Low Mr

Sulphydryl Compounds in Wheat Flour and Their Functional Importance

223

glutathione in flour, since values reported in the literature vary by several orders of magnitude,, 14. This is almost certainly due to methodological problems in measuring the compound in flour. Furthermore, a convenient and reliable method for measuring not only GSH itself, but also GSSG and PSSG, has not been available making it difficult to obtain a complete picture of the reactions that glutathione undergoes in flour and dough. Without the ability to measure GSH, GSSG and PSSG discretely, definitive conclusions cannot be drawn about the reactions of GSH in flour and dough and their significance. The recent establishment in our laboratory of a relatively rapid, straightforward, sensitive and accurate HPLC technique for measuring directly the contents of GSH, GSSG and PSSG individually in flour and dough,,1 represents an important methodolo§ical breakthrough. The method is based on that described by Reed and co-workers . It involves initial extraction of GSH and GSSG with ice cold 5% (w/v) perchloric acid (PCA). The use of PCA as extractant has several advantages. Firstly, because PCA is a strong acid, the low « 1 .0) pH during extraction suppresses the ionisation of the SH groups both on GSH and on flour proteins. This prevents SHISS interchange reactions and the consequent loss of free GSH and/or free GSSG through the formation of PSSG, which may occur when extracting under neutral or slightly alkaline conditions. This may have compromised the analyses in some previous studies. PCA is also a strong chaotrope, which ensures efficient extraction of the peptides and minimises their entrapment by or adsorption onto the flour proteins. Lastly, PCA is an efficient protein precipitant, which results in a convenient separation of GSH and GSSG from the flour proteins, and minimises any interference by proteins in later stages of the procedure. It also precludes the need to include a time consuming chromatographic or other type of protein separation steps in the clean-up procedure. After extraction with PCA, free SH groups on GSH molecules and any other SH compounds extracted are blocked by alkylation with iodoacetic acid (lAA) at pH 8.0. It is important that the lAA is immediately available to react with the SH groups when the pH of the extract is raised from pH25nm to approximately 14nm in this region. The latter was similar to film thicknesses obtained for films formed from LPC alone. Thirdly, initiation of lateral diffusion of fluorescent labelled puroindoline in the plane of the adsorbed layer was observed at R=1 .5. Finally, the amount of adsorbed puroindoline decreased sharply in the R value range 1 to 3 although there were still low levels of puroindoline present in the interfacial layer up to approximately R= l O (Figure 8). The mechanism responsible for the enhancement of foaming properties is still unclear. However, it is evident that the enhancement is observed under conditions where both puroindoline and LPC are present in the interfacial layer. In addition, the protein is known to bind the lipid analogue. It seems likely that the complex formed has enhanced surface properties and this could have important technological significance. It is quite possible that this complex is present in bread dough and may play an important role during proofing and baking. In addition, it may be possible to exploit proteins such as puroindoline, which possess lipid binding activity in the development of novel food formulations or for improvement of existing foods. For example, the presence of low levels of egg yolk lipids in separated egg white seriously impedes foaming properties. It may prove possible to selectively remove extraneous yolk lipid by introduction of low levels of puroindoline. An alternative application is protection of beer foam against lipid-induced destabilization. Preliminary results have revealed that low levels of puroindoline that comprise only 1% ( 1 O-201ig!ml) of the total protein load present in beer, can restore the foaming properties to beer adulterated with stearic acid, phospholipids or triglycerides64. Similar results have been obtained concerning the foaming properties of egg white proteins adulterated with oil65. The negative effect of the oil on foaming properties can be negated by the presence of small quantities of puroindoline. 4. 3.2. Role of proteins in the adsorption and spreading of lipids at air-water inteifaces. As previously discussed, adsorption of bilayer liposomes to an air-water interface is not spontaneous and some defect such as inverted micelles have to be created at the contact interface to facilitate the spreading of lipids. Such structures can be induced at the surface ofbilayers by peptides and proteinsl2, 66 Theoretically this can be caused by proteins which exhibit a high affinity for non bilayer lipids. Such proteins will induce a lateral segregation and a local concentration of non bilayer lipids which can favour spreading. Such mechanisms have been described in the case of pulmonary surfactant proteins. Pulmonary surfactant is a mixture of phospholipids and proteins, which helps the lungs expand by lowering the surface tension at the air/liquid interface in the alveoli67. The main phospholipid component of lung surfactant is DPPC which has the ability to greatly lower the surface tension. However, DPPC does not exhibit rapid adsorption and spreading27. Other unsaturated phospholipids (PC, PE, PG) and some specific proteins contribute to increasing greatly the spreading kinetics68-69. It is interesting to note that the most efficient lung surfactant proteins are low molecular weight amphipathic proteins. For example, SP-B is an amphiphilic basic protein of 79 residues containing 7 cysteines, and structural characteristics reminiscent of those ofwheat lipid binding proteins68. We can speculate how LTPs can facilitate the exchange of lipids between the monolayer and the underlayer of liposomes, and such a mechanism has been postulated to explain the spreading of lipids as a monolayer25. Furthermore, these proteins, which act

Nature and Functionality o/Wheat Lipids. Lipid Binding Proteins and Added Emulsifiers

257

only on the outer bilayer lipid leaflets may facilitate in some cases, the transbilayer movement of phospholipids from the inner to the outer membrane leaflet 70. This would serve to increase the yield of lipid transferred from the inner bilayer to the monolayer at the air-water interface26. We have recently shown that such mechanisms involving wheat LTP depend on the surface pressure of the lipid monolayer44.

4.4. Gluten Viscoelasticity and Interfacial Behaviour of Polar Lipids Previous studies on the organisation and dynamics of lipids in gluten using both freeze-fracture electron microscopy and phosphorus NMR have clearly show that no interactions occur between the gluten proteins and lipid organised in sma1\ vesicles18. However, phosphorus NMR has shown that the viscoelasticity influences the dynamics of vesicles in gluten network 1 8. Therefore, we can imagine that the viscoelastic dough network serves only control the expansion of gas bubbles stabilized by lipoprotein films during proofing and baking. This view is supported by breadmaking experiments carried out by adding lipids to defatted wheat flour of good and poor qualities7 1-72. The loaf volume-lipid content curves exhibit quite similar shapes with only translation towards higher volumes or higher lipid content. Furthermore by interchanging components between good and poor quality flours variations in the curves reflect only differences in the proteins. 5.CONCLUSIONS: POSSmLE WAYS FOR THE IMPROVEMENT OF WHEAT QUALITY THROUGH ENGINEERING OF LIPIDS AND LIPID-PROTEIN INTERACTIONS Although we have still a fragmented view of the role of lipids in breadmaking technology and even most of the previously described mechanisms are still speculative, the recent data described here allow us to think about possible ways for improving the breadmaking quality of wheat through manipulation of lipids, especially wheat lipids. The simple fact that adding surfactants in breadmaking is always necessary to obtain good bread crumb texture means that the polar lipid content and composition of wheat are not optimal. In the future, the challenge wi1\ be to find the means of improving the surface properties of wheat polar lipids. The genetic route is less easy for lipids than for proteins, since many enzymes and therefore many genes are involved in the synthesis of polar lipid molecules. The best way of modifYing lipid structure is certainly through use of enzymes and especia1\y hydrolytic enzymes. In this regard lipases are good candidates because they are able to generate polar lipids - monoglycerides- from non polar triglycerides and detergent-like molecules from phospholipids and glycolipids (lysophospholipids, galactosylmonoglycerides) which are good improvers of bread volume and texture. However, the main drawback in the use of such enzymes is that they also generate free fatty acids. These lipid components are known to be deleterious to the quality of cereal products so that it is necessary to limit their presence in wheat doughs. Fina11y, one of the best pathways would be to improve the functionality of polar lipids through lipid binding proteins. For example, proteins such as puroindolines can act synergistically with polar lipids to improve the stability of lipoprotein films or to prevent the destabilization of protein foams by non polar lipids. Since these proteins are encoded by a single or a limited number of genes, it is possible to introduce these proteins in breeding programs. Furthermore, the transgenic approach offers fascinating opportunities for manipulation of the genes coding for such proteins in order to improve their expression, change their localisation and their functionality using directed mutagenesis. Increasing the

258


content of these proteins could also be a way to improve the effect of commonly used bread surfactants. As shown in the case of beer foam64, this could also avoid the negative effect of free fatty acids generated by the use of lipases.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

PJ. Frazier, In 'Lipids in Cereal Technology' ed. P.l Barnes, Academic Press, New York, 1983, p. 189. l Nicolas and D. Drapron, in 'Lipids in Cereal technology', ed. PJ. Barnes, Academic Press, New York, 1983, p. 213. F . MacRitchie, in 'Lipids in Cereal technology', ed. P J . Barnes, Academic Press, New York, 1983, p. 165. D. Marion, in 'Cereal Chemistry and Technology: a Long Past and a Bright Future', ed. P.Feillet, IRTAC, Paris, 1992, p.57. D. Small, in Handbook of Lipid Research, Plenum Press, New York, 1986, vol. 4. V. Luzzati, in 'Biological Membranes', ed. D. Chapman, Academic Press, New York, 1968, p. 7 1 . SJ. Singer and G.L. Nicolson, Science, 1972, 175, 720. l M . Seddon, Biochim. Biophys. Acta, 1987,1031, 1 . G. Lindblom and L. Rilfors, Biochim. Biophys. Acta, 1989, 988, 22 1 . M.W. Tate, E.F. Eikenberry , D.C. Turner, E. Shyamsunder, and S.M. Gruner, Chem. Phys. Lipids, 199 1 , 57, 147. K Larsson and S . Puang-Ngern, in 'Advances in the Biochemistry and Physiology of Plant Lipids,' eds L.-A Appleqvist and C. Liljenberg, Elsevier, Amsterdam, 1979, p. 27. B. De Kruijff, P.R. Cullis, A.l Verkleij, MJ. Hope, CJ.A. Van Echteld, and T.F. Taraschi, in 'The Enzymes of Biological Membranes', ed. A.M. Martonosi, Plenum Press, New York, 1985, voU , p. 1 3 1 . S . Akoka , C . Tellier , C . Le Roux, and D . Marion, Chem. Phys. Lipids, 1988, 46,

43.

22.

T. Carlson, K Larsson, and Y. Miezis, Cereal Chem., 1978, 55, 168 T . Carlson, K Larsson, and S . Poovarodom, Cereal Chem., 1979, 56, 4 17. K Larsson, in 'Chemistry and Physics of Baking'. eds lM.V. Blanshard, PJ. Frazier, and T. Galliard, Royal Society of Chemistry Special Publication 56, London, 1986, p. 62. A. AI-Saleh, D. Marion, and DJ. Gallant, Food Microstruct., 1 986, 5, 1 3 1 . D. Marion, C. Le Roux, S. Akoka, C.Tellier, and D. Gallant, J. Cereal Sci., 1987, � 101. . D. Marion, C. Le Roux, C. Tellier, S. Akoka, D. Gallant, l Gueguen, Y. Popineau, and lP. Compoint, in 'Interactions in Protein Systems", eds KD. Schwenke and B. Raab, Springer Verlag, Berlin, 1989, p. 147 and p. 373. W.I. Vail and lG. Stollery, Biochim. Biophys. Acta, 1 979, 551, 74. E.W. Simon, in 'Dry Biological Systems', eds lH. Crowe and L.M. Crowe, Academic Press, New York, 1978, p. 205. WJ. Gordon-Kamm and P.L. Steponkus, Proc. Natl. Acad. Sci. U.S.A., 1984, 81,

23. 24.

F. MacRitchie and P.W. Gras, Cereal Chem., 1973, 50, 292. F.MacRitchie, J.Sci. Food Agric., 1977, 18, 53

14. 15. 16. 17. 18. 1 9. 20. 21.

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57.

S.B. Sorensen, L.M. Bech, T.B. Muldlberg, and K. Breddam, MBAA Technical Quater. 1993, 30, 136. M. Coke, P.I. Wilde, E.I. Russell, and D.C. Clark, J. Colloid Interface Sci. , 1 990, 138 489. Z.I. Lalchev, P.I. Wilde, and D.C. Clark, J. Colloid Interface Sci. , 1 994, 167, 80. D.C. Clark, P.l Wilde, D. BerginK-Martens, A Kokelaar, and APrins, in 'Food Colloids and Polymers: Structure and Dynamics', Royal Society of Chemistry Special Publication. 1993, p.354. D.C. Clark, M. Coke, A R Mackie, AC. Pinder, and D.R Wilson, J. Collaid Intetjace Sci. , 1 990, 138, 207. Z. Gan, RE. Angold, M.R. Williams, P.R Ellis, lG. Vaughan, and T. Galliard, J. Cereal Sci., 1990, 12, 1 5 . P.I. Wilde, D.C. Clark, and D.Marion, J. Agric. Food Chem. , 1 993, 41, 1 570. D.C. Clark, P.I. Wilde, and D. Marion, J. Inst. Brew., 1 994, 100, 23 . F. Husband, P.I. Wilde, D. Marion, and D.C. Clark, in 'Food Macromolecules and Colloids', eds E. Dickinson and D. Lorient, Royal Society of Chemistry, Cambridge, 1 995, p. 285. C.IA Van Echteld, B. De Kruijff, A.I. Verkleij, l Leunissen-Bijvelt, and l De Gier, Biochim. Biophys. Acta, 1982,692, 1 26. Rl King Rl in 'Pulmonary Surfactant' eds. B. Robertson, L.M.G.Van Golde and lJ.Batenburg, Elsevier, Amsterdam, 1984, p. 1 . S. Hawgood and lA Clements, J. Clin. Invest. , 1 990, 86, 1 . S. Hawgood, B.I. Benson, l Schilling, D. Damm, lA Clements, and RT. White, Proc. Natl. Acad Sci. U.S.A., 1 987, 84, 66. K.WA Wirtz, lAF. Op Den Kamp, and B. Roelofsen, in 'Progress in Lipid Protein Interactions' eds A Watts A and lJ.H.H.M. De Pont, Elsevier, Amsterdam, 1986, p. 22 1 . F. MacRitchie, J.Food Technol, 1 978, 13, 1 87. F. MacRitchie, Bakers Dig., 1980, 54, 10.

58. 59. 60. 61. 62. 63 . 64. 65. 66. 67. 68. 69. 70. 71. 72.

STARCH LIPIDS, STARCH GRANULE STRUCTURE AND PROPERTIES

William R. Morrison Department of Bioscience and Biotechnology University of Strathclyde Glasgow Gl l XW

1

INTRODUCTION

Historically, lipids in cereal starches have aroused little interest1.3 because they are minor components of the starch granules, and apparently inert For example, lipids in wheat starch are exceptionally resistant to oxidation and chlorination,4 they do not articipate � in any of the biochemical and physical processes that affect dough properties and they can be extracted efficiently only by using hot polar solvent systems that partially disrupt granule organisation.6- 1 1 However, recent studies have shown that the lipids occur as inclusion complexes with amylose, located in amorphous domains within the granules, which do modify starch gelatinisation and swelling properties. These studies have also led to a more detailed model of starch granule organisation, but important questions concerning the biosynthesis of the granules have still to be addressed. Although this paper is primarily concerned with wheat starch, there are many references to starches from barley, which is very similar to wheat but often provides a better choice of samples for study.

