Coffee: Recent Developments

COFFEE Recent Developments Edited by R.J. Clarke and O.G. Vitzthum b Blackwell Science # 2001 by Blackwell Science ...

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COFFEE Recent Developments Edited by R.J. Clarke and O.G. Vitzthum

b

Blackwell Science

# 2001 by Blackwell Science Ltd Editorial Offices: Osney Mead, Oxford OX2 0EL 25 John Street, London WC1N 2BS 23 Ainslie Place, Edinburgh EH3 6AJ 350 Main Street, Malden MA 02148 5018, USA 54 University Street, Carlton Victoria 3053, Australia 10, rue Casimir Delavigne 75006 Paris, France Other Editorial Offices: Blackwell Wissenschafts-Verlag GmbH KurfuÈrstendamm 57 10707 Berlin, Germany Blackwell Science KK MG Kodenmacho Building 7±10 Kodenmacho Nihombashi Chuo-ku, Tokyo 104, Japan Iowa State University Press A Blackwell Science Company 2121 S. State Avenue Ames, Iowa 50014-8300, USA The right of the Author to be identified as the Author of this Work has been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. First published 2001 Set in 9.5/11 Ehrhardt by DP Photosetting, Aylesbury, Bucks Printed and bound in Great Britain by MPG Books Ltd, Bodmin, Cornwall The Blackwell Science logo is a trade mark of Blackwell Science Ltd, registered at the United Kingdom Trade Marks Registry

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Marston Book Services Ltd PO Box 269 Abingdon Oxon OX14 4YN (Orders: Tel: 01235 465500 Fax: 01235 465555) USA and Canada Iowa State University Press A Blackwell Science Company 2121 S. State Avenue Ames, Iowa 50014-8300 (Orders: Tel: 800-862-6657 Fax: 515-292-3348 Web www.isupress.com email: [email protected] Australia Blackwell Science Pty Ltd 54 University Street Carlton, Victoria 3053 (Orders: Tel: 03 9347 0300 Fax: 03 9347 5001) A catalogue record for this title is available from the British Library ISBN 0-632-05553-7 Library of Congress Cataloging-in-Publication Data Coffee: recent developments/edited by R.J. Clarke and O.G. Vitzthum p. cm. Includes bibliographical references and index. ISBN 0-632-05553-7 1. Coffee. I. Clarke, R.J. (Ronald James) II. Vitzthum, O.G. TP645 .C65 2001 663'.93Ðdc21 00-049815 For further information on Blackwell Science, visit our website: www.blackwell-science.com

Contents Preface vii List of Contributors ix 1 Chemistry I: Non-volatile Compounds 1 1A Carbohydrates 1 A.G.W. Bradbury 1.1 Introduction 1 1.2 Green coffee 1 1.2.1 Low molecular weight carbohydrate 1 1.2.2 High molecular weight carbohydrate 3 1.3 Roast coffee 6 1.3.1 Low molecular weight carbohydrate 6 1.3.2 High molecular weight carbohydrate 7 1.4 Soluble coffee 8 1.4.1 Low molecular weight carbohydrate 8 1.4.2 High molecular weight carbohydrate 12 1.5 Reactions of carbohydrates on roasting 13 1.6 Functional properties of coffee carbohydrates 14 1.6.1 Role in soluble coffee processing 14 1.6.2 Foam 15 1.6.3 Coffee fiber 15 References 15 1B Acids in Coffee 18 H.H. Balzer 1.7 Quantitative data on organic acids in green coffee 18 1.8 Determination of organic acids in roasted coffee 19 1.9 Acid formation mechanisms 23 1.9.1 Acetic, formic, lactic, glycolic and other carbohydrate derived acids 23 1.9.2 Quinic acid 23 1.9.3 Citric and malic acid 25 1.9.4 Phosphoric acid 25 1.10 Acid increase on storage 26 1.11 Volatile acids 26 1.12 Acid content and sensory characteristics 27

1.12.1 Total acidity and sour taste 1.12.2 Acid content and acidity 1.12.3 Roast kinetics References 1C Lipids K. Speer and I. KoÈlling-Speer 1.13 Introduction 1.14 Coffee oil 1.14.1 Determination of total oil content 1.14.2 Isolation of coffee oil for detailed analysis 1.15 Fatty acids 1.15.1 Total fatty acids and fatty acids in triglycerides 1.15.2 Free fatty acids 1.16 Diterpenes in the lipid fraction of robusta and arabica coffees 1.16.1 Free diterpenes 1.16.2 Diterpene fatty acid esters 1.16.3 Diterpenes in the lipid fraction of roasted coffees 1.16.4 Diterpenes in coffee: health aspects 1.17 Sterols 1.18 Tocopherols 1.19 Other compounds 1.20 Coffee wax References 2 Chemistry II: Non-volatile Compounds, Part II S. Homma 2.1 Amino acids and Protein 2.1.1 Amino acids 2.1.2 Amino acid derivatives 2.1.3 Protein 2.2 Fate of chlorogenic acid derivatives during roasting 2.2.1 Quinic acid moiety 2.2.2 Cinnamic acid derivative moiety 2.3 Antioxidative compounds in coffee brew 2.3.1 Compounds occurring naturally in green beans

iii

27 28 29 30 33 33 33 33 34 34 34 35 36 38 38 39 41 41 42 44 45 46 50 50 50 51 51 54 54 56 57 57

iv

Contents

2.3.2

Effect of roasting on antioxidative activity 2.4 Colored macromolecular compounds 2.4.1 Characterization of colored polymers 2.4.2 Characterization of the zincchelating compounds in coffee brews References 3 Chemistry III: Volatile Compounds W. Grosch 3.1 Introduction 3.2 Methodology 3.2.1 Isolation of the volatile fraction 3.2.2 Screening for potent odorants 3.2.3 Enrichment and identification 3.2.4 Quantification 3.2.5 Aroma models and omission experiments 3.3 Raw coffee 3.3.1 First studies 3.3.2 Potent odorants 3.3.3 Content and OAVs of odorants 3.3.4 Contaminants causing off-flavour 3.4 Roasted coffee 3.4.1 Concentration of important odorants 3.4.2 Evaluation of key odorants 3.4.3 Arabica versus robusta coffee 3.4.4 Influence of degree of roast 3.4.5 Aroma changes during storage 3.5 Coffee brew 3.5.1 Extraction yield of potent odorants 3.6 Formation of odorants 3.6.1 Mono- and dicarbonyl compounds 3.6.2 Furanones 3.6.3 Alkylpyrazines 3.6.4 Phenols 3.6.5 Thiols 3.7 Conclusions References 4 Technology I: Roasting R. Eggers and A. Pietsch 4.1 Introduction 4.2 Roasting methods and their parameters 4.2.1 General

58 58 58 62 65 68 68 69 69 69 71 72 73 73 73 73 73 75 75 75 77 77 79 79 80 80 82 82 83 84 84 84 85 85 90 90 90 90

4.2.2 4.2.3 4.2.4 4.2.5

Conventional roasting Fluidized bed roasting Fast roasting Detection of optimum degree of roast 4.3 Bean behaviour during roasting 4.3.1 Bean temperature, mass and moisture 4.3.2 Swelling and structure 4.3.3 Decaffeinated coffee 4.4 Heat and mass transport 4.4.1 Complexity of the process 4.4.2 Specific heat of coffee 4.4.3 Thermal conductivity 4.4.4 Heat uptake of the bean 4.4.5 Temperature profiles in the bean 4.4.6 Heat transfer from gas to bean and overall heat transfer coefficient 4.5 Some aspects on future scientific research 4.6 Industrial roasting equipment 4.6.1 Traditional roasters 4.6.2 Fluidized beds 4.6.3 Packed bed roasting 4.6.4 Roasting with heated cooling gas 4.6.5 Technical data and capacities 4.6.6 Roaster patents 1986±99 References 5 Technology II: Decaffeination of coffee W. Heilmann 5.1 Introduction 5.2 Solvent decaffeination 5.3 Water decaffeination 5.4 Supercritical CO2 decaffeination 5.5 Liquid CO2 decaffeination 5.6 Decaffeination with fatty material 5.7 Special developments 5.7.1 In-home decaffeination 5.7.2 Coffee with adjusted caffeine content 5.8 Caffeine recovery from activated carbon 5.9 Economic aspects References 6 Technology III: Instant Coffee R.J. Clarke 6.1 Introduction 6.1.1 Instant coffee in the market place

91 91 92 92 93 93 94 95 96 96 96 97 97 98 99 100 101 101 101 104 104 104 104 107 108 108 109 110 113 118 118 119 119 119 119 122 123 125 125 125

Contents

v

6.1.2 6.1.3

New technology The legacy of Professor H.A.C. Thijssen 6.1.4 Legislation and standardization 6.2 Processing 6.2.1 General 6.2.2 Roasting/grinding 6.2.3 Extraction 6.2.4 Freeze concentration of extracts 6.2.5 Thermal concentration and volatile compound recovery 6.2.6 Volatile compound handling 6.2.7 Reverse osmosis 6.2.8 Spray drying and agglomeration 6.2.9 Freeze drying 6.2.10 Aromatisation 6.2.11 Spent grounds disposal 6.2.12 Grading, storage and blending of green coffees 6.2.13 Liquid extracts 6.3 Physical properties of volatile compounds 6.3.1 Important physical properties in relation to instant coffee processing 6.3.2 Tables of physical properties References 7 Technology IV: Beverage Preparation: Brewing Trends for the New Millennium M. Petracco 7.1 Introduction 7.2 Extraction methods 7.2.1 Decoction methods 7.2.2 Infusion methods 7.2.3 Pressure methods 7.3 Beverage characterization 7.3.1 Physical and chemical characteristics 7.3.2 Organoleptic characteristics 7.4 Modified coffee beverages 7.4.1 Coffee±milk admixtures 7.4.2 Canned coffee beverages 7.4.3 Flavoured coffee beverages References 8 Health Effects and Safety Considerations B. Schilter, C. Cavin, A. Tritscher and A. Constable 8.1 Introduction 8.2 Objectives and scope

125 125 126 126 126 127 127 128 129 130 130 130 131 132 132 132 132 133 133 137 137 140 140 141 143 144 145 151 151 157 160 160 161 161 162 165 165 165

8.3 8.4

Coffee Coffee 8.4.1 8.4.2 8.4.3 Coffee 8.5.1

consumption and cancer Human data Experimental data Conclusions 8.5 and cardiovascular disease Myocardial infarction or coronary death 8.5.2 Arrhythmias 8.5.3 Caffeine and blood pressure 8.5.4 Serum cholesterol 8.5.5 Serum homocysteine 8.5.6 Conclusions 8.6 Coffee and bone health 8.6.1 Calcium metabolism 8.6.2 Osteoporosis 8.6.3 Conclusions 8.7 Reproductive and developmental potentials of coffee and caffeine 8.7.1 Congenital malformations 8.7.2 Neurodevelopmental effects 8.7.3 Low birth weight, growth retardation and prematurity 8.7.4 Spontaneous abortion 8.7.5 Fertility 8.7.6 Conclusions 8.8 Emerging benefical health effects 8.8.1 Neuroactivity 8.8.2 Chemoprotection 8.9 Coffee consumption ± safety considerations 8.10 Conclusions References 9 Agronomy I: Coffee Breeding Practices H.A.M. Van der Vossen 9.1 Introduction 9.1.1 World production increase 9.1.2 Selection and breeding before 1985 9.1.3 New developments 9.2 Genetic resources 9.2.1 World collections 9.2.2 Species relationships 9.2.3 Conservation 9.3 Breeding 9.3.1 General objectives and strategies 9.3.2 Productivity 9.3.3 Quality 9.3.4 Resistance to coffee leaf rust 9.3.5 Resistance to coffee berry disease

166 166 166 167 168 169 169 170 170 171 172 172 173 173 173 174 174 174 175 175 176 177 177 177 177 178 178 179 179 184 184 184 184 185 186 186 186 188 189 189 189 191 192 193

vi

9.3.6 Resistance to other diseases 9.3.7 Resistance to nematodes 9.3.8 Resistance to insect pests 9.3.9 Drought tolerance 9.4 Propagation of new cultivars 9.4.1 Seeds 9.4.2 Clonal propagation Abbreviations References 10 Agronomy II: Developmental and Cell Biology M.R. Sondahl and T.W. Baumann 10.1 Overview 10.2 Organ development and the allocation of defense compounds 10.2.1 Introduction 10.2.2 The leaf 10.2.3 The fruit 10.3 Purine alkaloid formation in coffee cell cultures 10.3.1 Introduction 10.3.2 Callus culture 10.3.3 Suspension culture 10.4 New advances in cell and organ culture 10.4.1 Brief review of the literature 10.4.2 New advances 10.5 Coffee scale-up by micropropagation 10.5.1 Mass production of somatic embryos 10.5.2 Applications 10.6 Somaclonal variation and new breeding lines 10.6.1 Definitions and examples 10.6.2 Coffee somaclonal variation program 10.6.3 Commercialization of new varieties 10.7 Summary Abbreviations References 11 Agronomy III: Molecular Biology J.I. Stiles 11.1 Introduction

Contents

194 195 195 196 196 196 196 197 197

11.2 11.3 11.4 Appendix 1.1 1.2

202 202 202 202 203 205

1.3 1.4 1.5 Appendix 2.1

207 207 207 208 209 209 209 212 213 215 217 217 217

2.2 2.3 Appendix 3.1

218 219 220 220 224 224

Index

3.2

Coffee genes Transformation systems for coffee Prospects References 1 International Standards Organization (ISO) Glossary relating to coffee and its products Green coffee (guides and sampling procedures) Instant coffee (sampling procedures) Methods of test (chemical or physical) General comments 2 International Coffee Organization (ICO) The International Coffee Agreement 1994 2.1.1 Background 2.1.2 Priorities 2.1.3 Coffee development projects 2.1.4 Promotional activity 2.1.5 Involvement of the private sector 2.1.6 Statistics and information 2.1.7 Global research network on coffee 2.1.8 Economic studies and publications 2.1.9 Towards a new Agreement in 2001 Conclusions Statistical information 3 Units and Numerals Units 3.1.1 SI base units 3.1.2 Some derived SI units used in engineering 3.1.3 Some prefixes for SI units 3.1.4 Some conversions of SI and non-SI units References Numerals ± cardinal

225 230 231 233 235 235 235 235 235 236 238 238 238 238 238 238 239 239 239 239 239 239 240 242 242 242 242 243 243 245 245 246

Preface A considerable period of time has now passed since the publication of six volumes upon all the technical aspects of coffee (Chemistry ± Technology ± Physiology ± Agronomy ± Related Beverages ± Commercial and Technico-legal) by Clarke and Macrae (eds), 1985± 8; and indeed since other major comprehensive volumes on coffee by Clifford and Willson (eds), 1985; and by Sivetz and Desrosier in 1979. Sadly, Robert Macrae was to die at an early age in 1995, and his literary and scientific work is sorely missed. The present editors considered that the time is now well due for an update on the topics covered by these volumes. Since 1987, seven Colloquia, organised by the Association Scientifique Internationale du CafeÂ, on coffee, have been held, with some 500 papers published in their Proceedings. Accordingly, we have been fortunate enough to secure the contributions of more than 15 internationally respected coffee scientists and technologists around the world to provide our readers with generally new information in the various areas in which they are expert. We have arranged for three updating chapters on the non-volatile and volatile compounds present, including some new ones, but especially the important ones, now known more clearly, to determine flavour. There follows four updating chapters on coffee technology; one, specifically, on some new developments in roasting techniques; two others on instant coffee and decaffeination processing; whilst another reflects recent developments in home/catering beverage preparation, especially of the true Italian espresso type. The

physiological effects of coffee drinking continue to interest and concern both the scientific and general public, so that the numerous publications and investigations of the last decade are considered in a comprehensive chapter on coffee and health. Agronomic aspects continue also to show considerable progress in the areas of coffee plant breeding, by conventional and tissue culture techniques; and to show rapid developments in a detailed understanding of the genes present in the coffee plant, and their function, and so to the possibilities and actualities of genetic engineering for desired characteristics, for example caffeine-free. Finally, some other topics relevant to coffee, that is, recent activities of some international organisations, such as the International Coffee Organization, are reviewed in a lengthy Appendix. Any opinions expressed by our contributors are those of the contributors themselves, and do not necessarily reflect those of the editors. R.J. Clarke M.A. (Oxon), Ph.D(Hons), C.Eng., F.I.Chem.E, F.I.F.S.T. Consultant, Chichester, UK O.G. Vitzthum Honorary Professor, Technical University of Braunsweig Scientific Secretary, Association Scientifique Internationale du CafeÂ, Paris (ASIC) Formerly Head of Coffee Research, Kraft Jacobs Suchard, Bremen, Germany

vii

List of Contributors Dr Hartmut H. Balzer, Kraft Foods R and D Inc., Unterbibergerstrasse 15, D-81737, Munich, Germany (1B Acids in Coffee) Professor Thomas W. Baumann, Institute of Plant Biology, University of Zurich, Switzerland (10 Agronomy II: Developmental and Cell Biology) Dr Allan G.W. Bradbury, Kraft Foods R and D Inc., Unterbibergerstrasse 15, D-81737, Munich, Germany (1A Chemistry I: Non-volatile Compounds) Christophe Cavin, Nestle Research Center, Department of Quality and Safety Assurance, PO Box 44, Vers chez-les Blanc, CH1000 Lausanne 28, Switzerland (8 Health Effects and Safety Considerations) Dr Ronald J. Clarke, Ashby Cottage, Donnington, Chichester, West Sussex, PO20 7PW, UK (6 Technology III: Instant Coffee) Anne Constable, Nestle Research Center, Department of Quality and Safety Assurance, PO Box 44, Vers chez-les Blanc, CH1000 Lausanne 28, Switzerland (8 Health Effects and Safety Considerations) Mr C. Pablo R. Dubois, International Coffee Organization, 22 Berners Street, London, W1P 4DD, UK Professor Dr.-Ing. Rudolf Eggers, Technical University Hamburg-Harburg, Eissendorfer Strasse 38, D-21073 Hamburg, Germany (4 Technology I: Roasting) Professor Werner Grosch, Burgstrasse 3B, D-85604, Zermeding, Germany (3 Chemistry III: Volatile Compounds) Dr Wolfgang Heilmann, Hollerender Weg 50, D-28355 Bremen, Germany (5 Technology II: Decaffeination) Professor Selichi Homma, Ochanomizo University, Department of Nutrition and Food Science, Ohtsuka 2-1-1 Bunkyo-ko, Tokyo, 112-8610, Japan (2 Chemistry II: Non-volatile Compounds Part II) Dr Isbelle KoÈlling-Speer, Institut fuÈr Lebensmittelchemie, Facultat fuÈr Mathematik and Naturwissenschaften. Technische Universitat Dresden, D01062 Dresden, Germany (1C Lipids)

Marino Petracco, Illycaffe Spa, Via Flavia 110, 34147 Trieste, Italy (7 Technology IV: Beverage Preparation: Brewing Trends for the New Millennium) Dr.-Ing. Arne Pietsch, Eurotechnica IngenieurbuÈro GmbH, Bartemeide, Germany (4 Technology I: Roasting) Dr Benoit Schilter, NestleÂ Research Center, Department of Quality and Safety Assurance, PO Box 44, Vers-chez-les Blanc, CH1000 Lausanne 28, Switzerland (8 Health Effects and Safety Considerations) Dr Maro Sondahl, Fitolink Corporation, 6 Edinburgh Lane, Mount Laurel, 08054 NJ, USA (10 Agronomy II: Developmental and Cell Biology) Professor Karl Speer, Institut fuÈr Lebensmittelchemie, Facultat fuÈr Mathematik und Naturwissenschaften. Technische UniversitaÈt Dresden, D-01062 Dresden, Germany (1C Lipids) Dr John Stiles, Integrated Coffee, Technologies Inc., 4 Waterfront Plaza, Suite 575, 500 Ala Moana Boulevard, 96813 Honolulu, Hawaii, USA (11 Agronomy III: Molecular Biology) Angelika Tritscher, Nestle Research Center, Department of Quality and Safety Assurance, PO B ox 44, Vers-chez-les Blanc, CH1000 Lausanne 28, Switzerland (8 Health Effects and Safety Considerations) Dr Herbert Van der Vossen, Steenhuil 18, 1606 CA Venhuizen, The Netherlands (9 Agronomy I: Coffee Breeding Practices)

ix

Chapter 1

Chemistry I: Non-volatile Compounds 1A:

Carbohydrates

A.G.W. Bradbury Kraft Foods R and G Inc., Munich, Germany 1.1 INTRODUCTION

(1) Sucrose contents for the arabica samples varied from 6.25% to 8.45% and those for the robusta samples from 0.9 to 4.85% (apart from two low values of 0.9 and 1.25%, the robustas analyzed fell in the range given above). (2) Robustas contained more reducing sugars than arabicas. (3) Apart from sucrose and one robusta which contained a minimal content of maltose (0.01%), there was no evidence of other simple oligosaccharides such as the `flatulent sugars', raffinose or stachyose, in the green beans.

This review summarizes the literature in the field of coffee carbohydrate chemistry with an emphasis on work done since the chapter on the subject by Trugo (1985). The importance of the carbohydrate fraction in coffee is evident in its high content; on a dry weight basis, it constitutes about half of the green coffee bean. Low and high molecular weight carbohydrate components are present in green coffee; these both participate in the extensive chemical changes associated with coffee roasting. Carbohydrates are also present at a high level in roasted and soluble coffee products. In this chapter, the contents and properties of the carbohydrate fractions in green, roast and soluble coffees will be described.

It would be expected that the action of endogenous enzymes during ripening or processing would lead to the presence of monosaccharides derived from the polysaccharides and, indeed, small yields of arabinose were found in all coffees, and mannose was found in most coffees, although none of the samples contained free galactose. The sugar data described above was obtained mainly by use of HPLC or GC-based methods. However, the recent advent of ion exchange chromatography coupled with ampometric detection allows excellent resolution with low detection limits, and it is now the preferred technique for the analysis of sugars in coffee, particularly in commercial soluble products (see section 1.4.1). By use of this technique, Zapp (personal communication) obtained sucrose contents between 2.60% and 3.02% for three green Indonesian robustas coffees. He also obtained values of 6.6% for a dry processed arabica (Brazil) and 7.02% (New Guinea) and 6.5% (Mexico) for two wet processed arabica samples. It would be expected that sucrose content would increase with degree of ripening and this was apparent with defective coffee beans, where for both immatureblack and immature-green Brazilian beans, sucrose

1.2 GREEN COFFEE 1.2.1 Low molecular weight carbohydrate The principal low molecular weight carbohydrate or sugar in green coffee is sucrose; the monosaccharide content is relatively low. Published values show much variation among bean types, although, in general, arabican varieties tend to contain about twice as much sucrose as robustas. Most literature values for sucrose are in the range of approximately 2% to 5% for robusta beans and 5% to 8.5% for arabicas (summarized by Clifford 1985). Silwar and LuÈllman, (1988; LuÈllman & Silwar, 1989) used an HPLC-based method to determine the low molecular weight carbohydrate profile of 20 green coffee samples from 13 different producer countries) (Table 1.1). The key findings from their work can be summarized as follows: 1

2

Table 1.1 1988.) Coffee

Coffee: Recent Developments

Mono- and disaccharide content of green arabica and robusta coffees. (From Silwar & LuÈllman, Sucrose

Fructose

Glucose

Mannose

Arabinose

Rhamnose

Total

Arabica Colombia Colombia Salvador Brazil Brazil Kenya Kenya Tanzania Ethiopia Ethiopia New Guinea East India

8.20 8.30 7.30 6.65 6.30 8.45 7.05 7.55 6.30 6.25 7.70 6.50

0.15 0.07 0.02 0.15 0.15 0.02 0.03 0.20 0.40 0.25 0.07 0.04

< 0.01 0.30 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 0.45 0.40 0.45 < 0.01 < 0.01

ND ND ND 0.02 0.10 b-sitosterol > campesterol, while for the sterol esters the order is b-sitosterol > campesterol > stimasterol. Furthermore Picard et al. studied the individual fatty acids in the sterol esters, C18 , C16 and C18:1 were the main compounds, with a proportional distribution

42

Table 1.26


Sterols identified in coffee.

Sterols 4-Desmethylsterols Cholesterol* Campesterol* Stigmasterol* b-Sitosterol* 5-Avenasterol* Cholestanol* Campestanol* 24-Methylenecholesterol* Stigmastanol* = sitostanol 7-Stigmastenol* 7-Avenasterol* 7-Campesterol* 5,23-Stigmastadienol 5,24-Stigmastadienol = fucosterol Clerosterol* Brassicasterol 4-Methylsterols Citrostadienol* Cycloeucalenol* Obtusifoliol* Gramisterol* 24-Methylenelophenol 24-Ethylenelophenol 4a,24R-Dimethyl-5a-cholest-8-en-3b-ol 4a,24R-Dimethyl-5a-cholest-7-en-3b-ol 4a, 24R Methyl-5a-stigmast-8-en-3b-ol 4,4-Dimethylsterols Cycloartenol* 24-Methylenecycloartanol* Cycloartanol* Cyclobranol b-Amyrin

References N N N N N N N

I I I I I

I I

T T T T T T T T T T

P P P P P

P P

D D D D D

D D D

I N N N N

I I I

N N N N N

T T T T T T T

I I I I I

T T T

P P P P

M M M M M M M M M M M M M M M

F F F F F

S S S S S

F F

S S S S

F

S S S

P

P P

F F

S S

* Structural formula is given D = Duplatre et al. (1984), F = Frega et al. (1994), I = Itoh et al. (1973a,b), M = Mariani & Fedeli (1991), N = Nagasampagi et al. (1971), P = Picard et al. (1984), S = Speer et al. (1996), T = Tiscornia et al. (1979).

similar to that reported in triglycerides. Roasting the coffee beans hardly affected the amounts and the distribution of the sterols (Duplatre et al., 1984; Speer et al., 1996). No changes, either, have been observed after industrial steaming processes according to the Lendrich procedure (Speer et al., 1996). In order to examine the sterols in coffee infusions, a Scandinavian style coffee, an espresso and a filter coffee were analyzed. Cholesterol, campesterol, stigmasterol, b-sitosterol, stigmastanol, 5-avenasterol, 7-stigmastenol, 7-avenasterol, citrostadienol, gramisterol and cycloartenol and traces of 24-methylenecycloarte-

nol were identified and quantified in all the coffee infusions. As reported for the diterpenes, filter coffee obtained the lowest content of sterols (Fig. 1.27) (Speer et al., 1996).

1.18 TOCOPHEROLS The presence of tocopherols in coffee oil was described by Folstar et al. (1977) for the first time, a-tocopherol was clearly identified, while b- and g-tocopherol, not being separated by TLC and GC, were considered as

Chemistry I: Non-volatile Compounds: Lipids

Fig. 1.24 4-Desmethylsterols.

one group (Fig. 1.28). Cros et al. (1985) also determined total b-and g-tocopherol by HPLC. Folstar et al. (1977) found concentrations of a-tocopherol of 89 to 188 mg/kg oil, and values for b- plus g-tocopherol of 252±530 mg/kg oil. In 1988, Aoyama et al. analyzed a-, b- and g-toco-

43

Fig. 1.25

4-Methylsterols.

Fig. 1.26

4,4-Dimethylsterols.

pherols in different varieties of coffee beans. They were contained in a ratio of approximately 2:4:0.1, the total content being about 5.5 to 6.9 mg/100 g. The predominance of a-tocopherol is a prominent feature of coffee beans, in contrast to other vegetables and fruits. Ogawa et al. (1989) determined the contents of tocopherols by HPLC in 14 green coffee beans, their roasted beans and infusions, and in 38 instant coffees. The maximum of total tocopherols in the green coffee beans was 15.7 mg/100 g and the average was 11.9 mg/ 100 g. The contents of a- and b-tocopherol were 2.3 to 4.5 and 3.2 to 11.4 mg/100 g, respectively, g- and dtocopherol were not found. Roasting diminishes the content of a-tocopherol, b-tocopherol and total tocopherols to 79 to 100%, 84 to 100% and 83 to 99%, respectively. In Fig. 1.29 the HPLC chromatograms of tocopherols for a green arabica and robusta coffee ± using the method published by Coors (1984) for vegetable oils ± are presented. In the arabica coffee oil, values of 161 mg/kg a-tocopherol and 597 mg/kg b-tocopherol

44


Table 1.27 Distribution (%) of desmethylsterols in arabica and robusta coffees (30 samples) (Mariani & Fideli, 1991). Mean value Sterols Cholesterol Campesterol Stigmasterol b-Sitosterol 5-Avenasterol Campestanol 24-Methylenecholesterol Sitostanol 7-Stigmastenol 7-Avenasterol 7-Campesterol 5,23-Stigmastadienol 5,24-Stigmastadienol Clerosterol

Arabica

Robusta

Arabica

Robusta

0.2±0.4 14.7±17.0 20.5±23.8 46.7±53.8 1.6±4.1 0.2±0.6 0.0±0.4 1.4±2.8 0.9±4.5 1.2±2.1 0.4±1.2 0.2±0.5 0.0±0.4 0.2±0.8

0.1±0.3 15.5±18.8 20.0±26.7 40.6±50.7 5.1±12.6 0.1±0.3 1.5±2.4 0.5±1.2 0.1±0.8 0.2±0.6 0.1±0.6 0.1±2.0 0.0±0.3 0.5±1.0

0.3 15.8 21.9 51.6 2.7 0.4 0.2 2.0 2.2 1.5 0.6 0.3 0.1 0.5

0.2 16.9 23.1 45.4 9.1 0.2 1.9 0.8 0.2 0.4 0.2 0.5 0.0 0.7

were found; the robusta coffee oil contained 107 mg/kg a-tocopherol and 260 mg/kg b-tocopherol ± significantly higher values. By the use of GC-MS, gtocopherol has been detected in some robusta coffees (KoÈlling-Speer, unpublished data). Tocopherols have also been analysed in coffee brews. The contents of total tocopherols in coffee infusions and instant coffee solutions were determined as 0.003±0.013 and 0.001±0.013 mg/100 ml, respectively (Ogawa et al., 1989). Fig. 1.28

Structural formulae of tocopherols.

1.19 OTHER COMPOUNDS

Fig. 1.27 Contents of selected sterols in differently prepared coffee infusions.

Kaufmann & Sen Gupta (1964) identified squalene in the unsaponifiable matter of coffee oil. Furthermore, Folstar (1985) reported a number of both odd and even chain length alkanes in wax-free coffee oil as well as in coffee wax. In 1999, Kurt & Speer detected and isolated a new component with the molecular formula C19 H30 O2 . Its structure is similar to the known coffee diterpene cafestol. The most important differences are the absence of the furan ring and the location of one methyl group at the carbon atom C10 . The new component was named coffeadiol (Fig. 1.30).


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Fig. 1.31 Structural formulae of carbonic acid 5hydroxytryptamides (C-5-HT).

Fig. 1.29 HPLC chromatograms of tocopherols of green coffees. HPLC conditions: LiChrosorb Si 60, 5 mm, n-hexane/dioxan (94:6), detection: 295/330 nm.

Fig. 1.30 Structural formula of coffeadiol.

1.20 COFFEE WAX The surface of green coffee beans is covered by a thin wax layer. The wax content is generally defined as the material obtained from the unground beans by extraction with chlorinated solvents such as chloroform or dichloromethane. The amount of surface wax is between 0.2% and 0.3% of the total coffee lipids. Folstar (1985) observed that only 37% of the coffee wax is soluble in petroleum ether. Investigating the fatty acid composition, he reported a large difference between the fatty acids in the petroleum ether-soluble part of the wax and that of coffee oil. The relatively high percentage of saturated higher fatty acids found in coffee wax is significant. The first investigations into the wax composition of green arabica coffee beans were performed by Wurziger and his co-workers (Dickhaut, 1966; Harms, 1968). They isolated and identified three carbonic acid 5hydroxytryptamides (C-5-HT) (Fig. 1.31). Arachidic acid (n = 18), behenic acid (n = 20) and lignoceric acid

(n = 22) are combined with the primary amino group of 5-hydroxytryptamine. In addition, Folstar et al. (1979) reported the presence of stearic acid 5-hydroxytryptamide (n = 16), and later that of o-hydroxyarachidic acid 5-hydroxytryptamide (n = 18), and o-hydroxybehenic acid 5hydroxytryptamide (n = 20) (Folstar et al., 1980). All components were confirmed by KoÈnig & Sturm (1982). Arachidic acid and behenic acid 5-hydroxytryptamide predominate, the other amides are only minor constituents. The coffee wax and its constituents may not be digested adequately in certain susceptible individuals, and are considered responsible for their gastro-enteric reactions to coffee beverages (Lickint, 1931). Removal of the waxy layer by washing the beans with solvents or by steaming increases their wholesomeness (Behrens & Malorny, 1940; Wurziger, 1971a; Fintelmann & Haase, 1977; Corinaldesi et al. 1989). A steaming method was developed in 1933 (Lendrich et al.), and was subsequently improved several times, for example by Roselius et al. (1971). As main constituents of the wax, although poorly water-soluble, the C-5-HT were considered to be the ìrritating substances' (Wurziger, 1971b; RoÈsner et al., 1971). However, Fehlau and Netter (1990), studying the influence of coffee infusions on the gastric mucosa of rats, reported that the gastric irritating effect of C-5-HT was much less than that caused by comparable coffee infusions. Several working groups developed analytical methods for determining the contents of C-5-HT in green, roasted and variously treated coffees (Culmsee, 1975; Hubert et al., 1975; Kummer & BuÈrgin, 1976; Hunziker & Miserez, 1977, 1979; Studer & Traitler, 1982; Chiacchierini & Ruggeri, 1985; Lagana et al., 1989; Battini et al., 1989; Kele & Ohmacht, 1996). The total content in green arabica coffees ranged between 500 and 2370 mg/kg, whereas in robusta coffees levels of 565±1120 mg/kg were found (Maier, 1981b). In coffee

46

stored for 30 years, the total content varied between 30 and 625 mg/kg (Wurziger, 1973). Treatment of the coffee beans, for example by polishing, dewaxing and decaffeinating, led to a substantial reduction in C-5HT (Harms & Wurziger, 1968; Hunziker & Miserez, 1979; Folstar et al., 1979, 1980; van der Stegen & Noomen, 1977). Furthermore, C-5-HT is partly decomposed by roasting (Wurziger, 1972; Hunziker & Miserez, 1979). For normal roasted coffees the contents ranged from 600 to 1000 mg/kg. Viani & Horman (1975) proposed pathways for the thermal decomposition of C-5-HT. They identified a number of alkylindoles and alkylindanes after pyrolysis of pure C22 -5-HT. Folstar et al. (1980) detected 5-hydroxyindole, 3-methyl-5-hydroxyindole as well as several n-alkanes, n-alkanenitriles and n-alkaneacidamides. Considering 5-hydroxytryptamides to be present only in the waxy layer of the coffee beans and being reduced by the processes described above, Wurziger (1971b) suggested the amount of C-5-HT should be a measure for treated coffees. Since 1973 in Switzerland, roasted C-5-HT-reduced coffees are designated `low irritating' (in German: `reizarm'), when the content of C-5-HT is lower than 400 mg/kg (Anon, 1973). According to van der Stegen (1979), coffee brew may contain up to 2.3 mg C-5-HT per liter when the beverage is prepared by percolation of untreated beans. The C-5-HT could not be detected in beverages prepared by a filtration method, or by percolating dewaxed beans. Furthermore, the C-5-HT become of interest because of its antioxidant effects (Lehmann et al., 1968; Bertholet & Hirsbrunner, 1984).

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Viani, R. & Horman, I. (1975) Determination of trigonelline in coffee. In: Proceedings of the 7th ASIC Colloquium (Hamburg), pp. 273±8. ASIC, Paris, France. Vitzthum, O.G. (1976) Chemie und Bearbeitung des Kaffees. In: Kaffee und Coffein (ed. O. Eichler), pp. 3±64. Springer Verlag, Berlin, Heidelberg, New York. Wahlberg, I., Enzell, C.R. & Rowe, J.W. (1975) Ent-16-kauren19-ol from coffee. Phytochemistry, 14, 1677. Wajda, P. & Walczyk, D. (1978) Relationship between acid value of extracted fatty matter and age of green coffee beans. J. Sci. Food Agric., 29, 377±80. Wettstein, A., Spillmann, M. & Miescher, K. (1945) Zur Konstitution des Cafesterols 6. Mitt. Helv. Chim. Acta, 28, 1004±13. Weusten Van der Wouw, M.P.M.E., Katan, M.B., Viani, R. et al. (1994) Identity of the cholesterol-raising factor from boiled coffee and its effect on liver function enzymes. J. Lipid Res., 35, 721±33. White, D.R. (1995) Coffee adulteration and a multivariate approach to quality control. In: Proceedings of the 16th ASIC Colloquium (Kyoto), pp. 259±66, ASIC, Paris, France. Wilson, A.J., Petracco, M. & Illy, E. (1997) Some preliminary investigations of oil biosynthesis in the coffee fruit and its subsequent re-distribution within green and roasted beans. In: Proceedings of the 17th ASIC Colloquium (Nairobi), pp. 92±9. ASIC, Paris, France. Wurziger, J. (1963) L'huile du cafeÂ vert et du cafeÂ torreÂfieÂ. CafeÂ, Cacao, TheÂ, 7, 331±40. Wurziger, J. (1971a) Neuentdeckte Kaffee-Inhaltsstoffe. Ihre Bedeutung fuÈr die BekoÈmmlichkeit von KaffeegetraÈnken. Med. Heute, 22, 10±13. Wurziger, J. (1971b) CarbonsaÈure-5-hydroxy-tryptamide zur Beurteilung von frischen und bearbeiteten Kaffees. In: Proceedings of the 5th ASIC Colloquium (Lisbon), pp. 383±7. ASIC, Paris, France. Wurziger, J. (1972) CarbonsaÈuretryptamide oder aÈtherloÈsliche Extraktstoffe um Nachweis und zur Beurteilung von bearbeiteten bekoÈmmlichen RoÈstkaffees. Kaffee- und Tee-Markt, 22, 3±11. Wurziger, J. (1973) CarbonsaÈurehydroxytryptamide und Alkalifarbzahlen in Rohkaffees als analytische Hilfsmittel zur Beurteilung von RoÈstkaffee-Genuûwert und BekoÈmmlichkeit. In: Proceedings of the 6th ASIC Colloquium (Bogota), pp. 332±42. ASIC, Paris, France.

Chapter 2

Chemistry II: Non-volatile Compounds, Part II S. Homma Ochanomizu University Tokyo, Japan tidine were quantitatively determined in robusta and arabica beans. The sum of these minor amino acids is, on average, 2.8% of the total concentration of free amino acids for arabica green coffee, and 1.9% for robusta green coffee. Arnold and Ludwig (1996) determined amino acid content changes in green coffee beans after the processing in the post-harvest period, such as drying, fermentation and storage (1 to 3 months). Drying of freshly harvested coffee beans at different temperatures of 208C and 408C alters the content of free amino acids: the concentration of glutamic acid increased in all the samples by about 500 mg/kg dry basis (db), while aspartic acid decreased in five out of seven samples by 110 to 780 mg/kg (db). Hydrophobic amino acids such as valine, phenylalanine, leucine and isoleucine significantly increased for most of the samples by 50 mg/kg db. When the adhering pulp was removed from the coffee beans by fermentation (27 h at 20±308C, 32 h at 6±408C) before subsequent drying, the concentrations of most amino acids did not change significantly compared with drying alone. Steinhart and Luger (1995) investigated the effect of steam treatment on free and total amino acids in green coffee beans. The free amino acids decreased significantly during the steam treatment; in particular, glutamic acid, asparagine, arginine, leucine, phenylalanine, tryptophan and lysine decreased remarkably with the duration of steam treatment at 0.8 bar. The decrease in the total amount of free amino acids was greater in arabica than in robusta coffee beans. The protein-bound amino acids amounted to 95% of the original value after 1 hour of steaming and to 80±85% after 4 hours of steaming at 1008C with saturated steam. This decrease was observed in all arabica and robusta coffee beans. Therefore, the industrially steamed coffee bean is roasted with about 90% of the original protein-bound amino acids and only half of the original free amino acids.

The desirable color, aroma and taste of brewed coffee are formed by the roasting process that is applied to the green coffee beans. The major reactions involved that occur during roasting are the Maillard reaction and oxidative polymerization or degradation of phenolics compounds. This chapter refers to non-volatile components with potential to contribute to coffee brew quality, such as minor constituents and compounds with bitter-tasting, antioxidative and metal-chelating activities in green and roasted coffee beans.

2.1 AMINO ACIDS AND PROTEIN Amino acids are involved in the formation of flavor and color of coffee brew; both quantity and types of amino acids affect the intensity and quality of aroma. Since free amino acids in coffee beans are largely transformed by roasting, resulting in negligible amounts in the roasted coffee, the presence of free amino acids in coffee beans after the harvesting of coffee berries should be checked at each processing step of coffee beans.

2.1.1 Amino acids Arnold et al. (1994) developed analytical methods for the determination of free and total amino acid content in green coffee beans by extracting milled samples of green coffee beans with 5-sulphosalicylic acid solution and precolumn derivatization of amino acids in the extract with 9-fluorenylmethylchloroformate reagent. They showed that arabica and robusta coffee beans consisted of the same main and minor amino acids also reported by Macrae (1985) from the work of Thaler. The minor free amino acids such as ornithine, hydroxyproline, b-alanine, pipecolic acid and 3-methylhis50

Chemistry II: Non-volatile Compounds, Part II

2.1.2 Amino acid derivatives Minor constituents such as hydroxycinnamic acid and caffeoyl derivatives of amino acids have been isolated from green coffee beans. Caffeoyltryptophan, which has been identified in green robusta coffee samples (Morishita et al., 1987), has been detected in commercial coffee brands by mass spectroscopy (MS) and UV. According to Balyaya and Clifford (1995) caffeoylL-tryptophan and caffeoyl-L-tyrosine are only found in robusta coffees. The botanical and geographical distributions of p-coumaroyl-L-tryptophan (Murata et al., 1995), the content of which in green coffee beans is 30 mg/kg, need to be investigated. Unidentified components with characteristic absorption spectra in the range of 270±350 nm were observed by threedimensional HPLC analysis in green coffee beans, and it is generally speculated that they are caffeoyl, feruloyl, or p-coumaroyl derivatives (Clifford et al., 1989a). The systematic analyses of coffee beans provide data to assist botanical and geographical classification.

2.1.3 Protein Information relating to the protein and amino acid content of green coffee was fully reviewed by Macrae in 1985. Protein in coffee bean has been little investigated despite its involvement in flavor and color formations in the roasting process. Compared with free amino acids, protein-bound amino acids seem to be rather inert in the reactions during roasting, as shown in their rapid degradation in the roasting process. Nevertheless, recent reports suggest that proteins or peptides may contribute to the formation of aroma, bitter taste and metal-chelating compounds in coffee brews. Rogers et al. (1997) undertook biochemical and molecular characterization of the major storage protein in Coffea arabica bean endosperm by molecular genetic studies. The endosperms proteins were analyzed by two-dimensional electrophoresis and amino acid micro-sequencing. The principal bean storage protein has been characterized, and a full length 1706 bp cDNA coding for this protein has been identified. The protein bears a strong sequence homology to the 11S storage protein of soybean seeds, which supports the assumption of a storage function for this protein. This protein accounts for approximately 50% of total proteins in the endosperm, representing between 5% and 7% of coffee bean dry weight, and exists in vivo as a mature coffee flavor precursor of approximately 52 kilo daltons (kDa) (see also Chapter 11). Ludwig et al. (1995) investigated the reactivity of a

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coffee protein isolate. The protein isolate was prepared by aqueous extraction from the matured green beans of Columbian Coffea arabica, with a yield of 45% total coffee protein. The protein isolate consists of 15% albumin and 85% globulin, without color, taste or smell. The protein isolate contained small amounts of sugars such as galactose, arabinose, rhamnose and glucose. The molecular masses of the reduced protein monomers were in the range of 20±30 kD for major bands and 30±45 kD for minor bands, by SDS PAGE. The main bands were electrofocused between pH 5 and 7. The major amino acids were glutamine/glutamic acid (28.2 mol%), glycine (8.8%) and asparagine/ aspartic acid (7.8%). The major N-terminal amino acids were glycine (36%), glutamic acid (15%) and aspartic acid (10%). After heating at 2008C, simulating coffee roasting, the protein isolate showed a yellow to light brown color with a light roasted taste and smell. Almost half of the protein (45±46%) was dissolvable in water at room temperature. Amino acid decomposition was not strong, whereas peptides with a molecular mass smaller than 10 kD were formed.

(a) Reactivity of protein Protein in green coffee beans has been regarded as being rather labile in reactions occurring on heating, compared with free amino acids. The rapid decomposition of free amino acids in green coffee beans upon roasting supports the rapid reactivity of free amino acids. Nevertheless, recent report and communications suggest possible contributions of protein or peptide to the formation of aroma, bitter taste and metal-chelating compounds in coffee brew. Several workers have investigated the reactivity of the E-amino group of lysine, and sulfhydryl and methylthio groups on side chains of amino acids in the protein, compared with those same functional groups in the free amino acids present.

Reactivity of the lysine side chain According to Hofmann et al. (1999a,b), as well as ionic condensation reactions, mechanisms involving amineassociated oxidative carbohydrate fragmentation and free radical formation also produce colored compounds by the Maillard reaction, prior to the Amadori rearrangement. They designed a series of model experiments in order to test the reactivity of the E-amino group of lysine in protein. Color development in the thermal treatment of neutral aqueous solutions of alanine and carbohydrate

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degradation products at 958C was studied; glycolaldehyde was shown to lead to the most effective color development (Table 2.1) accompanied by intense radical formation. The radicals were also detected in heated mixtures of L-alanine and pentose or hexose, respectively, and were identified as 1,4-dialkylpyrazinium radical cations. Under the reaction conditions applied, glyoxal is formed as an early product in hexose/L-alanine mixtures prior to radical formation. Reductones then initiate radical formation upon reduction of glyoxal and/or glyoxal imines, formed upon reaction with amino acid, producing glycolaldehyde. The thermal treatment of neutral aqueous solutions of glucose and N-a-acetyl-L-lysine, a model substance of lysine side chains of proteins, generated 1,4-bis[5(acetylamino)-5-carboxyl-1-pentyl]-pyrazinium radical cations accompanied by intense browning development. The thermal treatment of neutral aqueous solutions of bovine serum albumin (0.05 mmol) and glycolaldehyde (1.25 mmol) at 958C for 5 minutes generated 1,4-bis[5-amino-5-carboxy-1-pentyl]-pyrazinium radical cation (CROSSPY) as a cross-linker leading to protein dimerization (Fig. 2.1). In order to verify the formation of CROSSPY in foods, wheat bread crust and roasted cocoa as well as coffee beans during intense non-enzymatic browning, were investigated by electron spin resonance (ESR). An intense radical was detected; it was identified as the protein-bound CROSSPY by comparison with the


Fig. 2.1 Structure of protein-cross-linking amino acid 1,4-bis(5-amino-5-carboxy-1-pentyl)pyrazinium radical cation (CROSSPY) (Hofmann, 1999b).

radical formed upon reaction of bovine serum albumin with glycolaldehyde. The E-amino group on the lysine of proteins participates in radical-associated non-enzymatic browning reactions during the thermal processing of foods.

Reactivity of sulfhydryl and methylthio groups Sulfur-containing amino acids in coffee beans are known to be involved in the formation of characteristic aroma compounds such as furan derivatives like 2furfurylthiol and 2-methyl-3-furanthiol during roasting (Grosch, 1995). Generally, the reactivity of sulfurcontaining amino acids has been considered to be

Table 2.1 Color development (CD) and radical formation in binary mixtures of L-alanine and carbohydrates and carbohydrate degradation products, respectively (Hofmann, 1999a). Carbonyl compound Glucose Xylose N-(1-deoxy-D-fructos-1-yl)-L-Alanine Glycolaldehyde Glyoxal Furan-2-carboxaldehyde Pyrrol-2-carboxaldehyde 2-Oxopropanal Butane-2,3-dione 5-(Hydroxymethyl)furan-2-carboxaldehyde Glycerinaldehyde 2-Hydroxy-3-butanone 1

CD factor1

Rel radical formation (%)

16 64 8 1024 128 1024 256 256 128 2 2 2

42 82 12 1003 43 03 03 03 NA NA NA NA

The CD factor was applied to compare the color intensities of the reaction mixtures, which were heated for 15 minutes at 958C. For EPR measurements the mixture was heated for 10 minutes at 958C. 3 For EPR measurements the mixture was heated for 2 minutes at 958C. NA: not analyzed. 2


greater when they are in the free form than when they are bound in protein. Rizzi (1999) compared reactivity during thermal treatment, between the free and bound forms (with Nacetyl or peptide bond) according to the amount of production of sulfur-containing compounds produced. Furfuryl alcohol or 5-methylfurfuryl alcohol was allowed to react with cysteine, methionine, and peptides with these sulfur amino acids and their N-acetyl derivatives in pH 4 acetate buffer solution at 1008C to simulate the protein reaction in the initial stage of coffee bean roasting. It was predicated that N-acetyl amino acids should be prone to net positive charge at the sulfur atom on the peptide, since they are uncharged molecules in pH 4 solution as amides. Nacetyl cysteine (AcCys) and furfuryl alcohol produced more 2-furfurylthiol (7.9% of total volatiles, TV) compared with that from free cysteine and furfuryl alcohol (trace of TV). In addition, similar reactions with 5-methylfurfuryl alcohol gave 5-methylfurfurylthiol (11% TV). Also, N-acetylmethionine with 2furfurylalcohol and 5-methylfurfuryl alcohol produced methyl furfuryl sulfide (1.4% TV) and 5-methylfurfuryl methyl sulfide (0.03% TV), respectively. As expected, the yield of methyl furfuryl sulfide was higher with N-acetylmethionine when compared with free methionine (0.05% TV). The cysteine tripeptide, glutathione, reacted with furfuryl alcohol to produce more 2-furfurylthiol (2.7% TV) than free cysteine, suggesting that protein-bound cysteine could function as a direct precursor of 2-furfurylthiol during heating of foods. The methionine dipeptide, glycyl-methionine, failed to generate methyl furfuryl sulfide from furfuryl alcohol, apparently as the value of its pK1 is greater than the 2.38 reported for methionine. A higher pK1 will lead to more net positive charge on the peptide at pH 4 and therefore reduced reactivity (nucleophilicity) at the sulfur atom. Coffee bean protein would provide biogenetically determined numbers and locations of sulfhydryl and methylthio residues, which could offer more control over aroma formation compared with reactions of free amino acids. Reducing sugars in coffee glycoprotein may be characteristically positioned during roasting to interact with basic and sulfur-containing amino acid residues on protein. Rizzi suggested that proteins might play a key role in aroma and melanoidin formation in coffee beans as well as in processed foods, because of the higher reactivity of E-amino, thiol or methylthio groups, and the higher content of protein compared to free amino acids.

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(b) Bitter tasting componds The bitter taste of coffee brew has always interested food chemists. Since caffeine tastes bitter, it has been regarded as the major contributor to the bitter taste of coffee brew. Nevertheless, on the basis of sensory evaluation of caffeine concentration in coffee brews, the caffeine concentration only accounts for some 10±30% of the bitter taste of coffee brews (Macrae, 1985). The simple fact that decaffeinated instant coffee also tastes bitter suggests that substances other than caffeine might be contributing to the bitter taste. Other bittertasting compounds in coffee brew considered as candidates are trigonelline, polyphenolic compounds such as chlorogenic acids and melanoidin or polymeric compounds. Ginz and Engelhardt (2000) have suggested that bitter tasting compounds might be formed by roasting protein. An 80±90% protein-rich isolate was prepared from green coffee beans. It was roasted in a model roaster, and the bitter tasting hot water extract was subsequently fractionated. The presence of the cyclic peptides cyclo(Pro-Val), cyclo(Pro-Pro), cyclo(ProLeu) and cyclo(Pro-Phe) was identified. The cyclic dipeptides isolated are diketopiperazines, which are known to taste bitter in beer (Gautschi et al., 1997) and in cocoa (Pickenhagen et al., 1975). The threshold concentration of these cyclic dipeptides for bitter taste is reported as 10±50 ppm, and a synergistic effect of the cyclic peptides with theobromine on the bitter taste has been reported in cocoa (Pickenhagen et al., 1975). It is anticipated that cyclic dipeptides with a bitter taste will soon be isolated from coffee brews. The roasting of coffee beans accelerates the Maillard reaction, as well as the oxidative polymerization of chlorogenic acids. Hofman (1999) identified bittertasting bispyrrolidino- and pyrrolidinohexose reductones formed by roasting an equimolar mixture of powdered glucose and L-proline at 1808C for 15 minutes. Sensory evaluation of the dry-heated mixture revealed a strong bitter taste, as already reported by Papst et al. (1984, 1985) using heated mixtures of sucrose and proline. The development of the bitter taste by roasting was drastically reduced by adding Lcysteine to the mixture of glucose and proline. Hofmann (1999) identified acetylformoin derived from hexose as the precursor of the aminohexose reductones, which reacted more easily with cysteine to form 7-hydroxy-4a,6-dimethyl-2H,3H,4aH-furo[2,3-b]thiazine than with L-proline to form the aminohexose reductones. The addition of L-cysteine was found to block the development of the bitter taste (Fig. 2.2).

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roasting on the production of characteristic flavors in roasted coffee beans. Compared with the caffeic acid moiety in chlorogenic acid, the quinic acid moiety might be considered rather inert for oxidative changes during the roasting process.

2.2.1 Quinic acid moiety

Fig. 2.2 Blocking of formation of bitter-tasting bispyrrolidinohexose reductone 1 and pyrrolidinohexose reductone 2, by formation of 7hydroxy-4a,6-dimethyl-2H,3H,4aH-furo[2,3-b] thiazine 4 upon reaction of acetylformoin 3 with Lcysteine (Hofmann 1999).

Fig. 2.3

(±)-Quinic acid occurs free or bound in the chlorogenic acids of green coffee beans, and the roasting makes bound (±)-quinic acid, free, producing stereoisomers and quinides. Scholz-BoÈttcher and Maier (1991) determined the isomers of quinic acid (Fig. 2.3) and quinides (Fig. 2.4) produced in roasted coffee beans of Salvador arabica. Using GC/MS, five further quinic acids and seven quinides were identified, including (+)-g-quinide.

Stereoisomers of quinic acid (from Schloz-BoÈttcher & Maier, 1991).

Hofmann (personal communication) has suggested that these aminohexose reductones may be present in roasted coffee beans. It follows that the bitter taste may be developed by the combination of caffeine, trigonelline and phenolic compounds originally present in green coffee beans with compounds formed by roasting, such as cyclic peptides and aminohexose reductones. The evidence for their presence in coffee brews and the degree of contribution to the bitter taste, are involved in their current work.

2.2 FATE OF CHLOROGENIC ACID DERIVATIVES DURING ROASTING Information relating to chlorogenic acids in green and roasted coffee beans was reviewed by Clifford in 1985. The fate of chlorogenic acids is still one of the major factors to be considered when assessing the effect of

Fig. 2.4 Quinic acid lactones (from Scholz-BoÈttcher & Maier, 1991).


55

g/kg green coffee dmb

The changes in the quinic acids and quinides contents during the roasting process are shown in Fig. 2.5. (+)Quinic acid slightly increases at the beginning of the roasting process and remains relatively constant up to higher degrees of roast. The amount of (+)-g-quinide increases in light to medium roasts and decreases slightly in dark roasts. In contrast to (+)-quinic acid and its quinide, all other stereoisomeric acids and lactones continuously increase with higher roasting temperature, and the increasing rate of each product differs. The amount of (+)-epi-quinic acid rises with an approximately linear increase at higher roasting degrees. (+)-epi-g-Quinide and (+)-epi-d-quinide show a strong increase at higher roasting loss. Only scyllo-quinic acid and its quinide were found in light roasted coffees, and nearly all stereoisomeric quinic acids and quinides were generated at medium degrees of roast. The higher the degree of roast, the higher the contents of the isomeric compounds. The major stereoisomers in roasted coffee are scyllo- and mesoquinic acid I for quinic acid and scyllo-d- and (+)-epig-quinide for quinides.

acid and corresponding lactones. The presence of feruloylquinic acid lactones was also reported (Wynnes et al., 1987). Both 3- and 4-caffeoylquinic acid-g-lactones were identified in roasted coffee beans by Bennat et al., (1994) (Fig. 2.6). The content of the caffeoylquinides in coffee beans after different degrees of roasting produced from green arabica coffee ranged from 1.5 to 3.5 g/kg dry matter. The formation of lactones reaches a maximum in medium roasted coffee. A higher degree of roasting reduces the contents, suggesting decomposition of chlorogenic acid lactones. Bennat et al. (1994) also considered that the content of the lactones in an instant coffee sample was very low, because of hydrolysis during the extraction process.

Fig. 2.6 Structure of 4-caffeoyl-g-quinide (from Bennat et al., 1994).

12 10 8 6 4

(+_)–quinic acid

2

(+_)–γ–quinide

0

0

2

4

6

8

10

12

14

16

ORV (%)

Fig. 2.5 Variation of the (+)-quinic acid and the (+)-g-quinide concentrations as a function of the roasting loss on a dry matter basis (ORV) (from ScholzBoÈttcher & Maier, 1991).

On the basis of observations made during the roasting of coffee beans, they proposed the possibility that quinic acids and quinides could be used as an indicator for the degree of roasting: they defined the degree of isomerization as the ratio between the sum of the isomeric quinic acids and quinides (neo-quinides as well as quinide No 1 excluded) and the sum of (+)quinic acid and its quinide. A correlation between the degree of isomerization and the roasting loss is obtained. The content of chlorogenic acids in green coffee beans decreases during the roasting, giving (-)-quinic

Schrader et al. (1996) developed an HPLC system to determine mono- and dicaffeoylquinic acids, corresponding lactones and feruloylquinic acids in roasted coffee with one chromatographic run. The levels of 3and 4-caffeoylquinic acid-g-lactones were found to be 2.1 and 1.0 g/kg dry matter, respectively. Keeping coffee brews at an elevated temperature of 808C reduced the amount of caffeoylquinic acid lactone to 60% of its initial value. The change in these chlorogenic acid lactones and quinic acid lactones is involved with the generation of a bitter, sour taste in a coffee brew after it has stood for some hours on a hot plate. The patent of Bradbury et al., (1998) refers to the hydrolysis of chlorogenic acid lactones into quinic acid and caffeic acid, as well as to the hydrolysis of quinic acid lactones. These acids contribute significantly to the increase in acidity in a coffee brew. Table 2.2 shows the change in organic acids with time in stored coffee brew. A standard coffee solution prepared from Colombian beans was stored at 608C, the development of acid levelled off after about 200 hours, and the pH dropped from about 4.9 to 4.5. The concentration of quinic acid increased by 14.8 mmol/kg while the quinic acid lactone concentration decreased by 12.2 mmol/kg, suggesting that

56

Table 2.2


Change in organic acids in stored coffee brew (from Bradbury et al., 1998).

Acid

Time (hours)

(g/kg) Quinic Acetic Glycolic Formic Malic Citric Phosphoric

0

2.5

8

24

72

120

7.8 3.15 1.14 2.0 2.09 6.6 1.44

8.7 3.6 1.29 2.10 2.19 6.9 1.50

8.7 3.6 1.23 2.13 2.16 6.9 1.53

9.0 3.6 1.25 2.19 2.40 6.9 1.59

9.9 3.9 1.23 2.22 2.22 6.9 1.71

10.8 3.9 1.32 2.28 2.19 6.9 1.83

these lactones represent the primary precursors to the acids which develop upon storage.

2.2.2 Cinnamic acid derivative moiety Chlorogenic acids in roasted coffee beans are probably first hydrolyzed to quinic and caffeic acids, which then undergo pyrolysis to form phenolic volatiles. For the cinnamic acid, decarboxylation producing vinylphenols such as 4-vinylguaiacol is a commonly reported mechanism. The quantity of 4-vinylphenols formed in roasted coffee beans is small relative to the amount of the chlorogenic acid consumed (Heinrich & Baltes, 1987a), suggesting a competitive reaction pathway. Rizzi & Boekley (1993) investigated the mechanism of the thermal decomposition of p-hydroxycinnamic acid derivatives for alternative reaction pathways. Each derivative was pyrolyzed at 2078C, and the residual amount of the derivatives and carbon dioxide evolved were determined to estimate the extent of initial vinylphenol production (Table 2.3). The ethyl acetate soluble reaction product was methylated, and analyzed Table 2.3

Thermal decomposition of cinnamic acids at 2078C (from Rizzi & Boekley, 1993).