2 CHEMICAL PROPERTIES

2.1 Amylose-Lipid Complexes in Starch Granules It has been known for some time that cereal starches contain small quantities of monoacyl lipids such as free fatty acids (FFA) 12 and lysophospholipids (LPL). 13'IS The fatty acid composition of the lipids is typically one-third saturated (palmitate > stearate) and two-thirds cis-unsaturated (linoleate > oleate > linolenate). 4-6.8. 1S Since FFA and LPL form inclusion complexes with amylose in which they are resistant to oxidation and to solvent extraction, it was naturally assumed that this was how they occurred in cereal starch granules,14. 19 although most properties of the starch granule lipids could be explained equally well if they were merely trapped in interstices within the granules. 17 Proof that amylose-lipid inclusion complexes do exist in native starch granules, and that they are not artefacts formed during starch isolation, was obtained eventually using 13C-CPIMAS-NMR, supplemented with other evidence. 20-23 Cis-unsaturated fatty acids and solvent-extracted starch-lipids are liquid at ambient temperature, and when mixed with dry amylose ( 1 :7) they do not give any solid-state cross-polarisation NMR signal. 2o.23 However, when an inclusion complex is formed the fatty acid chains are immobilised within the amylose helix, and they give a clear methylene carbon signal with a chemical shift of 3 1 ppm, while glucosyl C- l of am�lose gives a sharp peak at 1 03- 104 ppm characteristic of the V -helical conformation. 0.23 These features were found in spectra from non-waxy starches of barley,2° rice, maize, oats,22 and from the lintner residues of barley and wheat starches?1

262


It was also concluded from other evidence20 that lipid was not distributed uniformly throughout the amylose fraction, and that in all probability there are two types of amylose, namely lipid-complexed amylose (LAM) and lipid-free amylose (FAM) , For barley and wheat, LAM 7 x LPL content,20 and LPL can be taken as approximately 16.3 x starch phosphorus content 15 In the colorimetric assay for amylose24 that was used, apparent amylose (measured in the presence of starch lipids, which interfere with iodine binding) is the same as FAM, while the difference between total amylose (measured on delipidated starch) and apparent amylose is the same as LAM. It is generally assumed that the residue obtained on lintnerisation is from the acid resistant crystalline parts of amylopectin in a starch granule,25 and, by inference, that amylose is totally degraded, but in barley starches FAM and LAM are more resistant than 16) in the residue are derived from amylopectin?1 The shortest chains (CL amylopectin, while intermediate length chains (CL 46) are from FAM that has been partially hydrolysed and then retrograded into resistant double helices. The longest chains (CL 77- 1 30) are from the V-helical segments of LAM.21 The lintner residue of ball-milled wheat starch is comprised of similar residues from LAM and FAM.21 Many properties of amylose-lipid inclusion complexes (LAM) are quite different from those of water-soluble amorphous FAM.1.3 Amylose in the collapsed single helical (V-) conformation has six glucosyl residues per turn (with bulky ligands there are seven or eight), stabilised by hydrogen bonds between hydroxyl groups of adjacent glucos�l residues, i.e. 0-2 .... 0-3(2) and 0-2 . . . . 0-6(7), located on the outer surface of the helix. 6 The helix cavity is effectively a hydrophobic tube. The hydrocarbon chain of the fatty acid or lipid lies within the amylose helix, and is stabilised by van der Waals contacts with adjacent C(5)-hydrogens of amylose,27 but the polar ends of the lipid are not inside the helix cavity :3.27 Amylose complexes with most lipids are insoluble and amorphous (type I), but complexes with FFA and monoglycerides can be annealed into a semi-crystalline form (type II). Type I complexes, which are probably the form in most cereal starches20, generally dissociate at 94- 100°C when heated in water?8,29 Type II com�lexes, originally found in starches after gelatinisation/o dissociate at l 00_ 1 25°C.18,28,29,31, 2 Only the type II complexes give strong wide-angle X-ray diffraction patterns.29 =

=

=

=

2.2 Starch Isolation and Purification When studying the minor constituents of starches, such as lipids, it is essential to have very pure preparations5,11,33-38 to avoid misleading results due to artefacts and impurities_ It is well known that starch granules swell reversibly, according to their level of hydration, at temperatures well below the onset of gelatinisation. When this happens they can absorb monoacyl non-starch lipids (usually FFA, which can be confused with true starch lipids) and inorganic salts such as phosphates (phosphorus contentJ l,39 is then no longer an accurate measure of lipid phosphorus and hexose phosphate1 1,15). The granules can also retain traces of adsorbed proteins and other lipids which are normal non-starch components of the endosperm, Thus, the presence of diacylglycerolipids and triglycerides (which do not form inclusion complexes) reported in some starches is clear evidence of contamination with non-starch lipids.9 These considerations led to the recognition of two types of lipid artefacts that should be clearly distinguished from the true (internal or integral) starch lipids - namely, loosely associated non-starch lipids, and lipids absorbed into the surface layers of the granules,9 The nitrogen content of a starch sample is a useful index of gluten contamination (which also implies the presence of non-starch lipids), although well purified starches from soft wheat species still have small quantities of friabilins on their surface,40 In four purified wheat starches we found (per l 00g starch) 14- 1 9mg lipid N (from 592-794mg LPL), 3 .4-8.6mg surface N (from friabilins) and 1 3- 14mg integral N (from c, 80mg integral proteins which include granule-bound starch synthase),36


263

2.3 Amylose-Lipid Relationships Cereal starches are unusual, compared with root, tuber and pith starches, in that they contain monoacyl lipids (FFA and LPL) in amounts closely related to amylose (AM) content,IS In wheat, barley, rye and triticale the lipids are almost exclusively LPL, while in the other cereals they are comprised of characteristic proportions of FFA and LPL. IS In barley starches the fatty acid composition of the lipids becomes progressively more unsaturated as lipid content increases,41 but in wheat starches the fatty acid composition of the smaller granules (which contain more lipids) is more saturated than in the larger granules.42-44 In starches from wheat and barley harvested at various stages of grain development, both amylose and LPL contents increase with maturity, and this has given us a model of a large A-type starch granule that has gradients (from the hilum to the periphery) of increasing amounts of amylose and LPL.3S.4S-47 To extend our studies of the am lose-lipid relationship we also used starches from J F barley (normal x high-amylose) and maize (normal x waxy, normal x amylose 2 extenderi7 to obtain gene dosage effects, and starch from grain grown at different temperatures49.SO (the most important component of site/environmental variation affecting amylose and lipid contents, and starch properties). Different regressions were required to describe the amylose-lipid relationships in starches from mature diploid cereals, from waxy and non-waxy barleys, and also in starches taken at various stages of grain development,2o.s 1 In practice, this means that starches from waxy barleys and from all types of maize have variable amounts of amylose comprised of FAM and LAM in constant proportions, while non-waxy barley and wheat starches have an additional increment of FAM.sI Typical FAM and LAM contents are -4:)-()

• • • • • • • • •

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. .

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125 82.5 31.3 15.6 7.6

•

•

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3.9 1.95 0.96 0.48 0.24 0.12 0.06

MGDG concentration (lIg1ml)

•

Figure I. Binding reactivity of MG405 (culture supernatant) to wheat MGDG at various concentrations of the latter. The culture supernatant was diluted 1.' 20 with either 10% (v/v) FBS (e) or with 10% FBS containing 0. 05% Tween 20 (0). Resultsfor normal mouse serum (.) and 10% (v/v) FBS control (dotted line) are also shown.

0

3. 1.2 Effect of Detergent. The use of 0.05% (w/v) Tween 20 in antibody diluents has been reported to enhance antilipid antibody binding9, 10 . In contrast, we observed that it reduced the binding reactivity of our Mab substantially, with no antibody binding being detected at antigen concentrations of about 1 5.6 IlglmL and below (Fig. 1). This was probably a result of the lipid antigen's being washed away by the detergent, a widely reported observation in anti-lipid antibody immunoassaysll, 12. 3. 1.3 Effect ofIncubation Temperature, Time and Blocking Buffer. Incubation at 37° C enhanced antibody binding considerably compared with incubations carried out at lower temperatures (Fig. 2). The length of the incubation time of the Mab with MGDG had no effect on its binding reactivity. All buffers tested for blocking in the ELISA [ 1 % (w/v) BSA, 0.3 % (w/v) gelatin, 1 0 % (v/v) ABS, 1 0% (v/v) FBS and SuperBlockTM in PBS] produced reasonable results. Freshly made 1 0% (v/v) FBS and SuperBlockTM gave slightly better blocking (results not shown). 3. 1.4 Role of a Plasma Cofactor. When 0.3% (w/v) gelatin was used as antibody diluent and blocking agent to exclude any cofactor that may be present in bovine serum,


274

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0 20

Figure 2. Binding reactivity to MGDG of serial dilutions of MG405 culture supernatant at 3 7 'C (_), 20 'C (0) and 4 'C (.) .

o

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Reciprocal of cuHure supernatant dilution

the binding reactivity of the affinity purified MG405 was about 30% lower than that of the same Mab diluted in 10% (v/v) FBS. The addition of human plasma J32Glycoprotein I (J32GPI) to the 0.3% (w/v) gelatin diluent in the same assay produced only a small increase in antibody binding, and no inhibition was produced by J32GPI over a concentration range of 0- 1 00 IlgimL. 3.2

13C NMR Verification of MGG Structure

The carbon atom resonances in the NMR spectra (Figure 3a,b,c) were assigned by comparison with the chemical shifts given in the literature13• 1 4, together with those identified by the DEPT experiment (Figure 3c). Signals characteristic of those of fatty acid chains were absent from the MGG spectrum, which showed only those representing the anomeric carbon atom and galactosyVglycerol backbone carbon atoms (Figure 3b). It was concluded that the saponification had been carried out successfully and that the product was indeed MGG. 3.3

Antibody specificity

Pre-incubation of the Mab with lipid standards containing 1 6:0, 1 8:0, 1 8 : 1 , 1 8 : 2, and 1 8 : 3 fatty acids typical of those present in wheat MGDG, MGG, galactose and glycerol produced only a small degree (5- 1 8%) of inhibition. However, addition of MGDG inhibited antibody binding almost totally (99.8%) at high concentrations. In an experiment using purified phospholipids from a variety of animal, plant and microbial sources as antigen, no binding reactivity with these lipids was detected irrespective of their origin. 4 DISCUSSION Anti-glycolipid antibodies, like other antibodies directed against carbohydrate determinants, have generally been found to have low titre and affinity compared with anti-peptide antibodies. This low affinity, together with the amphipathic nature of the lipid


275

a)

I lSI

b)

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I 10

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10

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Figure 3. High resolution HC NMR spectra, showing a) The five main spectral regions for MGDG with signals at approximately (ppm): / 73 (C1, carboxyl carbon), 127-130 (acyl chain olefinic carbons), 104 (anomeric carbons), 62-69 (galactosyllglycerol backbone carbons), 14-34 (acyl chain aliphatic carbons); b) Signals for MGG at approximately (ppm): 106.4 (C-1, anomeric), 78. 6 (C-5), 76. 1 (C-3), 74. 3 (C-2), 74. 1 (C-6), 73.9 (C-4), 72. 1 (C-2'), 65.8 (C-J'), 64. 4 (C-3'); and c) DEPT spectrum (() 3;&14) showing signals from the three CH2 groups at 74. 1, 65.8 and 64. 4 ppm, respectively. =

276


antigen, makes it difficult to measure accurately small amounts of antibody in solid phase immunoassays, which require extensive washing to minimise unspecific binding of proteins. It is not surprising, therefore, that many different procedures have been reported in the literature for ELISA protocols for aGL detection6. Primary antibody incubations, for example, were carried out at room temperatureS, at 37°C with the use of PBS/Tween7, 10, or at 4°C for various time periods8, despite the fact that a recent workshop recommended overnight incubation at 4°C and discouraged the use of PBS/Tween for washing6. Our data also indicated that the inclusion of Tween 20 in the antibody diluent reduced the binding reactivity ofMG405 with MGDG. The greater reactivity observed for antigen-antibody binding at 37°C than at lower temperatures, however, is contrary to the workshop's recommendations. Early observations with anti-phospholipid antibodies (aPL) have led to proposals that temperature-dependent binding reactivity may be due to the physical state of the lipid antigen 1 S, 16. It may also be related to the properties of different antibody isotypes; IgM antibody, for example, was reported to fix complement most efficiently at 37°C, and IgG most efficiently at 4°CI7,18. Lockshin et aI., however, found that the binding of IgG type aPL was temperature dependent but not that of the IgM type l9. This is contrary to the results obtained here with MG405, which is of the IgM isotype, but its MGDG binding reactivity was found to be temperature dependent. It had been recognised for a number of years that the use of bovine serum-based diluents and blocking solutions greatly improves the discrimination between positive and negative samples for anticardiolipin antibodies2o,2 1 . Recent reports have indicated that some aPL bind to anionic phospholipids only in the presence of 132GPI22.24, a highly glycosylated, single chain polypeptide of 326 amino acids and of M, 50k2s. Although the inclusion of 132GPI at 5J.lglmL enhanced the binding of MG405 with MGDG, the degree of enhancement was small, suggesting that a factor other than 132 GPI may have been responsible for the somewhat reduced binding reactivity when FBS was replaced by gelatin in the modified ELISA. The Mab did not recognise 132GPI immobilised on ELISA plates, and neither did pre-incubation of the Mab with 132 GPI inhibit the antibody's binding to immobilised MGDG. MG405 therefore appears not to be an intrinsically low affinity antibody to 132GPI unlike aPL. Galactose, the non-reducing terminal residue of the Type II chain (Galpl�GlcNAcp I �R) and the Type II chain H structure (Fuca l �2Galpl �4GIcNAcPI �R) were identified as the antigenic determinants for some aGL 1 1 ,26 Other closely related structures, such as those based on the disaccharide Galpl �3GIcNAc, which may be carried on both 0- and N-linked oligosaccharide chains in glycoproteins and on both short chain and complex glycosphingolipids, were also established to be determinants for blood group specific antigens27. The complete inhibition produced by MGDG suggested that MG405 was directed against the whole MGDG molecule and not its carbohydrate (galactose residue or the galactosyVglycerol backbone) or lipid moieties. An alternative explanation for the inhibition result is that MGDG may be present in micellar form, which may present the actual epitope multivalently and which therefore binds better than the water soluble univalent MGG. This research has resulted in the development of a monoclonal antibody against MGDG, which has unique specificity characteristics amongst anti-polar lipid antibodies. Preliminary immunolocation studies have shown that the Mab binds to cytoplasmic


277

membranes in the developing wheat caryopsis. It is potentially useful in immunohisto chemistry and other studies.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 1 1. 12. 13. 14. 15. 16. 1 7. 18. 1 9. 20. 21. 22. 23.

A. H. Bloksma, Cereal Foods World, 1990, 35, 228. Z. Gan, R. E. Angold, M. R. Williams, P. R. Ellis, 1. G. Vaughanf and T. Galliard, J. Cereal Sci. , 1990, 12, 390. Z . Gan, P. R . Ellis, and 1 . D . Schofield, J. Cereal Sci., 1 995, 21, 2 1 5 . Z . Gan, P. R . Ellis, and 1. D . Schofield, J. Cereal Sci., 1 993, 18, 207. H. Ozawa, M. Kotani, I. Kawashima, and T. Tai, Biochim. Biophys. Acta, 1 992, 1 1 23, 1 84. D. M. Marcus, N. Latov, B. P. Hsi, and B. K. Gillard, J. Neuroimmunol., 1 989, 25, 255. T. Ariga, T. Yoshida, T. Mimori, and R. K. Yu, Clin. Exp. Immunol., 1 99 1 , 86, 483 . A. A. Llyas, F. A. Mithen, Z. W. Chen, and S. D.Cook, J. Neurol. Sci., 1 99 1 , 102, 67. H. M. Cheng, and S. F. Yap, J. Immunol. Methods, 1 988, 109, 253. M. H. Ravindranath, R. M. H. Ravindranath, D. Morton, M. C. Gaves, J. Immunol. Methods, 1 994, 169, 257. W. W. Young, 1. Portoukalian, and S. Hakamori, J. Bioi. Chem., 1 98 1 , 256, 1 0,967. E. N. Harris, A. E. Gharavi, B. M. Patel and G. R. V. Hughs, C/in. Exp. Immunol., 1 987, 68, 2 1 5 . S. R . Johns, D . R . Leslie, R . I . Willing, and D. G. Bishop, Aust. J. Chem., 1 977, 30, 823 . F. Adebodun, 1. Chung, B. Montez, E. Oldfield, and X. Shan,. Biochemistry, 1992, 31, 4502. 1. Rauch and A . S. Janoff, Proc. Natl. Acad Sci. USA ., 1 990, 87, 4 1 12. 1. Rauch, M. Tannenbaum, H. Tannenbaum, H. Ramelson, P. R. Cullis, C. P. S. Tilcock, M. 1. Hope, and A. S. Janoff, J. Bioi. Chem., 1 986, 261, 9672. B. D. Stollar and A. L. Sandberg, J. Immunol., 1966, 96, 755. A . L . Sandberg and B. D . Stollar, J. Immunol., 1 966, 96, 764. M. D. Lockshin, T. Qamar, R. A. Levy and M. P. Best, J. C/in. Immunol., 1 988, 8, 1 88- 1 92. S. Loizou, 1. D. McCrea, A. C. Rudge, R. Reynolds, C. C. Boyle and E. N. Harris, Clin. Exp. Immunol., 1985, 62, 738. A. E . Gharavi, E. N. Harris, R. A. Asherson and G. R . V . Hughs, Ann. Rheum Dis., 1 987, 46, I . H. P. McNeil, R. 1. Simpson, C. N. Chesterman and S. A. Krilis, Proc. Natl. Acad Sci. USA, 1 990, 87, 4 1 20. M. Galli, P. Comfurius, C. Maassen, H. C. Hemker, M. H. de Baets, P. 1. C. van Breda-Vriesman, T. Barbul, R. F. A. Zwaal and E. M. Bevers, Lancet, 1 990, 335, 1 544- 1 547.


278

24.

E. Matsuura, Y. Igarashi, M. Fujimoto, K. Ichikawa and T. Koike, T. Lancet,

1 990,

336, 1 77. 25.

1. Lozier, N. Takahashi and F. M. Putman, Proc. Natl. Acad Sci. USA, 1 984, 81,

26.

1. A. Benjamins, R. E. Callahan, I. N. Montgomery, D. M. Studzinski and C. A. Dyer, J. Neuroimmunol., 1 987, 14, 325. W. M. Watkins, Pure andAppl. Chem., 1 99 1 , 63, 56 1 .

3 640.

27.

ASPECTS ON THE FUNCTIONALITY OF DATEM IN BREADMAKING

N. O. Carr and P. 1. Frazier Dalgety pic Food Technology Centre Station Road Cambridge CB 1 2JN

1 INTRODUCTION The emulsifier DATEM (diacteyl tartaric esters of mono- and di- glycerides) has a critical role to play in modem bread production, particularly in high-volume, granary and wholemeal formulations, whereby a small addition (typically 0.35% flour weight basis) provides tolerance against dough collapse and atlows satisfactory loaf volume to be achieved. Thus, while DATEM generally can be shown to increase the volume of standard white bread, it is more usual to measure the quality of DATEM using the so-called "bang test" where its affects become exaggerated. Here dough from a high-volume recipe (that is dough prepared from an extended-proof and with a high yeast addition) is subject to a controlled degree of mechanical shock and this leads to dough collapse in the absence of good-quality DATEM. The inclusion of DATEM eliminates the requirement for hard fat in the Chorleywood Bread Process and it is likely that both hard fat and DATEM function through similar mechanisms. The present work has attempted to elucidate this mechanism using baking and analytical procedures on a number of commercial DATEMs and other fats and emulsifier systems. 2 METHODS For the "bang-test", dough (500g) was prepared using commercially-milled English flour with 5% (flour weight basis, fwb) bakers yeast, 2% (fwb) salt, 1 .6% (fwb) soy flour, 0.7% (fwb) bakery fat, 0.02% (fwb) ascorbic acid, and water equal to the Farinograph absorption at 500 BU. To avoid dispersion problems that can be encountered with certain fats, emulsifiers and fats were generalIy coated onto a small proportion of the flour (typicalIy 8% of the total flour) using chloroform, which was then evaporated. Dough was developed according to the Chorleywood Bread Process, divided into 8x60g pieces, moulded and prooved for 75min at 40°C. Four of the doughs were subject to "banging" by rolling a 76.5g balI down a 20cm ramp set at an angle of 55° in order to shock the loaf-tin. AlI dough pieces were then baked at 200°C for 20min. Loaf volume was determined using a standard rapeseed-displacement method. When defatted flour was evaluated, the "bang test" was not used was and standard white bread was made. Conditions were as above except: 2.5% yeast was used, bakery fat was omitted, dough' was divided into 4x1 20g pieces, proof time was 60min, and, obviously, the "banging" was avoided. Emulsifiers and

280


fats were obtained from Grindsted, Croda, Quest, AB Foods or Sigma. Where defatted flour was used, this was prepared by extracting flour four-times with light petroleum using approximately 1 1 of solvent for each 1 kg of flour. Analysis of emulsifiers on the basis of polarity used an HPLC either according to the protocol described by Carr et al I (termed Gradient 1 ) or according to the following gradient (termed Gradient 2): t=Omin, A= l OO%; t=7.5min, A=S5%, B=1 5%; t=9min, A=20%, B=52%, C=2S%; t=l l . 5min, A=30%, B=70%; t= 1 2 . 5min, A=1 00%, t= 1 5min, A= 1 00% . The composition of solvent A, B and C are as described by Carr et al. 1 The method utilises light-scattering detection and, while able to provide qualitative profiles, is unreliable for routine quantification. Pure DATEM ( l -palmitoyl-3-diacetyl tartaric ester of monoglyceride: I -DATEM), used in this method as a standard, was prepared by the esterification of I -palmitoyl glycerol with diacteyl tartaric anhydride? Preparative-scale quantitative fractionation of DATEM, used silica columns from Bond Elut. Material fractionated for evaluation in baking used a 1 09 column (-2.5g sample), conditioned with 40ml chloroform:acetic acid (9S:2). Using the same solvent, sample was applied in 40ml and, after applying a further 20ml, fraction FA was recovered. Fraction Fa was obtained using 1 40ml of this solvent while application of SOml chloroform:propan-2-01 (94:6) gave fraction Fe. Fraction FD was obtained by applying SOrnl methanol. A similar method was used to characterise a range of commercial DATEM samples, by quantifYing selected fractions. In this case, 0.5g columns were used conditioned with 2ml chloroform:propan-2-01 (9S :2). Sample (- 1 00mg) was applied in 3rnl of the same solvent and a neutral fraction was recovered using a further 3ml of the same solvent. A mid-polar fraction was obtained using 4ml chloroform:propan-2-01 (94:6), while a polar fraction was obtained using 4ml methanol. After solvent evaporation, emulsifier fractions could be determined gravimetrically. For assay of tartaric acid, glycerol and acetic acid, samples of DATEM (-0. 1 2g) were saponified in 0.42M sodium hydroxide ( 1 5ml) at 1 00°C for 60min. Sample pH was then adjusted to between 4-5 using 5M hydrochloric acid, and the total volume was made up to 25ml. After filtering, acids and glycerol were quantified using an organic-acid-analysis column (300x7.Smm, Aminex HPX-S7H) using 0.005M sulphuric acid at a flow rate of 6mllmin. Components were detected by refractive index.

3 STUDY OF COMMERCIAL DATEMS Fourteen samples of commercial DATEM were obtained from a number of suppliers including samples that were both liquid and solid (differential thermal analysis was used to confirm that the liquid samples were free of solids at proof temperature). Evaluation of these samples in baking at a low inclusion level of 0. 1 % (fwb) using the "bang-test" indicated a range offunctionalities (results not shown). However, the quality of the samples could not be related to the physical form of the material (i.e. whether liquid or solid) nor to the composition given by three analytical approaches. These approaches included: (i) qualitative profiling by HPLC on the basis of polarity, (ii), quantitative fractionation of the samples into neutral, mid-polar, and polar components and, (iii), assay of tartaric acid, acetic acid, glycerol, and fatty acid after hydrolysing the material. Some of the "fingerprint" chromatograms are shown in Figure 1 and this serves to illustrate that commercial DATEMs are complex and varied in composition. Moreover a range in the level of pure 1 DATEM within these samples was also indicated but was found not t o correlate with quality.

28 1


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Whole commercial OATEM

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Figure 1 Analysis ofa number of commercial DA TEMs andpure I-DA TEM (HPLC Gradient 2)

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2

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8

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Figure 2 Analysis offractions obtainedfrom a commercial DA TEM that subsequently were evaluated in baking (HPLC Gradient I)

Preparative fractionation was used to prepare four cuts from a selected commercial DATEM which were then evaluated in baking (whole commercial DATEM included at a level of 0.34%, fwb; fractions included according to the proportion present in whole commercial DATEM). The fractions obtained are shown in Figure 2 and the baking results are shown in Table 1 . Reconstituted DATEM was found to perform in a comparable way to the untreated sample, but no individual fraction was found to confer full functionality. Thus, commercial DATEM would seem to function as a consequence of the overall properties of the blend rather than because of presence of specific components (including pure I -DATEM). This conclusion is consistent with the findings obtained from assessing a range of commercial DATEMs.

Table 1 Influence of commercial DA TEMfractions on loaf volume and on tolerance of dough to col/apse on banging Sample

Loaf volume (ml)

DATEM-free Whole DATEM (0.34%) Fractions FA Fa Fc Fo (0.34%)

868 944 937

Loaf volume of "banged" dough (ml) 746 915 934

Fractions FA Fa (0.34% x 0.47) Fractions Fc Fo (0.34% x 0.53)

863 892

763 817

Fraction FA (0.34% x 0.32) Fraction Fa (0.34% x 0. 1 6) Fraction Fc (0.34% x 0.34) Fraction Fo (0.34% x 0. 1 8)

867 863 905 915

764 774 822 793

Note to Table: Inclusion levels shown in parentheses are expressed on fwb. Standard deviation

1%

282


Previous publications 3,4 have found evidence that bakery fat provides benefit only in the presence of "free" wheat lipid (i.e. lipid that can be extracted from flour by solvents such as light petroleum). In order to ascertain whether the requirement for DATEM was also dependent on free wheat lipid, bread was baked from untreated, defatted and reconstituted wheat flour in the presence and absence of commercial DATEM. Results are shown in Figure 3, where it can be seen that DATEM provides benefit only in the presence of free wheat lipid. Furthermore, results are most suggestive that free wheat lipid is a detrimental component of flour, and that commercial DATEM works by overcoming these detrimental properties. Intriguingly, commercial DATEM would seem to be itself a detrimental ingredient in the absence of the free lipid of wheat flour.

DATEM-free

DATEM 0.34% fwb Untreated flour

Reconstituted flour

Defatted flour

Figure 3 Assessment of commercial DA TFM (0. 34%fwb) in bqking using untreated, defatted and reconstituted wheatflour

4 STUDY OF OTHER FATS AND EMULSIFIERS A number of fats and emulsifiers were evaluated in baking at an inclusion level of 0.8% (fwb). The purpose of the work was to contrast function against structure, rather than to catalogue fats and emulsifiers comprehensively, and so this work attempted only to provide a crude screening. Thus, in the first instance only one dough was prepared (permitting eight loaves to be baked, of which four w.ere from "banged" doughs) and, with the exception of citric acid esters, only materials exhibiting some degree of functionality were re-tested. Arbitrarily the materials have been divided into five groups of functionality (strong, medium, low, none and deleterious), but because the work is based on limited experimentation it should be remembered that certain substances could have been grouped incorrectly. The high inclusion level was chosen so that materials exhibiting some degree of functionality could be identified more easily. Results of this work are given in Table 2. Interestingly, on the basis of "text-book" structure 5.6 emulsifiers of "strong" function are of a contrasting nature (i.e. DATEM, polysorbate 65 and certain sucrose esters have substantial differences in structure), while other emulsifiers share a similar theoretical


283

structure yet have dissimilar functions (in particular DATEM and citric acid esters). Furthermore, use of HPLC to fingerprint these samples, found some samples to be of similar profile but not of similar function (e.g. sorbitan mono-oleate and DATEM, results not shown). However, a characteristic that appeared of some use in discriminating between emulsifier-functionality was the HLB value (hydrophilic-lipophilic balance). Thus emulsifiers of strong function had cited HLB values between 9_1 3 ,5-7 those of medium function were either between 5-7 or were essentially hard fat (i.e. HLB 1 or less), while those of lower function had HLB values that were mostly outside these ranges. Notable exceptions to this pattern included polyglycerol esters (HLB 1 0) and citric acid esters (HLB 1 1) which might have been expected to provide strong function but, in practice, provided none.

Table 2 Relativefunction ofa range offats and emulsifiers in baking at an inclusion level of 0. 8% (fwb)

HLB value

EmulsifierlFat

Emulsifierljat

HLB value

No Function:

Strong Function: 13

Sucrose ester F 140

Acetic acid esters

3

9

Lactic acid esters

3

Sucrose ester F I lO

II

Citric acid esters

II

Polysorbate 65

II

Polyglycerol esters

10

Propan diol esters

2

Sorbitan monostearate

5 4

DATEM

Medium Function: 0

Sorbitan mono-oleate

Monopalmitin and mono-olein

7

Polysorbate 60

15

Calcium stearoyl lactylate

5

Polysorbate 80

15

Sodium stearoyl lactylate

7 7

Sucrose ester F20

Hard fat ?

Soy lecithin

2

(No addition)

Sucrose ester FlO Deleterious Function:

Low Function:

Hydrogenated soy lecithin ?

Hard fat ?

0

Egg lecithin

7

Glyceryl mono/di-stearate Sorbitan tristearate

7

3

3

Note to Table: Materials of "strong jUnction " gave a non-banged loaf volume (NBLV) in the

range 99-101% and a banged loafvolume (ELV) in the range 96-100%, relative to a good quality

commercial DATFM.

For the other groups the NBLV and BLV fell in the respective ranges:

"mediumjUnction " 97:t3% & 90:t3%, "lowjUnction " 95:t1 % & 86:t2%, "no jUnction " 90:t2% &

83:t3%, "deleterious jUnction " 81:t1 % & 80:t1 %. Materials marked with question-marks indicate s7 queried results. Where possible HLB values were obtained from published work. - No values could be found for pure monoglycerides and these were estimated by molecular formula. No reliable HLB value could be found for polyglycerol esters, egg lecithin or hydrogenated soy lecithin, and in these instances a value was estimated according to the dispersion-properties in water. 5-7

The HLB scale is based on an empirical measure of the ability of an emulsifier to stabilise oil in water, and arbitrarily has been set between 0-20.' Lipophilic emulsifiers are of low HLB and tend to promote water-in-oil emulsions. Hydrophilic emulsifiers are of high HLB and these tend to have good water-dispersion properties, promoting fat-solubilisation and foam-


284

stabilisation. An HLB of l O is, by definition, the mid-point between a hydrophilic and a lipophilic emulsifier and this is optimum for the stabilisation of oil in water. Since an HLB value of around l O is associated with emulsifiers of strong function in baking it is suggestive that such systems may work by producing oil-in-water emulsions. This being the case, one would have to assume that other factors have bearing on functionality in order to account for the anomalous results involving non-functional emulsifiers that are of an HLB around 1 0 (i.e. polyglycerol esters and citric acid esters). An aspect of the HLB scale is that when emulsifiers are blended they provide a system that gives an HLB value according to the overall average. Accordingly, blends were prepared containing various proportions of low-HLB-emulsifier (GMS of HLB 3 and sucrose ester F20 of HLB 2) and high-HLB-emulsifier (polysorbate 60 and polysorbate 80, both of HLB 1 5) to cover the HLB scale between the extremes of the emulsifiers used. In these instances, the low- and high- HLB-emulsifiers, that were non-functional when used alone at an inclusion level of 0.8% (fwb), became comparable to DATEM when used in blends giving an HLB value of around 10. Data from one such experiment, using various blends consisting of GMS and polysorbate 60, is shown in Figure 4.

950

I

�

'0 >

i...

900 850

___ Non-banged dough

800 750

-+- Banged dough

2

4

6

8

10

HLB value

12

14

16

Figure 4 Loaf volume of banged and non-banged doughs prepared using mixtures of

polysorbate 60 and GMS (glyceryl monoldi-stearate) providing a range of HLB values; emulsifier included at 0.8%fwb

These results, however, are not unequivocal because subsequent experimentation using incremental additions of polysorbate 60 or polysorbate 80 (i.e. the high-HLB-emulsifiers), showed that polysorbate gave similar functionality to that achieved with the blends containing polysorbate at the corresponding addition level (results not shown). Thus, the presence of low-HLB-emulsifier would appear to have had little influence on the functionality of the polysorbate in baking and, accordingly, the relationship shown in Figure 4 may have no direct bearing to the HLB scale. However, it is suggested that polysorbates used in isolation of low-HLB-emulsifier still show an optimum addition level because the native wheat lipids in this instance are participating as low-HLB-emulsifier. This explanation is preferred on the grounds that: (i) intuitively wheat lipids would be expected to perform as a low-HLB-emulsifier, (ii), DATEM does not show an optimum addition level like the polysorbates, and rather maintains functionality once it is beyond a threshold addition, which is consistent with the performance expected of mixing a high- and low HLB-emulsifier and, (iii), the pattern illustrated in Table 2 is consistent with the HLB scale having a bearing on emulsifier functionality.


285

5 CONCLUSIONS

From baking and analytical evaluation of a number of DATEMs and other emulsifiers, evidence has been obtained to indicate that DATEM functions as a consequence of the chemical properties of the overall blend rather than because the presence of specific components. The overall properties can be described reasonably well according to the HLB scale, although a number of exceptions have been found. While evidence is not unequivocal, it is thought that emulsifiers of high HLB can be blended either with low HLB-emulsifier, or with the endogenous lipids of flour, to provide a system with the same HLB as DATEM and, under such circumstances, similar functionality to DATEM is obtained. An HLB of around 1 0 would seem to be optimal for functionality and this, by definition, is also optimal for the stabilisation of oil in water. Work with defatted flour has indicated that only in the presence of flour lipid is there a requirement for DATEM and that DATEM may function by overcoming the deleterious properties of this lipid. Accordingly, the above findings suggest that DATEM, or other emulsifiers of a similar HLB, sequester native wheat lipid within emulsions thereby reducing the availability of wheat lipid to destabilise the gas/liquid interfaces of dough. In this way a more robust dough-structure is derived. Likewise, usage of bakery fat (providing similar although less pronounced benefit to DATEM) may also work by interacting with endogenous lipid and, thereby, retarding the migration of these lipids to the gas/liquid interfaces of dough.

Acknowledgements The authors are pleased to recognise the contributions of Prof N.W.R. Daniels, Forge, Mr D. Heavens and Dr T. Podgorski to this work.

Mr

C.D.

References 1 . N.O. Carr, N.O., N.W.R. Daniels, and PJ. Frazier, "Wheat End-Use Properties, Proceedings ofICC meeting", Helsinki, 1 989, p. 1 5 1 . 2. T A Podgorski, Dalgety Internal Report (GRU09/90) . 3 . OK Chung, Y. Pomeranz, K.F. Finney, M.D. Shogren and D. Carville, Cereal Chem., 1 980, 57, 1 06. 4. OK Chung, Y. Pomeranz, M.D. Shogren, K.F. Finney, and B .G. Howard, Cereal Chem. , 1 980, 57, I l l . 5 . Anon. Food Techno/., 1 988, 42, 174. 6. F.VK Young, C. Poot, E. Biernoth, N. Krog, LA O'Neill and N.GJ.Davidson, "The Lipid Handbook", eds. F.D. Gunstone, lL. Harwood and F.B. Padley, Chapman and Hall, London. 1 986, p. 1 8 1 . 7 . G . Schuster and W . F . Adams, Adv. Cereal Sci. & Technol. , 1 984, 6, 139.

CHANGES OF WHEAT FLOUR COMPONENTS INDUCED BY BREAD IMPROVER

M. Soral-Smietana, M. Rozad and A. Cielem«cka Centre for Agrotechnology and Veterinary Sciences, Polish Academy of Sciences, Tuwima 10, 10-81 7 Olsztyn, Poland,

1 INTRODUCTION Unified flour quality is the condition for obtaining good bakery products in automatized bakeries. Due to diversified agricultural conditions wheat grain has no unified quality parameters. Thus, flour has no fully reproducible technological properties, which unfavourably affects the quality of bakery products. Therefore, there are applied multicomponent bread improvers which act throughout the whole baking process, affect water absorption capacity of flour, improve dough structure, its texture and volume and elongate its freshness. Improvers are made of various substances, among others emulsifiers which combine the basic components of dough, sugars which ensure appropriate dough fermentation and crest colour, and ascorbic acid which permits dough to absorb the maximum amount of oxygen during kneading. There are two groups of emulsifiers: ionic (anion-active or kation-active) and non-ionic (mainly acylglycerols). Ionic compounds catalyse the hydrolysis of peptide bonds and strenghten gluten proteins. Anionic substances bind with their non-polar part to protein thus lowering its electric charge. This results in reduced electrostatic repulsion of particles and makes their association easier thus strenghtening dough structure. Non-ionic compounds (mono- and diacylglycerols) are used in the baking industry for catalysing of starch complexing. As a result, helix structure is formed from the linear fraction of amylose. Amylose helix is stabilized by the inbuilt hydrocarbon chain of fatty acid from emulsifier [1]. A study was made on the effect of Polish bread improver called AKO added to wheat bakery products during dough making and the range of its interactions with macrocomponents of wheat flour.