Cinnamic acid or ring derivative Cinnamic acid 3-Hydroxy 4-Hydroxy 3,4-Dihydroxy 3,4-Dihydroxy (K salt) 3-Hydroxy-4-methoxy 4-Hydroxy-3-methoxy 4-Methoxy 1 2

by GC/MS. Cinnamic acids with p-hydroxysubstituents, such as p-coumaric, caffeic and ferulic acids, were inclined to decomposition, disappearing completely in 45 minutes. The effect of a single phydroxy group is to accelerate decomposition relative to unsubstituted cinnamic acid (48%). p-Hydroxycinnamic acids which are further substituted with hydroxyl or methoxy groups are susceptible to relatively easy decomposition, for example caffeic and ferulic acids. Since the p-hydroxy derivatives exist in equilibrium with trienone isomers, they undergo easy loss of carbon dioxide, producing a 75±97% yield of carbon dioxide, indicating initially high yields of vinylphenols (Fig. 2.7). The final decomposition products of p-hydroxycinnamic acid were largely polymeric, resulting from rapid polymerization. The major products of cinnamic acid decomposition were vinylphenol dimers, that is 1,3-bis-arylbutenes with five isomeric forms depending on position and Z/E orientation of the olefinic bond.

Common name

p-Coumaric acid Caffeic acid Hesperitic acid Ferulic acid

Decomposition1 (%)

CO2 % yield2

48 24 > 99 > 99 Ð 0 > 99 22

Ð Ð 89 75 55 Ð 97 Ð

HPLC estimate of starting material lost after 45 minutes. Per cent of theoretical based on weight of BaCO3 isolated in 160 minutes (with the exception that ferulic acid was heated for 100 minutes).


57

Fig. 2.7 Proposed mechanism for cinnnamic acid decarboxylation (from Rizzi & Boekley, 1993).

2.3 ANTIOXIDATIVE COMPOUNDS IN COFFEE BREW Recent interest in the coffee beverage has been focused on the antioxidative activity of a roasted coffee brew (Turesky et al., 1993). This interest is not restricted to antioxidative usage in food systems, but also relates to the function that protects cells from oxidative damage in a biological system. The antioxidative activity in a coffee brew depends on natural constituents such as phenolic compounds, as well as reacted compounds formed by roasting (see also Chapter 8 on health and safety aspects).

2.3.1 Compounds occurring naturally in green beans Morishita and Kido (1995) have reported the potent contribution of chlorogenic acids to the antioxidative activity of coffee by using a 1, 1, diphenyl-2-picryl hydazil (DPPH) radical scavenging system and superoxide anion-mediated linoleic acid peroxidation system in vitro. Ohnishi et al. (1998) showed that DPPH radical scavenging activity of caffeoyltryptophan, a minor constituent in green coffee beans, increased dose-dependently at concentrations ranging from 1 to 50 mM. Caffeoyltryptophan inhibited the formation of conjugated diene from linoleic acid with the inhibitory activity in the increasing order of caffeic acid < 5-caffeoylquinic acid < caffeoyltryptophan < dla-tocopherol. They also examined the effects of caffeoyltryptophan on the in vitro haemolysis and

peroxidation of mouse erythrocytes induced by hydrogen peroxide. Caffeoyltryptophan exhibited strong inhibitory activities, suggesting that caffeoyltryptophan may be a natural antioxidant in the human diet and may intervene in toxicological processes mediated by radical mechanisms. Nakayama (1995) has shown that caffeic acid enhanced hydroxyl radical formation in the presence of transition metal ions such as Fe3 , Cu2 and Mn2 that cause oxidative damage, while caffeic acid esters showed protective effects in the absence of the metal ions. Devasagayam et al. (1996) showed caffeine at a millimolar concentration to be an effective inhibitor of oxidative damage to rat liver microsomes induced by the hydroxy radical, peroxy radical and singlet oxygen. It was speculated that caffeine might quench these reactive species, which suggests one more positive attribute from a daily intake of caffeine. According to Stadler et al. (1996a), caffeine and related methylxanthines were changed to the corresponding C-8 hydroxylated analogues as the major products of hydroxyl radical mediated attack. Further oxidation products of caffeine were found to be the N-1-, N-3and N-7-demethylated methylxanthine analogues, theobromine, paraxanthine and theophylline, respectively. It is generally recognized that hydrogen peroxide is detectable in roasted coffee beans. Since many factors may be involved in hydrogen peroxide formation in roasted coffee, caffeic, chlorogenic and quinic acids were pyrolyzed as model systems. The pyrolyzed caffeic acid catalyzed the highest levels of hydrogen peroxide formation over time in the presence of Mn2 (Table 2.4). The novel tetraoxygenated phenylindan isomers, 1,3-cis- and 1,3-trans-tetraoxygenated phenylindans, were identified as the major products in both the caffeic acid pyrolyzate at 2308C. The combined yield of both isomers was 3.6%. The acidcatalyzed cyclization of caffeic acid also gave a com-

Table 2.4 Hydrogen peroxide formation (micromolar) of pyrolyzed chlorogenic acids over time and effect of addition of Mn2 (from Stadler et al. 1996b). Caffeic acid Incubation time (min) 0 30 60

Chlorogenic acid

Quinic acid

-Mn2

+Mn2

-Mn2

+Mn2

±Mn2

+Mn2

11 + 1.3 33 + 2 64 + 2

15 + 1.0 104 + 2 205 + 5

6 + 1.2 10 + 3 17 + 1

16 + 2.2 24 + 1 41 + 2

3.8 + 0.8 6 + 0.5 3.4 + 0.3

11 + 0.5 15 + 3 18 + 0.7

58

bined yield of 5±6% for both isomers. The significant contribution of these indans to hydrogen peroxide formation has been demonstrated (Stadler et al., 1996b). Vacuum pyrolysis of rosmarinic, chlorogenic and caffeic acids at 2288C for 15 minutes raised their antioxidative activity in a rat liver membrane assay by 4-, 11-, and 460-fold, respectively (Guillot et al., 1996). The antioxidative components, 1,3-cis- and 1,3-transtetraoxygenated phenylindan isomers, were identified only in the caffeic acid pyrolyzates. These indan isomers were 8-fold larger in antioxidative activity than BHT. The potent reducing properties of the phenylindan isomers resulted in a pro-oxidative effect at a relatively high concentration in an ethyl linoleate peroxidation assay, and promoted the hydroxylation of 20 deoxyguanosine to produce 8-oxo-20 -deoxyguanosine. Although the existence of these phenylindans has not yet been confirmed in roasted coffee beans, a comparison of non-roasted, light-roasted, and dark-roasted coffee extracts showed a positive correlation between the degree of roasting and the inhibition of lipid peroxidation in rat liver membranes. The formation mechanism for the phenyl indans suggests the importance of a free carboxylic function in the dimerization process, and it is believed that these tricyclic dimers are formed by acid- or thermally catalyzed decarboxylation of caffeic acid monomers, which rapidly condense to form the phenylindan structure. Antioxidative activity of a coffee brew depends on the presence of compounds ranging from low to macromolecular, already present in raw green beans and those formed in roasting. The active compounds are not only phenolic compounds such as caffeoyltryptophan, caffeine and others naturally occurring in green coffee beans, but also more active compounds such as melanoidin and phenylindans, which might be produced by roasting. The mechanism of antioxidative action in coffee brew is complicated, and all of the antioxidative factors seem to be involved in chelation of transition metals, radical scavenging in chain reactions, trapping of active oxygen, and so on. It would be interesting to find a positive effect of coffee brew constituents on biological systems, in the view of the interets in the health aspects of coffee intake.

2.3.2 Effect of roasting on antioxidative activity An antioxidative effect of roasted coffee on processed foods was investigated by preparing cookies containing roasted coffee bean powder or another test antioxidant


with a 200 ppm level of iron. The change in lipid autoxidation was monitored by a storage test at 408C for 12 months. The coffee bean powder showed a strong antioxidative effect according to the peroxide value of the lipid fraction, while caffeic acid and rosemary extract were slightly less effective (Ochi et al., 1997). Nicoli et al. (1997) prepared hot water extracts from coffee beans after various degrees of roasting, and evaluated the antioxidative activity of the aqueous extracts by their chain-breaking activity and oxygen consumption properties. The chain-breaking activity was measured by croicin bleaching due to the presence of peroxy radicals, and the presence of an antioxidant slowed down the rate of bleaching. The highest antioxidative properties were found in the medium-dark roasted coffee brew (Fig. 2.8).

Fig. 2.8 Oxygen scavenging properties of coffee brews expressed as percentage of oxygen uptake/min per g dry matter (DM) as a function of the roasting time (from Nicoli et al., 1997).

2.4 COLORED MACROMOLECULAR COMPOUNDS 2.4.1 Characterization of colored polymers It has been generally recognized that phenolic polymerization and the Maillard reaction are the major reactions contributing to the formation of the colored polymer, melanoidin, in roasted coffee beans. Evidence of the Maillard reaction in the roasting of coffee beans has been reviewed by a number of authors (Dart & Nursten, 1985; Ho et al., 1993; Maier, 1993; Reinec-


59

cius, 1995). The formation of furans, pyrazines and aldehydes as aroma constituents supports the degradation of sugars by the Maillard reaction.

(a) Chemical characterization Melanoidin in a hot-water extract from roasted coffee bean has been analyzed by gel filtration column chromatography. The molecular mass of coffee melanoidin was estimated by HPLC using a protein PAK-125 column developed with water. Pullulan was used as the most suited standard markers, which are macromolecular linear polysaccharides. The molecular mass of the coffee melanoidin ranged between 3000 and more than 100 000 daltons, according to the degree of roasting and the coffee species, and increased with longer roasting time. The high molecular weight melanoidin increased in amount in the robusta samples compared to the arabica (Steinhart et al., 1989). The aqueous extract was charged on a Sephadex G-25 column, developed with water and four fractions were separated in order of molecular mass. The fractions were further separated into three or four bands by TLC on Sephadex, and developed with a mixture of 25% ammonia and 1-propanol. Each band was hydrolyzed and analyzed for its sugar composition which varied between the bands, mannose, arabinose, galactose and glucose being the major sugars, and rhamnose the minor (Steinhart & Packert, 1993). Nevertheless, such phenolics as chlorogenic acids are degraded by roasting green coffee beans. A significant quantity of the chlorogenic acid lost during roasting remains in an uncharacterized form. Leloup et al. (1995) monitored the fate of chlorogenic acids during a medium-slow-roasting process at 2408C by kinetic analysis. 5-Caffeoylquinic acid and dicaffeoylquinic acid decreased the most rapidly. With a short roasting time, dicaffeoylquinic acids (diCQA) are partly hydrolysed into caffeoylquinic acid and the caffeic moiety. 5-Caffeoylquinic acid isomers rapidly undergo esterification with carbohydrate and protein, producing bound chlorogenic acids (Fig. 2.9). With a longer roasting time, the phenolic and quinic moieties start rapidly degrading such diverse phenolic components as 4-vinyl catechol and catechol from the phenolic moiety, and slowly degrading hydroquinone, catechol, phenol and pyrogallol from the quinic moiety (Fig. 2.10). Maier (1993) has reviewed melanoidin and the nonvolatile compounds in coffee by referring to the investigations of the analysis of pyrolyzed melanoidin in coffee brew. Heinrich and Baltes (1987b) prepared seven fractions of melanoidins from roasted Robusta

Fig. 2.9 Comparison of esterified quinic and phenolic moieties during roasting (expressed in g/100 g green coffee, dry basis) (from Leloup et al., 1995).

coffee beans. These melanoidins were degraded by Curie point pyrolysis, which was monitored by highresolution GC/MS, to about 100 products, among which were found 33 phenols.

(b) Microbiological characterization It is generally recognized that a dialyzed coffee brew and the separated polymeric fractions show an absorption spectrum, consisting of the general absorption in the visible range characteristics of a model melanoidin (with sugar±amino acid), and a similar spectrum to that of chlorogenic and caffeic acids in the ultraviolet range. Therefore, melanoidin in brewed coffee is considered to be a mixture of both sugar and phenolic type melanoidins, or copolymers of sugar and phenolic type moieties. The microbiological decolorization of brown pigments in foods has been tested in order to categorize the chemical structure of these brown pigments. The fungus Paecilomyces canadensis NC-1 that can decolorize an instant coffee solution has been isolated from a glass bottle containing instant coffee of the freeze-dried type. This glass bottle was left open for 2 weeks. The fungus decolorized the instant coffee solution by 79% under optimal conditions. The decolorized coffee solution was analyzed by gel permeation chromatography, the absorbance being detected at 500 nm. The two chromatograms for the control solution and the decolorized one are compared in Fig. 2.11, which shows that the high molecular weight fractions were decolorized (Terasawa et al., 1994). This strain also decolorized black tea. Streptomyces werraensis TT14

60

Fig. 2.10 1995).


Suggested degradation mechanism of 3,5 dicaffeoylquinic acid during roasting (from Leloup et al.,

Fig. 2.11 Decolorization of coffee by Paecilomyces canadensis NC-1 analyzed by gel permeation chromatography, ____, control; . . . . ., decolorized coffee (from Terasawa et al. 1994).

(Murata et al., 1992) and Coriolus versicolor IFO 30340 (Aoshima et al., 1985) have also been screened from soil by the decolorization rate of model melanoidin prepared from glucose and glycine. Three microorganisms, S. werraensis TT14, C. versicolor IFO 30340 and P. canadensis NC-1, have been cultured to compare the decolorization rate of model brown pigments and brown-colored foods (Terasawa et al., 1996). The resulting decolorization rates are summarized in Tables 2.5 and 2.6. These data could be categorized by the decolorization rate of the brown pigment that differed significantly (Fig. 2.12). Paecilomyces canadensis NC-1 decolorized phenol type brown pigments and is unique in comparison with the other two microorganisms. It follows that the major brown pigment in brewed coffee can be considered to be of the phenol type. The metal-chelating affinity for the brown pigments could be applied to categorize these pigments. Either Zn(II), Cu(II) or Fe(II) was charged into a chelating Sepharose 6B column, and soy sauce, instant coffee and a model melanoidins prepared from glucose and


61

Table 2.5 Percentage of decrease in brown color intensity after microbial cultivation of synthetic brown pigments (from Terasawa et al. 1996). Synthetic brown pigment Glc-Gly1 Xyl-Gly1 Gal-Gly1 Glc-GABA1 Glc-Lys1 Glc-Trp1 Oxidized2 Reduced2 Catechin3 Chl3 Chl-Suc4 Caramel P Caramel N

S. werraensis TT 14 42.6 + 6.4 70.4 + 7.2 40.1 + 12.6 1.2 + 4.0 72.9 + 1.6 40.5 + 14.5* 46.6 + 3.9 29.1 + 16.3 5.8 + 22.2 796.2 + 10.8 733.6 + 13.4 737.4 + 7.5* 70.8 + 4.7*

C. versicolor IFO 30340 68.1 + 1.5* 63.0 + 4.4 70.7 + 2.1* 58.7 + 4.3* 51.0 + 12.2 74.1 + 27.3 71.1 + 1.5* 58.5 + 6.3* (84.7 + 11.7)5 7140.0 + 30.0 730.1 + 11.3 14.2 + 14.3 34.4 + 5.8

P. canadensis NC-1 36.9 + 5.3 58.3 + 1.0 35.9 + 0.3 32.9 + 11.0 28.4 + 4.7* 94.5 + 0.2 35.9 + 5.2 23.8 + 13.1 95.9 + 1.0* 32.1 + 3.0* 42.9 + 2.3* 2.2 + 36.4 47.7 + 13.6

Values are shown as mean + SD. 1 Sugar and amino acid model melanoidins. 2 The Glc±Gly melanoidin was oxidized with K3 [Fe(CN)6 ] and reduced with NaBH4 . 3 Catechin and chlorogenic acid were oxidized with KI03 . 4 Chlorogenic acid and sucrose were heated at 2208C for 40 min. 5 The pigment was adsorbed on the surface of the mycelia. 6 The decrease in brown color intensity was expressed as a percentage of the decrease in A500 vs the control after microbial cultivation for 10 days at 278C for P. canadensis NC-1, 5 days at 378C for S. werraensis TT 14, and 10 days at 278C for C. versicolor IFO 30340. * Significantly different from the decolorization rate of the other two microorganisms (P 0.05).

Table 2.6 Percentage of decrease in brown color intensity after microbial cultivation of browned foods (from Terasawa et al. 1996). Browned food Cane molasses Soy sauce Miso Caramel A Caramel B Dark beer Cola Barley tea Instant coffee Black tea Worcestershire sauce A Worcestershire sauce B Worcestershire sauce C Cocoa Chocolate

S. werraensis TT 14 4.0 + 9.1 728.7 + 16.9 751.4 + 13.5 72.3 + 9.1 15.8 + 6.5 26.0 + 6.2 54.8 + 15.4 20.3 + 4.0 7100.0 + 61.4 768.9 + 16.4 741.9 + 12.3* 724.2 + 13.0 748.0 + 26.7* 76.9 + 7.3 766.2 + 31.2*

C. versicolor IFO 30340

P. canadensis NC-1

72.5 + 4.7* 66.6 + 1.6* 64.7 + 2.3* 51.8 + 2.1 36.2 + 4.3 45.9 + 8.6* 40.8 + 15.9 60.0 + 8.3* 713.2 + 11.0 7109.0 + 32.7 61.8 + 9.2 68.1 + 5.6* 69.4 + 3.1 87.7 + 4.0 79.5 + 11.4

719.0 + 11.5 18.3 + 26.3 753.0 + 16.5 14.6 + 3.0* 25.7 + 4.5 2.6 + 7.3 46.2 + 13.0 27.6 + 10.3 61.8 + 7.7* 58.1 + 8.4* 60.0 + 10.2 33.3 + 35.3 64.5 + 7.0 82.3 + 21.6 86.8 + 4.4

Values are shown as mean + SD. 1 The decrease in brown color intensity was expressed as a percentage of the decrease in A500 vs the control after microbial cultivation for 10 days at 278C for P. canadensis NC-1, 5 days at 378C for S. werraensis TT 14, and 10 days at 278C for C. versicolor IFO 30340. * Significantly different from decolorization rate of the other two microorganisms (P 0.05).

62


C. versicolor Model melanoidins Cane molasses Soy sauce Miso Dark beer Barley tea Worcestershire sauce B Model caramel N Chocolate

Glc–Lys melanoidin

S. werraensis

Commercial caramel A Cocoa Cola Xyl–Gly melanoidin

Glc–Trp melanoidin Worcestershire sauce A and C Phenol-type model pigments Instant coffee Black tea

P. canadensis

Fig. 2.12 Categorization of synthetic brown pigments and browned foods by statistical significance of microbial decolorization (from Terasawa et al., 1996).

glycine were chromatographed in these metal-chelating Sepharose columns. The brown pigment of soy sauce is typical of melanoidin formed by the reaction of a reducing sugar with amino acids and peptides. The model and soy sauce melanoidins showed weak affinity for the Fe(II) column, while the coffee pigment showed strong affinity, being eluted with EDTA. Column chromatography of instant coffee in the Fe(II)-chelating Sepharose 6B column supports the proposition that the major brown-colored components of brewed coffee are of the phenol type (Homma & Murata, 1995).

2.4.2 Characterization of the zincchelating compounds in coffee brews (a) Effect of coffee intake on the biological availability of minerals The effect of brewed coffee on mineral nutrition has recently been noticed. Brewed coffee has metalchelating activity which results in a trace element deficiency in those known geographical areas of malnutrition, where coffee is consumed in a large quantity (MuÄnoz et al., 1988). In addition, studies on non-heme iron absorption by humans (Morck et al., 1983) and by rats (Greger & Emery, 1987; Brown et al., 1990) have

been reported showing the result of a reduction in the non-heme iron availability. Using suckling rats fed on MnSO4 and polyphenol containing beverages such as tea, coffee and red and white wines, little effect on manganese absorption (91.7%) has been reported in the animal study by Fraile & Flynn (1992). Tannic acid, which was tested in the same series of animal studies, slightly reduced the absorption of manganese from 91.0% to 77.0%, suggesting that binding of manganese to the beverage polyphenols may be weaker than that to tannic acid. Mueller et al. (1997) have estimated that approximately 5% of the 19 mg dry matter of aluminum in ground coffee was transferred into the coffee brew. However, the metal-chelating activity also contributes to the antioxidative activity in food and biological systems. This activity depends on phenolics as well as on the Maillard reaction products formed by roasting coffee beans. It suggests a significant difference in the binding activity of metal ions to phenolics and the other chelators in beverages. Most of the test samples of coffee used in these biological studies were warm- or hot-water extracts that were preliminarily determined as tannin and phenolics. The modified vanillin±HCl assay (Burns, 1971) is the accepted method for determining condensed tannin in foods, based on the reaction between vanillin and the resorcinol group in flavanols and flavanoids. A disadvantage of the Folin-Ciocalteau reagent for total phenolics is that it detects all phenolic groups, including proteins and reducing substances such as ascorbic acid and reductones that are formed by the Maillard reaction. Brune et al. (1991) have developed a spectrophotometric assay to determine the iron-binding phenolic compounds in foods. An iron (III)-containing reagent is added to a dimethylformamide (50%) extract of the food sample, resulting in the development of colors due to Fe±galloyl and Fe±catechol complexes. The different absorbance maxima were separately determined at two wavelengths. Further fractionation and chemical characterization of phenolics and other active compounds with metal binding activity in brewed coffee are anticipated.

(b) Separation of the zinc-chelating compounds from instant coffee There have been a few studies on the separation of these metal-chelating compounds in roasted coffee. Asakura et al. (1990) have shown that the ligands present in instant coffee that bind zinc(II) are acidic in nature and have a molecular size of less than 5000 Da


by paper electrophoresis. Evidence that coffee pigments bind copper and iron in vitro has been reported by Homma et al. (1986). Tetramethylmurexide (TMM), a chelating titration reagent for Zn(II), has been used to determine the free Zn(II) in an assay system containing coffee constituents. This assay system used a pH 5.0 hexamine buffer (10 mM) with 50 mM ZnCl2 , 0.05% of a sample, and 10 mM KCl (Homma & Murata, 1995). Low molecular weight compounds such as citric acid for Zn(II) (NakamuraTakada et al., 1994) and aspartic acid for Fe(II) (Sekiguchi et al., 1994) have been isolated from instant coffee as chelators, and the Scatchard plot for the Zn(II)±citric acid complex gave 3.50 6 10ÿ8 [M] for the dissociation constant. These low molecular weight compounds were isolated as ligands because the metalbinding ability of a test sample was shown by the amount of bound zinc per gram of the sample at each fractionation step. Work on separating aspartic acid has demonstrated it to be an enantiomorphic mixture that might have been produced during roasting, the relative amount of aspartic acid to the total amount of free amino acids being much higher in instant coffee than in regular brewed coffee. It is expected that a fractionation method will be developed to separate macromolecular ligands with a dissociation constant less than or of the order of 10.ÿ6 The Zn(II)-chelating compounds have been isolated from instant coffee by coagulation with ZnCl2 , and after being dissolved in 1% ammonia they were purified by passage through successive ion-exchange (Amberlite IRA-410 and IR-120) columns followed by chromatography on cellulose columns and development in a mixture of 1% ammonia and n-propanol (Fig. 2.13). Ap is the purified chelated Zn(II) complex, which is subsequently separable into six different fractions, ApI±IV (as in Fig. 2.13), V and VI, with increasing molecular size but different zinc contents. During this separation procedure Zn(II) was released from the Zn(II)±coffee complex with a large dissociation constant, resulting in the selection of a complex with a small dissociation constant. The yield from instant coffee of the active compounds, Ap-III, that was finally separated in the cellulose column, was 0.3± 0.4%. The Ap-III was a brown amorphous powder, soluble in water. Its molecular weight was estimated to be about 48 000 by HPLC, using proteins as standard markers. The apparent dissociation constants for Zn(II) measured from a Scatchard plot were 1.82 6 10ÿ9 and 1.13 6 10ÿ7 [M], and the numbers of binding sites were 1.05 and 1.98, respectively. This active compound also showed chelating activity for

63

A470 32.0 30.0 28.0 15.0 n –PrOH:1%NH =5:2 3

Zn (ppm)

Ap–III

A470

3:2

1:1

10.0 Zn (ppm)

Ap–I–2 5.0

Ap–IV

Ap–I–1 Ap–II–1 Ap–II–2 0

100

200

2.0 1.0 300

400

500

600 Fr.

Fig. 2.13 Cellulose column chromatography of sample Ap (from Homma & Murata, 1995).

Cu(II) with apparent dissociation constants of 3.33 6 10ÿ9 and 2.67 6 10ÿ7 [M] and numbers of binding sites of 1.6 and 4.0, respectively. This active compound was dissociated into its subunits by treatment with EDTA and was polymerized by further exposure to Zn(II), resulting in the migration of the Ap-III fraction to the Ap-IV fraction which has a larger molecular mass than Ap-III on cellulose column chromatography (Homma & Murata, 1995). Iron (II)-chelating compounds have been also separated from instant coffee by the same procedure as that used for the Zn(II)±coffee complex. The yields of Ap-III (MW 36 000 Da) and Ap-IV (MW 50 000 Da) from instant coffee were 0.11% and 0.05%, respectively. The dissociation constant of Ap-III for iron(II), which was determined by the dialysis equilibrium method, was 5.56 6 10ÿ6 . The value of the dissociation constant of Ap-III for the Fe (II)±coffee complex is thus larger than that for the Zn(II)±coffee complex. Since iron was detected in the Fe(II)±coffee complex, most of the strong binding sites in the ApIII fraction had already been combined with iron, and the binding sites measured seem to have easily dissociated Fe(II) during the fractionation process. Further exposure of the Ap-III fraction to Fe(II) resulted in migration to the Ap-IV fraction on cellulose column chromatography. The results of gel permeation HPLC also support the proposal that Ap-III was converted to Ap-IV. Chelators such as EDTA, o-phenanthroline and bipyridyl did not release iron from Ap-III. EDTA was involved in the formation of complexes with ApIII, while o-phenanthroline and bipyridyl were involved in the migration from Ap-III to Ap-IV on

64

cellulose column chromatography (Homma & Murata, 1995).

(c) General properties of the zinc-chelating compound The chemical formula of the Zn(II)-chelating compound was experimentally determined to be C16 H21 O9 N3 , while its chemical composition was found to be 30.4% phenolics by colorimetry with the Folin-Denis method, using chlorogenic acid as the calibration standard, and 3% sugar and 4% amino acids. The nitrogen content of more than 10% is indicative of the involvement of protein through the Maillard reaction in the formation of the zinc-chelating polymer. If phytate is involved in the formation of the active compounds during roasting of green coffee beans, a strong contribution to chelation from the phosphorous group would be expected (McKenzie 1984). However, phosphorous was hardly detectable by the modified Bartlett method. The Zn-chelating compound Ap-III was found to be antioxidative toward linoleic acid by measuring the peroxide formed with ammonium thiocyanate-FeCl2 . The less the chelating metal in Ap-III, the greater its antioxidative properties. Hydrogenation of Ap-III reduced the antioxidative activity and Zn-chelating activity by half. Olefinic moieties such as enol and enaminol in the structure seem to have been involved in both these activities (Homma et al., 1997). The formation of metal-chelating compounds during the roasting process was monitored, and the Zn(II)chelating activity increased with increasing degree of roasting of the green beans. The chelating activity of brewed coffee was found to be greater in regular coffee than in instant coffee (Homma, 1999). The constituents of brewed coffee that formed brown compounds with Zn(II)-chelating activity were investigated by a model system. Model systems were prepared with one or mixtures of two to four combinations of chlorogenic acid, sucrose, bovine serum albumin and cellulose, and roasted at 2008C for 30 minutes. The Zn-chelating ability per gram of a sample was found to be highest in the model prepared with chlorogenic acid only, and lowest in the model using all four compounds (Homma & Murata, 1995; Homma et al., 1997).