2 EXPERIMENTAL

The aim of the study was to investigate the effect of 2% addition (in relation to wheat flour) of a complex bread improver AKO. For experimental baking (250 g dough) there was used improver containing glucose, maltodextrin, emulsifier,


287

ascorbic acid and wheat flour. The effect of AKO's action on the rheological properties and structure of dough were characterized. Transformation range of wheat flour components was determined by means of: -studies on protein fractions changes during dough making; -estimation of fatty acids of free lipids of flour and dough; -characterizing of wheat starch isolated from dough following fermenation and from bakery products after 1, 24, 48 and 72 h storage.

2.1. Material Commercial wheat flour (chemical composition in Table 1, technological characteristics in Table 2) was investigated.

Table 1 Chemical composition of commercial wheat flour Chemical composition of wheat flour

Moisture Ash Proteins [%NxS.7] Free lipids Fractions of free lipids: neutral glycolipids phospholipids

13.47 % 0.53 % d.m. 10.13 % d.m. 1 . 14 % d.m. 19.5 % 63.1 % 18.4 %

Table 2 Technological properties of wheat flour Gluten content Deliquescence Number of sedimentation Falling number Water absorption capacity (500 B.U.)

24.3 % 12.2 mm 22 408 57.7 %

2.2. Analytical methods

2.2. 1. Proteins. Sedimentation test was performed acc. to Zeleny. Protein fractions were separated acc. to Osborne with the following solvents: redistilled water, O.5M NaO, 70% �HsOH, O.5M CH3COOH at 5°C. Nitrogen content was

288


determined ace, to Kjeldahl. 2.2.2. Lipids. Quantitative analyses of free lipids followed cold extraction with petroleum ether. Fatty acids composition was determined by the GLC method following methylation. 2.2.3. Starch. Native starch was isolated with 0.5% NaCI [2], and with distilled water during the technological process [3]. Amylose-lipids complexing index was determined as the ability to bind iodine by amylose made avalilable during gelatinisation in 1 M NaOH at 50"C for 1 h [4]. Absorbance was measured at A 600 nm. Complexing index was determined from the equation: CI

=

[(A., - A.)/A.,] x 100

(1)

where A., - absorbance of amylose-iodine complex (control) A. - absorbance of amylose-iodine complex (sample) Gelatinisation degree was calculated based on digestion with bacterial a-amylase and measurement of colour complex with iodine at A 625 nm [5] from the following equation: GD%

=

[(a-b)/a] x 100

(2)

where a - absorbance of total starch fraction b - absorbance of nonavailable starch fraction. 2.2. 4. Scanning electron microscopy. Samples of wheat dough (2x3 mm) were

fixed in 2.5% glutaraldehyde solution in 0.1 M phosphate buffer (pH 7.4) at 4°C for 24 h. Following washing out with distilled water samples were dehydrated in acetone series, and dried by CO2 at critical point, sprinkled with carbon and gold, and with analysed by SEM.

3 RESULTS AND DISCUSSION 3.1 Protein-Lipid Interactions vs. Structure Forming Analysis of chemical composition (Table 1) of commercial wheat flour revealed that among native lipids the ratio of neutral to polar lipids was 1 :4. In the polar fraction, in tum, glycolipids made over 75% which indicates some additional action toward aggregation of technologically weak gluten proteins (Table 2) with glycolipids during dough kneading [6]. Joining of glycolipids with glutenins is considered to be mediated by hydrogen and electrostatic bonds [7]. Fatty acids


289

composition of free lipids of wheat flour is dominated by C18:2 (Table 3). Complex improver AKO brings in mainly C18 and C16 which interact with wheat proteins during dough making. This is illustarted by SEM micrograms of fibrous structures of gluten network (Figure 1). At the same time joining of native lipids of wheat flour with the improver added resulted in a clear transformation of unsaturated into saturated fatty acids and levelling of their ratio to 1 : 1 (Table 3). Especially evident quantitative changes were found for linoleic and stearic acids. Addition of AKO improver to wheat flour of weak baking qualities resulted in quantitative levelling of prolamins and glutelins ratio (Table 4). From the technological point of view, the phenomenon is advantageous as rheological properties of gluten depend on the prolamins to glutelins proportion and on hydrophobicity of prolamins [8]. On the other hand, increase in gluten strenght is accompanied by an increase of the protein fraction with the highest molecular weight. Also dough kneading in oxygen access favours oxygen polymerization of protein fractions ofvery high molecular weight. Moreover, the possibility of forming ionic bonds by dipolar ions affects rheological properties of wheat proteins. Thus, the use of ionic emulsifiers for wheat flour strongly affects the structure of proteins and the dough made [8].

Table 3 Changes in main fatty acids of the material studied. Material

Fatty acid composition [%J CI6

CI8

CI8:I

CI8:2

CI8:]

Wheat flour AKO improver Control dough

18.8 29.2 21.5

0.8

13.3 1.2 22.8

62.3 2.4 47 8

3.8 0.1 3.8

Dough with AKO improver

26.2

14.0

39.0

2.4

61.3 1.8 15.9

.

Table 4 Protein characteristics and changes in protein fractions Protein content [%N d.m.

Flour Control dough Dough with AKO

x

5. 7J

Total

Albumins

Globulins

Prolamins

Glutelins

Residue

10.1

2.2

1 .2

2.6

1.8

2.7

9.3

1.2

0.6

1.0

2.6

3.9

9.1

1.4

0.8

2.2

2.3

2.4

290

Figure 1


SEM micrograms ofstructure ofcontrol dough (a) and dough with AKO improver (b)

3.2 Amylose-Lipid Interactions vs. Limiting of Starch Retrogradation

Changes in physical and functional properties of starch depend on the interactions of the molecule caused by such environment factors as water content, pH, temperature and others (Figure 2). Transformation dynamics of hyper-molecular structure and interactions between starch and other food constituents depends on the mobility of amorphic phase of a given system. And so, water, acting as a plastifier, lowers the temperature of changes and affects the kinetics of phase transformations and reactivity of starch [9]. These conditions are ensured by, among others, dough forming and baking during which amylose released from starch molecules can crystallize already in the first hours following termination of the process. This accounts for forming of helical inclusion amylose-lipids complexes at increase in starch gelatinisation degree (Table 5). Inbuilding of 12 to 18 carbon atoms into monoglyceride chains is the most effective in watered environment at 60"C [ 10]. Mono- and polyunsaturated acids play a significant role in complexing of starch with lipids [4]. Their potential for joining amylose helix depends on geometrical isomerism of cis- and trans- chains of fatty acids [10, 1 1]. It appears interesting that the complexes formed during baking are the most active for up to 24 h and next they gradually dissociate (Table 5). Hence, amylose concentration decreases and its molecule elasticity diminishes as a result of adsorptive and helical inclusion interactions between amylose and lipids which gives a chance of slowing down the retrogradation rate of starch and its fractions.


Table

S

291

Effect of baking improver on starch-lipid interactions and changes of gelatinisation degree

Starch isolated from:

Complexing index· control

Wheat flour

with AKO

Degree of gelatinisation control

/%}

with AKO

19.9

0

Wheat dough: 0.8 0

before fermentation after fermentation Wheat bread:

1.1 1.2

2.8 2.2

19.9 6.9

1 h after baking

1.4

55.3

2.2 8.6 9.2

2.8 19.4 9.2 6.4

69. 1

24 h after baking 48 h after baking 72 h after baking

57.0 66.6 68.9

69.4 55.5 55.8

*Complexing index calculated against starch of wheat flour

Figure 2

SEM micrograms of wheat starch isolated from control dough (a) and dough with AKO improver (b)

292


3.3 Conclusions Native lipids of wheat flour with the improver added were transformed from unsaturated into saturated fatty acid and approached 1 : 1 ratio. Evident quantitatve changes were found for linoleic and stearic acids. Addition of the improver to wheat flour of weak baking qualities resulted in quantitative levelling of prolamins and glutelins ratio of fibrous structure of gluten which formed network. The helical inclusion amylose-lipid complexes are formed after baking at increase in starch gelatinisation degree, These complexes are the most active for up to 24h after baking and next they gradually dissociate. References

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

N, Krog, Cereal Chern ., 1981, 58, 158. J. Whattam and H, Cornell, Cereal Chern., 1991, 68, 152. R. D. Dragsdorf, Cereal Chern., 1980, 57, 3 10. M. Soral-Smietana, Acta Acad. Agricult. Techn. Olst., Technologia Alirnentorurn, 1992, 24B, 3. H. Tsuge, E . Tsaumi, N . Ohtani and A . Nakazima, Starch/Starke, 1992, 44, 29. F. Bekes, U. Zawistowska, R. R. Zillman and W. Bushuk, Cereal Chern ., 1986, 63, 327. R. C. Hoseney, K. F. Finney and Y. Pomeranz, Cereal Chern ., 1970, 47, 135. H. D. Belitz, R. Kieffer, W. Seilmeier and H. Wieser, Cereal Chern ., 1986, 63, 336. D. E. Rogers, K. J, Zeleznak, C. S. Lai and R. C. Hoseney, Cereal Chern., 1988, 65, 398. T. Riisom, N. Krog and J. Eriksen, J. Cereal Sci., 1984, 2, 105. A . C. Eliasson and N. Krog, 1 Cereal Sci., 1985, 3, 239.


EXPERIMENTAL AND CONCEPTUAL PROBLEMS IN THE RHEOLOGICAL CHARACTERIZATION OF WHEAT FLOUR DOUGHS

E. B. Bagley" F. R. Dintzis" and Sumana Chakrabartib "National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 N. University St. , Peoria, IL 61604, USA. bKrafi General Foods, 801 Waukegan Road, Glenview, IL 60025, USA

1 INTRODUCTION

Wheat flour doughs, as viscoelastic materials, have attracted the attention of numerous eminent scientists over the years. In addition to the intellectual challenge of determining and understanding the complex behavior of these common materials, there are practical reasons for investigating the flow properties of doughs. In evaluating new wheat varieties it is important to measure and understand the effects of such factors as growing conditions and varietal effects on processing and final product characteristics. Our ability to develop new processing methods will benefit enormously from increased sophistication of process engineering calculations. These calculations in tum require both experimental data input and an understanding of the relevant constitutive equations applicable to describing wheat flour dough behavior. Experience gained in the polymer industry has shown the value of rheological data for quality control purposes and for evaluation of effects of other ingredients, for instance additives. In the food industry rheological information is of value in minimizing the costs associated with the use of texture panels in evaluation of food textural properties. It has been evident from the earliest days of the scientific approach to evaluation of dough properties that wheat flour doughs were complex indeed. Schofield and Scott Blair in 19321 recognized that time during which stress was applied to a dough is as important as the magnitude of the stress itself. In 1970 Tschoegl et al.2 noted that "Wheat flour doughs are subjected to considerable deformation during the make-up and baking process" and emphasized that "few attempts have been made to describe the large deformation behavior of doughs in terms of fundamental quantities. " They found that dough properties depended on a variety of factors including rest period, mixing time, mixing atmosphere, flour variety, etc. Smith et al. 3 looked at dynamic methodology as a method to determine viscoelastic properties under small deformation or at short observational times, determining that the dynamic shear modulus was dependent on strain amplitude, frequency, and time. It is not feasible here to even attempt a review of the many other fine contributions made to our understanding of dough behavior. It is worth noting, with no attempt or pretence of claiming completeness, some recent general reviews, for example Faridi and Faubion,4 "Fundamentals of Dough Rheology" and "Dough Rheology and Baked Product Texture, lIS and the book by Blanshard et al. ,6 "Chemistry and Physics of Baking. "

296

Wheat Structure, Biochemistry and Functionality In recent years it has been recognized that for complete characterization of a

rheologically complex material testing in more than one deformational mode is required . Measurement in simple shear is perhaps the most commonly used rheological testing mode, but extensional flows have attracted a great deal of interest in polymer work since extensional or elongational flows are basic to such processes as spinning as discussed by Petrie . 7 Thus, in addition to a shear viscosity it would be most useful to have information on the extensional viscosity of a dough. The chain of events reported in this manuscript started with the recognition that uniaxial compression of dough discs

in an Instron Universal Testing Machine was imposing on the sample a biaxial

deformation in the two directions perpendicular to the applied force. This is the same

type of deformation that is generated in a Chopin Alveograph and the same as that imposed during bubble expansion which occurs during the baking of doughs. As normally operated, however, the Chopin Alveograph does not yield rheological data which can be converted to absolute units of stress (force per unit area) and strain. In contrast, the Instron compressional data can be quantified so that the stresses and strains can be determined during the compressional experiment.

From such data an

extensional , as opposed to a shear, viscosity can be determined. Such absolute data can then be examined in detail and fitted to appropriate constitutive relationships .

Actual

compressional data obtained on doughs have been examined in this way and were found to be quite well represented by the Upper Convected Maxwell Model (UCMM)8 yielding two parameters, a viscosity and a relaxation time . Figure

1 , taken from Bagley et al. 8 shows the biaxial extensional viscosity of a hard

wheat flour dough plotted against extensional rate .

The lines are computed from the

UCMM for two choices of the parameters , shear viscosity and relaxation time.

5.0 �------------------------------�-_--_ ----' _ -_.� . . _ _ _ _ _ . ��_.. 4.9

.- 4.8 II)

; 4.7

Q..

.� 4.6 , I- 4.5

-

g> 4.4

...J

4.3

4.2

.

,

· · • ·

jt.

, , , ,

..

Exptl }..

}..

= =

15.1

S, 1} = 3.8

26.4 S, 1} = 6.6

• •

104

Pc

104

Pc

• •

S S

4. 1 +-----�----�---4--+---� - 1 .3 - 1 .5 - 1 .4 - 1 .7 - 1 .6 - 1 .8 - 1 .9 Log

t (5-1)

Figure 1 Logarithmic plot of the biaxial extensional viscosity versus strain rate for a hard wheat flour dough. The experimental points were obtained by uniaxial compression of the dough and the solid and dotted lines were computed using the Upper Convected Maxwell Model with parameters as shown. (Bagley et al. 8)

297

Rheological Properties and Functionality o/Wheat Flour Doughs

An obvious question was whether or not the shear viscosity evaluated in fitting this compressional data agreed with an actual direct measurement of a viscosity in shear. The clear need was to determine a shear viscosity for the dough independently and to compare with the one computed from the UCMM. confmnation of the value of the model .

This would provide independent

A second question was why only one single

relaxation time did such a good job in fitting the compressional data since doughs are known to have a broad distribution of relaxation times.

The work below addresses

these questions in more detail.

1.1 Determination of the Shear Viscosity of Doughs Attempts to determine shear viscosity directly in a cone-and-plate rheometer failed because sample rolled out of the gap before a steady-state condition was reached. Bloksma and Nieman9 maintain this occurs at a total applied shear strain of about 20 units . The problem can be avoided by measuring the transient build-up of viscosity at various shear rates. The mirror image of the viscosity/time plots yield the steady-state values for viscosity/shear rate plots (e.g. , Gleissle and MukherjeelO) . Figure

2

shows the mirror image of the transient viscosity/time plot determined

using a Mechanical Spectrometer in the cone-and-plate mode . The viscosity values are shear rate dependent but appear to be approaching a constant viscosity level, the

1 0 6 ,-------------__________________________________--, Len 89

Cone and Plate •

0.5 Hours

� 1 .4

· 2. 1 + 2.7 Capillary, UR = 40 + 3.5

Shear Rate (S-I )

Figure 2 Apparent viscosity of a dough prepared from a wheat flour (Len 89) versus shear rate. Cone-and-plate data shown are the mirror images· of the logarithmic plots of transient viscosity versus time measurements (Gleissle and MukherjeeIO). Capillary data were obtained using a pressure driven capillary rheometer.


298

Newtonian viscosity, at the lowest shear rates. However, for the same flour, viscosity values for preparations of different ages were found to vary by as much as half a decade . In addition to this difficulty there was also the uncertainty as to whether or not the "mirror method " did, in fact, give the "correct" shear viscosity values for these doughs.

An additional independent determination of the shear viscosity was needed.

The tried and true extrusion methodology was adopted to provide an alternate approach to the measurement of the shear viscosity .

In this method nitrogen pressure

is applied to force material to flow from a reservoir through a capillary die of length L and radius R.

The value of LlR chosen was large in the hope of minimizing effects

of pressure losses in the barrel of the viscometer in calculating the viscosity. given applied nitrogen pressure P the output rate, Q, in cc/sec is determined.

For a Values

of Q for a range of applied pressures are found and the results converted to shear stress/shear rate or viscosity/shear rate plots.

As can be seen from Figure 2, there is

a gap between the cone-and-plate and the extrusion data.

Further, the slope of the

extrusion plot is different from that of the other data. Data such as that shown in Figure 2 left unanswered a number of questions including the critical one as to whether or not the LlR value is high enough to mask the effects of flow within the barrel.

Such effects within the barrel are accounted for by

the "end corrections " which describe quantitatively the pressure drops within the barrel as material accelerates towards the die (capillary) . the effective length of the capillary as (L

+

The end correction term, e, gives

eR) .

The shear stress, T, corrected for pressure losses within the barrel, is given by T

=

PRl2(L + eR)

. . . . . . . . . . . . . . . . . . (1)

The apparent shear rate , -Y is computed as 1'.

=

4Qf7rR3 . . . . . . . . . . . . . . . . . . . . . . . . . . . (2)

and then the apparent viscosity is 'Y/.

=

7rPR4/8Q(L + eR) . . . . . . . . . . . . . . . . (3)

If e is small enough, or the length of the capillary large enough, then eR can be neglected and the expression for viscosity reduces to 7rPR4/8LQ, the well-known Poiseuille relation.

(The correction to the computation of shear rate for the non

Newtonian character of the material, the Rabinowitsch correction, is readily made but need not concern us here . ) Equation P

=

(1)

2T(LlR)

can be rearranged to give

+

2Te . . . . . . . . . . . . . . . . . (4)

so that at constant shear rate a plot of pressure needed to attain that shear rate against the length to radius ratio of the die should be linear.

From such plots the end

correction e can be determined. These end corrections can be very large indeed for wheat flour doughs and even for gluten alone as is illustrated in Table

1.

These values of e were computed from data published by Kieffer et al . l l and indicate

299


Table 1 End Corrections for a Dough and for the Co"esponding Gluten, Calculated from Data Given by Kieffer et al. 11

Calculated from Data in Kieffer et al Lebensmittel U ntersuchung und Forschung -

.,

-

Dough (no additives) Shear Rate (S·1 ) 3.6 7 18 36 71 1 75

-

-

-

-

,

(1 982)

Gluten

End Correction -

-

-

-

-

-

-

-

- 1 38 - 1 29 - 1 23 - 1 66 - 1 70 - 206

-

-

-

-

-

-

-

-

-

-

-

-

- 63 .7 - 5 1 .3 - 52 7 . - 69 .3 - 7 1 .2 - 1 09 5 .

Capillary Diameter 0.76 mm Viscometer Length 150 mm

that the die lengths used in obtaining Figure 2 were not long enough. Clearly, a more detailed investigation of these end corrections for wheat flour dough systems was needed. An additional, and quite fascinating, feature of the Kieffer data given in Table 1 is the absolute size of the end corrections.

For many synthetic high polymers and for

polymer solutions values of e above the range 10 to 15 are rare. Kieffer's high values of e for doughs are not unique; similar high values have been reported by others, including ourselves. Note, however, that for Kieffer's experiments the value of eR is 206x.38

mm or 78 mm, more than half the length of the viscometer barrel, which is

The physical significance of such large values needs to be carefully 150 mm! considered, particularly with regards to the reliability and significance of data obtained when more

than half of the barrel charge has been extruded.

1.2 Extensional Viscosities from Capillary Flow Experiments Another reason for such capillary end correction studies was that from the end correction data information on extensional flow properties of the dough can be determined. This was first proposed by Cogswell some years ago and a more recent theoretical analysis by Binding12 has revived interest in the approach. Figure 3 illustrates the physical processes going on during extrusion from a reservoir through a capillary die. A pressure is applied at the top of the reservoir and material is forced through the tube at the bottom.

Along the center line material is

being subjected to an extensional deformation. Material off the center line undergoes increasing proportion of shear deformation as the distance from the center line is increased. Pressure in this flow process drops from P to atmospheric in passing down through the barrel and the capillary .

From the pressure drop in the barrel an

300


extensional viscosity is computed following Binding. capillary the shear viscosity is determined.

From the pressure drop in the

This extrusion experiment, in principle ,

completely characterizes the shear and extensional rheological behavior of a material . Figure

4

indicates how the separation between pressure drop in the barrel and

pressure drop in the capillary is made experimentally. obtained for a number of dies of different

LlR values.

Pressure/shear rate plots are From these plots, the pressure

required to give a given shear rate is determined for each plotted as P versus

LlR

as in Figure

LlR value and the results are

4.

The value o f the end correction e i s determined from the intercept o f these linear

T

�PC

=

PA

HtH

PEN - PEX L -' ..

..

+- 2 R o

. - - - PEN

..,..-- - - - PEX +- 2 R

Die Swell

=

RlRo

Figure 3 Diagrammatic illustration offlow of a viscoelastic fluid from a reservoir

(ba"el) through a capillary or die of radius Ro and length L. The total applied pressure is PA, and the pressure at the die exit is PEl{' usually atmospheric pressure. From pressure drops in the ba"el and capillary the extensional and shear viscosities, respectively, can be evaluated.

LlR plots

O.

on the P

=

0 axis.

The pressure drop in the barrel is the pressure at

LlR

=

Knowing this value the pressure drop in the capillary can be determined . Following

Bindings analysis then both the extensional and shear viscosities can be found . Preliminary experiments with a number of different spring and winter varieties gave results shown in Table

2.

It appears that the ratio of extensional to shear viscosity is

high for the spring and low for the winter varieties. However, in attempting to replicate data, experimental problems became very evident.

Specifically, as illustrated in Figure

4,

it can be hard to get data to the

precision needed to obtain good values either of the intercept at

LlR

=

0

or of the

slope of the plot (which gives the true shear stress at the capillary wall and hence the shear viscosity) .

It is not enough to simply fit the data statistically; one needs good

straight line plots of high precision and accuracy such as can often be obtained with synthetic polymers . For doughs, however, two problems exist that are normally absent


301

in synthetic polymer systems. First, dough samples change properties significantly as they age so the effects of sample age are of major concern. In consequence of this aging effect, it becomes necessary that each set of points at a given LlR is obtained with one batch of dough. A fresh batch is made up (and appropriately aged) for the next set of points at a new LlR. It is extremely difficult to prepare the replicate batches to the desired level of precision to yield acceptable values of either the slopes of the plots (which give the true shear stress) or the intercept at LlR (from which the calculation of extensional viscosity is made).

Bagel Flour 2-22,27-1 993 R = 0.053cm

p

(psi) 1000

400 8 "

800 •

-40

-20

o

20

40

(UR)

60

80

100

Figure 4 Pressure versus capillary (L/R) for a bagel dough (no yeast) for two shear rates. The potential data variability is indicated by the two lines fitted to the lower shear rate line, this variability leading to significant uncertainty in intercepts at both LlR 0 and P O. =

=

1.3 Sample Heterogeneity In establishing our ability to replicate data, to determine variability from laboratory to laboratory and to explore for time variations, extrusion experiments were conducted using both a gas pressure driven rheometer and a Rosand piston driven rheometer. For both instruments large and unexpected data fluctuations were observed. In the case of the Rosand piston-driven rheometer data were obtained in the form of pressure versus time at constant output rate. In the gas-driven rheometer, output rate was monitored

302


as a function of time at constant applied gas pressure . Surprisingly large fluctuations in output rate at constant pressure were observed as illustrated in Figure barrel used.

5

where output rate is plotted against volume of sample in the

(Using this volume measure is equivalent to using time as the abscissa . )

Such large fluctuations make i t impossible to obtain end corrections o r barrel pressure drops to the precision needed for adequate characterization of dough properties for applications such as quality control. Equivalent variations in pressure at constant output rate were found with the Rosand viscometer. fluctuations appear to

For a given flour and a given mix the

be quite random and the magnitude of the fluctuations seems to

be approximately the same for replicate mixes.

However, a detailed and careful

statistical study of the fluctuations has not at this time been carried out.

Table 2 Values of the Ratio of 1 and k, the Parameters Computedfrom Experimentally Determined Shear and Elongational Viscosities, Obtained for a Number of Spring and Winter Wheat Varieties. Protein Levelsfor the Particular Samples are also Tabulated.

11 s = kyn-l

11 E = ,R £ t-l

Spring Wheats Marshall (#354)

Protein % Ilk

Protein % Ilk

1 4.9 1 09

ROC�

Guard (#31 8)

Stoa (#304)

Butte (#350)

Len (#31 5)

1 6.5 157

17.1 215

1 5.5 231

1 9.5 417

Winter Wheats

(#403

Newton (#446)

Yolo (#41 1 )

Arkan (#433)

Chisholm (#421 )

1 7.6 30

1 3.4 32

7.8 36

1 4.5 80

1 2.7 1 61

That these variations are associated with sample heterogeneity was conflrmed by direct visual observation of the actual dough filament extrudates .

One could see

signiflcant fluctuations in extrudate diameter as the filaments emerged from the dies. An obvious way to make the samples less heterogeneous is to overmix them.

In

preparing dough samples the usual standard mixing procedures were employed in which water levels were adjusted and samples mixed to a peak in the Brabender mixing unit of

500

Brabender Units.

fluctuations seen in Figure

When samples were deliberately overmixed the output rate

5 disappeared,

and output rates which varied little with time

(or volume extruded) were observed (Figure

6).

303


LEN-92-7

P = 60psi; UR = 49.4; R = 0.1 52em

0.1 0 0.09 0.08 U CD

�

.2-

�

II:

�

0

u::

0.07 0.06 0.05 0.04 0.03 0.02 0.01 0

60 40 Volume used (ee)

20

0

100

80

Figure 5 Variations inflow rate during capillary extrusion at constant appliedpressure plotted against total volume of sample extrudedfrom the barrel (which is equivalent to time). Flour used is a LEN mixed to 500 BU. 21 ·C, UR

=

48.4 R

=

LEN-92b-33

0.052em (ovmix 40 min 400BU) P

=

250psi

500 .------,

400

300

200

1 00

I

I��ooo ooo r

1

- 1 64 ± 3 - - 1 70 ± 2 ---------..

O �--_.--_.----._--r_--��_.-

o

20

40

60

80

1 00

Volume used (ee)

Figure 6 Variations in flow rate during capillary extrusion of a LEN dough that has been overmixed. (Compare with Figure 5.) The variations in output rate are shown for the initial portion, last portion and total curve and it is evident that overmixing has reduced the fluctuations to something of the order of 1 % .


304

Thus with ovennixing, the output rate at constant pressure is constant, during the time that the barrel is being emptied, to within about 1 % as is seen on the Figure

6.

Such experimental precision i s needed t o obtain adequately precise end correction results and to reduce the variability of P versus

LlR plots illustrated in Figure 4.

Such

improved data precision is absolutely essential to obtain data good enough to differentiate among varieties and to compute " good" extensional and shear viscosities. The problem is that it is not the ovennixed doughs we really want to characterize . There are two obvious ways to proceed.

If the objective is shifted to answering the

question concerning the degree of heterogeneity of a dough mixed to the "optimum" level of 500 BU, then the fluctuations observed in these extrusion studies could be used to evaluate quantitatively the degree of sample heterogeneity. One could also use other methods involving very small samples of the dough, for instance cone-and-plate viscometry, and running a number of different samples.

The results from a series of

samples would, no doubt, fluctuate and these fluctuations could again be analyzed to provide a quantitative measure of sample heterogeneity . On the other hand , if the objective remains characterization of the shear and extensional viscosities through the capillary viscometer approach, then larger samples need to be used.

If samples are large enough, a better approximation to the " true "

output rate at constant pressure for the sample could be obtained .

This would lead to

end correction plots with less variability and hence more data precision.

Larger

samples would, for capillary viscometry , mean larger radii dies and this in tum means larger reservoirs and the approach could rapidly become unwieldy .

An alternate approach to using a "batch" type rheometer, where the dies or capillaries are being fed from a reservoir of limited capacity, is to feed the dies continuously from an extruder.

This approach has been successfully applied by, for

example, Senouci and Smith13.14 and Bhattacharya and coworkers . 15 There are, naturally , problems with the use of extruders , particularly if large radius dies are to used.

Operation at high shear rate can be very profligate of material and

the method is obviously unsuited to experiments where only small quantities of material are available.

Further, output rate has to be controlled and monitored . The control of

output rate can be done in various ways, for example, by changing the rate of rotation of the screw.

This will affect the amount of work and the rate of work input into the

dough and thus alter the properties of the material being investigated . One can operate with a starved screw13 or control output through the die by taking material off a side stream. 15 For an extrusion process measurements of the type discussed above may be very useful for process control or for monitoring of product properties.

However, it is not

clear whether or not the extrusion data (say through a slit die) can be obtained with the precision and accuracy desirable for a scientific investigation of such issues as how best to

evaluate protein quality and how protein quality relates

environmental effects, etc.

to

varietal

and/or

Some of the factors affecting extrusion studies and some

aspects of experimental errors involved in such studies have been discussed by Padmanabhan and Bhattacharya. 16 Frequently in published work general trends can be readily seen but often the data are plotted on log-log scales and scatter on such scales and the corresponding variations in the absolute values of the quantities being measured can be for many purposes unacceptably large.

Rheological Properties and Functionality o/ Wheat Flour Doughs

305

1.4 Relaxation Times The measurement and interpretation of relaxation times in wheat flour doughs represent another area of challenge. data for the dough of Figure this is a wide variation.

1

are

The two relaxation times chosen for fitting the

15.1

and

26.4 seconds.

Percentagewise, of course,

Further, even a cursory look at dough behavior reveals that

much longer relaxation effects can be present indicating relaxation processes occupying hundreds or even thousands of seconds. 17 Nevertheless, the values from Figure

20

s) were not out of line with values, which were in the range

Frazier et al. 18

12-50

1

(

-

s, reported by

This later reference is of especial interest because some effects of

protein level, wheat variety , and work input during dough preparation are described though it should be remarked that the method used by Frazier et al. to evaluate relaxation time is arbitrary, yet nevertheless effective for their needs. One approach to measuring the distribution of relaxation times for polymers is to measure dynamic properties over a decade or two of frequency and to do this at a variety of temperatures. Master curves can then be drawn, using the Williams-Landel Ferry shift factor. These master curves extend over numerous decades of time and thus a picture of the relaxation times and relaxation time distribution can be obtained . For doughs , as for most biopolymers, the concept of measuring over a range of temperatures just is not workable, since the properties of doughs change irreversibly with temperature .

Dough heated to 80°C is quite a different substance than dough at

room temperature . 19 The fact remains , though, that there is sufficient evidence in the literature to conclude that it would be valuable to have a better insight into the relaxation times,

and

hence

relaxation processes,

of doughs .

More detailed

investigations of, and theoretical and molecular analyses of, relaxation behavior of doughs could be most informative .

2

CONCLUDING COMMENTS

In spite of excellent contributions from many cereal scientists over the years , the problems associated with determination of the rheological properties of wheat flour doughs continue to provide ongoing challenges.

Theoretical considerations make it

clear that testing of such complex materials as wheat flour dough requires application of a variety of testing modes, two common testing modes being extension and simple shear.

Having obtained the rheological data, a critical subsequent step is to describe

the material behavior mathematically through the use of an appropriate constitutive equation or model.

Such a model can be used in a variety of ways, for example in

engineering calculations or to provide parameters that can be checked through independent experimental measurements to confirm both the value of the model and the validity of the experimental data. The dependence of dough properties on such factors as time , work input, and rate of work input coupled with the extreme sensitivity of dough properties to water level and biological activity (e .g. , activity due to the presence of specific enzymes) complicate the task of obtaining reliable and meaningful data. The difference between " reliable" and "meaningful " can be illustrated with reference to the heterogeneity of wheat flour doughs mixed to the normal level of

500 BU:

The data obtained on such

a mixture fluctuates over a range determined by the degree of heterogeneity and the sample size. These fluctuations can be minimized by overmixing the sample, but while


306

the data so produced are reliable they are not particularly meaningful because the market for overmixed dough is pretty limited though interest in the mixing process is high! 20. 21 This paper was to describe the experimental and conceptual problems arising in the rheological characterization of wheat flour doughs. It is essential to recognize first of all that any experimental method chosen (say to measure a shear viscosity) will yield numbers of some sort. It is necessary somehow to obtain independent checks of these numbers. To obtain these independent checks is not easy . The experimental problems can be very frustrating, as in the case of sample "roll-out" from a cone-and-plate viscometer. Methods developed with materials other than doughs, for example the Gleissle mirror image methodology, may or may not be valid for doughs. The applicability of a particular methodology needs to be confirmed. Other problems may arise unexpectedly, as for instance the effect of sample heterogeneity arising with doughs mixed to an "optimum" level. Such experimental problems give rise to conceptual problems. Heterogeneity can be overcome by making measurements on samples large enough so the heterogeneity is not observable, for instance by continuous extrusion using large dies. But how are the effects of extrusion processing on dough properties to be taken into account for a material so sensitive to moisture level, time, and shear history? How should we treat the question of relaxation times of doughs when certain data are well fitted with a single relaxation time when we know that there is a very broad distribution of relaxation times (from tenths of seconds to thousands of seconds) that can be significant as seen in stress relaxation experiments? What molecular and structural features exist in doughs to give rise to this broad distribution of relaxation times? How do we obtain a broad spectrum of modulus\time plots when time-temperature superposition methods cannot be used with a temperature sensitive material such as dough? These and numerous other questions remain to provide opportunities for advances both in our understanding of dough behavior and protein quality and in our ability to apply our knowledge to improve processing procedures and final product quality. Among ways available for the cereal scientist to exploit these opportunities one can include the application of the sophisticated computational methodology currently available today. These methods can be used to examine rheological data in the light of a variety of constitutive equations and rheological models. There also seem to be opportunities to apply some of the newer concepts of fractals, chaos and advances in treatment of non-linear systems in general, to examine in detail such effects as the output rate fluctuations observed during dough extrusion at constant pressure. Not least among the opportunities is the chance and need to obtain more experimental rheological information on well characterized flour systems to more fully delineate the behavior of wheat flour doughs. ACKNOWLEDGMENTS : Thanks are due to F. Alaksiewicz (NCAURIUSDA) and to R . Tames (Kraft General Foods) for first-class technical support.

References 1.

R. K. Schofield and G. W. Scott Blair, 'The Relationship between Viscosity, Elasticity ,and Plastic Strength of Soft Materials as Illustrated by some Mechanical Properties of Doughs' , Proc. Royal Soc. (London) , 1 932, A 1 3 8 , 707-7 1 8 .

Rheological Propenies and Functionality of Wheat Flour Doughs

2. 3.