(d) Chemical composition of the zinc-chelating compound The chemical structure of the active compound Ap-III has been investigated by characterizing the products


formed through such degradative reactions on the ApIII sample as alkaline fusion (3508C) and alkaline decomposition (2508C) in glycerol (Homma et al., 1997). Alkaline fusion of the active compound Ap-III yielded about 11% of ether-soluble compounds, the major ones being low molecular weight polyphenols such as pyrogallol (2.16%), protocatechuic acid (3.56%), catechol (2.16%), and p-hydroxybenzoic acid (0.76%). Table 2.7 shows tentatively characterized compounds in the acidic and basic fractions produced by alkaline fusion of Ap-III. The acidic fraction shows the presence of polyphenolics, benzoic acid and its derivatives, and carboxylic acids with 4±5 carbon chains. The basic fraction shows the presence of amides, which is indicative of the involvement of sugar and protein in the formation of Ap-III. Alkaline degradation in glycerol gave similar chromatographic patterns by LC-MS to those for alkaline fusion. Some peaks by LC-MS from the acidic fraction showed the connection of two adjacent benzene rings. The oxidative degradation of Ap-III with KMnO4 NaIO4 gave different HPLC patterns after a prior treatment by methylation. This shows that Ap-III contained a hydroxy group and a carboxy group which could be methylated. Degradation with NaClO2 (O'Neil & Selvendran, 1980) yielded pyrogallol and caffeic acid, which is indicative of the presence of benzene rings connected by an ether bond. The degradative reactions other than alkaline fusion gave fewer ether-soluble compounds than alkaline fusion, and similar phenolics were determined in the degradation products. This shows that the benzene rings involved in Ap-III were connected by strong bonds which alkaline fusion could release to the greatest effect to produce phenolics. Heinrich and Baltes (1987b) characterized pyrolyzed products of separated melanoidin in coffee brew by Curie point pyrolysis, monitored by high-resolution GC/MS, as described earlier. Comparing two independent analyses of degraded compounds from coffee melanoidins, five phenolics were found to be common to both the melanoidins separated on the basis of molecular size and on Zn(II)-chelation, respectively (Table 2.7): common phenolics are phenol, o-, m- and p-cresols, and 4-ethylphenol. Although a few nitrogeneous compounds were characterized, most of them, except for amino acids, were unknown. Most of the degraded compounds were tentatively characterized by libraries of GC MS and LC MS. Phenolic structures seem to predominate in the polymerization structures of coffee melanoidin. Cilliers and Singleton (1989, 1991) characterized oxidation products of caffeic acid


65

Table 2.7 Degradation products of coffee melanoidin by Curie point pyrolysis, alkaline fusion and alkaline degradation in glycerol, and a combination of these (from Heinrich & Baltes, 1987b; Homma et al. 1997). Curie point pyrolysis 2-Ethylphenol 3-Ethylphenol 2,3-Dimethylphenol 2,5-Dimethylphenol 2,6-Dimethylphenol 3,4-Dimethylphenol Ethylmethylphenol 3(4)-Hydroxyacetophenone 2-Hydroxyphenylacetate

Pyrocatechol 3-Methylpyrocatechol 4-Methylpyrocatechol 3(4)-Ethylpyrocatechol 3-Hydroxybenzaldehyde Hydroquinone Methylhydroquinone 4-Methylguaiacol Ethylguaiacol

Curie point pyrolysis, alkaline fusion and alkaline degradation Phenol p-Cresol 4-Ethylphenol o-Cresol Alkaline fusion and alkaline degradation Catechol Protocatechuic acid Pyrogallol Dihydroxyethylbenzene 2,4-Dimethylphenol Floroglucinol Butylated hydroxyanisol Butylated hydroxytoluene Trimethylbenzene Methylpropylbenzene Diethyl-propylbenzene 3,5-Dimethoxyacetophenone Benzoic acid 2-Methylbenzoic acid 3-Methylbenzoic acid 2,3-Dimethylbenzoic acid

m-Cresol

3-Hydroxybenzoic acid 2-Hydroxy-4-methylbenzoic acid 3-Methoxybenzoic acid Acetone Phenylbutanone Phenylbutanedione Diphenylbutanedione 1-Hydroxy-2-propanone 1-(1-Methylethoxy)-propane 2,2-Dimethyl-3-octanone 2-Ethoxybutane 1-(1-Methylethoxy)-butane Formic acid Acetic acid Propanoic acid 2-Hydoxypropanoic acid

in a model system at pH 8.5 and room temperature. The controlling factor in the rate of autoxidation was shown to be phenolate anion concentrations. The products were found to be specific oligomers of caffeic acid formed by reactions involving the side chain of at least one of the caffeic acid units. They were analogous to natural lignans and neolignans bridged with dioxane, furan, or cyclohexene between the caffeic units. The development of novel methods to cleave specific polymers of the parent phenolics from which these degraded phenolics were derived, is expected.

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of aluminium from tea and coffee. Z. Lebensm. Unters.-Forsch., 205, 170±73. Munoz, L.D., Lonnerdal, B., Keen, C.L. & Dewey, K.G. (1988) Coffee intake during pregnancy and lactation in rats: maternal and pup hematological parameters and liver iron, zinc and copper concentration. Am. J. Clin. Nutr., 48, 645±51. Murata, M., Okada, H. & Homma, S. (1995) Hydroxycinnnamic acid derivatives and p-coumaroyl-(L)-tryptophan, a novel hydroxycinnamic acid derivative, from coffee bean. Biosci. Biotech. Biochem., 59, 1887±90. Murata, M., Terasawa, N. & Homma S. (1992) Screening of microorganisms to decolorize a model melanoidin and the chemical properties of a microbially treated melanoidin. Biosci. Biotech. Biochem., 56, 1182±7. Nakamura-Takada, Y., Shata, H., Minao, M. et al. (1994) Isolation of zinc-chelating compound from instant coffee by the tetramethyl murexide method. Z. Lebensm. Wiss. Technol., 27, 115±18. Nakayama, T. (1995) Protective effect of caffeic acid esters against H2 O2 -induced cell damages. Antioxidant activities of chlorogenic acids. In: Proceedings of the 16th ASIC Colloquium (Kyoto), pp. 119±24. ASIC, Paris, France. Nicoli, M.C., Manzocco, L. & Lerici, C.R. (1997) Antioxidant properties of coffee brews in relation to the roasting degree. Lebensm.-Wiss. u.-Technol., 30, 292±7. Ochi, T., Aoyama, M., Maruyama, T. & Niiya, I. (1997) Effects of various antioxidative substances on cookies containing iron. J. Jap. Soc. Nutr. Food Sci. (Nippon Eiyo Shokuryo Gakkaishi), 50, 231±6. Ohnishi, M., Morishita, H., Toda, S., Yase, Y. & Kido, R. (1998) Inhibition in vitro of linoleic acid peroxidation and haemolysis by caffeoyltryptophan. Phytochemistry, 47, 1215±18. O'Neil, M.A. & Selvendran, R.R. (1980) Glycoproteins from the cell wall of Phaseolus. Biochem. J., 187, 53±63. Papst, H.M.E., Ledl, F. & Belitz, H.-D. (1984) Bitterstoffe beim Erhitzen von Proline und Saccharose. Z. Lebensm. Unters.Forsch., 178, 356±60. Papst, H.M.E., Ledl, F. & Belitz, H.-D. (1985) Bitterstoffe beim Erhitzen von Saccharose, Maltose und Proline. Z. Lebensm. Unters.-Forsch., 181, 386±90. Pickenhagen, W., Dietrich, P., Keil B., Polonsky, J., Nouaille, F. & Lederer, E. (1975) Identification of the bitter principle of cocoa. Helv. Chim. Acta, 58, 1078±86. Reineccius, G.A. (1995) The Maillard reaction and coffee flavor. In: Proceedings of the 16th ASIC Colloquium (Kyoto), pp. 249± 57. ASIC, Paris, France. Rizzi, G.P. (1999) Formation of sulfur-containing volatiles under coffee roasting conditions In: Proceedings of the 217th ACS National Meeting in Anaheim. Division of Agriculatural and Food Chemistry No. 048. American Chemical Society. Rizzi, G.P. & Boekley, L.J. (1993) Flavor chemistry based on the thermally-induced decarboxylation of p-hydroxycinnamic acids. In: Food Flavors, Ingredients and Composition (ed. Charalambous), pp. 663±70. Elsevier Science Publishers, Amsterdam, Netherlands.

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Rogers, W.J., Bezard, G., Deshayes, A., Petiard, V. & Marraccini, P. (1997). An 11s-type storage protein from Coffea arabica L. endosperm: biochemical characterization, promoter function and expression during grain maturation. In: Proceedings of the 17th ASIC Colloquium (Nairobi), pp. 161±8. ASIC, Paris, France. Scholz-BoÈttcher, B.M. & Maier, H.G. (1991) Isomers of quinic acid and quinides in roasted coffee: indicators for the degree of roast? In: Procceedings of the 14th ASIC Colloquim (San Francisco), pp. 220±29. ASIC, Paris, France. Schrader, K., Kiehne, A., Engelhardt, U.H. & Maier, H.G. (1996) Determination of chlorogenic acids with lactones in roasted coffee. J. Sci. Food Agric., 71, 392±8. Sekiguchi N., Yata M., Murata M. & Homma, S. (1994) Identification of iron-binding compound in instant coffee. Nippon Nogeikagaku Kaishi, 68, 821±7. Stadler, R.H. Richoz, J., Turesky, R.J., Welti, D.H. & Fay, L.B. (1996a) Oxidation of caffeine and related methylxanthines in ascorbate and polyphenol-driven Fenton-type oxidations. Free Rad. Res., 24, 225±40. Stadler, R.H., Welti, D.H., Staempfli, A.A. & Fay, L.B. (1996b) Thermal decomposition of caffeic acid in model systems: identification of novel tetraoxygenated phenylindan isomers and their stability in aqueous solution. J. Agric. Food Chem., 44, 898±905. Steinhart, H. & Luger A. (1995) Amino acid pattern of steam treated coffee. In: Proceedings of the 16th ASIC Colloquium (Kyoto), pp. 278±85. ASIC, Paris, France. Steinhart, H., Moller, A. & Kletschkus, H. (1989) New aspects in the analysis of melanoidins in coffee with liquid chromatography. In: Proceedings of the 13th ASIC Colloquium (Paipa), pp. 197±205. ASIC, Paris, France. Steinhart, H. and Packert, A. (1993) Melanoidins in coffee. Separation and characterization by different chromatographic procedures. In: Proceedings of the 15th ASIC Colloquium (Montpellier), pp. 593±600. ASIC, Paris, France. Terasawa, N., Murata, M. & Homma, S. (1994) Isolation of a fungus to decolorize coffee. Biosci. Biotech. Biochem., 58, 2093± 5. Terasawa, N., Murata, M. & Homma, S. (1996) Comparison of brown pigments in foods by microbial decolorization. J. Food Sci., 61, 669±72. Turesky, R.J., Stadler, R.H. & Leong-Morgenthaler, P.M. (1993) The pro- and antioxidative effects of coffee and its impact on health. In: Proceedings of the 15th ASIC, Colloquium (Montpellier), pp. 426±32. ASIC, Paris, France. Wynnes, K.N., Fumilari, M., Boublic, J.H., Drummer, O.H., Rae, I.D. & Funder, J.W. (1987) Isolation of opiate receptor ligands in coffee. Clin. Exper. Pharmacol. Physiol., 14, 785±90.

Chapter 3

Chemistry III: Volatile Compounds W. Grosch Deutsche ForschungsansaÈtlt fuÈr Lebensmittelchemie Garching, Germany 3.1 INTRODUCTION

Table 3.1 Volatile compounds in coffee.

Besides its stimulatory effect, coffee is appreciated and/or consumed for its pleasing aroma, which is the result of roasting. It is not surprising, therefore, that numerous investigations have been carried out to identify the volatile compounds which evoke this pleasing aroma for most people, assessable directly through the nostrils of the nose, or as the odour element of the overall flavour on drinking a brew. Reichstein and Staudinger carried out the first exhaustive research in the years 1920±30. They isolated a yellow-coloured oil from large quantities of roasted ground coffee and identified more than 29 volatile substances by the preparation of derivatives and measurements of physical constants (Reichstein & Staudinger, 1926, 1950, 1955). The authors maintained that not a single one of the compounds identified cause the coffee aroma. However, they emphasised that a highly diluted aqueous solution of 2-furfurylthiol èxhales a pleasant note indicative of coffee' (Reichstein & Staudinger, 1955). Progress in instrumental analysis, particularly highresolution gas chromatography (HRGC) and mass spectrometry, has shown that the volatile fraction of roasted coffee consists of a great multiplicity of compounds. More than 800 volatile compounds with a wide variety of functional groups have been identified (Table 3.1). In the first place this progress is due to the work of Gianturco et al. (1963, 1964, 1966), Bondarovich et al. (1967), Goldman et al. (1967), Stoll et al. (1967), Friedel et al. (1971), Vitzthum & Werkhoff (1974a,b, 1975, 1976), Tressl et al. (1978a,b, 1981), Tressl & Silwar (1981), and Silwar et al. (1987). Details of these studies have been reviewed by Dart and Nursten (1985), Flament (1989, 1991) as well as by Nijssen et al. (1996). As the lists of volatile compounds increased in length, the question arose whether all of them, or which

Class of compound

Number

Hydrocarbons Alcohols Aldehydes Ketones Carboxylic acids Esters Pyrazines Pyrroles Pyridines Other bases (e.g. quinoxalines, indoles) Sulphur compounds Furanes Phenols Oxazoles Others

80 24 37 85 28 33 86 66 20 52 100 126 49 35 20 Total

841

Source: Nijssen et al. (1996).

of them, contribute to the aroma, and can be considered as potential odorants, or odoriferous compounds, especially in coffee brews, but also from the dry roast and ground product. A first approach to combine instrumental results with sensory properties of the volatile compounds was undertaken by Rothe and Thomas (1963). They suggested that only those compounds, the concentrations of which surpass their odour thresholds are odour active in food. However, the determination of activity values (OAVs, ratio of odour threshold of a compound to its odour threshold) for all of the volatiles in coffee would be very laborious because concentration and odour threshold data must be determined for the large number of compounds listed in Table 3.1. The first practicable methods to convert the results of instrumental analysis of the volatiles into sensory 68

Chemistry III: Volatile Compounds

69

data were CHARM analysis (Acree et al. 1984; Acree, 1993) and aroma extract dilution analysis (AEDA; Schmid & Grosch, 1986; Ullrich & Grosch, 1987). In both procedures serial dilutions of an extract containing the volatile fraction of a food are analysed by gas chromatography/olfactometry (GCO). The application of AEDA by Holscher et al. (1990) and Blank et al. (1992) to ground roasted coffee and brew was the starting point for the identification and quantification of the compounds contributing to the aroma. Grosch (1998a) and Vitzthum (1999) have reviewed these new developments in coffee flavour research. The aim of this chapter is to present the characterising impact flavour compounds of raw and roasted coffee as well as those of the coffee brew. Changes in the composition of relevant odorants during storage of roasted coffee and identification of those causing off flavours are additional topics of this chapter. However, first the state of the art in the methodology of aroma analysis needs to be considered. Furthermore, reaction routes which lead to the development of important odorants in the roasting process will be discussed.

Table 3.2

3.2 METHODOLOGY Roasted coffee contains a very complex mixture of volatile compounds, the concentrations of which vary over a broad range. Because of this, the identification and quantification of the aroma-active compounds is a difficult task. As will be discussed, the procedures listed in Table 3.2 have proved to be successful.

3.2.1 Isolation of the volatile fraction The first step, preparation of a coffee extract containing the volatile compounds, has to be performed under mild conditions. The temperature, in particular, is a critical point, due to the instability of, for example, thiols and disulphides (Guth et al., 1995). In roasted coffee some of the 2-furfurylthiol and other thiols are linked by disulphide bonds to cysteine and cysteinecontaining peptides and proteins (see the section on the formation of odorants). The increase of 2-furfurylthiol, which was observed when the volatiles from a coffee brew were isolated by combined steam distillation extraction (SDE) according to Likens and Nickerson (Grosch et al., 1994), was most likely caused by a reduction in the corresponding disulphides. To avoid the formation of artefacts, the temperature during isolation of the volatiles may not exceed 508C for a longer period as in SDE. Therefore, distillation has to

Step

Outline of aroma analysis. Procedure

I

Extraction of the coffee sample with a solvent, e.g. diethyl ether; distillation of the extract in vacuum

II

Separation of the extract by high-resolution gas chromatography (HRGC) and localisation of potent odorants by aroma extract dilution analysis (AEDA) or CHARM analysis

III

Detection of highly volatile potent odorants by gas chromatography-olfactometry of static headspace samples (GCOH)

IV

Enrichment of potent odorants by separation of the volatile compounds in neutral/basic and acidic compounds, by column chromatography and by multidimensional gas chromatography (MDGC)

V

Identification of the potent odorants by comparison of their HRGC and mass spectrometric (MS) data and odour quality with the corresponding properties of authentic substances

VI

Quantification of potent odorants and calculation of their odour activity values (OAVs)

VII

Preparation of a synthetic blend of the potent odorants on the basis of the quantitative data obtained in step VI. Critical comparison of the aroma profile of the synthetic blend, denoted aroma model, with that of the original

VIII

Comparison of the overall odour of the aroma model with that of models in which one or more components are omitted (omission experiments)

be carried out in vacuum, for example by using the new technique of solvent assisted flavour evaporation (Engel et al., 1999) which has been applied to coffee brews (Mayer & Grosch, 2000).

3.2.2 Screening for potent odorants According to Table 3.2, in the next step of analysis the coffee extract is separated by HRGC and the effluent from the capillary column is examined by sniffing. This procedure is denoted gas chromatography-olfactometry (GCO). However, the number of odorants detectable by GCO depends not only on the odour thresholds of the volatile compounds, but also on the parameters that are arbitrarily selected, such as the amount of coffee sampled, the dilution of the volatile fraction by the solvent and the sample size analysed by HRGC. Consequently, one GCO run alone is usually

70

insufficient to distinguish between the potent odorants that contribute strongly to the aroma and those odorants that are only components of the background aroma or that are insignificant. In AEDA and CHARM analysis, therefore, the extract is diluted with a solvent, for example as a series of 1+1 (v/v) dilutions, and each dilution is analysed by GCO. In the case of AEDA, the result is expressed as a flavour dilution (FD) factor (Grosch, 1993), which is the ratio of the concentration of the odorant in the initial extract to its concentration in the most dilute extract in which odour was detected by GCO. Consequently, the FD factor is a relative measure and is proportional to the OAV of the compound in air (Grosch, 1994). CHARM analysis constructs chromatographic peaks, the areas of which are proportional to the amount of chemical in the extract (Acree, 1993). The primary difference between the two methods is that CHARM analysis measures the dilution value over the entire time the compounds elute, whereas AEDA simply determines the maximum dilution value detected (Acree, 1993). Holscher et al. (1990), Blank et al. (1992) and Grosch et al. (1996) performed AEDA of roasted arabica coffee. Figure 3.1 illustrates a FD-chromatogram, which was obtained in these studies, as an example. Altogether, 38 odorants with FD factors 16 were found. Among them, numbers 14, 17 and 35, smelling catty/ roasty, earthy/roasty and boiled apple-like, respectively, appeared with the highest FD factor of 2048. The highly volatile odorants are not perceivable by AEDA or CHARM analysis because they get lost during concentration of the aroma extract or are masked in the gas chromatogram by the solvent peak. To overcome this limitation, AEDA or CHARM analysis has to be completed by GCOH of decreasing headspace volumes (step III in Table 3.2). In the example presented in Table 3.3, the procedure was started with a headspace volume of 25 ml. GCOH revealed 22 odorants. Then the headspace volume was reduced in a series of steps to reveal the most potent odorants. GCOH of the 0.4 ml volume indicated only six odorants (numbers 5, 8, 9, 11, 12 and 14 in Table 3.3) and after reduction to 0.2 ml, only 2,3pentanedione (number 8) was found. According to these results, number 8 was the most potent, highly volatile odorant in this sample of medium roasted arabica coffee. As thiols might be adsorbed, the surface of the glassware used in GCOH has to be deactivated, for example by treatment with a silyl reagent (Semmelroch


Fig. 3.1 FD Chromatogram of odorants isolated from medium roasted arabica coffee (Blank et al., 1992) 1 2,3-Butanedione (1)a 2 3-Methylbutanal (2) 3 2,3-Pentanedione (1) 4 3-Methyl-2-buten-1-thiol (3) 5 2-Methyl-3-furanthiol (3) 6 2-Furfurylthiol (1) 7 2-/3-Methylbutanoic acid (1) 8 Methional (4) 9 Unknown 10 2,3,5-Trimethylthiazole (5) 11 Trimethylpyrazine (6) 12 Unknown 13 3-Mercapto-3-methyl-1-butanol (3) 14 3-Mercapto-3-methylbutyl formate (3) 15 2-Methoxy-3-isopropylpyrazine (7) 16 5-Ethyl-2,4-dimethylthiazole (5) 17 2-Ethyl-3,5-dimethylpyrazine (6) 18 Phenylacetaldehyde (8) 19 2-Ethenyl-3,5-dimethylpyrazine (9) 20 Linalool (8) 21 2,3-Diethyl-5-methylpyrazine (10) 22 3,4-Dimethyl-2-cyclopentenol-1-one (11) 23 Guaiacol (1) 24 4-Hydroxy-2,5-dimethyl-3(2H)-furanone (12) 25 3-Isobutyl-2-methoxypyrazine (13) 26 2-Ethenyl-3-ethyl-5-methylpyrazine (9) 27 6,7-Dihydro-5-methyl-5H-cyclopentrapyrazine (14) 28 (E)-2-Nonenal (15) 29 2-(or 5-)Ethyl-4-hydroxy-5-(or 2-)methyl-3(2H)-furanoneb (12) 30 3-Hydroxy-4,5-dimethyl-2(5H)-furanone (16) 31 4-Ethylguaiacol (17) 32 p-Anisaldehyde (16) 33 5-Ethyl-3-hydroxy-4-methyl-2(5H)-furanone (16) 34 4-Vinylguaiacol (1) 35 (E)-b-Damascenone (3) 36 Unknown 37 Bis(2-methyl-3-furyl)disulphide (18) 38 Vanillin (19) a The reference for the first identification in roasted coffee is numbered in brackets: 1, Reichstein & Staudinger (1926); 2, Zlatkis & Sivetz (1960); 3, Holscher et al. (1990); 4, Silwar et al. (1987); 5, Vitzthum & Werkhoff (1974a); 6, Goldman et al. (1967); 7, Becker et al. (1988); 8, Stoll et al. (1967); 9, Czerny et al. (1996); 10, Bondarovich et al. (1967); 11, Gianturco et al. (1963); 12, Tressl et al. (1987b); 13, Friedel et al. (1971); 14, Vitzthum & Werkhoff (1975); 15, Parliment et al. (1973); 16, Blank et al. (1992); 17, Gianturco et al. (1966); 18, Tressl & Silwar (1981); 19, Clements & Deatherage (1957). b Of the two tautomeric forms, only the 5-ethyl-2-methyl isomer is odour-active (BruÈele et al., 1995).


Table 3.3

71

GCOH of ground roasted arabica coffee1

No

Odorant

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Acetaldehyde (1)5 Methanethiol (1)5 Propanal (2)5 Methylpropanal (3)5 2,3-Butanedione 3-Methylbutanal 2-Methylbutanal 2,3-Pentanedione 3-Methyl-2-buten-1-thiol 2-Methyl-3-furanthiol Methional 2-Furfurylthiol Unknown 3-Mercapto-3-methylbutyl formate 2-Ethyl-3,5-dimethylpyrazine Guaiacol 2-Ethyl-3,5-dimethylpyrazine 2,3-Diethyl-5-methylpyrazine 2-Ethenyl-3-ethyl-5-methylpyrazine 2-lsobutyl-3-methoxypyrazine Unknown (E)-b-Damascenone

Rl2

Volume3 (ml)

FD Factor4

< 500 < 500 &500 &500 580 653 662 697 822 870 906 911 986 1022 1086 1092 1107 1155 1182 1186 1225 1400

1 5 5 5 0.4 2 5 0.2 0.4 1 0.4 0.4 25 0.4 1 2 1 1 2 1 25 5

25 5 5 5 62.5 12.5 5 125 62.5 25 62.5 62.5 1 62.5 25 12.5 25 25 12.5 25 1 5

Source: Semmelroch & Grosch (1995). 1 The sample (100 mg) was placed into a vessel (volume 250 ml), sealed with a septum and then held at room temperature. 2 RI, retention index on a non-polar capillary (RTX-5). 3 Lowest headspace volume required to perceive the odorant at the sniffing port. 4 The highest headspace volume (25 ml) was equated to an FD factor of 1. The FD factor values of the other odorants were calculated on this basis. 5 Odorants numbers 1±4 were only detected by GCOH, the others also by AEDA (see Fig. 3.1). Numbers 1±4 were identified in coffee for the first time by (1) Reichstein & Staudinger (1926); (2) Prescott et al. (1937); (3) Rhoades (1958).

& Grosch, 1995). A limitation of GCOH is that polar odorants, such as 4-hydroxy-2,5-dimethyl-3(2H)-furanone, are not detected, although they are important contributors to the coffee aroma (see the section on the evaluation of key odorants). Therefore, GCOH cannot replace AEDA or CHARM analysis.

3.2.3 Enrichment and identification The screening for potent odorants is not corrected for the losses of odorants during the isolation procedure. Consequently, the identification experiments should be focused not only on compounds with the highest dilution value, but also on those perceived at lower dilutions, particularly in the 50±100-fold dilution range (Grosch, 1993). In the case of roasted coffee, the identification experiments were focused on the 38

odorants with FD factors in the range 16 to 2048 (Fig. 3.1). Altogether the chemical structures of 35 odorants were established. The list of authors (legend of Fig. 3.1) who had detected these compounds for the first time in roasted coffee reveals that six odorants (numbers 1, 3, 6, 7, 23 and 34 in Fig. 3.1) had been already identified by Reichstein & Staudinger (1926) in their classical study. Nine odorants (numbers 4, 5, 13, 14, 19, 26, 30, 33 and 35) were concealed in the gas chromatogram by large peaks of odourless volatiles, and were only detectable by GCO analysis of the coffee extract (Holscher et al., 1990; Blank et al., 1992). Therefore, they had to be enriched before their identification by HRGC-MS was successful, by a procedure such as will now be described. After separation of the acids and furanones the aroma extract was chromatrographed on a silica gel

72

column and each fraction was examined by GCO to establish the position of the analyte (Blank et al., 1992). This procedure was helpful for the identification of 3isopropyl-2-methoxypyrazine, 2,3-diethyl-5-methylpyrazine and (E)-b-damascenone. However, it was not sufficient for 3-mercapto-3-methylbutyl formate. Therefore, the fraction containing this analyte was further purified by HPLC on silica gel. It was then possible to identify this thiol on the basis of the criteria outlined in Table 3.2 (step V). As reported by Czerny et al. (1996) a special procedure was necessary for identification of the pyrazines, numbers 19 and 26 (Fig. 3.1). Multidimensional gas chromatography (MDGC; Weber et al., 1995) is a new procedure for the enrichment of trace amounts of volatile compounds. After separation of the extract on a polar precolumn, the cut of the effluent containing the analyte is cryofocused with liquid nitrogen and then transferred to the non-polar main column, which is combined with a mass spectrometer and a sniffing port. Mayer et al. (1999) analysed arabica coffees of different provenance and degree of roast using MDGC. As mentioned above, odorants are often concealed in the gas chromatogram by major volatile compounds that do not contribute to the aroma. To avoid misidentification it is necessary to compare, by GCO, the odour quality of the analyte with that of the authentic sample at approximately equal levels. Only when there is agreement in the sensorial properties, in addition to GC and MS data is the analyte, which has been perceived by GCO in the volatile fraction, correctly identified.

3.2.4 Quantification As discussed above, the results of the dilution experiments are not corrected for losses of the odorants during the isolation and concentration steps. Furthermore, in AEDA and CHARM analysis the odorants are completely volatilised and then evaluated by GCO, whereas the volatility of the aroma compounds in ground coffee and in the coffee beverage depends on their binding to non-volatile constituents, and on their solubility in water, respectively. To indicate which of the compounds revealed by the dilution experiments might be involved in the aroma, quantification of the potent odorants and calculation of their OAVs is the next step of the analytical procedure (Table 3.2, step VI). Because of the complexity of the volatile fraction of coffee, and the large differences in concentration,


volatility and reactivity of the odorants, it is not possible to quantify them precisely (error < 15%) by using conventional methods. Losses during the clean-up of the analyte as well as during adsorption in gas chromatography (Blank et al., 1992), which are often overlooked, may lead to incorrect results. However, precise quantitative measurements of the odorants can be performed by the use of stable isotopomers of the analytes as internal standards (see examples in Fig. 3.2) in the so-called `stable isotope dilution assays'. Losses occurring during isolation and purification of the analyte are corrected because the corresponding isotopomer has identical chemical and physical properties apart from a small isotope effect that can be ignored.

Fig. 3.2 Isotopomers of potent coffee odorants used as internal standards in isotope dilution assays. Position of labeling with carbon-13 (&) or deuterium (.). The number in brackets refers to the odorant (see Table 3.6) which was quantified on the basis of the standard.

The precision of stable dilution assays has been confirmed in model experiments (Schieberle & Grosch, 1987; Guth & Grosch, 1990). Although after clean-up the yield of some analytes was lower than 10%, the quantification was correct as the standards showed equal yields. In contrast to coffee brews, direct addition of internal standards to a solid coffee sample is not practicable, because errors may result from incomplete extraction. As reported by Semmelroch et al. (1995), Semmelroch & Grosch (1996) and Mayer et al. (1999),


a number of solvents have been used to extract the odorants in high yields. The extracts were than spiked with the labelled internal standards. 2-Methyl-3-furanthiol and 3-methyl-2-buten-1thiol, which belong to the potent odorants of coffee (numbers 4 and 5 in Fig. 3.1) are difficult to quantify due to the instability of the former (Hofmann et al., 1996) and the very low concentration of the latter (see Table 3.6). However, an exact determination is possible when, after addition of the corresponding labelled internal standards, the thiols are trapped by a reaction with p-hydroxymercuribenzoic acid (Darriet et al., 1995). After extraction of the derivatives with a phosphate buffer, the analytes and their standards are liberated by addition of excess cysteine and then quantified by a dynamic headspace procedure (Kerscher & Grosch, 1998; Mayer et al., 1999). With a few exceptions, the quantitative data discussed in the following sections were obtained by the application of stable isotope dilution assays.

3.2.5 Aroma models and omission experiments In the dilution experiments the odour impact of the volatiles is evaluated separately. Interactions of the odorants, which in most cases are characterised by inhibition and suppression (Acree, 1993), are abolished. Therefore, the question of which compound among the potent odorants actually contributes to the aroma has to be answered. To detect these odorants, synthetic mixtures (aroma models) were prepared on the basis of quantitative data for roasted coffee (Czerny et al., 1999) and brew (Mayer et al., in press) (step VII in Table 3.2). Then omission experiments were performed as triangle tests (step VIII in Table 3.2). The results are discussed in the section on the evaluation of key odorants.

3.3 RAW COFFEE 3.3.1 First studies A review of the literature on volatiles of green coffee led to a list of some 230 compounds (Holscher & Steinhart, 1995). To gain an insight into the odour-active volatiles, which may contribute to the characteristic `green peas' aroma, Vitzthum et al. (1976) were the first to analyse the volatile fraction of raw coffee by GCO. They identified four 3-alkyl-2-methoxypyrazines and concluded that 2-methoxy-3-isopropylpyrazine and

73

the corresponding isobutyl-derivative are involved in the aroma.

3.3.2 Potent odorants Table 3.4 summarises the results of recent studies in which the odorants of raw coffee were analysed by GCO (Holscher & Steinhart, 1995) and AEDA (Czerny & Grosch, 2000). AEDA revealed 21 odorants, of which 3-isobutyl-2-methoxypyrazine and 2-methoxy-3,5-dimethylpyrazine appeared with the highest FD factors. Nine of the odorants detected by AEDA (numbers, 1, 3, 6, 8, 10, 11 and 13±15 in Table 3.4) were also detected by Holscher & Steinhart (1995). In addition, they found phenylacetaldehyde, (E)-bdamascenone and a further five odorants (numbers 22, 23, and 25±27) which are known oxidation products of unsaturated fatty acids.