307

N. W. Tschoegl, J. A. Rinde, and T. L. Smith, 'Rheological Properties of Wheat Flour Doughs' , I. - "Method for determining the large deformation and rupture properties in simple tension" , J. Sci. Fd. Agric. , 1970, 21, 64-70. J. R. Smith, T. L. Smith, and N. W. Tschoegl, Rheol. Acta, 1970, Band 9, Heft

2, 239-252. H. Faridi and J. M. Faubion, Editors, 'Fundamentals of Dough Rheology ', American Assoc. of Cereal Chemists, 1986, St. Paul, Minnesota. 5 . H. Faridi and J. M. Faubion, Editors, 'Dough Rheology and Baked Product Texture' , Van Nostrand Reinhold, New York, 1990. 6. J.M.V. Blanshard, P. J. Frazier, and T. Galliard, 'Chemistry and Physics of Baking' , Royal Soc. of Chemistry, Burlington House, London, 1986, WIV OBN. 7. C.J.S. Petrie, 'Elongational Flows - Aspects of the Behavior of Model Elasticoviscous Fluids' , Pitman Publishing Ltd . , London, 1979, WC2B 5Pa. 8. E. B. Bagley, D. D. Christianson, and J. A. Martindale, 'Uniaxial Compression of a Hard Wheat Flour Dough: Data Analysis Using the Upper Convected Maxwell Model' , J. Texture Stud. , 1988, 19, 289-305 . 9. A. H. Bloksma and W. Nieman, 'The Effect of Temperature ori the Rheological Properties of Some Wheat Flour Doughs' , J. Texture Stud. , 1975, 6, 343-361 . 10. W. Gleissle and D. Mukherjee, "Measurement of transient and steady-state shear and normal stresses in filled polymers" , Progress and Trends in Rheology II, Proceedings of the Second Conf. of European Rheologists, Prague, June 17-20, 1986, Edited by H. Giesekus and M. F. Hibberd, Springer-Verlag, New York,

4.

1988. 1 1 . R. Kieffer, J. Kim, M. Kempf, H-D Belitz, J. 12. 13. 14. 15.

Lehman, a. Sprossler, and E. Best, 'Untersuchung Rheologischer Eigenschaften von Teig und Kleber aus Weizenmehl durch Capillarviscosimetrie' , Z Lebensm Unters Forsch, 1982, 174, 216-22 1 . D. M . Binding, 'An Approximate Analysis for Contraction and Converging Flows' , J. Non-Newton. Fluid Mech. , 1988, 27, 173-189. A. Senouci and A. C. Smith, 'An Experimental Study of Food Melt Rheology. I. Shear Viscosity Using a Slit Die Viscometer and a Capillary Rheometer' , Rheol. Acta, 1988, 27 , 546-554. A. Senouci and A. C. Smith, 'An Experimental Study of Food Melt Rheology. II. End Pressure Effects' , Rheol. Acta, 1988, 27, 649-655 . K . Seethamraju, M . Bhattacharya, U. R. Vaidya, and R. G. Fulcher, 'Rheology and Morphology of Starch/Synthetic Polymer Blends' , Rheol. Acta, 1994, 33, 553-

567. 16. M. Padmanabhan and M. Bhattacharya, 'Flow Behavior and Exit Pressures of Com Meal Under High-Shear-High-Temperature Extrusion Conditions Using a Slit Die',

J. Rheol. , 1991 , 35(3) , 315-343.

17. E. a. Bagley and D. D. Christianson, 'Stress Relaxation of Chemically Leavened Dough-Data Reduction Using the BKZ Elastic Fluid Theory' , J. Rheol. , 1987, 31(5), 405-413. 18. P. J. Frazier, N.W.R. Daniels, and P.W.R. Eggitt, 'Rheology and the Continuous Breadmaking Process' , Proc. of Symp. on Rheology of Wheat Products. Cereal Chem. , 1975, 52(3) , l06r-130r. 19. J. D. Schofield, R. C. Bottomley, M. F. Timms, and M. R. Booth, 'The Effect of Heat on Wheat Gluten and the Involvement of Sulphydryl-Disulpbide Interchange Reactions' , J. Cereal Sci. , 1983, I , 241-253.

308


20. K. Okada, Y. Negishi, and S. Nagao, 'Factors Affecting Dough Breakdown in Mixing ' , Cereal Chem. , 1987, 64(6) , 428-434 . 2 1 . G. Danno and R. C. Hoseney, 'Changes in Flour Proteins During Dough Mixing ' , Cereal Chem. , 1982, 59(4) , 249-253 .

PHYSICAL FACTORS DETERMINING GAS CELL STABILITY IN A DOUGH DURING BREAD MAKING

T van Vliet Department of Food Science Wageningen Agricultural University P.O. Box

8129 6700 EV Wageningen

The Netherlands

1

INTRODUCTION

A good quality bread should have a high gas volume and a fine, regular crumb structure. During mixing of bread dough a small amount of air is entrapped , in the form of small spherical gas cells. The number of gas cells is between with a diameter of about

10-100

1012

and

1014

m-3

",m. During fermentation and baking some of these

gas cells will grow, initially due to carbon dioxide produced by the yeast and later mainly due to the temperature increase and water evaporation. I The number of visible gas cells in the crumb of bread is about between

10-4

and

10-2

1010

per m3 solid material which is only

of the estimated number of gas cells in dough after mixing .

Obviously a large number of gas cells have disappeared (or became invisible) because they were physically unstable or did not grow out. Physical instability processes that play a crucial role during the bread-making process are Ostwald ripening and coales cence.2 Ostwald ripening or disproportionation is the growth of large gas bubbles at the expense of smaller ones due to the higher overpressure (Laplace pressure) of the gas in the small gas cells resulting in a higher gas concentration in the vicinity of these cells. It causes diffusion of gas towards larger cells. Coalescence of gas cells is due to rupture of the dough film between them. It is the main instability mechanism at the end of the tin proof and during baking and extensive coalescence then would result in a irregular and coarse crumb structure. Copious coalescence leads to contact of gas ceUs with the outside air, hence to a large loss of bread volume. Another process that is important for obtaining a regular crumb structure is that the gas cells growing out do so at roughly equal rate. If only surface properties would be involved the lower Laplace pressure in the large gas ceUs would cause that the gas produced diffuses preferentially to these gas cells, resulting in an irregular coarse crumb structure. In this paper we will discuss the various physical parameters that may play a part in the stability of a bread dough against disproportionation and coalescence and for obtaining equal growth of gas cels during fermentation and baking.


310

2

OSTWALD

RIPENING

Ostwald ripening is caused by the gas pressure difference between gas cells of different size. Due to the curvature of the gas-liquid interface a pressure difference

tlP (the so

called Laplace pressure) exists over this interface, which is given by: tl p

where -y is the interfacial tension and

R

2 .1.

=

R

(1)

the radius of the gas cell This excess pressure

results in an enhanced equilibrium gas concentration around a gas cell, which is higher around a small gas cel than around a large one. This concentration difference results in transport of gas through the liquid mass by diffusion from the small to the large gas cells. The end result will be the disappearence of the small gas cells. An order of magnitude calculation on the shrinkage rate of the small gas cells due to Oswald ripening can be made by using the de Vries equation, which reads:3

4 R TDSy Ph

where

'I

is the bubble radius at time t,

constant (8. 3 1

J . KI . mol-I), T

the dough (about 10-9 m2 • S-I),

t

(2)

'0

temperature

the initial radius (5-50 I'm) , R the gas (K), D diffusion coefficient of the gas in

S solubility of the diffusing gas (about 0.43 mmol . m-3

• Pa-I for CO and 2 % of it for N gas), -y the surface tension (about 40 m N · m-I), P 2 2 atmospheric pressure (lOS Pa) and h the average thickness between the small and the much larger surrounding gas cells. Due to the increasing difference in the Laplace pressure the disappearence of a smal gas cell is a self accelerating process. According to Eq. 2 gas cells with a diameter of 10 to 20 I'm would disappear within a minute for

h

is 100 I'm. For larger ones it may take an order of magnitude longer. On average it

will take about half an hour before the yeast has produced enough carbon dioxide for the liquid dough phase to become saturated . During that time most small gas cells would have disappeared already. Ostwald ripening in dough may be retarded or stopped for several reasons: - The Laplace pressure in large gas cells is smaller than in small ones and so the driving force for Ostwald ripening will decrease after the disappearence of the smallest cells. - During shrinkage of a gas cell its gas-liquid interface decreases and, depending on the properties of the surface active material present in the surface, its surface concen tration will increase. Such an increase results in a lowering of -y and thereby in the gas cell considered . Shrinkage of a gas cell will stop if the decrease in

R

tlP of

is off-set

by a decrease in -y. The response of the surface tension is expressed in the surface dilational modulus Ed == d-yldlnA , where A is the surface area. 4,s If Ed > 0, shrinkage of the gas cell is slowed down and if

Ed

�

Ih-y, it stops. However, due to the

viscoelasticity of the gas dough surface, the modulus will be smaller at longer times scales; consequently this mechanism would not stop Oswald ripening in dough. It does cause a decrease in the shrinkage rate of the small bubbles, to an extent that can be affected by the action of emulsifiers added to bread dough . 6 However, as long as

Ed

-- +

(3)

U is not simple. I1P acts over an area 7rIf while U acts u(r) depends on the biaxial strain E and on biaxial strain rate E, and both decrease with Estimation of the relevant

as a first approximation on a spherical shell around the gas cell. However, increasing distance from the gas cell surface. For an isolated gas cell;

(4) where

r is the distance from the centre of the gas cell and 'Y the surface tension. For a r, E and E can be calculated .7 The relation between u(r) and E and between u(r) and E is known from experiments, and the effec tive u acting on the gas cell can thus be estimated by a numerical approximation for an isolated gas cell. It results in u is about 0.4 · u(R) where u(R) is the biaxial stress in

given gas production the relation between

,

the dough directly adjacent to the gas cell. In reality however, gas cells are not isolated

and the

u(r) developing around a gas cell will interfere with those around adjacent u and u(R) to become larger than 0.4.

cells. This would cause the ratio between

Arbitrarily we have chosen a ratio of volume fraction. As said above

I1P

0.5,

although it will actually depend on the gas

equals to 2'YIR. Shrinking of gas cells in a dough would cert

ainly lead to a lowering of 'Y while, in principle, 'Y around a growing gas cell would become somewhat higher. However, due to the low strain rates involved

5

•

(E

of about

l O-4f the increase in 'Y will be very small and insignificant compared to the other

factors. The decrease in 'Y for a shrinking gas cell will be much more substantial, as the relative rates of shrinking of the interface around these gas cells are much higher than the relative expansion rates around the larger ones. Taking into account that dRl is positive and dR. is negative and assuming that initially 'Y is the same for the small and the large gas cells, formula

3

dO l dRl

can be rewritten as: _

� Rf

After multiplication of both side by

after some rearrangements:

>

dos dRs

+

� R;

_

2� Rs dRs

(l/(UIUJ and making use of dE

(5) =

dR/R) one obtains

312


For not too small e it is often found that dlnol de is independent of e . 7 After multiplica tion of both sides by u.R1> taking u

[

]

=

0.5

u(R) and by making use of de,

=

-

(RI3/R,3) . del one obtains some rearrangements and taking into account that dlnu, is negative, the next formula:

dlna ( R) l _ R; a ( Rs) de R{ a ( R1 ) For u(R,)

=

0 and

(d')'/dR)

=

0

7,

+

2 Y I Rs Rl a ( R1 ) Rs

formula

dlna ( R ) de

In the derivation of formula

(

>

7

>

Rs] Rl

[

reduces to.

2 Y IRs Rl Rs + a ( R1 ) Rs Rl

_

2_ R l � a ( R1) Rs dR

_

]

(7)

(8)

effects due to varying strain rate are neglected. Due to

the smaller R, the strain rate by which the continuous phase around the smaller gas cell is compressed is higher than the strain rate by which the larger gas cell is expanded . This makes that dlnu(R,)/de and dlnu(RJ/de are not the same as was implicitely assumed in deriving formula

7.

In the latter stage, however, a correction for this effect

would be rather small as the dependency of dlnu(R) on the strain rate is much smaller than that on the strain .6,7 As follows from the derivation of equation

7

the dependency of ')' on R for the

small gas cell leads to an extra term {-2RI/U(RJR,} ' {d')'/dR} at the right hand side. Because d')' and dR are both negative, d')'/dR is positive, which implies that the crite rion expressed by formula

7

will be fullfilled easier if d')'/dR is larger. Consequently

surface rheological effects will enhance the retarding effect on Ostwald ripening due to bulk rheology and will help to stop it completely, even after the bulk rheological properties have become the main factor.

3

EQUAL GROWTH OF GAS CELLS

In the absence of bulk stresses growth of a gas cells will occur if the gas concentration in the material adjacent to the cell is higher than the gas concentration in equilibrium with its Laplace pressure. In viscoelastic materials the gas concentration for growth has to be higher, due to the extra resistance against (further) biaxial extension. The amount of dissolved gas must be high enough to compensate for stress u. For u

=

0.5 · u(R)

!:lP and the opposing biaxial

equal growth of gas cells with the same size due to gas

production in their surrounding would occur if:

�(l:. a ( R ) + 2 :1.. ) dR 2 R If ')' is independent of R and using deB

=

> 0

(dRIR) the next criterion is obtained :

(9)

313

Rheological Properties and Functionality of Wheat Flour Doughs dIna

de

( R)

2_ � _

>

a

( 10 )

( R) R

This equation is equal to equation 8 for R, RI• The criterion for obtaining equal growth of gas cells of similar size thus depends on the ratio of the Laplace pressure 2"(/R over the biaxial stress and on the strain hardening of the continuous phase. The criterion is fulfilled easier if the extent of strain hardening is stronger and/or the biaxial stress is higher. An estimate by Kokelaar et al.8 of the values of the Laplace pressure and of u(R) during breadmaking indicate that for most cultivars u(R) would become larger than the Laplace pressure already during the first proof, but the precise stage at which this would be the case depends on the cultivar. For gas cells of different sizes, the analysis is somewhat more complicated. Due to the higher Laplace pressure in small gas cells than in large cells, gas formed in the continuous mass between various gas cells will diffuse preferentially to the larger cells. To obtain preferential growth of the small gas cells, the large overpressure in these cells has to be compensated by a larger biaxial stress around the larger cells. This will be the case if the condition given by formula 3 is fulfilled. The only difference with the analysis for Ostwald ripening is that in this case both gas cells grow at a low strain rate, so d"(/dR will be very small and can be neglected. The end result is: =

dIna

de

( R)

[1 -

R; R{

which is equal to formula 7 for d"(/dR

a

a

( Ra ) ( R1 )

=

1

>

2y IRs

a

( R1 )

[

Rl Rs

+

Rs Rl

J

( 11 )

O.

3 COALESCENCE Coalescence of gas cells involves rupture of the dough film between them. It will be primarily important after the transformation of the dough from a foam with spherical gas cells into one with polyhedral ones. For a very high-volume bread this transition may occur at the end of the fermentation stage, in other cases it will be during baking. During growth of the gas cells the dough films between them are extended biaxially.1 The stability of these films against biaxial extension will determine the stability of the dough against coalescence. Two mechanism may cause rupture of the dough films. The first is due to the development of weak spots caused by accidental local thinning and the second is due to a too small rupture strain of the film . Van Vliet et al.1 derived criteria that relate the stability of dough films between two gas cells against local thinning and so against rupture, to the relative increase of the biaxial stress u (dlnu )over the accompanying increase of the biaxial strain e (de). If this ratio is greater than 2 a thinner, which implies a relatively more extended, part of the dough film will have a higher resistance towards further extension than the thicker, i . e. less extended parts. However, since dough is a viscoelastic material and because the biaxial strain rate of the dough around a larger gas cell will be smaller than that around a small gas cell (at constant gas production), a correction has to be made. This results in the next condition:


314

( 12 )

where Eb is the biaxial strain rate. The factor alnE/ae corrects for the dependence of the biaxial tensile strain rate of the dough around the gas cells on the biaxial strain of the dough, its value depends on the stage of the bread-making process. During fermentation the quotient is about -3, while during baking it was observed to vary between + 1 .5 and +2. 1 ; hence, the criterion given by formula 1 2 is more strict at the end of the fermentation stage than during the baking stage.7 Often, the transition of a foam structure with spherical gas cells to one with polyhedrical cells occurs only during the baking stage. If this is the case lower requirements are set to the strain hardening properties of the dough and vice versa. Kokelaar showed that the variation in strain hardening among cultivars at 20 °C is also present at a 55 °C.6 At the end of the baking stage the dough films between two gas cells may become as thin as the size of a starch granules. 10 The film then can not be considered anymore as a homogeneous dough film but will behave more as a hydrated gluten (+ other soluble components) film containing starch particles. Also hydrated gluten films exhibit strain hardening both at 20 and at 55 °C . 6, 1 1 The second mechanism of film rupture occurs if the biaxial fracture strain (the biaxial "extensibility") of the dough is too small. That there is a relation between the baking behaviour of a bread and the extent to which a dough film can be extended before it breaks was shown by de Bruijne et al. 12 They also illustrate that for obtaining a good relation it is essential that the extension experiments are done at the relevant (low) rate of elongation . The high elongation rates in instruments like the extensograph and the alveograph seriously limit their value for determining rupture strain. Moreo ver, in the extensograph and in the experiments done by de Bruijne the dominant deformation was uniaxial extension in stead of biaxial. How seriously this affects the applicabilty of their results is not known.

4 GENERAL DISCUSSION The discussion given above show that for all the three physical mechanisms important for obtaining a good quality bread, more than one physical parameter determines the bread-making potential of a dough. A summary of the relevant physical parameters is in table 1 . These physical parameters should be determined at the relevant low strain rates and the right (large) strains. As discussed above, not all three mechanism are equally important during each stage of bread-making. Kokelaar did not observe variation in the surface tension or the surface dilational modulus among various (4) wheat cultivars . 6 This would imply that the variation in baking behaviour among the cultivars studied was due to variation in bulk rheological properties. In accordance with this, both in the study of Kokel� and that of Janssenll the ranking of the bread-making performance could be done on basis of bulk rheologi cal properties. However, for definitive conclusions more research has to be done. Surface rheological properties can be affected by the addition of emulsifiers and so they will clearly affect the disproportionation process and with that bread-making performance.6,1 3 Emulsifiers did not affect strain hardening properties.