3.3.3 Content and OAVs of odorants More information about the compounds contributing to the characteristic smell of raw coffee was obtained by quantification and calculation of OAVs using odour threshold values of the compounds on cellulose (Czerny & Grosch, in press). The results shown in Table 3.5 reveal that 3-isobutyl-2-methyoxypyrazine with an OAV of 490 is the predominant odorant of raw coffee. The concentration of this pyrazine with a pealike smell (97 mg/kg) agrees with the value reported by Holscher & Steinhart (1995) and is near the range of 50 to 70 mg/kg reported by Spadone & Liardon (1988). However, the high concentrations reported for 4vinylguaiacol (2.3 to 7.5 mg/kg; Spadone & Liardon, 1988), (E)-2-nonenal (280 mg/kg) and (E)-b-damascenone (90 mg/kg; Holscher & Steinhart, 1995) are not confirmed by the data presented in Table 3.5. Most likely, the conventional quantitative methods used by Spadone & Liardon (1988) and Holscher & Steinhart (1995) are not suitable for an accurate determination of odorants numbers 8, 11 and 13 in raw coffee. 2-Methoxy-3,5-dimethylpyrazine, with an earthy odour, is the second important odorant on the basis of OAV (Table 3.5). With odour threshold values of 0.4 ng/l (water) and 6 ng/l (cellulose) it belongs to the most odour-active volatiles which have been detected in food. 3-Isopropyl-2-methoxypyrazine reached an OAV of only 23 (Table 3.5). As its odour quality is very similar to that of the isobutyl derivative, its contribution to the aroma of raw coffee in comparison to the latter might be small. However, Becker et al. (1988) found that a pea-like off-flavour of some batches of

74


Table 3.4

Potent odorants in raw coffee.

No.

Compound

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

n-Hexanal Butyric acid 2-/3-Methylbutyric acid Ethyl 2-methylbutyrate Ethyl 3-methylbutyrate Methional Pentanoic acid 1-Octen-3-one 2-Methoxy-3,5-dimethylpyrazine 2-Methoxy-3-isopropylpyrazine Linalool 3-Hydroxy-4,5-dimethyl-2(5H)-furanone (Sotolon) (Z)-2-Nonenal (E)-2-Nonenal 3-Isobutyl-2-methoxypyrazine Unknown (RI on DB-5: 1248)4 Unknown (RI on DB-5: 1259)4 4-Ethylguaiacol 4-Vinylguaiacol Vanillin Unknown (RI on FFAP: 2068)4 Nonanal (E,Z)-2,6-Nonadienal Phenylacetaldehyde (E,E)-2,4-Nonadienal (E,Z)-2,4-Decadienal (E,E)-2,4-Decadienal (E)-b-Damascenone

FD factor1

GCO2

16 16 32 256 256 64 16 16 512 128 16 64 64 128 4096 256 256 64 64 128 64

+ +++ +++ + +++ +++ +++ +++ +++

First identification3 1 2 3 1 1 3 2 3 4 5 1 4 3 6 5 4 5 6

++ +++ +++ ++ + +++ +++

6 3 5 7 3 6 6

Source: Holscher & Steinhart (1995), Czerny & Grosch (in press). 1 Flavour dilution (FD) factor. 2 Odour intensity in GCO: +, weak; ++, strong; +++, very strong (Holscher & Steinhart, 1995). 3 References: (1) Guyot et al. (1983); (2) WoÈhrmann et al.. (1997); (3) Holscher & Steinhart (1995); (4) Czerny & Grosch, (in press); (5) Vitzthum et al. (1976); (6) Spadone et al. (1990); (7) Spadone & Liardon (1988). 4 Retention index (RI) on capillary DB-5 or FFAP.

roasted East African coffee was caused by unusual high concentrations of 3-isopropyl-2-methoxypyrazine. This off-flavour, which is also called `potato taste', is most likely caused by the interaction of insects and bacteria (Bouyjou et al., 1999). The variegated coffee bug (Antestiopis orbitales) and other insects inflict wounds on the unripe coffee cherries so that methoxypyrazine-producing bacteria can penetrate them. An increase in ethyl 2-methylbutyrate and ethyl 3methylbutyrate (numbers 1 and 2 in Table 3.5) of up to 37 and 345 mg/kg, respectively, indicates an uncontrolled fermentation of raw beans (Bade-Wegner et al., 1997). In addition, cyclohexanoic acid ethylester has been detected in over-fermented beans (Bade-Wegner et al., 1997). Concentrations of 10 to 20 mg/kg were

responsible for the fruity, silage-like off-flavour due to the very low odour threshold of 0.01 mg/kg determined for the ester dissolved in a coffee brew (Bade-Wegner et al., 1997). An off-flavour reminiscent of rotten fish has been detected in immature green beans (Illy & Viani, 1995). 4-Heptenal, an autoxidation product of linolenic acid, was identified by Full et al. (1999) as a key odorant of this aroma defect. The data in Table 3.5 confirm the assumption of Vitzthum et al. (1976) that methoxypyrazines are stable during roasting. Other odorants like methional, sotolon and the phenol numbers 10 to 12 increase greatly during this process. Also, (E)-b-damascenone appears in a concentration of 255 mg/kg (Table 3.5).


75

Table 3.5 Concentrations, odour thresholds and odour activity values (OAVs) of potent odorants in raw and medium roasted arabica coffee. Concentration (mg/kg) No.

Compound

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

Ethyl 2-methylbutyrate Ethyl 3-methylbutyrate Methional 2-Methoxy-3,5-dimethylpyrazine 3-Isopropyl-2-methoxypyrazine 3-Hydroxy-4,5-dimethyl-2(5H)-furanone (Z)-2-Nonenal (E)-2-Nonenal 3-Isobutyl-2-methoxypyrazine 4-Ethylguaiacol 4-Vinylguaiacol Vanillin (E)-b-Damascenone

Raw coffee 2.4 22 22 0.5 2.3 0.7 < 0.3 12 97 21 117 82 < 0.3

Roasted coffee 3.9 14 213 1.1 2.4 1870 < 0.3 19 97 4060 39000 3290 255

Odour threshold1 OAV in raw coffee2 0.5 0.6 9 0.006 0.1 2.1 ND 15 0.2 35 80 100 0.15

4.8 37 2.4 83 23 5 cups/day). An average caffeine concentration of 60 to 85 mg of caffeine per cup has been assumed for instant and roasted and ground coffees, respectively.

8.4 COFFEE AND CANCER 8.4.1 Human data Numerous epidemiological studies have focused on the relationship between coffee consumption and cancer incidence at various sites. Thorough reviews on coffee and cancer have been published recently (World Health Organisation International Agency for Research on Cancer (WHO/IARC, 1991; Nehling & Debry, 1996). Overall, there is no conclusive evidence that coffee drinking represents a significant risk for the development of cancer in humans. For example, in a large study of almost 43 000 people conducted in Norway (Stensvold & Jacobsen, 1994), a country where the per capita coffee consumption is among the largest in the world, there was no association between coffee drinking and the overall risk of cancer.

(a) Cancers of the sexual organs Breast cancer The review of seven case studies by IARC (WHO/ IARC, 1991) did not reveal any association between breast cancer risk and the consumption of coffee. Recent studies further support that coffee intake is not related to breast cancer (Folsom et al., 1993; Smith et al., 1994; Tavani et al., 1998) in both pre- and postmenopausal women.

Ovarian cancer A weak positive association between coffee consumption and ovarian cancer has been seen in case-control studies. Only one reported a dose±response relationship and in most of the studies, the effect was not significant (Nehling & Debry, 1996; WHO/IARC,


1991). In its review IARC concluded that the evidence for an association between coffee drinking and ovarian cancer is inadequate, although the data indicate a marginal but significant increase in relative risk (WHO/IARC, 1991). In another review of the same body of literature, it was concluded that coffee consumption is unlikely to increase the risk of ovarian cancer (Leviton, 1990). A more recent study did not identify any association between coffee consumption and ovarian cancer (Polychronopoulou et al., 1993).

Prostate cancer Recent studies have not found any association between coffee consumption and prostate cancer (Jain et al., 1998; Hsieh et al., 1999).

(b) Cancers of the urinary tract Kidney, urinary tract The aetiology of renal cancer is still largely undefined (Nehling & Debry, 1996; Tavani & La Vechia, 1997). Available information indicates that renal cancer is unlikely to be associated with coffee consumption (WHO/IARC, 1991; Nehling & Debry, 1996; Tavani & La Vechia, 1997). A similar conclusion can be drawn for cancers of the urinary tract (WHO/IARC, 1991; Nehling & Debry, 1996).

Bladder cancer The epidemiological data regarding coffee consumption and bladder cancer have been equivocal. Many studies have suggested the possibility that coffee consumption could be a risk factor for bladder cancer, although many others found no such correlation (WHO/IARC, 1991; Nehling & Debry, 1996; Bruemmer et al., 1997; Donato et al., 1997; Probert et al., 1998). The association between coffee consumption and bladder cancer is often weak and only a few studies reported a dose±response relationship (WHO/IARC, 1991; Nehling & Debry, 1996). In its review of 1991, WHO/IARC concluded that the data are consistent with a weak positive relationship between coffee consumption and occurrence of bladder cancer (WHO/ IARC, 1991). It is important to note, however, that strong confounding factors such as smoking, dietary habits and occupation are well known for bladder cancer and it is recognised that they could have significant impacts on the data regarding coffee (Viscoli et al., 1993; Nehling & Debry, 1996). From two comprehensive overviews (Viscoli et al., 1993; Nehling &

Health Effects and Safety Considerations

Debry, 1996), it appears that coffee consumption is unlikely to be an important risk factor for bladder cancer in humans.

(c) Cancers of the gastrointestinal tract Oesophageal and gastric cancers The relationship between coffee consumption and cancers of mouth, pharynx, oesophagus and stomach has been addressed in several studies. Overall, no association was found (WHO/IARC, 1991; Nehling & Debry, 1996), in fact, some protective effects have been sometimes suggested (Inoue et al., 1998).

Pancreatic cancer Numerous studies have examined the potential link between coffee drinking and the risk of pancreatic cancer. Although most of the studies do not support any association, some have raised the possibility of a weak increase in pancreatic cancer for heavy coffee drinkers (WHO/IARC, 1991). Further studies and more recent analysis of the aetiological factors for pancreatic cancer have revealed that the weak effects of coffee, if any, are likely to be related to confounding factors such as smoking and therefore coffee consumption is not considered to represent a significant risk factor for cancer of the pancreas (Nehling & Debry, 1996; Silverman et al., 1998; Weiderpass et al., 1998). In a recent case control study, a U-shaped relationship was found between the level of coffee consumption and the risk for pancreatic cancer (Nishi et al., 1996). These authors concluded that as compared to abstinence, small to moderate amounts of coffee might prevent pancreatic cancer, whereas large amounts may increase the risk of developing this disease. The U-shaped dose±response effect was confirmed through a meta-analysis involving 14 studies published between 1981 and 1993 (Nishi et al., 1996). The lowest relative risk of pancreatic cancer was found at low consumption levels ranging from one to four cups a day.

Colorectal cancer The relationship between coffee consumption and the incidence of colorectal cancer has been addressed in numerous studies conducted in different geographical areas (WHO/IARC, 1991; Nehling & Debry, 1996). The data are inconsistent, but many case-control studies have revealed an inverse (protective) association between coffee drinking and the risk of colorectal

167

cancer. In its review of 1991, WHO/IARC concluded that although it is not possible to exclude bias and confounding as the source of the apparent inverse association, the collective evidence is compatible with a protective effect. A meta-analysis of coffee consumption and risk of colorectal cancer was published recently (Giovannucci, 1998). The results from 12 case-control studies showed an inverse association between coffee consumption and risk of colorectal cancer, while five cohort studies did not support a positive or negative link. Although definitive conclusions cannot be drawn because of inconsistencies between case-control and cohort studies, this metaanalysis strongly suggests a lower risk of colorectal cancer associated with a substantial consumption of coffee (> 4 cups a day).

8.4.2 Experimental data (a) Mutagenic and antimutagenic effects Many studies have addressed the mutagenic effects of coffee, using various biological test systems such as bacteria, yeast, fungi, mammalian cells and whole animal (WHO/IARC, 1991; Nehling & Debry, 1994a). Overall, relatively high concentrations of coffee have been shown to be slightly mutagenic in in vitro systems such as bacteria, fungi and mammalian cells. In bacterial assays coffee is particularly mutagenic to strains sensitive to oxidative mutagens (electrophiles). Most of the mutagenicity of coffee is abolished by the addition of exogenous detoxification systems such as liver extracts, catalase or peroxidases, implying that hydrogen peroxide (H2 O2 ) plays a key role in mediating coffee genotoxicity (Nagao et al., 1986). The formation of H2 O2 and the pro-oxidant activity of coffee in vitro has been attributed to polyphenolic thermal degradation products of chlorogenic and caffeic acid which reduce atmospheric oxygen in the presence of transition metals. The health significance of the mutagenic activity of coffee should be interpreted with caution since the in vitro assays used do not reflect adequately conditions present in physiological situations. In particular, the oxygen tension and the concentration of iron, two major players in the production of H2 O2 , are much higher in experimental assays than in the body. Furthermore, organisms possess efficient oxidant detoxifying mechanisms as well as repair systems. In this context, it is important to note that in contrast to the results of the in vitro studies, in vivo experiments in

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rodents have not shown any evidence of mutagenicity (Nehling & Debry, 1994a). Depending upon the end-point measured, the mechanism of oxidation and the concentration range of the compound tested, dietary phenolic compounds can act either as pro-oxidants or antioxidants. The antioxidant activity of coffee has been demonstrated in in vitro systems (Stadler et al., 1994, 1995). It was shown that instant coffee and its polyphenolics which catalyse H2 O2 formation and mutagenicity also exhibit potent antioxidant and antimutagenic activity as evidenced by the protective effect of coffee against t-butylhydroperoxide-challenged cells (Stadler et al., 1994). Other in vitro studies have documented that coffee or polyphenolic-rich coffee fractions protect against the mutagenicity of several carcinogenic compounds such as heterocyclic amines (Obana et al., 1986) or nitrosating agents (Stich et al., 1982) as well as counteract the effects of UV-radiation (Obana et al., 1986). Studies conducted in in vivo test systems confirm the antimutagenic effects of coffee. For example, instant and roasted ground coffees were reported to protect mice against the genotoxic actions of various carcinogenic chemicals (Abraham, 1991). In summary, based on an overall review of the in vitro and in vivo mutagenicity data of coffee, and taking into consideration the mechanisms involved, it appears that in the amounts usually consumed by humans, coffee is unlikely to produce any genetic damage (Nehling & Debry, 1994a; Nehling & Debry, 1996). The possibility of protective, antimutagenic effects has gained experimental support.

(b) Experimental carcinogenicity data The carcinogenic potential of coffee has been investigated in several long-term animal bioassays. Feeding high levels of coffee as part of the diet did not produce tumours in either rats or mice (Nehling & Debry, 1996). On the contrary, some studies reported that instant coffee resulted in a decreased incidence of spontaneous tumours (Stadler et al., 1990). Other studies have shown that coffee or coffee constituents protect against the action of well-known carcinogens such as nitrosamines (Nishikawa et al., 1986) or 1,2dimethylhydrazine (Gershbein, 1994). Several studies have demonstrated that green as well as roasted coffees inhibit the development of 7,12-dimethylbenz[a]anthracene-induced carcinogenesis at various tissue sites in different animal cancer models (Huggett et al., 1997).


(c) Mechanistic information A number of coffee components have been identified as being potentially responsible for the chemoprotective effects of coffee. As discussed above, several coffee constituents have been shown to possess strong antioxidant properties resulting in significant antimutagenic activity (Stadler et al., 1994, 1995; Abraham, 1991). Among others, caffeine, polyphenols including chlorogenic acid derivatives and degradation products such as caffeic acid and phenylindans as well as melanoidins have been documented to exhibit antioxidant activities. There is increasing evidence that oxidative damage may be involved in various pathological processes including cancer and that antioxidants may be protective. Therefore antioxidant activity could be a key mechanism involved in the chemoprotective effects of coffee on cancer development. Other potential mechanisms of chemoprotection have emerged from experimental investigations. For example, the coffee-specific diterpenes cafestol and kahweol (C + K) have been reported to be anticarcinogenic in several laboratory animals (Huggett et al., 1997). Experimental evidence has indicated that this protective activity may be related to the ability of C + K to induce detoxifying enzymes such as glutathione S-transferases (Schilter et al., 1996; Huggett et al., 1997). Recently it has been suggested that besides a stimulation of detoxification processes, a reduction of carcinogen activation could also play an important role in the chemoprotective effects of C + K. With respect to the hepatocarcinogen aflatoxin B1 (AFB1), C + K was shown to decrease the expression of AFB1-activating cytochrome P450s in the rat liver and to strongly induce glutathione S-transferase subunit Yc2 which efficiently detoxifies aflatoxin 8,9epoxide, the most genotoxic metabolite of aflatoxin B1 (Cavin et al., 1998). Further studies are necessary to address the significance of these effects with regards to human chemoprotection.

8.4.3 Conclusions There is still debate on the potential impact of coffee drinking on human cancer. Based on the available data, there is currently no conclusive evidence that moderate coffee consumption (up to five cups a day) represents a risk for the development of cancer. The American Cancer Society did not find any evidence to recommend against moderate coffee intake (American Cancer Society, 1996). The most consistent effect observed in epidemiological studies is a potential, but not yet


demonstrated, protection against colorectal cancer and maybe other types of cancers. Experimental data and mechanistic information are compatible with such a possibility.

8.5 COFFEE AND CARDIOVASCULAR DISEASE The potential association between coffee consumption and cardiovascular disease has been highly debated and is not yet totally clarified. In humans, studies have focused on the potential link between coffee consumption and recognised endpoints of cardiovascular disease such as myocardial infarction and arrhythmias. Many reports have also investigated the possible effects of coffee on known cardiovascular risk factors such as hypertension, elevated blood cholesterol, and more recently, increased blood homocysteine.

8.5.1 Myocardial infarction or coronary death During the past two decades, reports addressing the potential relationship between coffee consumption and heart disease have provided conflicting results suggesting positive, negative or no effects. Part of the reported negative effects of coffee on cardiovascular disease can be attributed to the consumption of boiled coffee which is known to increase blood cholesterol (see Section 8.5.4). In addition, some of the data have been difficult to interpret because of the presence of important confounding factors which were often not taken into account, particularly in the earlier studies. Among the possible confounders are very strong risk factors for heart disease such as stress, cigarette smoking, alcohol consumption, dietary habits and sedentary lifestyle. Over time, methodologies have improved and adjustments for major confounding factors have been introduced. However, heavy coffee consumption seems to be directly associated with the lifestyle risk factors of cardiovascular disease (Debry, 1994) and therefore, it is always difficult to conclude that observed effects are specifically related to coffee drinking and not to residual confounding factors. The link between coffee consumption and myocardial infarction has been assessed in many prospective surveys (cohort studies) as well as in casecontrol studies. Most of the cohort studies did not find any correlation between moderate coffee drinking and myocardial infarction, while the absence of a link to heavy coffee consumption (more than five cups a day)

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is less clear (Debry, 1994). In a meta-analysis including 11 prospective studies and involving a total of 143 030 people, it was concluded that there is no association between coffee consumption (at any level) and coronary heart disease (Myers, 1992). In this metaanalysis, case-control studies were excluded due to the potential for the introduction of serious uncontrolled bias such as problems in selecting appropriate controls and in estimating coffee intake. The conclusion was more ambiguous in another report presenting a meta-analysis of 22 studies (8 casecontrol and 14 cohort studies) of coffee use and myocardial infarction or coronary death (Greenland, 1993). Although from the case-control studies an increased risk is suggested, a more heterogenous trend was obtained from the cohort studies. Many studies involved in this analysis failed to account for strong confounding factors such as cigarette smoking. Based on this meta-analysis, the author concluded that an increased risk of coronary heart disease is unlikely at five cups of coffee a day, but cannot be ruled out at ten cups a day. Over the past few years, the potential association between coffee drinking and myocardial infarction has still been a matter of debate. For example, a study found a higher risk of myocardial infarction in women consuming more than five cups of coffee a day (Palmer et al., 1995). However, most of the new studies do not provide any evidence of a link between coffee and myocardial infarction. The Scottish Heart Health Study found the prevalence of coronary heart disease to be highest among those who abstain from coffee drinking and the lowest amongst those who drink five or more cups a day (Brown et al., 1993). In a follow-up study, a small benefit of coffee consumption was still observed among men (Woodward & Tunstall-Pedoe, 1999). In the US Nurses Study involving over 80 000 women, after adjustment for important cardiovascular risk factors such as smoking and age, there was no evidence for an association between coffee consumption and risk of coronary heart disease (Willett et al., 1996). In 1990, Tverdal et al. reported that coffee intake was related to death from coronary heart disease in men and women. After 6 years' follow-up, a slight increased risk was found only in subjects who drank nine or more cups a day (Stensvold et al., 1996). In a recent case-control study, neither caffeinated nor decaffeinated coffee was associated with the risk of myocardial infarction, even for those drinking more than four cups a day (Sesso et al., 1999). In summary, there is no evidence supporting a health relevant association between moderate coffee

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consumption (up to five cups a day) and the occurrence of myocardial infarction or coronary death. A slight increased risk associated with higher coffee consumption cannot be ruled out, although data have to be interpreted with caution since heavy coffee drinking may just be an indicator of a high risk lifestyle for cardiovascular disease. A different conclusion has to be drawn for boiled coffee, which has been associated with an elevated risk for cardiovascular disease.

8.5.2 Arrhythmias Experimental, epidemiological and clinical studies have addressed the possible effects of coffee on heart rate. Because of its known pharmacological activity on cardiac tissue, caffeine has been the focus of many studies on arrhythmia. Studies include normal subjects, patients with pre-existing arrhythmias and patients with a recent history of myocardial infarction. Overall, the results of the available studies are ambiguous, although most of them suggest that usual, moderate amounts of coffee or caffeine do not affect cardiac rhythm. In a review on this subject (Myers, 1991), it was judged that caffeine ingestion at levels equivalent of up to five or six cups of coffee a day does not affect the severity or frequency of cardiac arrhythmias in healthy subjects, patients with coronary heart disease or persons with known ventricular ectopy. Such a conclusion was further confirmed in an epidemiological study of 128 934 people (Klatsky et al., 1993) which did not find any influence of coffee consumption on death attributed to cardiac arrhythmias. Further studies have confirmed that moderate caffeine is unlikely to affect heart rate in both normal people and patients with heart disease (Newby et al., 1996; Arciero et al., 1998; Daniels et al., 1998; Myers, 1998).

8.5.3 Caffeine and blood pressure The potential effects of coffee on blood pressure are still a matter of controversy and debate (Debry, 1994; Jee et al., 1999). It has been shown in animal models and in humans that caffeine can interfere with purinergic receptors and can therefore antagonise the vasodilatating effect of adenosine (Debry, 1994). This pharmacological effect increases peripheral vascular resistance and may therefore induce hypertension. Although less plausible, a caffeine-dependent stimulation of the sympathetic nervous system activity resulting in increased plasma norepinephrine has also been proposed as a possible trigger for high blood pressure (Debry, 1994).


The effects of caffeine on blood pressure have been extensively investigated in humans through various experimental designs. Several different types of clinical studies have been conducted involving acute caffeine dosing in the presence or absence of stress, or chronic exposure to caffeine. Data on both normotensive and hypertensive human populations are available. In addition, several epidemiological studies have addressed the relationship between coffee consumption and blood pressure in the general population.

(a) Acute dosing The effects of acute caffeine/coffee ingestion on blood pressure have been reviewed (Green et al., 1996; Myers, 1988, 1998). Either no effects or a small and transient rise within the first few hours following the dosing has been observed. Increases in blood pressure (up to 10±15 mm Hg) were found, mostly in studies involving caffeine-naõÈve individuals, where caffeine was restricted for variable periods of time before dosing. In habitual caffeine users, little or no change was found. It is well documented that although caffeine may raise blood pressure after a period of abstention, tolerance then develops with multiple exposure and blood pressure returns to the baseline level in 2 to 3 days. Abstinence from caffeine for periods as short as 24 hours may lead to a partial loss of tolerance to caffeine.

(b) Acute dosing and stress Physical and mental stress is known to increase blood pressure. The possible potentiating effects of acute caffeine dosing on the blood pressure increases induced by various types of stress have been investigated in many studies. In a review of 27 studies, it was concluded that, overall, stress plus acute dosing of caffeine cause small increases in blood pressure in caffeinenaõÈve individuals (Green et al., 1996). In the research setting, these effects have been found to be additive (Green et al., 1996; Myers, 1998).

(c) Chronic exposure The studies on repeated/chronic exposure to caffeine are in agreement with those using acute dosing (Green et al., 1996; Myers, 1988, 1998). Many of them did not find any effects on blood pressure while some reported a small increase. When an increase was found, the magnitude of the effect was much less than in the acute dosing studies (Myers, 1998). In many of the repeated/


chronic exposure studies, a complete or partial tolerance to caffeine was induced (Myers, 1998). In a recent meta-analysis of 11 controlled clinical trials in which the effects of long-term coffee drinking on blood pressure was assessed, a small increase of 2.4 and 1.2 mm Hg were found, respectively, for systolic and diastolic pressure (Jee et al., 1999). Compared to other factors known to affect blood pressure on a daily basis, the clinical significance of the increases, if any, resulting from caffeine ingestion has been considered to be minimal (Green et al., 1996; Myers, 1998). It is considered that 24-hour ambulatory blood pressure is the most accurate measure of blood pressure status. Recent studies applying this approach have provided inconsistent data with increases, decreases or no effect on blood pressure (Green et al., 1996; Myers, 1998). Overall, these studies support previous results suggesting that chronic caffeine exposure has modest or no effects on blood pressure (Myers, 1998).

(d) Studies in hypertensive people The potential effects of caffeine or coffee drinking on blood pressure have also been investigated in hypertensive subjects. For example, 2 weeks of caffeine use versus placebo were compared in treated hypertensive patients (Eggertsen et al., 1993). No effect of caffeine was found on ambulatory blood pressure. Similarly, in untreated patients with borderline hypertension, caffeine use over 2 weeks had no effect on ambulatory blood pressure (MacDonald et al., 1991).

(e) Epidemiological studies The potential link between coffee consumption and blood pressure in the general population has been addressed in several epidemiological studies. The results are variable and inconsistent (Green et al., 1996; Myers, 1988, 1998) and they suffer from methodological limitations. Reports have indicated no association, positive associations, and inverse relationships with systolic and/or diastolic blood pressure (Green et al., 1996). One study found a curvilinear association (Stensvold et al., 1989), with abstainers and high users (more than nine cups) showing no difference in blood pressure, but with those taking one to four cups per day showing a slight rise.

(f) Summary Single doses of caffeine corresponding to several cups of coffee produce a small increase in blood pressure,

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mainly in caffeine-naõÈve individuals. The increases found are considered to be within the physiological range which could be observed during common activities such as conversation (Myers, 1998). Caffeine tolerance develops after 1 to 3 days of repeated exposure. Epidemiological surveys have provided inconsistent results which preclude drawing definitive conclusions. Overall, they do not support a major role of caffeine in inducing hypertension. Stress and psychological tension, which are known to increase blood pressure, may be correlated with higher coffee consumption. Therefore coffee may not be the cause of the effects observed in some studies.

8.5.4 Serum cholesterol Population studies regarding the influence of coffee consumption on serum cholesterol have provided conflicting results. For example, in a review article, it was found that in approximately two-thirds of the studies evaluated, coffee consumption was associated with an increase in serum cholesterol concentration (Thelle et al., 1987). In only some studies was this effect dose-dependent. Further analysis of available information revealed that the correlation between coffee and serum cholesterol was mostly restricted to studies conducted in Scandinavia, but was much less consistent in studies from USA or other European regions (Ugert & Katan, 1997). High consumption of boiled coffee (decanted without filtering), a brew particular to Scandinavian countries, has been clearly associated with elevated levels of serum cholesterol (Ugert & Katan, 1997). In this region, a relation between boiled coffee consumption and coronary heart disease was also identified (Tverdal et al., 1990). In Scandinavia, a substantial percentage of the decline in serum cholesterol over the years has been attributed to the switch from boiled to filtered coffee (Tuomilehto & Pietinen, 1991), leading to a reduction in cardiovascular disease (Tverdal et al., 1990; Johansson et al., 1996; Stensvold et al., 1996). Subsequent epidemiological and controlled clinical studies have further confirmed that the hypercholesterolaemic effect of coffee was dependent on the method of preparation of the coffee brew. For example, in contrast to boiled coffee, the consumption of filtered coffee has no significant effect on serum cholesterol levels (Van Dusseldorp et al., 1991) whereas Turkish style coffee appears to increase serum cholesterol (Kark et al., 1985). A series of clinical trials has found that the hypercholesterolaemic agents are present in the lipid

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fraction of boiled coffee. In addition, the major causative factors were identified as the diterpenes cafestol and kahweol, which are present mainly as fatty acid esters (Weusten-Van der Wouw et al., 1994; Ugert & Katan, 1997). These investigations demonstrated a dose response and reversible effect of cafestol and kahweol on increasing serum cholesterol concentrations. Chemical analysis of the diterpenes in different coffee brews has shown that Scandinavian-type boiled coffee, Turkish and cafetiere coffee contain the highest amounts of cafestol and kahweol, while instant and filtered coffee contain negligible amounts and espresso intermediate amounts (Ugert et al., 1995; Ugert & Katan, 1997). In summary, only substantial amounts of coffee containing high levels of the diterpenes cafestol and kahweol, such as boiled coffee, have been shown to consistently raise serum cholesterol levels. Filtering the brew or using other types of coffee prevent any hypercholesterolaemic effect.

8.5.5 Serum homocysteine For more than 20 years, moderately elevated serum concentrations of total homocysteine have been correlated with an increased risk of atherosclerotic cardiovascular disease and peripheral vascular disease (Meleady & Graham, 1999). Recently, it has been suggested that the association may be causal (Meleady & Graham, 1999). The mechanisms involved are still unclear. Experimental evidence has indicated that homocysteine may promote vascular damage through oxidative stress (Meleady & Graham, 1999). Only a few studies have explored the lifestyle factors determining homocysteine blood concentrations. The most important factors identified up to now have been a low intake of fruits, vegetables, folic acid, vitamin B12 and vitamin B6 . Cigarette smoking and age also play an important role. Recently, three studies have suggested a potential link between heavy coffee consumption and higher plasma total homocysteine (Nygard et al., 1997a; 1998; Oshaug et al., 1998; Stolzenberg-Solomon et al., 1999). For example, Nygard et al. (1997a, 1998) reported a direct and dose-dependent association between coffee consumption and blood homocysteine in a population in Norway. This effect was particularly pronounced in subjects drinking nine or more cups of coffee a day. No effect was found with decaffeinated coffee or caffeinated tea. In contrast, no significant association was found between coffee consumption or caffeine and total blood homocysteine in a sample of the


atherosclerotic risk in communities study (Nieto & Comstock, 1997). One controlled intervention clinical study is available where several sources of bias present in previous studies were eliminated. It was observed that the consumption of 1 litre of strong unfiltered boiled coffee every day for 2 weeks was associated with a 10% increase in mean plasma total homocysteine concentration (Grubben et al., 2000). However, this extreme coffee intake may affect diet composition and other factors which may influence plasma homocysteine (Vollset et al., 2000). The association between coffee consumption and total blood homocysteine is an unexpected finding and deserves further confirmation. Furthermore, there are no plausible mechanisms proposed yet. Since several other lifestyle factors are likely to play a major role, it is still unclear whether the coffee effect found in the observational studies is real or whether it results from residual confounders such as smoking or other unmeasured or unidentified factors. The authors of the Norwegian study acknowledged the possibility that residual confounders with vitamins, especially folate, could be responsible for their finding (Nygard et al., 1997b). Furthermore, the health significance of the coffee effects is difficult to interpret. In the Norwegian study (Nygard et al., 1997a), it was observed that elevated plasma homocysteine levels were correlated with coffee intake mainly in people with low to intermediate homocysteinemia. Therefore the link between the coffee-dependent increase in homocysteine and overall cardiovascular risk within the general population may not be straightforward to establish. In summary, a slight increase in blood homocysteine in heavy coffee drinkers has been shown in several studies. The direct implication of coffee and the health significance of such an effect have still to be demonstrated.

8.5.6 Conclusions The potential relationship between coffee drinking and cardiovascular disease has been the subject of much debate and investigation. It is important to note that there are many other dietary and lifestyle factors which are known to have a greater impact on cardiovascular disease. Some of these factors are associated with coffee consumption and may explain some of the coffee effects reported. Overall, except for brews rich in diterpenes such as boiled coffee, the data show that moderate coffee consumption is not a causal factor in the development of cardiovascular disease.


8.6 COFFEE AND BONE HEALTH Osteoporosis is a chronic degenerative bone disease that affects mainly, but not exclusively, postmenopausal women, in which demineralisation (calcium loss) of bones leads to an increased likelihood of fracture. It has a complex aetiology that includes genetic, physiological and environmental contributors. Among factors, oestrogen deficiency, smoking, heavy alcohol consumption, lack of exercise, obesity and inadequate nutrition are believed to play significant roles in the development of this disease. Of the nutritional factors, low calcium intake throughout life is believed to be most important, although low intakes of other minerals and poor vitamin D status have also been implicated. During recent decades the number of osteoporotic fractures has increased in industrialised countries. Since this increment cannot be solely explained by an increased life expectancy, other aetiological factors have been intensively examined, particularly those related to nutrition and lifestyle. Experimental data obtained in animals and humans have suggested that caffeine may affect calcium metabolism. The potential role of caffeine, mainly through coffee consumption, as a contributing factor for bone loss in humans has received a lot of attention. In recent years numerous studies have reported on caffeine consumption as a possible risk factor for osteoporosis.

8.6.1 Calcium metabolism Caffeine has been shown to increase the urinary excretion of calcium in experimental animals (Debry, 1994). In humans, several studies have also suggested that caffeine may negatively influence calcium balance, particularly in women, but the data are inconsistent (Debry, 1994). An initial study reported a small but significant negative effect of caffeine intake on calcium economy in 168 premenopausal women (Heaney & Recker, 1982). This effect was, however, no longer significant when dietary calcium intake was considered. Other studies indicated that caffeine induces a significant acute calcium diuresis (Massey & Wise, 1984; Debry, 1994; Heaney, 1998). However, subsequent investigation suggested that the increase in calcium excretion was followed by a reduction in excretion, resulting in a net negative effect on calcium balance lower than previously thought (Kynast-Gales & Massey, 1994). The effect of caffeine on calcium metabolism was recently addressed in a double-blind, randomised,

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placebo-controlled, cross-over metabolic study. The administration of 400 mg of caffeine over 19 days did not produce any effect on total 24 hours calcium loss (Heaney & Recker, 1994; Barger-Lux & Heaney, 1995). However, a small negative balance effect was detected due to a slight decrease in calcium absorption efficiency. Importantly, the calcium intake of the women enrolled in these studies was significantly lower than current recommendations. Therefore, these data indicate that caffeine may lead to a small negative calcium balance when dietary calcium intake is inadequate. This has been confirmed in another study showing that caffeine only produced observable effects on calcium metabolism in women consuming less than 600 mg of calcium daily (Massey et al., 1994). New dietary reference intakes for calcium intakes in adults range from 1000 to 1300 mg/day according to lifestage. Overall, the magnitude of the caffeine effect on calcium balance is low and it has been estimated that it could be offset simply by the addition of 1 to 2 tablespoons of milk to a cup of coffee (Barger-Lux & Heaney, 1995). In summary, it is currently thought that at standard recommended calcium intake, caffeine is unlikely to have harmful effects on calcium bone economy. In the most recent US RDA, it was stated that the available evidence does not warrant a specific calcium intake recommendation for people with a different caffeine intake.

8.6.2 Osteoporosis Studies addressing the potential link between caffeine consumption and the risk of osteoporosis have given contradictory results (Debry, 1994; Heaney, 1998). Comparison and interpretation of the studies are complicated by the variety of both bone-related measurements (e.g. fracture risk, bone density, bone mass) and caffeine intake estimations. Furthermore, in many studies, variables known to affect bone loss such as calcium intake, smoking, body weight, physical activity and hormone replacement therapy were not or could not be adequately controlled for. This aspect is of particular importance for calcium intake, which is likely to be inversely correlated with caffeine exposure (Heaney, 1998). Although some epidemiological studies have suggested that caffeine may slightly increase the risk of fracture or may decrease bone density, the majority of the reports available failed to find any effects from caffeine (Debry, 1994; Heaney, 1998). A review of 23

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observational studies indicated that five showed a negative effect of caffeine on bone health, two a partial effect and 16 showed no effect (Heaney, 1998). Two studies proposed a negative effect only for women whose dietary intake of calcium is below the recommendations (Barrett-Connor et al., 1994; Harris & Dawson-Hughes, 1994). In a recent study which was designed to minimise confounding variables no association could be found between caffeine intake and bone loss in 138 healthy postmenopausal women (Lloyd et al., 1997). The potential effects of caffeine on bone health have also been evaluated in women who are still in the period of bone gain. Caffeine did not affect the rate of gain in spinal bones in women of 30 years age or less (Packard & Recker, 1996). Another study revealed that dietary caffeine intake at levels presently consumed by American teenage women was not correlated with total bone mineral gain or hip bone density at age 18 (Lloyd et al., 1998).

8.6.3 Conclusions Based on current available literature, there is no evidence that moderate caffeine intake through coffee consumption has a harmful effect on bone health in normal healthy individuals ingesting recommended levels of calcium. Caffeine and coffee consumption are therefore unlikely to be important risk factors for osteoporosis. Furthermore, data indicate that the small effects of caffeine in women with calcium deficiency may be counteracted by increased calcium intake, for example through milk.

8.7 REPRODUCTIVE AND DEVELOPMENTAL POTENTIALS OF COFFEE AND CAFFEINE Since caffeine was shown to be teratogenic in animal models, safety concerns were raised regarding coffee drinking during pregnancy. It is well documented that caffeine metabolism is slower in pregnant women, resulting in longer and possibly higher exposures. Consequently, there have been a number of animal and human studies addressing the potential effects of coffee/caffeine on various reproductive and developmental outcomes such as teratogenicity (congenital malformations), neurodevelopment, low birth weight (growth retardation and prematurity), spontaneous abortion (miscarriage) and fertility parameters.


8.7.1 Congenital malformations (a) Human data Several epidemiological studies have examined the association between caffeine ingestion and congenital malformations (Nehling & Debry, 1994b; Brent, 1998). Most of these studies do not support any link between caffeine intake and teratogenicity (Nehling & Debry, 1994b; Brent, 1998). In a review of 14 publications addressing the relationships between caffeine or coffee consumption and congenital malformations, only three mentioned potential teratogenic effects (Nehling & Debry, 1994b), while the other 11 failed to provide evidence for an association. Jacobson et al. (1981) reported three cases of extrodactyly in children born from mothers consuming high amounts of coffee (8 to 25 cups/day). Unless an increased incidence of such a malformation is observed and confirmed in other controlled, large-scale epidemiological studies, this report cannot be appropriately interpreted. In a Japanese study, the rate of many different types of congenital malformations, including chromosomal abnormalities, was found to be about twice as high in the coffee drinkers than in non-drinkers (Furuhashi et al., 1985), suggesting that coffee may have teratogenic and mutagenic effects. This outcome is surprising since most of the well-documented teratogens are known to produce a specific pattern of teratogenicity and not a wide variety of different malformations. In addition there is no evidence that coffee is mutagenic in vivo. Hidden bias has been considered to be the most probable explanation for these data (Narod et al., 1991). In the third study, it was suggested that drinking more than eight cups of coffee a day during pregnancy was weakly associated with an increased frequency of congenital malformations (BorleÂe et al., 1978). However, this study involved only a small number of cases and did not account for important confounding factors such as tobacco consumption. Furthermore, the statistical analysis was questionable (Nehling & Debry, 1994b). Overall, there is no evidence to implicate moderate coffee or caffeine consumption in the aetiology of human congenital malformations.

(b) Animal data Contrary to the human data, dose-dependent teratogenic effects have been observed in various animal models including mice, rats, rabbits and monkeys (Nehling & Debry, 1994b; Brent, 1998). Most of these effects were observed with very high doses of caffeine,


which resulted in maternal toxicity. Such doses are not achievable through normal coffee consumption in humans. In addition, the mode of administration of caffeine plays a major role on the final outcome (Nehling & Debry, 1994b). Teratogenic effects in animals have been principally observed in studies using a daily administration of caffeine as single high doses (injection, subcutaneous or gavage) resulting in high plasma concentrations. Exposing the animals through multiple fractionated administrations or dietary feeding generally failed to produce any effects or required much higher doses to be active. The experimental designs of most of the animal studies showing teratogenic effects do not reflect the pattern of human caffeine exposure through coffee consumption. The relevance of these data to humans is therefore difficult to evaluate.

8.7.2 Neurodevelopmental effects It is well known that caffeine is a stimulant because of its neuropharmacological properties. Neuropharmacological agents may produce subtle neurochemical or behavioural effects on developing organisms at doses which do not induce any overt toxicity. Therefore the potential neurodevelopmental effects resulting from either pre- or postnatal exposure to caffeine have been investigated in both humans and animal models (Nehling & Debry, 1994b; 1994c).

(a) Human data Limited information is available regarding the potential influence of caffeine intake by pregnant women on the function of the newborn nervous system. The consequences on neurodevelopment resulting from maternal caffeine consumption during pregnancy were investigated by following about 500 children from birth to 7 years of age. The prenatal caffeine exposure did not influence neurobehavioral outcomes and the suckling reflex in the first 2 days of life (Barr & Streissguth, 1991) and no effects on cognitive and motor development could be observed at 8 months of age (Streissguth et al., 1980; Barr et al., 1984). In addition, no effects on intelligence quotient at 4 and 7 years or on motor ability at 4 years and on vigilance at 7 years were found (Barr & Streissguth, 1991). In one study, no physiological or neurobehavioral disturbances were observed in infants who had measurable caffeine plasma concentrations at birth (Dumas et al., 1982) although in another, increased visual arousal and nervousness were linked to salivary

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caffeine concentration in 1 to 2-day-old babies (Emory et al., 1988). A withdrawal type syndrome has been observed in babies from mothers exposed to very high levels of caffeine (Nehling & Debry, 1994b). In addition, some studies have suggested a possible role of caffeine on respiratory control function and neonatal apnea (Toubas et al., 1986) and therefore the issue of caffeine exposure and sudden infant death syndrome has been raised. In a recent retrospective epidemiological study, an increased risk for sudden infant death syndrome was associated with heavy maternal caffeine ingestion (Ford et al., 1998) although no dose response was found. Since such a link has not been described before, further analysis is required, taking into consideration all possible known risk factors which could play a confounding role. In this context it is important to note that caffeine has been used safely in clinical settings to treat apnea in premature babies (Nehling & Debry, 1994c).

(b) Animal data The administration of moderate to high doses of caffeine or coffee to female rodents during gestation has been documented to significantly alter the brain neurochemistry and composition of the neonatal pups (Nehling & Debry, 1994b; Brent, 1998). In addition, pre- and perinatal caffeine exposure have been shown to affect sleep control and behaviour in the offspring. For example, in the offspring of dams exposed to moderate to high doses of caffeine during gestation and/or lactation, increased locomotion and spontaneous activities were observed later in life (Nehling & Debry, 1994b). Effects on learning abilities have also been found. In general, long-term behavioural disturbances in rodents have only been observed at high levels of maternal caffeine exposure, which induce other effects such as delayed growth. The doses involved are unlikely to be achievable in the human situation.

8.7.3 Low birth weight, growth retardation and prematurity Low birth weight is often defined as < 2500 g. Low birth weight may be the result of a shortened gestational period (prematurity) or the consequence of intrauterine growth retardation resulting in a `small for age' infant. Many medical, social and lifestyle factors are known to influence birth weight, some of them being directly correlated with coffee consumption. Therefore the interpretation of the literature in this

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field is particularly difficult. Maternal caffeine or coffee ingestion as a risk factor for delivering low birth weight infants has been extensively investigated. Generally, there appears to be no relationship between caffeine intake during pregnancy and the probability of premature delivery in humans (Hinds et al., 1996). The epidemiological literature on maternal coffee or caffeine intake during gestation and the risk factor for low birth weight or `small for age' babies is conflicting. Some studies did not find any evidence of association while others found a direct correlation (Brent, 1998; Narod et al., 1991; Nehling & Debry, 1994b). In studies showing a link, effects were observed at relatively high dose of caffeine, often > 300 mg/day, although some studies suggested effects at lower levels of exposure. The standard reported decrease in birth weights ranged from 70 to 121 g (Narod et al., 1991). More recent studies have also provided contradictory data. For example, in a study conducted in Brazil, the proportion of mothers who delivered babies with intrauterine growth retardation increased according to coffee consumption (Rondo et al., 1996). In a Japanese study, coffee was not identified as a risk factor for low birth weight (Maruoka et al., 1998). The likelihood for a low birth weight baby to suffer from further health problems later in life is thought to be dependent upon the cause of the low birth weight. There is no evidence reported on potential long-term adverse consequences resulting from coffee- or caffeine-induced low birth weight. The inconsistency of the literature regarding maternal caffeine ingestion and low birth weight has been thought to result from the difficulty of establishing small effects or their absence. Furthermore, the direct relation between coffee intake and other agents documented to adversely affect fetal growth such as smoking and alcohol consumption complicates the interpretation of the data in this area of research. Smoking is thought to induce foetal hypoxia through an increase in blood carboxyhemoglobin and a reduction in placental blood flow (Nehling & Debry, 1994c). These effects trigger an adaptive tissue response. Caffeine, by blocking adenosine receptors, could inhibit the normal adaptive cellular response to hypoxia and therefore may potentiate the effects of smoking. Reports have suggested that the effects of caffeine on birth weight were stronger in or even restricted to smokers (Nehling & Debry, 1994b,c). In a recent prospective study (Cook et al., 1996) the relationship of fetal growth to caffeine intake and blood caffeine concentrations during pregnancy was investigated. In


smokers, caffeine consumption was inversely related to birth weight. Smokers were found to consume more caffeine than non-smokers. However, blood caffeine concentrations were lower in smokers than in nonsmokers due to the stimulation of caffeine metabolism by tobacco consumption. No relation was found between blood caffeine concentrations during pregnancy and birth weight. These data support the key role played by cigarette smoking as a confounder in the studies addressing the association between prenatal caffeine exposure and birth weight.

8.7.4 Spontaneous abortion The relationship between maternal coffee or caffeine ingestion in pregnancy and the risk of spontaneous abortion has been extensively investigated. The data are conflicting. Several studies have suggested an association while others did not observe any effects (Narod et al., 1991; Nehling & Debry, 1994b,c; Hinds et al., 1996; Brent, 1998). Recent studies found either no effect or a slight increase in spontaneous abortion associated with coffee or caffeine consumption. In one study, neither total estimated caffeine nor individual caffeinated beverage consumption during the first trimester of pregnancy was associated with an appreciable increase in risk for spontaneous abortion (Fenster et al., 1997). Another study found a modest increased risk of clinically recognised spontaneous abortion when caffeine intake exceeds 300 mg per day (Dlugosz et al., 1996). Data have been reported on the association between maternal serum paraxanthine, the primary caffeine metabolite, and the risk of spontaneous abortion (Klebanoff et al., 1999). It was found that only extremely high serum paraxanthine concentrations corresponding to a consumption of more than six cups/day were associated with spontaneous abortion. Confounding factors and bias may have played an important role in the association between caffeine or coffee consumption and spontaneous abortion reported in some articles. For example, it is known that nausea in pregnancy is associated with food aversion and is likely to result in a reduction of coffee or caffeine consumption. Furthermore, it is documented that nausea is associated with a decrease in spontaneous abortion (Stein & Susser, 1991). Therefore, it was postulated that a pregnancy with a higher probability of a viable outcome might increase nausea and in consequence decrease caffeine ingestion. Based on this hypothesis, it appears that studies addressing fetal loss and caffeine which do not account for nausea are likely to over-


estimate the risk of caffeine exposure. Most of the studies available do not have any information on nausea incidence. One study reported that the risk of spontaneous abortion for heavy caffeine consumers varied according to whether there was nausea during pregnancy (Fenster et al., 1991).

8.7.5 Fertility Animal studies on the effects of caffeine on fertility and reproduction in animals are limited. High doses have been shown to produce an increased time to pregnancy in rodents, suggesting a possible effect of coffee or caffeine on delaying fertility. The literature regarding coffee or caffeine consumption and human fertility is controversial and inconsistent. Some studies observed a reduction in fertility associated with coffee intake, sometimes in a dose-dependent way (Wilcox et al., 1988), others found no effects, even in heavy drinkers (Narod et al., 1991; Nehling & Debry, 1994c; Bolumar et al., 1997). In a large study conducted in Denmark involving 10 886 women (Olsen et al., 1991), a delayed time of conception (subfecundity) was found, but only in smokers consuming high doses of coffee (> 8 cups). In a recent American study, it was found that high levels of caffeine consumption (> 300 mg/day) may result in delayed conception in women who do not smoke cigarettes (Stanton & Gray, 1995). In a recent study involving a large random European population, subfecundity was associated with a high caffeine intake (> 500 mg/day) in fertile women (Bolumar et al., 1997). This effect was stronger in smokers. At the highest level of intake, the time leading to the first pregnancy was increased by 11%. In this study, several but not all important confounding factors were adjusted for and the caffeine exposure determinations were relatively accurate. Based on their data, the authors stated that caffeine, among others, can be considered a weak risk factor that probably reduces fecundity by a certain fraction, but without being a sufficient cause of infertility. Delayed conception is relatively common and many factors, including exercise, stress, nutrition, lifestyle and social influences, may be involved. In many studies, most of these confounding factors were not or could not be adjusted for. In addition, the question of the mechanism involved in the potential effects of coffee or caffeine on fertility is not answered. In conclusion, there is no solid evidence linking moderate coffee consumption and adverse effects on fertility parameters.

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8.7.6 Conclusions Caffeine, mostly through coffee consumption, has been implicated in several types of developmental and reproductive adverse events. Based on the literature, it appears that caffeine at levels lower than 300 mg/day is unlikely to produce in humans any effects on reproductive and developmental health outcomes. The information currently available does not allow the accurate estimate of the effects of higher levels of exposure.

8.8 EMERGING BENEFICIAL HEALTH EFFECTS Coffee has recently been shown to have positive health effects. Although not yet proven and requiring substantial confirmation through well controlled epidemiological studies, taking into account all possible bias and confounders, these emerging beneficial effects were considered worth discussing.

8.8.1 Neuroactivity Coffee consumption has been perceived to have a positive influence on human behaviour. Caffeine appears to be the key coffee constituent responsible for these effects. For example, caffeine has been documented to increase alertness, to improve performance on vigilance tasks and to reduce fatigue (Smith, 1998). A beneficial effect which has emerged in the area of coffee-related neuroactivity is the potential preventive influence of caffeine on suicide and depression. A strong inverse association has been reported between coffee intake and risk of suicide in a prospective study involving 128 933 people (Klatsky et al., 1993). A dosedependent relationship was observed and those consuming more than six cups of coffee per day showed a 5-fold lower risk of suicide than nonconsumers. Another prospective study showed the relative risk of suicide in women consuming two to three cups of coffee per day to be about 3-fold lower than in non-coffee drinkers (Kawachi et al., 1996). This association could be spurious if depressed patients avoid caffeine either spontaneously or through the advice of health professionals. However, the neuroactive property of caffeine could explain a preventive effect of caffeine. Compared to placebo, experimental administration of caffeine has been shown to increase the subjective feelings of well-being, social disposition, self-confidence, energy and motivation at work

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(Griffiths et al., 1990). In a psychiatric setting, reports suggest that patients' experience improved mood (Furlong, 1975) and decreased irritability (Stephenson, 1977) following administration of caffeine. Although no population-based prospective studies on coffee or caffeine intake and depression have been reported, a cross-sectional study of Japanese medical students found that high intake of caffeine was associated with fewer depressive symptoms among female but not male students (Mino et al., 1990).

8.8.2 Chemoprotection Epidemiological evidence indicates that coffee consumption may be protective against certain types of cancers such as colon cancer (Giovannucci, 1998). Experimental data suggest that this chemoprotective effect could be related to the presence in coffee of both strong antioxidants and components able to stimulate chemical detoxification processes (see Section 8.4). Since these coffee components act through relatively general mechanisms, other chemoprotective effects can be expected. Recently, several epidemiological studies have observed that coffee consumption significantly reduced the risk of developing liver disease (cirrhosis) induced by alcohol ingestion (Klatsky et al., 1992, 1993; Corrao et al., 1994). In the first report on that topic, it was observed that persons who drank four or more cups of coffee a day had a 5-times lower risk of developing alcoholic cirrhosis than non-coffee drinkers (Klatsky et al., 1992). In a recent study, the beneficial effect of coffee on alcoholic cirrhosis was further confirmed (Collaborative GESIA group, in press). In addition, this study addressed the joint action of coffee consumption and hepatic viral risk factors of cirrhosis on the resulting risk of developing the disease. Coffee was found to antagonise the promoting effects of hepatitis B and C infection on cirrhosis development, suggesting a protective effect of coffee on non-alcoholic cirrhosis. Further work is required to demonstrate clearly this chemopreventive effect. For instance, it has to be clarified whether the inverse association between coffee intake and cirrhosis observed in epidemiological studies is real or whether it is a consequence of coffee aversion in patients developing severe cirrhosis. Furthermore, the mechanism of action has to be established. A plausible mechanism refers to the presence in coffee of potentially protective factors including antioxidants and detoxification stimulating agents. Evidence excludes caffeine as playing a key role


(Collaborative GESIA Group, in press; Klatsky et al., 1992). Coffee consumption has been repeatedly found in clinical and epidemiological studies to reduce the levels of serum g-glutamyltransferase, a marker of hepatobiliary diseases (Nilssen & Forde, 1994; Ugert & Katan, 1997; Tanaka et al., 1998). This effect further supports a potentially more general protective effect of coffee on the liver. The coffee-specific diterpenes cafestol and kahweol have recently been shown to reduce serum levels of g-glutamyltransferase and to modulate the levels of other common indicators of liver function (Ugert et al., 1995; Ugert & Katan, 1997) and detoxification (Schilter et al., 1996; Cavin et al., 1998). Other recent data suggest that coffee and caffeine may possess broader chemoprotective properties. The potential development of late radiation-related tissue complications is an important dose-limiting consideration in clinical radiotherapy treatment to control tumours. An epidemiological study has found that patients consuming caffeine-containing beverages such as coffee at the time of their radiotherapy against cervical cancer had significantly decreased incidence of severe late radiation injury (Stelzer et al., 1994).

8.9 COFFEE CONSUMPTION – SAFETY CONSIDERATIONS Traditional foods are thought to be safe on the basis of long-term experience, even though these foods may contain inherent toxicants. With respect to toxic potential, food is presumed safe unless a significant risk has been identified in humans. However, it is also acknowledged that the innocuity of food is not strictly demonstrated without the provision of a fully documented history of safe use in humans based on specific data. For most traditional foods, such data are not available. Coffee has been consumed for over 1000 years by many human beings. There has been no evidence that coffee intake is associated with clearly identified adverse health effects. Therefore coffee should be considered as a traditional food with a long history of safe use. In contrast to most other traditional foods, coffee and coffee components have been the subject of many extensive scientific investigations in both animal models and humans. Epidemiological studies addressing the potential adverse effects of moderate coffee consumption (3 to 5 cups/day) on various key health outcomes including cancers, cardiovascular disease, osteoporosis and developmental effects have been largely inconsistent.


Where present, the effects were usually weak and not dose-dependent. Plausible mechanisms are often missing. Based on this literature, it appears that, in general, moderate coffee consumption ranging from 3 to 5 cups per day is unlikely to be of any health concern. While most of the human data converge to show that moderate coffee consumption is safe, the information presently available does not allow accurate evaluation of the risk associated with higher levels of consumption. It is important to note that in the situation of high intake, residual confounding factors may significantly bias the data. Many studies have indirectly seen that heavy coffee consumption was directly related to lifestyles known to be important risk factors for vascular diseases, malignancies and developmental adverse effects. A recent study (Leviton et al., 1994) indicated that heavy coffee drinkers were more likely to smoke and less likely to take vitamin supplements and to consume a healthy diet (high vegetable, high vitamin, high fibre, low fat). The authors of this study proposed that heavy coffee drinkers may be at increased risk for a number of diseases not because of coffee consumption per se, but because of other aspects of their lives. Assuming an average caffeine concentration of 60 to 85 mg per cup of instant and roasted and ground coffees, respectively (Barone & Roberts, 1996), moderate coffee consumption as defined above corresponds to a caffeine exposure ranging from 180 to 425 mg. This is in the range of what has been considered safe by many authors cited in the present paper. Some developmental studies have sometimes suggested slight effects at lower doses, so it is prudent to advise pregnant women to stay at the lower level of the safe range in order to account for the remaining uncertainty. Safe levels of exposure to the cholesterol-raising diterpenes cafestol and kahweol have not been officially established. However, based on an average cafestol concentration of 1 mg/cup (0 to 3.1 mg/cup) and using clinical data on the effects of the diterpenes on blood cholesterol, the consumption of five cups per day of espresso coffee has been considered to have negligible hypercholesterolemic effects (Ugert et al., 1995). Comparison of this figure used as a safe level of exposure with cafestol occurrence data (Ugert et al., 1995; Ugert & Katan, 1997) reveals that, except for boiled Turkish and French press coffee, up to five cups of coffee a day are unlikely to have any appreciable effects on blood cholesterol.

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8.10 CONCLUSIONS The potential health effects of coffee consumption have been extensively investigated in animal models and in human studies. Overall, the available information indicates that moderate consumption, corresponding to three to five cups of average strength coffee per day, is safe for human health. The data do not allow an accurate evaluation of the potential risk at higher consumption levels.