315

Table 1 Relevant physical parameters during bread-making i n relation to the various mechanism discussed above + relevant, - not relevant, ? relevance not clear but probably small. 6,8 Physical Parameter

Disproportionation

Equal growth gas cells

Coalescence

-

?

+

+

?

Biaxial stress

+

+

Strain hardening

+

+

Surface tension

+

Surface dilational modulus

Fracture strain (in biaxial extension)

-

-

-

+ +

References 1. 2. 3. 4. 5. 6. 7. 8.

9. 10. 11.

12.

13.

A.H. Bloksma, Cereal Food World, 1990, 35, 228. T . van Vliet, A.M. Ianssen, A.H. Bloksma and P . Walstra, J. Texture Stud. , 1992, 23 439. A.I. de Vries, Recueil Trav. Chim. , 1958, 77, 209. I. Lucassen, in 'Anionic Surfactants: Physical Chemistry of Surfactant Action' (E.H. Lucassen-Reinders, ed.) Dekker, New York, 198 1 , p. 2 17. P. Walstra, in 'Foams: Physics, Chemistry and Structure' (A.I. Wilson, ed.) Springer-Verlag, London, 1989, p. 1. 1.1. Kokelaar, 'Physics of Breadmaking, ' Ph D Thesis, Wageningen Agricultural University, Wageningen, The Netherlands, 1994. T van Vliet, A.M. Ianssen, A.H. Bloksma and P. Walstra, J. Texture Stud. , 1992, 23, 439. 1.1. Kokelaar, T. van Vliet and A. Prins, in 'Food Colloids and Polymers: Stability and Mechanical Properties, (E. Dickinson and P. Walstra, eds.), Royal Soc. Chem. ,Cambridge, 1993, p. 272. T. van Vliet and 1.1. Kokellku , in 'Progress and Trends in Rheology' , Proc. Fourth Eur. Rheol. Conf. (C. Gallegos, ed.), Steinkopff, Darmstadt, 1994, p. 201 . R.M. Sandstedt, L . Schaumberg and I . Fleming, Cereal Chem. , 1954, 31, 43 A.M. Ianssen, 'Obelisk and Katepwa Wheat Gluten, A Study of Factors Determi ning Bread Making Performance, Ph.D. Thesis, State university Groningen, Groningen, The Netherlands. D.W. de Bruijne, I de Loof and A. van Eulem, in 'Rheology of Food, Pharma ceutical and Biological Materials with General Rheology' (R.E. Carter, ed), Elsevier Aplied Sci . , 1990, p. 269. 1.1. Kokelaar, T. van Vliet and A. Prins, in 'Food Macromolecules and Colloids' (E. Dickinson and D. Lorient, eds.), Royal Soc. Chem. , Cambridge, 1995, p. 277.

STRAIN HARDENING EXTENSION

&

DOUGH GAS CELL WALL FAILURE IN BIAXIAL

BJ. Dobraszczyk RHM Technology Ltd.

The Lord Rank Centre Lincoln Road

High Wycombe HP12 3QR

1. INTRODUCTION There has long been a conviction amongst bakers that baking performance is in some way related to the rheological properties of the dough, probably due to the common practice amongst bakers of stretching the dough by hand. Although this is a very subjective method of measuring rheology, it tells us something about the sort of rheological measurements we should be making in order to predict baking performance. Correlations between rheological measurements on dough and baking performance have often produced inconsistent and conflicting results. One reason may be that the rheological instruments measure dough properties at different stages of development: the torque recording mixers, such as the Farinograph, provide information on the short-term transient molecular changes in dough rheology during mixing, whilst devices such as the Extensograph and Alveograph, which measure force against extension, measure dough properties at some time after mixing, and would be expected to reveal the more permanent structural changes occuring in dough as a result of mixing. Secondly, most rheological measurements are performed at rates and conditions very different from those experienced by the dough during baking. For example, rates of expansion in proving doughs have been calculated as approximately 5 x lO-4s-l, compared with measuring rates in rheological tests several orders of magnitude greaterl . Conventional rheological tests also usually operate at small strains in the order of up to 1 % , whilst strain in gas cell expansion during proof is expected to be in the region of several hundred percent. Furthermore, many rheological tests are carried out in shear, whilst most large-strain deformations in dough (Le. mixing, sheeting and baking) are extensional in nature. Therefore, any predictive tests on dough should be carried out under conditions close to those of baking expansion. The Alveograph was considered to approximate these conditions best since it can, with certain modifications, be operated at rates and strains close to those observed during baking expansion, and can be used to obtain fundamental tensile rheological properties under conditions close to those of baking expansion. Bloksma2 proposed equations from which Alveograph data could be converted directly into rheological units of stress, strain and strain rate from a precise knowledge of the bubble height, pressure and inflation rate with time.

Rheological Properties and Functionality o/Wheat Flour Doughs

317

2. MATERIALS & METHODS Doughs were mixed on a Brabender Docorder 300g bowl at 150rpm to peak: work input. Two flours were used: (1) a standard commercial Chorleywood Baking Process (CBP) white flour, and (2) a soft non-breadmaking wheat cultivar, Apollo. The resulting dough was tested according to standard Alveograph procedures3. Dough temperature was monitored by inserting a thermocouple probe into one of the dough samples. Each dough piece was placed in the Alveograph head in turn and compressed to a thickness of 2.5mm. The sample was then inflated at a given flow rate, and bubble height and pressure were monitored throughout inflation. Test baking was performed on the doughs used in these experiments, using 56g dough in a micro-bake tin. Proof height was measured after 60 minutes, and the loaves were baked for 20 minutes at 225 °C in a Simon rotary oven. Loaf volumes were measured by seed displacement. The Alveograph was modified as follows. Pressure change during inflation was measured using an electronic pressure transducer (Furness Controls Ltd.), with a maximum range of 20 in. water (5kPa) and a resolution of 0. 1 in. water. Pressure readings during inflation were recorded via the analogue input of a BBC microcomputer. Bubble height was recorded using a laser ranger (Electronic Automation Ltd.) with a sensitivity of 0.5mm, and the output fed into the RS423 digital input of the BBC microcomputer. Inflation rate was controlled using compressed air and flow regulators and measured using a flow meter. Flow rate was varied between 10 and 2000 cm3/min, corresponding to maximum strain rates of 1x1Q-3s-1 to 2x lO-ls-l, the lower limit approaching rates of baking expansion. The pressure and height data were transformed directly into stress and strain on the BBC microcomputer using the equations derived by Bloksma3 (see Appendix 1).

3. RESULTS & DISCUSSION Data obtained from the Alveograph are normally presented as pressure against time as the bubble inflates. Figure 1a shows pressure and bubble height versus time as the bubble inflates and Figure 1b shows the transformation of this data into a true stress-strain curve. (a) W 0: :;) (f) (f) W 0: (1-

////

-

---

-

-

-

(b)

,-- RUPTURE i PO I NT -"

" ....

BUBBLE i PRESSUR�

BUBBLE HE I GHT

(f) (f) W 0: t (f)

-

W I

T I ME

STRA I N

Figure l.(a) Typical pressure and height versus time trace for an inflating dough bubble; (b) true stress-strain curve derived from Figure J (a).


318

The stress-strain curve showed two interesting features. Firstly, the stress increased throughout inflation and showed no inflection at the peak: in pressure, showing that this peak: has no significance in terms of stress. Secondly, the stress-strain data exhibited considerable curvature up to failure, indicating that the modulus (stiffness) of the dough increased with inflation. This phenomenon is known as strain hardening, and is typical of materials which need to stretch to large deformations, such as polymers drawn into fibres or inflated into thin films. Strain hardening is essential to maintain stability against failure in any large stretching operation. When stretching occurs, there are bound to be some areas which are thinner than others. While the material is thick this is of little importance, but as it becomes thinner on stretching, the stress in the thinner areas increases in relation to the surrounding material . This would normally cause fracture as the higher stresses tend to favour increased thinning, leading to catastrophic failure. Strain hardening locally increases resistance to stress in these thinner regions, stabilising any regions of incipient localised thinning and allowing much greater deformations before failure. Plotting typical Alveograph stress-strain results on a log-log graph shows the data fit well to a straight line (Figure 2a) , indicating a power-law relationship in the form of: (1)

where u=stress, f = strain, n =strain hardening index and k =constant. 3 . 1 Stability of Deformation During Stretching

During large stretching of materials, the cross-sectional area changes in a non-uniform manner along the length of the sample, and eventually the material thins locally where the stress is highest. The point at which thinning occurs is called yield and is generally defined by a maximum in force on a force-extension plot. Once the material reaches this point, any further extension occurs at a lower force, making it easier for increased thinning to occur, making failure inevitable on further stretching. Hence yield defines a point of tensile instability. For bubble inflation we can calculate the tensile force from the bubble pressure and radius. Experimental data for a CBP dough are shown in Figure 2b, (a)

( bJ dF

<Jl <Jl W 0: IUJ

8-.l

"

SLOPE

=

( n)

LOG . STRA I N

0

W U 0:

0 LL

BUBBLE RUPTURE

EXTENS I ON

Figure 2(a) Power-law fit to stress-strain data; (b) Force-extension plot for the polar region of the Alveograph bubble showing point of tensile instability at dF= O.

319


where the tensile force is plotted against the extension (LlLo) at the polar region of the Alveograph bubble. The onset of unstable thinning begins at the load maximum in tension (dF =O), or point of load instability: this point is defined in terms of stress (0") and strain (E) in the Considere criterion of instability in tension\ or:

rur =

dE

0" (at dF = O)

(2)

The stress 0" at the point of instability is equal to the slope of the true stress-strain curve at that point. For a material whose stress-strain curve follows a power law, the Considere instability criterion can be substituted into equation (1) to give a simple relationship between the strain hardening index (n) and the critical strain (fcriJ at which instability occurs:

(3) This is an important relationship: it shows that the strain at instability and subsequent failure are defined only by the strain hardening properties of the material. The higher the value of n, the greater the strain hardening and the greater the strain at which instability occurs. In terms of bubble inflation, this means that bubbles will inflate to a larger volume before failure occurs. Thus, the strain hardening properties of the dough will be critical in determining the limit of expansion. This has been verified experimentally for a large number of doughs inflated in the Alveograph under a range of conditions3 (Figure 3), where the strain hardening index (n) showed a good correlation (R2 =O.762) with the bubble failure strain, or the final volume at rupture. 3.2 Rate Dependence of Failure The Considere criterion assumes the material is insensitive to changes in strain rate. Extensive published work has shown that dough is viscoelastic, i.e. its stiffness depends on the rate at which it is tested. Changing inflation rates from 10-2000 cm3/min in the Alveograph showed that dough rheological properties are sensitive to the rate of inflation. 2.5 z

-

:

0.26 0.24 0.22

0

°

C

-- --y

z 0.2 + 0.36 · 01.14 Harvest 1 (n=13) Harvest 2 (0=31)

• o

...

'il

.. ...

1

1 .5

2

0'0

�

Two rollers gap

0.20 0. 1 8

1-

.

�

- �

"I

329

. r = +

r = 0 0.83

o

8. • • . ' . 0' 00

t% �

2.5

.. "CP' .. - ... +I... .. .Jl

ka (s·l)

3

�

3.5

0

0

Two rollers gap 4 50

100

150

. . .' . ' . ..;0

0.74

III B. lI"'"

.

0

o

.... " .... (5:J

0�

0

,. .. 0 0

000

0

0

0

••

0

200

W

250

0.32 0.30 0.28 0.26 0.24

1 - - - - o y - 0.2 + 2.78 1O.. W • Harv... 1 (0-13) Harv... 2 (0-3 1)

I

0.34

300

0.22 0.20 0.18 350

Figure 3: Dough Band Thickness (in biscuit process, just after rolling) versus relaxation rate constant after LSF and versus W. Regressions have been calculated with the results of harvest 1 and 2 . 3 . 2 Correlation w i t h biscuit tests 3.2.1 Biscuit dimensions. The best correlations were obtained with relaxation following LSF, but not BI, and with W from the Alveograph (see table 2). For packaging, constancy of biscuit dimensions is very important. We observed that the lengths and the widths of biscuits were negatively correlated (r=-0.85 and -0.90 for harvest I and 2, respectively). It is an indication that these dimensions are probably mainly dependent on dough viscoelastic properties: following stamping, the sample length will decrease along the rolling direction and its width will increase, as a consequence of strain recovery. 3.2.2 Thickness of the dough after rolling. This parameter seems to be closely related to the relaxation rate constant following LSF and the same exponential relationship appears to hold for the two groups of flours, which are very different (figure 3). For doughs having marked elastic properties (long relaxation times) the effect of ka is strong, but, at high values of ka, dough thickness tends towards a limit. We can explain this result in terms of elastic recovery: dough, compressed during roiling, stores temporarily mechanical energy, inducing partial strain recovery. However, a high value of ka corresponds to a fast relaxation process and, therefore, to a low level of stored energy and then to a vanishing strain recovery phenomenon. Accordingly, the rollers'gap (0.2 cm) may be used for the asymptotic value of dough thickness as shown on figure 3. The same type of result is observed for biscuit thickness (see table 2) but, in addition, this parameter depends on oven rise. However, a highly significant linear correlation (r=+0.74, n=44) is observed between biscuit and dough band thicknesses: this confirms that dough viscoelasticity plays here a major role in biscuit dimensions. A linear correlation was also observed between W and dough thickness, mainly for harvest 2 (see figure 3). It is interesting to note that the extrapolated dough thickness corresponding to W=O is just found equal to the rollers'gap. 3.2.3 Density is an important quality parameter for biscuits, in particular for predicting crispness. It was found positively related to LSF indices (initial compression force Fi and t I a), but also to P from the Alveograph test (see table 2).

3.3 Correlations with bread baking tests 3.3.1 Volumes. In general, bread volumes are not well correlated with rheological parameters (see table 2). For the European test (YOE) there was only a low correlation with LSF (ka and Fi) but it was not observed with both sets of samples. French bread loaf volume (YOB) is correlated with the relaxation rate following BI (k) and with the AlvtDgr.pl parameters (G, and, less, W and P/L). Pan bread loaf volume (YOM) is significantly related.


330

80 ,-__,--,__-,__-,__-,__-,__-,__,-__-,__-,____,-__,-__,-__-,__-, 80

�

E

� c

:E Ol

�

75

70 65

� 60 " -5 00

j

•

55

1 .2

---0

0 8

0

-

Harvest 1 (0=23) Harvesl 2 (n=31)

o 0 / 00 � 0 0 0

�oor/' 0� o

50

45

�

..

r = +

0.83

o"",,0

0

o

•

0 .5' ClJ ....... 0 0 o """"o

o

1 .4

1.6

1 .8

k

2 2.2 (5.1 )

2.4

r

2.6

100

o

D

•

75

0.73

70 65

60

0

,,!

0 0

r = .

1 50

=

0.7S

200

55

""-

e c .......

50

0

W

250

300

350

Figure 4: Length of bread dough after moulding (in French bread baking) versus relaxation rate constantfollowing BI (k) and versus W.

to the flow behaviour index na deduced from stress relaxation following LSF. However, a higher correlation coefficient is obtained with G. The better correlation observed in this case may be tentatively attributed to more tightly defined baking conditions, due to sugar addition (fem1entation) and to the presence of tin walls (dough leavening and oven rise). 3 .3 .2 Length of Bread Dough After Stretching (EXT) . A high positive linear correlation is observed between the rate constant of stress relaxation following BI (k) and the length of bread dough samples issuing from the long-roller (see figure 4). However, in contrast to the results shown on figure 3, there is not a unique relationship for both sets of flours. An increase in k corresponds to a faster relaxation phenomenon: as the elastic energy stored into the dough is dissipated more rapidly, the recovery is smaller and, therefore, the final length is larger. In bubble inflation, PoIPmand the half-relaxation times t l give also rather high correlation coefficients. These results show the prevalence of the viscoelastic properties on the extensibility of bread dough during mOUlding. However, while relaxation following BI is well related to bread dough length, this is not the case with LSF: even though these two measurements imply a biaxial deformation, they don't provide the same information. A significant negative correlation coefficient is also observed with W: however, it is lower than with k (figure 4). The length of bread dough after stretching may be used as an unambiguous evaluation of dough extensibility during the process: it is interesting to note that there is no relation with G (IrIIROXIDE

In bread dough peroxidases can act without the addition of hydrogenperoxide. This indicates that ij� is present in the dough at sufficient amounts or that it is generated as a result of the peroxidase reaction, thus giving a continuous cycle of use and generation of this cosubstrate. The generation of hydrogenperoxide is thought to proceed through the following sequence of reactions (see figure 2).

The formation of substrate radicals reaction with oxygen can lead to formation of hydrogenperoxide. When these reactions are occuring, catalytic amounts of Ii� present in the dough are sufficient to get the cycle started and explains why no hydrogenperoxide has to be added together with the peroxidase. However, in a normal dough the amount of free oxygen is limited due to the presence of yeast which consumes most of the oxygen.

WHFAT FLOUR PEROXIDASE

Wheat flour contains peroxidase that can cross-link phenolic constituents like ferulic acid and vanillic acid. However, the pH optimum of the wheat enzyme is reported to be considerably lower than the pH of bread dough. In order to prove this, the pH dependence of several peroxidases was investigated in comparison with that of wheat flour peroxidase. The results are shown in figures 3a and 3b .

•

PEROXIDASE + H202

�

COMPOUND I + AH2

-.

COMPOUND II + AH.

COMPOUND II + AH2

�

PEROXIDASE + AH.

AH. + AH.

COMPOUND I

A + AH2

In presence of 02 : AH. + 02 + H+

---.

AH2 + 02-

02- + 02- + 2H+

--..

H202 + 02

I

Conclusion: only catalytic amounts of peroxide a re required

Figure 2. Sequence of hydrogenperoxide generating reactions.

354


(a) Spec.

�l

act. (Ulmg)

Spec. act.

�I

Peroxidase 1

Spec. act. (Ulmg)

Wheat flour peroxidase

pH

pH

(U/mgl

relativ• •ctivity (%) 120

Peroxidase 2

100

80 80

...,

20

03

03

6.'

Normalised activities

POX 1 • wheat tIour POX • POX 2 • PQX 3 • '---

� pH

pH

(b) WHEAT FLOUR PEROXIDASE

PEROXIDASE

Spec. acl. (Ulmgl

spec. act (Ulmgl

3

[I �I �I'� --- . .I �1 � l i�lr pH

.aTS

PEROXIDASE 1

Spec. act. IUlmg)

°3

pH

•

5

pH

NORMALISED ACTIVITIES

•

7

1

4

5

pH

•

7

Figure 3. pH dependence of 4 different peroxidases determined on guaiacol (3a) and ABTS (3b).

As

can

be seen in this figure, the pH optimum of wheat flour peroxidase is clearly lower than that of the

other three enzymes when guaiacol is used as a substrate. This could explain why wheat flour peroxidase is not active in bread dough and why added peroxidases from other sources than wheat flour may have a beneficial effect on dough strengthening. However, when ABTS is used as a substrate, the pH dependence of all four peroxidases is more or less the same. This indicates that the pH dependence of the different

peroxidases is also substrate dependent. When it is not clear on which component peroxidases are acting in a dough, it is difficult to draw the conclusion that wheat flour peroxidase is not active due to its apparent lower pH optimum.


355

ANALYSIS OF OXIDISING E'iZYMES

The analysis of glucose oxidase, sulfhydryl-oxidase and Iipoxygenase actvities are straight forward and well documented. The reason is that these enzymes catalyse well defined reactions acting on also well defined substrates. For peroxidase the situation is completely different. This enzyme catalyses the oxidation of a wide variety of compounds. This is the reason that many different assay methods have been developed for this enzyme. In table II the activity of a number of different peroxidases on a range of synthetic substrates is shown. Although the table is not completed yet, it will be clear that there are large differences in the specificities of several peroxidases. AAP and ABTS serve as hydrogen donors and this process results in a colour change of these compounds. DMABlMB1H are oxidatively coupled by peroxidase in presence of Ii� to form a deep purple compound. Guaiacol has been used for more than 30 years in the vegetable processing industries in order to determine the peroxidase activity as a measure for the blanching efficiency.

Table II. Enzymatic activities of peroxidases from several sources. Activities in !lmol/minlmg (Schenkels and van Oort, 1 994). ABTS

GUAIACOL

PYROGALLOL *

AAP

DMABI MB1H

Peroxidase I

1 49.3

325.9

1377

39 1 .5

452.5

lPeroxidase II

0.03

0.02

n.d.

0.027

n.d.

Peroxidase III

0.093

0.008

n.d.

0.13

0.2

Peroxidase IV

42 1 1

401.9

1 1 34

1 735.7

134

Iperoxidase V

0.016

0.055

n.d.

n.d.

0.03

1Peroxidase VI

526.4

1 8.5

356.4

13.3

0.46

lPeroxidase VII

6.3

n.d.

n.d

3.69

n.d.

1Peroxidase VIII

130.7

n.d.

972

288.1

n.d.

iENZVME

SUBSlRATE

Arbltrary UnIts, smce specifiC ii6SOfj)flon coettIclent ** In most assays no linear kinetics. ABTS

=

AAP DMABlMB1H

=

=

IS

not known.

2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulphonic acid) 4-aminoantipyrine 3-(dimethylamino)benzoic acid/3-methyl-2-benzothiazolinone hydrazone

Pyrogallol is one of the oldest POX substrates and is hardly used anymore because of the inferior kinetics of the POX reaction. There are more POX substrates available (e.g. o-dianisidine, 3,3-dimethoxybenzidine or o-phenylene-diamine) but many of them are hardly being used because of the harmful (carcinogenic) character of the compounds. However, the use of such enzyme activities upon application of peroxidases in breadmaking is very difficult. The mentioned substrates have no relation with the actual substrates which may be encountered by peroxidase in dough and there is no correlation between these activities and the performances of the enzymes mentioned in dough. Therefore, a relevant assay is required.

RlNcnOOAL PIROXIDASE ASSAY

In order to set up such an assay, use is made of soluble wheat pentosans, since this is one of the structures peroxidase may encounter in a dough. The rate of viscosity increase may be taken as a measure of peroxidase activity. This is shown in figure 4.

356


"0 Q)

.!!!

nI

E .... 0 c:

0.�

� III 0 (.)

.!!! >

Peroxidase 1 (ABTS Un its)

1 00

21 U +

80

14

•

1

60

11 U •

9U

40

-+

7 U

20 0

U

•

0

50

1 00

1 50

Time (sec)

200

250

Figure 4. Viscosity increase of pentosan solution due to oxidative gelation catalysed by peroxidases. A relatively high dose level of peroxidase gives an immediate and fast viscosity increase. Upon lowering the peroxidase concentration the rate of increase is going down, just as the final level of viscosity that is reached after a certain time interval. Furthennore, a lag phase is introduced with using lower peroxidase levels. The rate of viscosity increase may be correlated to the effects of peroxidases in dough strengthening. ACllON OF PIROXIDASES IN WHEAT DOUGH

Although the mechanism of the peroxidase reaction in dough is not completely understood yet, there are several theories describing the reactions that may be catalysed by these enzymes leading to the experienced dough strengthening. I n the first model (figure 5a) the gelation of wheat pentosans (arabinoxylans) is thought to proceed through the peroxidase catalysed dimerisation of ferulic acid groups which are esterified to the pentosans (Geissmann and Neukom, 1 973). This gelation is supposed to be responsible for the observed dough strengthening. The second model (figure 5b) is almost comparable and describes the oxidative coupling of pentosans (through ferul ic acid) to cysteine or tyrosine side chains of proteins (Hoseney and Faubion, 1 98 1 ). Although the gelation type of effects of peroxidase are well documented, it is still not clear whether peroxidases act on these carbohydrate structures in the dough, especially since a positive effect of a mixture of xylanases and peroxidases is found. As can be seen in figure 6, peroxidase is not able to gelate pentosans anymore when xylanase has (partly) hydrolysed these carbohydrates. Pentosans without added xylanase immediately gelate in presence of peroxidase and hydrogenperoxide. This can be seen by the prompt and strong viscosity increase. When xylanase can react on the pentosans prior to peroxidase action, the viscosity increase is much lower and after a short period the viscosity even decreases. Upon longer pre-incubation with xylanase there is no viscosity increase anymore. From these experiments it may be concluded that peroxidase is not acting on the soluble pentosans in wheat dough. However, since xylanases are also able to produce more soluble pentosans in a dough by converting insoluble hemicellulose, there may be sufficient pentosans present in a dough for peroxidase to work on. Model studies with peroxidase, glutathion and cysteine have indicated that sulfhydryl groups may be involved (Matheis et aI., 1987) in the peroxidase reaction and that the dough strengthening effects of peroxidases can be explained by an action of this enzyme on proteins. Peroxidase catalysed reaction results in a decreased amount of lysine recovered from proteins after acid hydrolysis. The peroxidases, or the quinones fonned by peroxidase, oxidatively deaminated Iysyl residues to fonn lysyl-aldehydes. This results in the formation of dimers, trimers and higher protein polymers, as was revealed by gel filtration (Stahmann, 1 977). These aggregates were not dissociated by detergents which means that covalent bonds were fonned.


(a) �,,"

� � -

>

o

4

6

sunflower seed

8

(%)

o

4

linseed

(%)

6

Figure 5 The effect ofadding sunflower seed and linseed on the volume of baking tests

Non-Starch Polysaccharides and Enzymic Improvement of Bread Quality The addition of 4

375

%, 6 % and 8 % sunflower seed to the dough decreased gradually

the volume of baking tests. The seeds dilute the gluten and break the structure of it resulting in less gas retention ability and volume comparing to the control. There are big pores around the seeds, the texture of crumb is uneven.

The addition of 4 %, and 6 % linseed to the dough produced more pronounced decrease in the volume of baking tests. Because of the grains of the linseed are smaller

than that of the sunflower seed, in the same weight % there are more seed. Consequently, the gluten structure is more interrupted, the gas retention ability and volume is poorer, the porosity of the crumb is more uneven comparing to the baking tests made with sunflower seed (Figure 5).

4. CONCLUSION

As it was shown, most of the investigated additives have an unfavourable effect on the structure of gluten network, on thc volume and porosity of baking tests, and on the texture of bread crumb. The "non wheat dough components" such as oilseeds and oat bran, dilute the gluten and interrupt the structure of dough resulting in less gas retention ability and poorer volume comparing to the control samples. But by means of the rheological methods which was shown above, we can find optimum concentration ranges or combinations of these additives and dough improvers in the industrial practice. On the other hand, the sensory properties of such baked products containing these additives are well accepted, and their biological value due to the increase of unsaturated fatty acids and dietary fibre content has improved.

References H. 1. G. Wutzel and W. F. Wutzel, "Functional Properties of Food Proteins" Ed.: R. Usztity, METE, Budapest, 1988, p. 1 54. 2. Hungarian Standard 6369/6-73. 3. Hungarian Standard 6369/8-71 .

1.

Subject Index

Arabinoxylan dough properties and baking performance, 37 1 -375 flour milling fractions, 368-370 Bread bread making quality and emulsifiers, DATEM, 279-285 bread making quality of durum wheat, 160-166 effect of bran on bread making quality, 37 1 -375 gliadins and bread making quality, 1 73-179, 1 80- 1 83 glutenin polymer properties and bread making quality, 1 46- 1 52 high Mr glutenin subunits and bread making quality, quality relationships, 1 22- 1 23, 1 25- 1 26, 146- 1 52, 160- 1 66, 173-1 79, 1 801 83 improvement by oxido-reductases, 350-360, 361 -367 improvement by xylanases, 343-349 improver mix, 286-292 lipids and lipid-binding proteins, 245260 low Mr glutenin subunits and bread making quality, 1 25- 1 26 rheological assessment of bread making quality, 309, 3 1 6, 323 use of transgenic wheats to characterise determinants of bread making quality, 1 99, 206 Dough Alveograph measurements, 1 53, 3 1 6 microstructure, 332-334 quality assessment through dough rheology, 309, 3 1 6, 323

rheological characterisation, 295-308, 3 1 6-322 rheology and gas cell stability, 3093 1 5, 3 1 6-322 strain hardening and gas cell wall failure, 3 1 6-322 Durum wheat, 1 53- 1 58 bread making quality and gluten proteins, 1 60- 1 66 translocation lines, l BUIRS, in T. durum, 1 53-158 Emulsifiers bread improver, 286-292 bread making quality, 279-285 DATEM, 279-285 Glutathione effect of dough mixing, 227-230, 235240 flour, 224, 225-227, 235-240 measurement, 22 1 , 235 protein bound, 221 -232, 235-240 rheological effects, 240-24 1 Gluten, 1 06 (see also Proteins) genetic engineering, 1 99 relationships with bread making quality in durum wheat, 1 60- 166 rheology during baking, 1 06- 1 1 1 Grain fracture characteristics, 3 1 -35 macrostructure and properties, 19, 25 image analysis, 1 9-20 mechanical/fracture properties, 25-30 quality relationships, 20-23 sprouted grain/alpha-amylase, 22-23 protein synthesis and deposition, 44-49 quality relationships, 3 1 -35 sample preparation methods, 37-43 Triticum durum, 1 46, 1 53, 1 60

378


Grain (continued) Triticum tauschii, 1 39 Tritordeum, 1 67- 1 72 ultrastructure and properties, 3 1 , 37, 44 environmental effects, 44-49

conformational stability, 1 1 9- 1 2 1 , 136 glycosylation, 74-77, 79-83 protein engineering, 2 1 1 , 2 1 6 quality relationships, 1 22- 1 23 , 1 251 26, 1 46- 1 52, 1 60- 1 66, 1 731 79, 1 80- 1 83 transgenic wheats, 1 99, 206 Triticum tauschii, 1 39- 1 45 Tritordeum, 1 67- 1 72 lipid binding, 249 lipid transfer proteins (LTP), 249 low Mr glutenin subunits, 79, 85, 1 1 7 glycosylation, 79-83 mutated gliadins, 1 23- 1 24 protein engineering, 1 99 quality relationships, 1 25- 1 26 Triticum tauschii, 1 39- 1 45 Tritordeum, 1 67 - 1 72 molecular biology, 1 99 pathogenesis-related, 1 84 puroindolines, 250 relationships with bread making quality in durum wheat, 1 60- 1 66 structure, 53, 63, 70, 74, 79, 85, 90, 1 17 cysteine residues, 1 17- 1 22 domain structure, 53-60, 1 1 7- 1 22 primary and secondary, 53-60, 1 1 71 22 �-tums and �-spiral. 55-60 Triticum tauschii, 1 39- 1 45 Tritordeum, 1 67- 1 72

Lipids and lipid-binding proteins, 245260 emulsifiers in bread making, 279-285 foam stability, 245-260 functionality, 245-260 glycolipids, 27 1 -278 monoclonal antibodies, 27 1 -278 starch, 26 1 -270 Mixograph, 1 46 Pasta, 1 46 Proteins anti-fungal, 1 84 capillary electrophoretic analysis, 1 28133 cysteine and glutathione mixed disulphides, 221 -233, 235-24 1 fractionation and analysis, 90, 1 1 2, 1 28 genetic engineering of gluten proteins, 1 99 gliadins, 57-60, 63 capillary electrophoretic analysis, 1 28- 1 3 1 disulphide structure, 63-69 dot-blot assay for in gluten-free dietary products, 1 89 protein engineering and expression in E. coli, 2 1 5 quality relationships, 1 73 - 1 79, 1 801 83 glutenin polymers, 90 composition, 92 dough mixing changes, 96-98 fractionation, 90-92 heat effects, 1 02- 1 04 rheological properties, 95, 99- 1 05 quality relationships, 1 46- 1 52 structure, 93-95 high Mr glutenin subunits, 54-60, 7073, 74, 79� 92, 1 1 7, 146- 1 52 capillary electrophoretic analysis, 1 3 1 - 1 32

Quality, 1 - 1 5 assessment methods, 1 -9 emulsifier effects, 279-285 environmental effects, 1 1 - 1 3 gliadin relationships, 1 73- 1 79, 1 80- 1 83 glutenin polymer property relationships, 1 46- 1 52 high Mr glutenin subunit relationships, 1 22- 1 23, 1 25- 1 26, 1 46- 1 52, 1 601 66, 1 73 - 1 79, 1 80- 1 83 improvement through genetic engineering, 1 99 improver, 286-292 lipids and lipid-binding proteins, 245260, 286-292

Subject Index

Quality (continued) low Mr subunit relationships, 1 17, 1251 26, 146- 1 52 malting and brewing, 192 modelling and prediction, 1 3 rheological assessment, 3 16-322, 32333 1 , 336-338 storage protein alleles, 9- 1 3, 1 17- 1 27 translocation lines, l BUIRS, in T. durum, 1 53-158 Rheology biscuit dough, 336-338 dough rheological characterisation, 295, 309, 3 1 6, 323, 336 dough rheology and gas cell stability, 309-3 1 5

379

enzymes and dough rheology, 361 -367 glutathione and dough rheology, 240241 gluten effect of xylanases, 347 quality assessment through dough rheology, 309, 3 16, 323, 336 rheological properties, 95, 99- 105 rheology during baking, 106- 1 1 1 Starch, 261 -270 isolation, 262 lipids, 26 1 -263 physical properties, 263-265 structure, 265-268 Transgenic wheats, 199, 206

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Geoarchaeology: Exploration, Environments, Resources (Geological Society Special Publication, No. 165) (Geological Society Special Publication, No. 165)

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Wheat Structure, Biochemistry and Functionality (Special Publication)

Carbohydrate Chemistry and Biochemistry: Structure and mechanism

Carbohydrate Chemistry and Biochemistry: Structure and mechanism

Palaeozoic Reefs and Bioaccumulations: Climatic and Evolutionary Controls - Special Publication no 275 (Geological Society Special Publication)

Cytochrome P450: Structure, Mechanism, and Biochemistry

Fjord Systems and Archives : Special Publication 344

Endotoxins: Structure, Function and Recognition (Subcellular Biochemistry)

Food Macromolecules and Colloids (Special Publication)

Mine Water Hydrogeology and Geochemistry (Special Publication)

The Gluten Proteins (Special Publication)

Fishes and the Break-up of Pangea - Special Publication no 295 (Special Publication)

Devonian Events and Correlations : Special Publication no 278 (Geological Society Special Publication)

Non-Marine Permian Biostratigraphy and Biochronology - Special Publication No. 265 (Geological Society Special Publication)

Wheat Antioxidants

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Geoarchaeology: Exploration, Environments, Resources (Geological Society Special Publication, No. 165) (Geological Society Special Publication, No. 165)

Communicating Environmental Geoscience - Special Publication no 305 (Geological Society Special Publication) (No. 305)

The Evolving Continents: Understanding Processes of Continental Growth - Special Publication 338 (Geological Society Special Publication)

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Building Stone Decay: From Diagnosis to Conservation - Special Publication no 271 (Geological Society Special Publication)

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