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Packard, P.T. & Recker, R.R. (1996) Caffeine does not affect the rate of gain in spine bone in young women. Osteoporosis Int., 6, 149±52. Palmer, J.R., Rosenberg, L., Sowmya, R. & Shapiro, S. (1995) Coffee consumption and myocardial infarction in women. Am. J. Epidemiol., 141, 724±31. Polychronopoulou, A., Tzonou, A., Hsieh, C. C. et al. (1993) Reproductive variables, tobacco, ethanol, coffee and somatometry as risk factors for ovarian cancer. Int. J. Cancer, 55, 402± 407. Probert, J.L., Persad, R.A., Greenwood, R.P., Gillatt, D.A. & Smith, P.J.B. (1998) Epidemiology of transitional cell carcinoma of the bladder: profile of an urban population in the south-west of England. Br. J. Urol., 82, 660±66. Rondo, P.H.C., Rodrigues, L.C. & Tomkins, A.M. (1996) Coffee consumption and intrauterine growth retardation in Brazil. Eur. J. Clin. Nutr., 50, 705±709. Schilter, B., Perrin, I., Cavin, C. & Huggett, A.C. (1996) Placental gluthatione S-transferase (GST-P) induction as a potential mechanism for the anti-carcinogenic effect of the coffeespecific components cafestol and kahweol. Carcinogenesis, 17, 2377±84. Sesso, H.D., Gaziano, J.M., Buring, J.E. & Hennekens, C.H. (1999). Coffee and tea intake and the risk of myocardial infarction. Am. J. Epidemiol., 149, 162±7. Silverman, D.T., Swanson, C.A., Gridley, G. et al. (1998) Dietary and nutritional factors and pancreatic cancer: a case-control study based on direct interviews. J. Nat. Cancer Inst., 90, 1710±19. Smith, A. (1998) Effects of caffeine on human behavior. International Life Sciences Institute (ILSI) North America Publishers, Washington, DC. Smith, S.J., Deacon, J.M. & Chilvers, C.E.D. (1994) Alcohol, smoking, passive smoking and caffeine in relation to breast cancer in young women. Br. J. Cancer, 70, 112±19. Stadler, R., Bexter, A., WuÈrzner, H.P. & H. LuginbuÈhl (1990) A carcinogenicity study of infant coffee in swiss mice. Food Chem. Tox., 28, 829±30. Stadler, R.H., Richoz, J., Turesky, R.J., Welti, D.H. & Fay, L.B. (1995) Oxidation of caffeine and related methylxanthines in ascorbate and polyphenol-driven Fenton-type oxidations. Free Rad. Res., 24, 225±40. Stadler, R.H., Turesky, R.J., MuÈller, O., Markovic, J. & LeongMorgenthaler P.-M. (1994) The inhibitory effects of coffee on radical-mediated oxidation and mutagenicity. Mut. Res., 308, 177±90. Stanton, C.K. & Gray, R.H. (1995) Effects of caffeine consumption on delayed conception. Am. J. Epidemiol., 142, 1322± 9. Stein, Z. & Susser, M. (1991) Miscarriage, caffeine and the epiphenomena of pregnancy: the causal model. Epidemiology, 2, 163±7. Stelzer, K.J., Koh, W.J., Kurtz, H., Greer, B.E. & Griffin, T.W. (1994) Caffeine consumption is associated with decreased severe late toxicity after radiation to the pelvis. Int. J. Radiat. Oncol. Biol. Phys., 30, 411±17. Stensvold, I. & Jacobsen, B.J. (1994) Coffee and cancer: a


prospective study of 43 000 Norwegian men and women. Cancer Causes Contr., 5, 401±408. Stensvold, I., Tverdal, A. & Foss, O.P. (1989) The effects of coffee on blood lipids and blood pressure: results from a Norwegian cross-sectional study, men and women, 40±42 years. J. Clin. Epidemiol., 42, 877±84. Stensvold, I., Tverdal, A. & Jacobsen, B. (1996) Cohort study of coffee intake and death fron coronary heart disease over 12 years. Br. Med. J., 312, 544±5. Stephenson, P.E. (1977) Physiologic and psychotropic effects of caffeine in man. J. Am. Dietetic. Assoc., 71, 240±44. Stich, H.F., Risin, M.P. & Bryson, L. (1982) Inhibition of mutagenicity of a model nitrosation reaction by naturally occuring phenolics, coffee and tea. Mut. Res., 259, 307±24. Stolzenberg-Solomon, R.Z., Miller E.R., Maguire M.G., Selhub, J. & Appel, L.J. (1999) Association of dietary protein intake and coffee consumption with serum homocysteine concentrations in an older population. Am. J. Clin. Nutr., 69, 467±75. Streissguth, A.P., Barr, H.M., Martin, D.C. & Herman, C.S. (1980) Effects of maternal alcohol, nicotine, and caffeine use during pregnancy on infant mental and motor development at eight months. Alcoholism Clin. Exp. Res., 4, 152±64. Tanaka, K., Tokunaga, S., Kono, S. et al. (1998) Coffee consumption and decreased serum gamma-glutamyltransferase and aminotransferase activities among male alcohol drinkers. Int. J. Epidemiol., 27, 438±43. Tavani, A. & La Vechia, C. (1997) Epidemiology of renal-cell carcinoma. J. Nephrol., 10, 93±106. Tavani, A., Pregnolato, A., La Vecchia, C., Favero, A. & Franceschi, S. (1998) Coffee consumption and the risk of breast cancer. Eur. J. Cancer Prevent., 7, 77±82. Thelle, D.S., Heyden, S. & Fodor, J.G. (1987) Coffee and cholesterol in epidemiological and experimental studies. Artherosclerosis, 67, 97±103. Toubas, P.L., Duke, J.C., McCaffree, M.A., Mattice, C.D., Bendell, D. & Orr, W.C. (1986) Effects of maternal smoking and caffeine habits on infantile apnea: a retrospective study. Pediatrics, 78, 159±63. Tuomilehto, J. & Pietinen, P. (1991) Coffee and cardiovascular disease. Cardiovasc. Risk Factors, 1, 165±73. Tverdal, A., Stensvold, I., Solvoll, K., Foss, O.P., Lund-Larsen, P. & Bjartweit, K. (1990) Coffee consumption and death from coronary heart disease and mortality in middle-aged Norwegian men and women. Br. Med. J., 300, 566±9. Ugert, R. & Katan, M.B. (1997) The cholesterol-raising factor from coffee beans. Ann. Rev. Nutr., 17, 305±24. Ugert, R., Van der weg, G., Kosmeijer-Schuil, T.G., Van Bovenkamp, P., Hovenier, R. & Katan, M. (1995) Levels of the cholesterol-elevating diterpenes cafestol and kahweol in various coffee brews. J. Agr. Food Chem., 43, 2167±72. Van Dusseldorp, M., Katan, M., Van Vliet, T., Demacker, P.N. M. & Stalenhoef, A. (1991) Cholesterol-raising factor from boiled coffee does not pass a paper filter. Arterioscler. Thromb., 11, 586±93. Viscoli, C.M., Lachs, M.S. & Horwitz, R.I. (1993) Bladder cancer and coffee drinking: a summary of case-control research. Lancet, 341, 1432±7.


Vollset, S.E., Nygard, O., Refsum, H. & Ueland P.M. (2000) Coffee and homocysteine. Am. Clin. Nutr., 71, 403±404. Weiderpass, E., Partanen, T., Kaaks, R. et al. (1998) Occurrence, trends and environmental etiology of pancreatic cancer. Scand. J. Work Environ. Health, 24, 165±74. Weusten-Van der Wouw, M.P.M.E., Katan, M., Viani, R. et al. (1994) Identity of the cholesterol-raising factor from boiled coffee and its effects on liver function enzymes. J. Lipids Res., 35, 721±33. Wilcox, A., Weinberg, C. & Baird, D. (1988) Caffeinated beverages and decreased fertility. Lancet, 2, 1453±6. Willett, W.C., Stampfer, M.J., Manson, J.E. et al. (1996) Coffee consumption and coronary heart disease in women, a ten-year follow-up. J. Am. Med. Assoc., 276, 458±62.

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Woodward M. and Tunstall-Pedoe, H. (1999) Coffee and tea consumption in the Scottish heart health study follow-up: conflicting relations with coronary risk factors, coronary disease, and all cause mortality. J. Epidemiol. Community Health, 53, 481±7. World Health Organisation International Agency for Research on Cancer (WHO/IARC) (1991) Coffee, Tea, Mate, Methylxanthines and Methylglyoxal. In: IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, 51, 47±206. IARC, Lyon, France.

Chapter 9

Agronomy I: Coffee Breeding Practices Herbert A.M. Van der Vossen Plant Breeding & Seed Consultant Venhuizen, the Netherlands 9.1 INTRODUCTION 9.1.1 World production increase World coffee production ± about 70% arabica (Coffea arabica) and 30% robusta (C. canephora) coffees ± continues to show large annual fluctuations, but has generally increased by about 14% over the past 15 years: from 5.2 million tonnes per year averaged over the years 1980±84, to 5.9 million over the period 1995± 9 (ICO, 1990±99). There was a very low production of 4.8 million t in 1986±7 and a record of 6.4 million t in the 1998±9 crop year. As usual, the tremendous variation in annual coffee output by the leading producer Brazil ± from 19% to 33% of world coffee production, mainly as a result of recurring abiotic calamities (frosts and droughts) ± had a major impact on fluctuations in world coffee supplies and market prices. About 61% of the world coffee was produced in Latin America, 18% in Africa and 21% in Asia during the 1998±9 crop year. Of particular interest is the accelerated expansion of robusta coffee production in Vietnam and Indonesia to more than 400 000 t. Brazil produced in that year some 300 000 t robusta coffee in addition to its 1.8 million t arabica crop and is rapidly overtaking the traditionally leading robusta producers Ivory Coast and Uganda. India has almost doubled its annual production during the last decade and may soon reach an annual output of 300 000 t of high quality arabica (40%) and robusta (60%) coffees.

9.1.2 Selection and breeding before 1985 Reviews on the history and progress of selection and breeding for arabica and robusta coffees until the mid1980s have been presented by Van der Vossen (1985), Carvalho (1988), Charrier & Berthaud (1988) and Wrigley (1988). Bettencourt & Rodrigues (1988) produced a review specifically on disease resistance

breeding and Cambrony (1988) on interspecific hybridisation in coffee. The following summary of major advances in breeding and variety development, as reviewed by these authors, may serve as a useful background to the remaining paragraphs of this chapter. Much of the world coffee is still produced by traditional cultivars released some five to eight decades ago from relatively simple selection and breeding programmes and generally multiplied by seed. Cultivars of the self-pollinating arabica coffee are truebreeding lines from single-plant selections in growers' fields, or from progenies of simple crosses and backcrosses; while those of the outbreeding robusta are open-pollinated cultivars produced from selected seedling and bi- or polyclonal gardens. Clonal robusta cultivars have found limited application so far, except in plantation coffee in Indonesia and the Ivory Coast, largely because the logistics of mass propagation and distribution are too complex and expensive for smallholder production systems, which dominate coffee production. Yield, plant vigour and quality have been the main selection criteria in both coffee types, but host resistance to the destructive coffee leaf rust (CLR) disease has already been given high priority in arabica coffee breeding in India since the 1920s. Breeding programmes with systematic crossing designs and statistically laid out field trials implemented during the last 30 years have provided opportunities for biometrical genetic analyses of yield and other agronomic characters. There is considerable evidence of predominantly additive genetic variance for almost all components of yield, quality and other quantitative traits, except sometimes the complex factor yield itself, both for arabica as well as robusta coffee. This should facilitate the estimation of parental breeding values from relatively simple progeny testing and increase selection progress. Hybrid vigour for yield noticed in crosses between parents of different origins

184

Agronomy I: Coffee Breeding Practices

appears to be the result of accumulation of complementary polygenes dispersed over subpopulations. Some breeding programmes in robusta coffee (e.g. in the Ivory Coast) have already adopted methods of reciprocal recurrent selection with distinct subpopulations to increase chances of producing genotypes superior in yield, quality and other important traits. In arabica coffee, disease resistance is a breeding objective of the highest priority. Efforts to obtain durable resistance to CLR have had a long history of initial successes followed by disappointments because of the repeated appearance of new virulent races of the rust fungus, but some lines of the cultivar Catimor (selected from crosses between Caturra and Hibrido de Timor) have shown complete resistance in most countries. These results were obtained by breeding plans normally applied to self-pollinating crops, including recombination crosses followed by backcrossing, inbreeding and pedigree selection. A similar plan was initially also applied in a breeding programme in Kenya to obtain resistance to coffee berry disease (CBD), which turned out to be controlled by a few major genes but nevertheless also durable. The change of breeding strategies to produce F1 hybrid (seed) cultivars instead of clones or true breeding lines was partly inspired by the confirmation of transgressive hybrid vigour in genetically divergent crosses in arabica coffee. Other advantages were chances of earlier introduction of cultivars with resistance to both CBD and CLR, as well as several other desirable agronomic characters. Interspecific hybridization has played a significant role in coffee, such as crosses between arabica and robusta coffee with the objective of introgressing disease resistance into arabica (for example the cultivar Icatu in Brazil) or improved liquor quality into robusta coffees (for example the variety Arabusta in the Ivory Coast). Other examples of interspecific hybridization leading to successful cultivars in arabica and in robusta coffee can be found in India.

9.1.3 New developments Modern arabica cultivars with higher yield potential and resistance to important diseases (CLR and CBD) have started to replace traditional varieties on a large scale in several countries: for example Catimor and Sarchimor types of cultivars in Colombia, Brazil, Central American countries and India, Icatu in Brazil, Java in Cameroon, Ruiru II in Kenya and Ababuna in Ethiopia (the latter two being F1 hybrids). In robusta

185

coffee the release of new cultivars from advanced selection programmes is taking place more gradually, such as the BP and SA clones in Indonesia, the BR (seed) cultivars in India, the IF clones in the Ivory Coast and the cultivar Apoata in Brazil. The high expectations of the Arabusta programme in the Ivory Coast have not been fulfilled, because of persistent problems of genetic instability and low fertility. On the other hand, the `C 6 R' variety of India, which arose from a cross between C. congensis and C. canephora, has proved to be a success as a productive and stable robusta coffee with superior bean and liquor characteristics. The breakdown of resistance to CLR in Catimor lines in India, the sudden reappearance of a wilt disease (tracheomycosis) in robusta coffee in DR Congo and Uganda, increasing nematode problems in arabica coffee in Central America and the arrival of the coffee berry borer in Colombia (1988) and India (1990) are just a few examples of new challenges to national coffee industries, which need to be met also by innovative selection and breeding programmes. Plant biotechnology has evolved, particularly during the past decade, into an applied science providing powerful additional tools for plant breeding with the potential of increasing selection efficiency and creating new approaches to hitherto unattainable objectives. This so-called molecular breeding has basically two main applications of plant biotechnology: molecular markers and transgenic plants. In coffee, molecular marker technology has already been implemented in germplasm characterisation and management, detecting genetically divergent breeding subpopulations (for example to predict hybrid vigour), establishing gene introgression from related species and molecular marker-assisted selection (Lashermes et al., 1996a, 1997a; Charrier & Eskes, 1997). Generally, successful genetic transformation is still limited to characters controlled by major genes for which gene isolation and transfer is relatively easy. Techniques of regenerating plants from in vitro micropropagation and somatic embryogenesis are by now well established for various coffee species and transgenic coffee plants have been produced already, for example with insect resistance and with caffeinefree beans (see Chapters 10 and 11). However, lack of adequate legislation in some countries for proprietary rights and biosafety, as well as a negative public perception of biotechnology, can be temporary obstacles to the introduction and unrestricted cultivation of transgenic coffee cultivars. While formerly, breeding programmes were often

186

carried out in relative isolation at the various coffee research centres, the 1990s saw increased networking between a number of them and also with renowned agricultural research centres in Europe and the USA to implement collaborative research projects on genetic resources, resistance breeding and plant biotechnology.

9.2 GENETIC RESOURCES 9.2.1 World collections The first systematic efforts to collect Coffea arabica germplasm by an FAO mission to Ethiopia in 1964 had the real intention of international collaboration and the resulting 623 accessions were distributed worldwide to several coffee research centres (Meyer et al., 1968). A second expedition to the south-western highlands of Ethiopia mounted by the IRD (ex ORSTOM) in 1966 produced another 70 new accessions of C. arabica. The IRD (ex ORSTOM), sometimes in collaboration with IPGRI, made several collecting expeditions for a large number of other Coffea species between 1960 and 1985 to important centres of genetic diversity in Guinea, the Ivory Coast, Central Africa, Cameroon, Congo, East Africa and Madagascar. Most of these accessions are maintained as base field collections in the Ivory Coast and Madagascar (Berthaud & Charrier, 1988). More recent efforts of coffee germplasm collection include a mission to Yemen for C. arabica (Eskes & Mukred, 1989) and one to north-west Tanzania for C. canephora (Nyange & Marandu, 1997). There is a considerable amount of ex situ germplasm of C. arabica collected and maintained in Ethiopia since 1966 (Bellachev, 1997). Altogether about 100 species (taxa) of the genus Coffea have been identified so far (Bridson & Verdcourt, 1988). They are without exception indigenous to the forests of tropical Africa and all are diploid (2n = 22) species (C. canephora, C. congensis, C. liberica, C. eugenioides, C. stenophylla, C. racemosa, C. zanguebariae, etc. and some 50 taxa belonging to the section Mascarocoffea) except the allotetraploid (2n = 44) C. arabica, which has its origin in the highland forests of south-west Ethiopia. Progressive crop improvement requires easy access to intra- and interspecific genetic variation. The monophyletic origin of all Coffea species and general absence of strong interspecific crossing barriers (Charrier & Berthaud, 1985; Charrier & Eskes, 1997) provide opportunities to exploit as well the genetic variation of several other species for the purpose of


introgressing agronomically and biochemically interesting characters into the two species of commercial value, C. arabica and C. canephora. The three main base collections in Ethiopia, the Ivory Coast and Madagascar are, therefore, of vital importance to coffee breeding in general and should receive adequate support for maintenance and further systematic exploration for new germplasm by an internationally recognised network organization for the conservation and study of coffee genetic resources with the participation of all major coffee producing countries. IPGRI in conjunction with IRD, CIRAD and the African Coffee Research Network (ACRN) has taken initiatives in that direction (Guarino et al., 1995; Ngategize, 1997), but free exchange of coffee germplasm between producing countries will continue to be restricted until the formal establishment of a network, such as the one already existing for cocoa (Eskes et al., 1998). Hamon et al. (1998) proposed strategies to manage such large germplasm collections more efficiently and increase their accessibility by identification of core collections, which are representative of specifically desired genetic diversity without undue duplication. The identification procedure involves the application of so-called principal component score strategies. Dulloo et al. (1998) have described strategies for in situ conservation of Coffea species. In addition to the three earlier mentioned base collections, many research centres in coffee-producing countries maintain duplicate field collections of cultivated and wild coffee germplasm as genetic resources for their breeding programmes. Table 9.1 presents an overview of location, type and size of major field collections of coffee germplasm in the world. Accessions often contain several genotypes, especially when originally collected as a seed sample from wild coffee trees. Published reports on coffee germplasm collections sometimes give rather inflated numbers, because the distinction between an accession and a genotype is not always clearly made. In Table 9.1 adjustments have been made for number of accessions, whenever such information was available.

9.2.2 Species relationships Various recent studies of genetic diversity and phylogenetic relationships within the genus Coffea have applied molecular marker technologies to chloroplast and nuclear DNA extracted from several coffee species (Orozco-Castillo et al., 1994, 1996; Lashermes et al., 1995, 1996b, 1997a, 1999a; Cros et al., 1998). These molecular markers have advantages over the


Table 9.1

187

Field collections of coffee genetic resources. Germplasm of

Country

Organisation

Base collections Ethlopia BCRI/E EIAR/JARC

Location

Coffea arabica

Choche Jimma, Gera

6 6 6

Coffea canephora

Other species

Mascarocoffea

Total number of accessions 800 2000

Ivory Coast

CNRA

Divo Man

6

Madagascar

FOFIFA

Kianjavato Ilaka-Est Sahambavy

6

Work collections Costa Rica CATIE

Turrialba

6

6

[24]

6 6

1200 200 6

[7]

800 1200 350 1700

Brazil

IAC

Campinas

6

6

[9]

310

Colombia

CENICAFE

Chinchina

6

6

[10]

980

Kenya

CRF

Ruiru

6

[5]

500

Tanzania

TARO

Lyamungu

6

6

[10]

300

Cameroon

IRAD

Foumbot, Santa Nkoumvane

6

6

[3]

100 50

Indonesia

ICCRI

Jember, etc.

6

6

6

1000

India

CCRI

Chickmagalur

6

6

[18]

360

Note: [24] = number of other species. Sources: Dulloo et al. (1998); IPGRI Directory of coffee germplasm collections (1999). 6 = available at location.

morphological and biochemical characters measured in conventional taxonomic analyses, because they are more polymorphic and unaffected by environmental influences. Some of the main conclusions can be summarized as follows: . The results based on molecular markers produce dendrograms of relationships of coffee species very similar to those of conventional taxonomic studies. They confirm a fairly recent African origin of the genus Coffea and subsequent ecological differentiation into numerous species, which are clustered in groups of genetic relationships corresponding to geographic regions (Table 9.2). However, the process of differentiation has not yet progressed into a stage of strong genetic barriers, as is shown for instance by successful interspecific hybridization between the Mascarocoffea and other groups of mainland Africa. The earlier taxonomic

classifications of the genus Coffea into sections and subsections have, therefore, been abolished. . There is molecular evidence for fairly close genetic relations between the genera Coffea and Psilanthus, as distinguished by Bridson & Verdcourt (1988) to suggest taxonomic revision into a single genus, Coffea. This is supported also by Couturon et al. (1998), who achieved viable hybrids from crosses between P. ebracteolatus (at the tetraploid level) and C. arabica. . Species very closely related or identical to C. eugenioides and to C. canephora (or C. congensis) are indeed the most likely maternal and paternal progenitors, respectively, of the allotetraploid C. arabica. Raina et al. (1998) arrived at similar conclusions in a cytogenetic study of C. arabica using genomic and fluorescent in situ hybridization techniques. Segregation analysis with molecular (RFLP) markers confirmed earlier cytogenic evi-

188

Table 9.2


Geographic origin of some Coffea species in Africa.

Species C. arabica C. eugenioides C. canephora C. congensis C. liberica C. humilis C. stenophylla C. brevipes C. racemosa C. salvatrix C. zanguebariae C. fadenil Mascarocoffea (50 species)

West

G 6 6 6

Central I (Atlantic)

Central II

C 6 6 6

6 C 6 6

Ethiopla

East

Madagascar

6

6

6 6 6 6

6

Note: G = Guinean, C = Congolese subpopulations of C. canephora. Adapted from Berthaud & Charrier (1988). 6 = available at location.

dence for regular disomic meiotic behaviour in C. arabica, probably under the control of pairing regulating genes (Lashermes et al., 2000a). . The existence of two subgroups of partial genetic differentiation within germplasm of C. arabica was established by an analysis with molecular (RAPD) markers (Lashermes et al., 1996c) and also by a multivariate analysis of phenotypic characters (Montagnon & Bouharmont, 1996): (a) all accessions collected in Ethiopia, west of the Rift Valley (Kaffa, Illubabor, Wollega) and (b) accessions collected east of the Rift Valley (Sidamo, Hararge) and the cultivated varieties. In this perspective it would appear that the coffee cultivated in the Yemen, from where almost all cultivated varieties of C. arabica derive, had its origin in Ethiopia east of the Rift Valley. . The existence of genetically diverse subpopulations (Congolese and Guinean) has also been confirmed within C. canephora by means of characterization with morphological, biochemical (isozymes) and molecular markers (Berthaud, 1985; Leroy et al., 1993). . Genetic variation of C. arabica populations is much enhanced by introgressive breeding with C. canephora genotypes. The arabica-like variety Hibrido de Timor is derived from a natural cross between C. arabica and C. canephora. Molecular studies with AFLP technology indicated that the genetic variation in Catimor and Sarchimor lines (derivatives

of crosses with Hibrido de Timor) was almost double that observed in traditional arabica cultivars or accessions from Ethiopia (Lashermes et al., 2000b).

9.2.3 Conservation In coffee there have been no alternatives so far to ex situ field collections for long-term germplasm conservation, because coffee seeds are recalcitrant and conventional methods of seed storage cannot extend viability beyond 2 to 3 years (Van der Vossen, 1985). Field collections require expensive resources of land, qualified staff and upkeep, while there is also a risk of losing valuable germplasm due to diseases and pests, as well as to poor adaptation of certain species to the local environment. By applying slow-growth conditions to in vitro cultured explants (zygotic embryos, apical meristem or nodal cuttings) and repeated subculturing, Dussert et al. (1997a) were able to conserve a core collection of coffee germplasm for about 3 years. However, there appeared to be some risk of genetic drift (random loss of genetic variability), since accessions differed considerably in their survival rates after several subcultures. Such methods of in vitro conservation have great advantages for germplasm distribution (less volume during shipping and simple quarantine procedures), but appear unsuitable for long-term germplasm conservation.


Encouraging results of cryopreservation techniques (storage under liquid nitrogen at -1968C) applied to coffee seeds have been reported recently (Dussert et al., 1997b). Expanding on methods developed by Abdelnour-Esquivel et al. (1992) for zygotic coffee embryos, Dussert et al. (1997c) succeeded in retrieving high rates of viable embryos from cryopreserved whole seeds of C. arabica. Rather low rates of normal seedling development after initially high seed germination indicated damage to the endosperm during the freezing and thawing stages of the process. However, in vitro cultured embryos, excised from the seeds after cryopreservation, survived and produced a very high rate of normal seedlings. Specific conditions of seed dehydration and precooling before and rapid thawing after cryopreservation, as well as the Coffea species, are all important factors influencing the retrieval rate of viable seedlings after storage (Dussert et al., 1998, 1999). Much additional experimental work may still be needed to solve remaining problems, but there is little doubt at this stage that cryopreservation opens interesting perspectives for long-term conservation of coffee germplasm in seed gene banks.

9.3 BREEDING

189

Table 9.3

Major selection criteria in coffee.

Criteria

Priorities Arabica

Robusta

3 3 3 3

3 3 3 2

Quality Bean size and shape Liquor quality Caffeine content

3 3 1

1 2 2

Host resistance to diseases Coffee leaf rust Coffee berry disease (Africa only) Other diseases

3 3 1

1 Ð 1

Host resistance to pests Nematodes Leaf miners Coffee berry borer Stem borers

3 2 2 2

1 1 2 1

Drought tolerance

1

1

Productivity Yield: kg per plant and per hectare Yield stability Plant vigour Compact plant type (short internodes)

Note: 1 = low, 3 = high breeding priority.

9.3.1 General objectives and strategies Arabica and robusta coffee breeding programmes have the same main objective of developing new cultivars, which have the potential of yielding optimum economic returns to coffee growers. An overview of selection criteria applied in coffee breeding (Table 9.3) indicates equal importance for factors of productivity in both species, but higher priority for bean size and liquor quality, as well as for host resistance to major diseases and pests in arabica coffee. Variations in the circumstances of climate, soil, biotic and abiotic stresses, cropping systems, socio-economic factors, market dynamics and consumer preferences further define priorities of selection criteria applied in specific programmes. Methods applied in breeding and variety propagation depend primarily on the mating systems of arabica (inbreeding) and robusta (outbreeding) coffee. Outlines of coffee breeding schemes have been discussed in detail elsewhere (Van der Vossen, 1985; Charrier & Berthaud, 1988) and Table 9.4 presents just a summary of actual methods implemented in various coffee research centres, together with examples of released cultivars. Four basic methods of breeding and selection

can be distinguished in each of the two species. These are listed in order of increasing complexity from line or mass selection to intra-and interspecific hybridization, the application depending on breeding objectives and intended output.

9.3.2 Productivity Some coffee breeding centres now emphasize hybrid varieties as the best strategy for further and more rapid increases of plant productivity. In arabica coffee, 30 to 60% heterosis in yield over the better parent has been observed in Ethiopia (Ameha, 1990), Cameroon (Cilas et al., 1998) and Central America (Bertrand et al., 1997), which confirms earlier reports from Kenya (Walyaro, 1983) and India (Srinivasan & Vishveshwara, 1978). Coffee hybrids were also found to have greater yield stability over location and time (fewer genotype 6 environment interaction effects). Chances of substantial (transgressive) hybrid vigour are increased by combining parents selected from genetically diverse subpopulations, such as crosses between

Varieties, clones

Varieties, clones (distinction of two sub-populations)

Arabica variety, tertaploid robusta genotypes C. congensis accession, robusta genotypes

(6) Family and clonal selection

(7) Reciprocal recurrent selection

(8a) Interspecific hybridization (arabica 6 robusta), family and clonal selection

(8b) Interspecific hybridization (C. congensis 6 robusta), backcrossing to robusta and family selection

Local or introduced varieties and accessions

Arabica varieties, tetraploid/diploid robusta genotypes

(4) Interspecific hybridization (arabica 6 robusta), backgrossing and pedigree selection

Robusta (5) Mass selection (individual plants)

Composite hybrid F1 hybrid F1 clone

Crossing and selfing

Varieties/ accessions, pedigrees of crosses

(3) Intraspecific F1 hybrids

Crossing, backcrossing, sibmating

Crossing and OP

Bi-parental crossing for inter-group combining ability tests and intra-group recombination; + doubled heploids

OP (half-sib families)

OP (open pollination)


Line


Varieties

(2) Pedigree selection after hybridization (sometimes also backgrossing)

OP variety

Synthetic hybrids (poly-clonal gardens) clone

Seed

Cuttings

Seed

C 6 variety (India)

Arabusta (Ivory Coast)

In progress (Ivory Coast, France)

Cuttings or somatic embryogenesis Seed F1 hybrid

In progress (Ivory Coast, France)

Seed

Synthetic hybrids (bi-clonal gardens) clone

BR sel 2 (India), SA and BP selections (Indonesia) IF 126, 202, 461 clones (Ivory Coast) BP39, BP42 (Indonesia)

Apoata (Brazil), S274 (India) Nemaya (C. America)

Icatu (Brazil), S2828 (India)

Ruiru II (Kenya) Ababuna (Ethiopia) in progress: Catimor 6 Et (C. America)

Catuai, Tupi (Brazil), Catimor Sarchimor (Costa Rica), S795 (India) Colombia (Colombia)

Caturra (Brazil), Kents (India) SL28 (Kenya), Java (Cameroon)

Examples

Seed

Seed

Seed

Seed (hand pollination) som. embryogenesis

Seed

Seed

Popagation by

Synthetic variety (bi-, polyclonal gardens) clone

OP variety

Line

Line

Selfing

Variety

Output

Arabica (1) Pure line selection

Breeding system

Source populations

Method

Table 9.4 Summary of methods applied in coffee breeding.


common cultivars and Ethiopian accessions (Lashermes et al., 1996c). In robusta coffee, experimental evidence for marked hybrid vigour for yield in progenies from interpopulation crosses has been reported from the large breeding programme based on methods of reciprocal recurrent selection implemented in the Ivory Coast since 1985 (Leroy et al., 1993, 1994, 1997; Montagnon et al., 1998a, 1998b). Yields of more than 40% above those of the best commercial clones were recorded in some progenies of intergroup (Congolese 6 Guinean) biparental crosses. Genetic variance for (early) yield and most other characters was considerable and mainly due to general combining ability (additive gene effects) in these trials, as well as in breeding trials including crosses with doubled haploids (Lashermes et al., 1994a). The presence of sufficient (predominantly additive) genetic variance and the possibility of exploiting transgressive hybrid vigour of interpopulation crosses provide ample opportunity for considerable selection progress for higher yields per tree in arabica and robusta coffee. Plant vigour was found to be highly correlated with yields in the aforementioned selection trials. This may have a physiological background, since coffee fruits are strong assimilate-accepting sinks requiring each at least 20 cm2 leaf area (half an arabica leaf) for support without affecting vegetative growth (Cannell, 1985). Vigorous trees will have a high rate of new shoot and leaf production to sustain a heavy crop. Coffee yields per unit area can be increased considerably by closespaced planting systems. However, strong plant vigour and large canopies will lead to early between-tree competition for light and, consequently, to reduced flower initiation and yields. The character short internode length of the compact arabica variety Caturra (single dominant gene Ct, reducing internode length by about 50%) has provided the opportunity of combining vigour with high density planting to increase productivity per hectare while avoiding early yield reduction due to mutual shading. It has been widely applied in variety development in arabica coffee (Table 9.4: cvs Catuai, Catimor, Colombia). F1 hybrids with one parent of the CtCt genotype will also show the required compact growth (Table 9.4: Ruiru II; Catimor 6 Et). The recent confirmation of `dwarf' mutants with short internodes (Kumar et al., 1994) in robusta coffee and also in the C 6 R (ex cross between C. congensis and C. canephora) variety (Srinivasan, 1996) opens the way for similar developments in robusta variety development.

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9.3.3 Quality Selection for bean size and cup quality has generally received much attention in arabica coffee breeding, particularly in countries producing mild (washed) coffee types, because the quality of new disease resistant cultivars should be at least equal to that of the traditional cultivars in order to uphold the country's reputation and position in the world coffee market. This was obviously achieved in Kenya with the CBD and CLR resistant hybrid cultivar Ruiru II (Njoroge et al., 1990) and in Colombia with the CLR resistant cultivar Colombia (Moreno et al., 1995), as judged by international coffee tasting panels. Most components of coffee quality show considerable (additive) genetic variation, but they are also affected by environmental factors (Walyaro, 1997). Rigorous standardization of pre- and post-harvest practices, bean grading and cup tasting applied in these two breeding programmes contributed to increased selection progress and helped to overcome the initially negative effects on quality due to introgression of disease resistances from exotic germplasm. Verification of the quality of new cultivars in widely different environments (for example climate, altitude and shade) appears necessary because of significant genotype 6 environment interaction effects reported for bean size and some components of liquor quality (Mawardi & Hulupi, 1995; Guyot et al., 1996; Agwanda et al., 1997). The CLR resistant Icatu cultivars released in 1992 in Brazil are also similar in bean size and liquor quality to traditional cultivars according to Brazilian quality standards for unwashed coffees (Fazuoli et al., 1999). This was achieved by a long-term breeding programme of interspecific hybridisation (robusta 6 arabica) started in 1950 and followed by repeated backcrossing to arabica and pedigree selection (Carvalho, 1988). Moschetto et al., (1996) have presented the results of studies on genetic variation in bean size and cup quality for robusta coffee grown in the Ivory Coast. Conclusions on the inheritance of quality characters are similar to those made for arabica coffee. Genotypes of the Congolese group within C. canephora were generally better in cup quality than those of the Guinean group, meaning milder body and acidity, lower bitterness and fewer undesirable aromas, but of course still far below the standards of an average arabica coffee. On the other hand, the cup quality of some Arabusta and Congusta coffees came close to arabica. The Arabusta programme had to be abandoned because of low and irregular yields (Charmetant et al., 1991; Yapo, 1995). However, the Congusta material appears to hold much promise to

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improving cup quality for robusta-like coffee production, as was shown by the C 6 R variety developed in India from a `Congusta' hybrid backcrossed once to robusta coffee followed by full-sib family selection (Srinivasan, 1996; Srinivasan et al., 1999). This variety combines quality close to arabica coffee with compact growth, good productivity and adaptation to low altitudes. Caffeine content (about twice as high in robusta as in arabica coffee) is a quantitative character with a high heritability (Montagnon et al., 1998c). Barre et al. (1998) deduced from interspecific crosses between the caffeine-free C. pseudozanguebariae and C. liberica var dewevrei that presence or absence of caffeine is under control of one major gene with the double recessive genotype conditioning absence. Unfortunately, absence of caffeine is linked to the presence of a heteroside diterpine causing bitterness, which is also under the control of one major (codominant) gene. Molecular markers for these characteristics could accelerate the search for a genotype lacking both caffeine and the bitter taste, as an alternative to transgenic caffeine-free coffee plants proposed by Moisyadi et al., (1999; see also Chapter 11). On the other hand, coffee is drunk mainly for its stimulating properties derived from the caffeine it contains and moderate coffee drinking does not pose health hazards to most people (see Chapter 8). Caffeine-free cultivars may, therefore, attain only limited prominence in coffee cultivation considering the demand for decaffeinated coffees, which is small (10% of total coffee consumption) and unlikely to increase much in the foreseeable future.

9.3.4 Resistance to coffee leaf rust Coffee leaf rust (Hemileia vastatrix) has spread to all other coffee-producing countries between 1970 (Brazil) and 1986 (PNG and Jamaica), reaching the Central American countries after 1976 and Colombia in 1983 (Carvalho et al., 1989). The relevance of durable host resistance to CLR can be deduced from its economic damage to world arabica coffee production, which has been estimated at US$1±2 billion per year due to crop losses (20 to 25%) and the need to apply cultural and chemical control measures (10% of production costs). For an authoritative review on research for host resistance and breeding of CLR-resistant arabica cultivars reference is made to Eskes (1989). Resistance to CLR is conditioned primarily by a number of major (SH) genes and coffee genotypes are classified in resistance groups according to their interaction with physiological races of the rust pathogen. Some


genotypes with relevance to resistance breeding in arabica coffee and also used as differentials, to test the virulence of rust races, are presented in Table 9.5. The host resistance of Catimor lines, which is based on major genes (SH6±SH9 + ?) originating from C. canephora, has continued to provide adequate protection against CLR epidemics during the past 15 years in countries where arabica coffee is grown under relatively cool climatic conditions (high altitudes) and the number of physiological races present usually remains limited, for example in Colombia, Central American countries, Kenya, Tanzania and PNG. Race II is usually the first one to appear and represents 58% of all isolates tested for virulence from 32 countries, followed by race I (14%), III (9%) and XV (4%). Races II, I and XV had been isolated on susceptible arabica cultivars in Brazil by 1974. However, several more races were subsequently found in the selection fields of the IAC (Instituto AgronoÃmico at Campinas), which emphasised the necessity of developing new cultivars with A type resistance (Carvalho et al., 1989). India has had a long history of arabica coffee breeding dominated by the repeated occurrence of new physiological races of CLR (Carvalho et al., 1989), probably due to the warmer and wetter climatic conditions in major coffee growing areas. CLR infection was noticed on Catimor (Cauvery) trees within a few years after the onset of large-scale planting in 1985 (Srinivasan et al., 1999). Some trees remained free of infection, others were severely infected and proved to be susceptible even to race II, but a number of them had rather mild symptoms of rust infection. By 1993 nine new physiological races had been identified, including races with complex virulence capable of overcoming all four resistance genes (SH6±SH9) but generally showing lower aggressiveness. This has brought the total number of races to 39, of which 30 are present in India (Rodrigues et al., 1993). Cauvery appears to be a mix of A type (resistant to all races), different R type resistance groups (which could have given rise to new virulence of the pathogen), and also some segregants of the E group. Inoculation tests at the Coffee Research Centre (CIFC-Oeiras) in Portugal confirmed that none of these new races could infect Catimor lines with A type resistance, which would indicate the presence of yet other, still unidentified SH genes. Rust infection on Catimor plants in other Asian countries (for example the Philippines) and even in Colombia has been reported recently, but the virulence of the rust isolates still remains to be confirmed (VaÂrzea & Rodrigues, personal communication) On the other hand, Indian arabica derivatives of


Table 9.5

193

Important differentials for the identification of races of coffee leaf rust. Differential

Host resistance

Group

Variety/cross

Clone

Genotype

Origin of genes

A R R-1 R-2 R-3 R-4

HDT (Hibrido de Timor) HOT (Hbrido de Timor) M. Novo 6 HW 26 M. Novo 6 HW 26 M. Novo 6 HW 26 Caturra 6 HdT 1343/269 S12Kaffa S12Kaffa S12Kaffa S4Agaro S288-23 S353-4/5 Kents KP532-31 Geisha Dilla & Alghe Bourbon

832/1 1343/269 H420/10 H420/2 H419/20 H440/7 635/2 134/4 635/3 110/5 33/1 34/13 32/1 1006/10 87/1 128/2 63/1

SH 5.6.7.8.9 + ? SH 6 Sh 5.6.7.9 SH 5.8 SH 5.6.9 SH 5.6 SH 4 SH 1.4 SH 1.4.5 SH 4.5 SH 3.5 SH 2.3.5 SH 2.5 SH 1.2.5 SH 1.5 SH 1 SH 5

C. canephora C. canephora C. canephora C. canephora C. canephora C. canephora C. arabica ex Ethiopia C. arabica ex Ethiopia C. arabica ex Ethiopia C. arabica ex Ethiopia C. liberica ex India C. liberica ex India C. arabica ex India C. arabica ex Tanzania C. arabica ex Ethiopia C. arabica ex Ethiopia C. arabica

I W J G H D L C E

Note: HW 26 = Caturra 6 HdT 832/1. Adapted from Bettencourt & Rodrigues (1988).

Devamachi (a spontaneous robusta 6 arabica hybrid similar to Hibrido de timor) and a selections like S2828, which was developed from interspecific (robusta 6 arabica) hybridization followed by backcrossing to arabica and pedigree selection, have shown continued high field resistance to CLR in combination with good yields and satisfactory quality (Srinivasan et al., 1999). The nature of the resistance has still to be confirmed, but could be similar to the Catimor-derived (for example Tupi, Obata) and Icatu cultivars from Brazil, of which certain selections appear to have durable resistance to CLR based on major as well as minor genes (Carvalho et al. 1989; Carvalho & Fazuoli, 1993; Fazuoli et al., 1999). Castillo & Alvarado (1997) also found incomplete resistance to CLR in a Catimor line. Nevertheless, incomplete resistance to CLR in certain Catimor and Icatu lines was shown to be race-specific and, therefore, unlikely to provide durable resistance (Eskes et al., 1990). Molecular markers linked to SH and other genes conditioning race-specific and non-specific resistance to CLR should increase selection efficiency for durable resistance in arabica coffee, particularly in regard to gene pyramiding (accumulating several resistance genes in one genotype). Initial work based on RAPD markers shows the existence of considerable polymorphisms between some rust differentials (Santa Ram

& Sreenath, 1999) and further work in collaboration with national and international institutes on molecular marker-assisted selection in CLR (and other important characters) may commence soon (Sreenath & Naidu, 1999). Resistance to CLR in robusta coffee is usually a secondary character of selection and based on individual plant, clone or family scores for field infection. Robustas of the Congolese group are generally much more resistant than those of the Guinean group (Montagnon et al., 1994; Leroy et al., 1997).

9.3.5 Resistance to coffee berry disease Coffee berry disease is caused by the fungus Colletotrichum coffeanum, renamed C. kahawae (Waller et al., 1993). It can be a devastating anthracnose of developing berries in arabica coffee in Africa and is particularly serious at high altitudes (Van der Graaff, 1992; Masaba & Waller, 1992). Coffee berry disease may cause crop losses of 50 to 80% in years favourable to a severe disease epidemic (prolonged wet and cool weather). Control by frequent fungicide sprays is expensive (30 to 40% of total production costs), not always effective and usually beyond the means of the smallholder coffee growers. Economic damage to arabica coffee production in Africa due to CBD alone (crop losses plus costs

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of control) is estimated at about US $ 300 to 500 million per year. Breeding programmes initiated some 30 years ago in Kenya and Ethiopia have been successful in developing new cultivars with high and apparently durable resistance to CBD (Van der Vossen, 1997). In Kenya more than 10 000 ha have been planted so far with the composite hybrid Ruiru II, which combines resistance to CBD and CLR with compact growth, high yields and quality similar to the standard Kenyan cultivars. In Ethiopia, farmers' acceptance of CBD-resistant hybrids like Ababuna is higher than of the earlier released lines, because of better agronomic performance, but data on actual area planted to CBDresistant cultivars are unavailable. A breeding programme in Tanzania, which is making use of CBD resistance found in Hibrido de Timor (clone CIFC 1343) and Rume Sudan, has reached the stage of multi-locational testing of clones derived from multiple crosses and the first release of CBD (and CLR) resistant clonal cultivars has been envisaged within a few years time (Nyange et al., 1999). However, these cultivars may not yet meet the cup quality standards of typical Tanzanian mild arabica coffees. Selection work in Cameroon, which concentrated on screening a large number of arabica varieties and also accessions of Ethiopian (Et) origin for agronomic characteristics and disease resistance, produced the CBD resistant cultivar Java by 1980 (Bouharmont, 1994). A subsequent breeding programme indicated considerable hybrid vigour for crosses involving some Et accessions as one parent, but also the poor combining ability of cultivar Java (Cilas et al., 1998). Host resistance to CBD appears to be mainly conditioned by three major genes (dominant R, codominant T and recessive k genes) according to evidence produced in Kenya, although breeders in Ethiopia claim additive effects of several recessive genes instead (Walyaro, 1997; Bellachev, 1997). Part of this discrepancy could be explained by the differences in germplasm and also the inoculation tests used in the inheritance studies. In Ethiopia the inheritance studies were mainly based on results from inoculation tests on detached berries. Gichuru et al., (1999) confirmed the absence of histological and chemical resistance mechanisms in detached berries, commonly expressed in attached berries or hypocotyl stems of CBD resistant varieties. Support for the hypothesis of major gene resistance has come from the recent application of molecular marker technology by Agwanda et al., (1997) and Cristancho (1999), who found closely linked RAPD markers for the T gene conditioning CBD


resistance in progenitor Hibrido de Timor and its derivatives such as Catimor. Only HdT1343 (progenitor of Colombian Catimor lines) carries the T gene, while Catimor lines derived from HdT832 (for example CIFC-Oeiras and Brazil) are all CBD susceptible (Van der Vossen, 1997). Rovelli et al., (1999) reported the detection of polymorphic microsatellites in arabica coffee, which offers prospects of developing useful molecular markers for other CBD resistance genes. This would enable breeders to reconfirm the genetic basis for CBD resistance and to ensure effective host resistance by gene pyramiding. It would also enable breeders outside Africa to verify or introgress CBD resistance in their breeding stock as pre-emptive action in case CBD inadvertently becomes a pathogen in their region. This strategy was followed for CLR by the Colombian breeders several years before it arrived in their country (Castillo, 1989). Reports of the existence of race-specific interactions between the CBD pathogen and arabica genotypes by Rodrigues et al., (1992) and VaÂrzea et al., (1999) could not be confirmed in extensive studies by Bella Manga et al., (1997) and Bella Manga (1999), which included a large number of isolates of C. kahawae collected from CBD susceptible and resistant arabica cultivars and accessions in several African countries. The genetic diversity of pathogen populations, as evaluated by VCG (vegetative compatibility groups) and RAPD molecular markers, was relatively narrow, but it was possible to distinguish two subgroups, one from East Africa and one from the Cameroon. Certain isolates from the Cameroon were also more pathogenic than those from East Africa. However, pathogenicity tests revealed insignificant pathogen±host interactions despite considerable variation in aggressiveness of isolates and in levels of host resistance. Omondi et al., (1997), in a study with CBD isolates from Kenya, produced comparable results. Variation in aggressiveness among CBD isolates was also found in Ethiopia (Derso, 1999). It can be concluded that the available host resistance is not (yet) threatened by race specification in the CBD pathogen, but levels of host resistance required for adequate crop protection may vary between different geographical areas.

9.3.6 Resistance to other diseases Several other fungal and bacterial diseases may affect coffee (Wrigley, 1988; Anon, 1997), but very few of these have been targeted in breeding programmes, notwithstanding considerable economic damage in certain coffee producing countries, because useful host


resistance could not be detected in available coffee germplasm. For instance, black rot (Koleroga noxia) is the second most important disease after CLR in India (Bhat et al., 1995), coffee leaf scorch caused by the bacterium Xylella fastidiosa has become a problem in some coffee regions in Brazil (Beretta et al., 1996) and bacterial blight (Pseudomonas syringae pv garcae) can be severe in a few areas in Kenya (Kairu, 1997). Fusarium wilt disease or tracheomycosis (Fusarium xylarioides) has been causing severe losses of robusta coffee in north-eastern DR Congo and south-western Uganda since the 1980s (Flood & Brayford, 1997; Birikunzira & Hakiza, 1997). The exact cause of this reemergence is still unknown, but it was noted that especially old and rather neglected coffee plots were severely affected. Some of the Ugandan robusta clonal cultivars have remained resistant and renewed selection for host resistance within robusta germplasm could, therefore, be rewarding.

9.3.7 Resistance to nematodes Root-knot (Meloidogyne spp.) and root-lesion (Pratylenchus spp.) nematodes can cause considerable economic damage to arabica coffee in Brazil (Carvalho, 1988), Central America (Anzueto et al., 1991), India (Anon, 1997) and Indonesia (Mawardi & Soenaryo, 1988). They are usually a minor problem in East Africa, provided a build-up of nematodes in nurseries is avoided (Mitchell, 1988). Host resistance to both types of endoparasitic nematodes is present in germplasm of robusta coffee and selections have been widely used as rootstock for arabica cultivars in problem areas, such as Nemaya in Central America (Anzueto et al., 1991) and Apoata in Brazil (Carvalho & Fazuoli, 1993). More recently, Apoata has been recommended also as a suitable cultivar for expansion of robusta coffee production in the State of Sao Paulo, on account of its good yield potential and resistance to CLR (MedinaFilho et al., 1999). According to research work in Costa Rica, Guatemala and El Salvador two groups of Meloidogyne species can be distinguished: (a) those forming egg masses inside the roots and causing intensive gall formation but less root destruction, such as M. exigua and M. arabicida, and (b) those forming egg masses outside the roots and causing fewer and smaller galls but high root damage, such as M. incognita, M. arenaria and M. javanica (Bertrand et al., 1995). Host resistance to the first group was found in a Catimor line from Colombia, while a few arabica accessions from Ethiopia showed resistance to the second group of nematodes. The

195

resistance in each case appeared to be conditioned by one or two dominant major genes. Resistance to both groups simultaneously was found in several robusta plants (major and minor genes). All arabica germplasm is susceptible to Pratylenchus species, but some robusta genotypes are tolerant. Coffee fields in Central America are often infested with root-knot and root-lesion nematodes at the same time and the two populations are antagonistically related. Introduction of a cultivar resistant to one type only would run the risk of an escalation of the other (Bertrand et al., 1998). It is, therefore, essential to rely on broad spectrum resistance to parasitic nematodes, initially by using suitable robusta rootstock (for example Nemaya) or in the long term by developing new arabica (hybrid) cultivars with resistance to major Meloidogyne as well as Pratylenchus species. A recently started collaborative research project aims at developing molecular markers linked to resistance genes to enhance selection for such nematode resistance (Lashermes et al., 1999a).

9.3.8 Resistance to insect pests Several hundred insect species have been described as minor or major coffee pests (Wrigley, 1988). Integrated pest management (IPM) ± early warning systems in combination with cultural, biological and chemical control ± has been successfully applied to a number of important coffee pests (Bardner, 1985). The arrival of the coffee berry borer (Hypothenemus hampei) in Colombia in 1988 (Bustillo et al., 1995) and in India (Bheemaya et al., 1996; Anon, 1997) gave a new impetus to the development of effective methods of IPM for the control of this most damaging insect pest in coffee, based on specific parasitoids and entomopathogens. The identification and synthesis of a male sex pheromone of the coffee white stem borer (Xylotrechus quadripes) offers promising perspectives of biological control of this important pest in arabica coffee in India (Hall et al., 1998; Jayarama et al., 1998). Host resistance to the leaf miner Perileucoptera coffeella, a severe coffee pest in Brazil, was found in the species Coffea stenophylla and C. racemosa, but only the resistance of the latter was successfully introgressed into arabica coffee (Carvalho, 1988). This resistance is conditioned by two complementary dominant genes (Guerreiro Filho et al., 1999). So far, no other cases of useful host resistance to important insect pests have been detected in Coffea germplasm. However, the successful regeneration of transgenic coffee plants expressing resistance to leaf miners (P. coffeella and Leucoptera spp. based on Bt genes (Leroy et al., 1999;

196

see Chapter 11) could be the start of molecular breeding for resistance to important coffee pests, especially the endocarpic insects (Guerreiro Filho et al., 1998).

9.3.9 Drought tolerance Arabica coffee is generally more tolerant to water stress than robusta, at least partly as the result of a more extensive and deeper root system. However, there are also large differences in drought tolerance between genotypes of the same species. Some of the East-African cultivars (for example SL28) appear to be the best genotypes available within arabica germplasm, because of an exceptionally well developed root system, outstanding plant vigour and an ability to retain their leaves under water stress (Van der Vossen & Browning, 1978). Such genotypes must have evolved in a long process of domestication, lasting some 12 to 15 centuries, from shade-adapted trees occurring in the understorey of the highland forests in Ethiopia to the dry and unshaded conditions first in Yemen and eventually in East Africa. Wilting of coffee plants during a prolonged dry spell in Kenya always started much later in plots of SL28, than in plots planted with introduced arabica cultivars or accessions of Ethiopian origin. In Brazil, cultivars like Caturra and Mundo Novo were also found to be more tolerant to drought than Ethiopian germplasm (Carvalho, 1988). Selection for more drought tolerant robusta coffee should emphasise depth and extent of root system, as well as leaf retention under stress conditions. The Indian C 6 R cultivar appears to have better drought tolerance, but it is unlikely that robusta-like genotypes would be found with better drought tolerance than arabicas. Carvalho (1988) mentions C. racemosa as a good progenitor for drought tolerance.

9.4 PROPAGATION OF NEW CULTIVARS 9.4.1 Seeds Propagation by seeds continues to be the preferred practice for new coffee cultivars in most countries (Table 9.4). When the output of a breeding programme consists of pure lines, as in the case of arabica coffee, spatially isolated seed gardens are established for lowcost seed multiplication and distribution. Examples are Brazil (Fazuoli et al., 1999), India (Srinivasan, 1996) and the Cameroon (Bouharmont, 1994). In Colombia, a


number of Catimor lines are multiplied in separate seed gardens and the cultivar Colombia consists of a synthetic seed mix from selected lines (Moreno, 1994). Seed multiplication of F1 hybrid arabica cultivars is a logistically complex operation involving hand pollination of previously emasculated and bagged flowers (Opile & Agwanda, 1993). Experience with the Kenyan hybrid cultivar Ruiru II shows that large-scale seed multiplication is technically feasible and cheaper than clonal propagation. However, the national output would probably be improved considerably by decentralisation into smaller seed production units (Van der Vossen, 1997). Male sterility conditioned by one recessive gene has been detected in arabica accessions of Ethiopian origin (Mazzafera et al., 1989; Dufour et al., 1997), which provides opportunities of reducing costs of seed production. This gene could become a suitable object of molecular breeding (MAS and even gene transformation) to obtain male-sterile female parents for seed production within a much shorter period of time than would be required with conventional methods of introgressive breeding. Seeds of robusta coffee cultivars are produced in strictly isolated seed gardens planted with selected seedling populations (for example cultivars Apoata, Nemaya and S274). Synthetic hybrid seeds require gardens planted with (preferably two) clones of known combining ability (Charmetant et al., 1990; Montagnon et al., 1998a). Self-incompatibility ensures cross-pollination. Such `clone hybrids' are not yet very uniform due to heterozygosity of the parent clones, but genetically uniform robusta hybrid seeds may eventually be realised with parents developed from doubled haploids (Lashermes et al., 1994b).

9.4.2 Clonal propagation Conventional methods of clonal propagation are about ten times more expensive than multiplication by seed (Montagnon et al., 1998a). Clonal robusta cultivars found limited application in large-scale coffee plantations, where the increased yield potential can be fully exploited and the higher initial costs are quickly recovered (Charrier & Berthaud, 1988). Clonal propagation of hybrid arabica cultivars is even more demanding of logistic and technical resources, due to slower rates of multiplication and hardening-off problems in the cooler and dryer environments of arabica coffee cultivation. Of all the in vitro regeneration systems tried out in coffee (Carneiro, 1997) the induction of high frequency somatic embryogenesis in a liquid medium (Zamarripa


et al., 1991; Berthouly & Michaux-FerrieÁre, 1996) appears to be very promising for efficient mass propagation. It is being implemented in arabica coffee in Central America to multiply Catimor 6 Et hybrids as an alternative to hybrid seed production (Etienne et al., 1997a, b) and in Uganda to multiply elite robusta clones (Berthouly et al., 1995). Coffee plants raised from rooted cuttings tend to be shallower rooting, in the absence of a tap root, than seedlings and consequently are less tolerant of prolonged spells of dry weather. A major advantage of plants raised from somatic embryogenesis is their similarity to seedlings with respect to the root system. Deshayes et al., (1999) claim that costs of producing robusta coffee plants through somatic embryogenesis are comparable to conventional rooted cuttings, in other words, still considerably more expensive than hybrid seed production.

ABBREVIATIONS ACRN AFLP BCRI/E CATIE CBD CCRI CENICAFE CIFC CIRAD CLR CNRA CRF FOFIFA IAC IAR/JARC ICO ICCRI IPGRI IPM IRAD IRD

African Coffee Research Network Amplified fragment length polymorphism Biodiversity Conservation and Research Institute, Ethiopia Centro AgronoÂmico Tropical de Investigacion y EnsenÄanza, Costa Rica Coffee berry disease Central Coffee Research Institute, India Centro Nacional de Investigaciones de CafeÂ, Colombia Centro d'InvestigacËao das Ferrugens do Cafeiero, Portugal Centre de CoopeÂration Internationale en Recherche Agronomique pour le DeÂveloppement, France Coffee leaf rust Centre National de Recherche Agronomique, CoÃte d'lvoire Coffee Research Foundation, Kenya Centre National de Recherche Agronomique AppliqueÂe au DeÂveloppement Rural, Madagascar Instituto AgronoÃmico de Campinas, Brazil Institute of Agricultural Research/Jimma Agricultural Research Centre, Ethiopia International Coffee Organization, London Indonesian Coffee and Cocoa Research Institute International Plant Genetic Resources Institute, Rome Integrated pest management Institut de Recherche Agronomique et DeÂveloppement, Cameroun Institut de Recherche pour le DeÂveloppement (ex ORSTOM), France

197

RAPD RFLP TARO VCG

Random amplified polymorphic DNA Restriction fragment length polymorphisms Tanzanian Agricultural Research Organization Vegetative incompatibility

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& Eskes, A.B. (1996) Studies on the effect of genotype on cup quality of Coffea canephora. Trop. Sci., 36, 18±31. Ngategize, P.K. (1997) The African Coffee Research Network: prospects and challenges into the next millennium. In: Proceedings of the 17th ASIC Colliqum (Nairobi), pp. 36±5. ASIC, Paris, France. Njoroge, S.M., Morales, A.F., Kari, P.E. & Owuor, J.B.O. (1990) Comparative evaluation of the flavour qualities of Ruiru II and SL28 cultivars of Kenya arabica coffee. Kenya Coffee, 55, 843± 9. Nyange, N.E., Kipokola, T.P., Mtenga, D.J., Kilambo, D.J., Swai, F.B. & Charmetant, P. (1999) Creation and selection of Coffea arabica hybrids in Tanzania. In: Proceedings of the 18th ASIC Colloquium (Helsinki), pp. 356±62. ASIC, Paris, France. Nyange, N.E. & Marandu, E.F. (1997) Improvement of Coffea canephora germplasm in Tanzania: exploration and collection of new robusta material from farmers' plots. In: Proceedings of the 17th ASIC Colloquium (Nairobi), pp. 502±505. ASIC, Paris, France. Omondi, C.O., Hindorf, H., Welz, H.G., Saucke, D., Ayiecho, P.O. & Mwang'ombe, A.W. (1997) Genetic diversity among isolates of Colletotrichum kahawae causing coffee berry disease. In: Proceedings of the 17th ASIC Colloquium (Nairobi), pp. 800± 804. ASIC, Paris, France. Opile, W.R. & Agwanda, C.O. (1993) Propagation and distribution of cultivar Ruiru Il: a review. Kenya Coffee, 58, 1496±508. Orozco-Castillo, C., Chalmers, K.J., Powell, W. & Waugh, R. (1996) RAPD and organelle specific PCR re-affirms taxonomic relationships within the genus Coffea. Plant Cell Rep., 15, 337± 41. Orozco-Castillo, C., Chalmers, K.J., Waugh, R. & Powell, W. (1994) Detection of genetic diversity and selective gene introgression in coffee using RAPD markers. Theoret. Appl. Genet., 87, 934±40. Raina, S.N., Mukai, Y. & Yamamoto, M. (1998). In situ hybridization identifies the diploid. progenitor species of Coffea arabica (Rubiaceae). Theoret. Appl. Genet., 97, 1204±209. Rodrigues Jr, C.J., VaÂrzea, V.M., Godinho, I.L., Palma, S. & Rato, R.C. (1993) New physiologic races of Hemileia vastatrix. In: Proceedings of the 15th ASIC Colloquium (Montpellier), pp. 318±321. ASIC, Paris, France. Rodrigues Jr, C.J., VaÂrzea, V.M. & Medeiros, E.F. (1992) Evidence for the existence of physiological races of Colletotrichum coffeanum Noack sensu Hindorf. Kenya Coffee, 57, 1417±20. Rovelli, P., Mettulio, R., Antony, F., Anzueto, F., Lashermes, P. & Graziosi, G. (1999) Polymorphic microsatellites in Coffea arabica. In: Proceedings of the 18th ASIC Colloquium (Helsinki), pp. 344±347. ASIC, Paris, France. Santa Ram, A. & Sreenath, H.L. (1999) Genetic fingerprinting of coffee leaf rust differentials with RAPD markers. In: Proceedings of the 3rd International Seminar on Biotechnology in the Coffee Agro-industry, Londrina, Brazil (in press). Sreenath, H.L. & Naidu, R. (1999) Coffee biotechnology research in India ± potential progress and future thrust areas. In: Proceedings of the 18th ASIC Colloquium (Helsinki), pp. 281± 94. ASIC, Paris, France.

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Chapter 10

Agronomy II: Developmental and Cell Biology M.R. Sondahl Fitolink Corporation Mount Laurel, USA T.W. Baumann Institute of Plant Biology University of Zurich, Switzerland 10.1 OVERVIEW The genus Coffea was proposed by Linnaeus in 1735, who later described the species Coffea arabica in 1753, presently known as the variety Typica. Coffea belongs to the Rubiaceae family, which includes more than 500 genera and about 800 species (Bridson & Verdcourt 1988). The Genus Coffea has about 100 species (Charrier & Berthaud 1985), but commercial production relies only on two species, C. arabica and C. canephora, which represent about 70% and 30% of the total coffee market, respectively. Arabica coffee is an isolated species in the genus Coffea because of its amphidiploid and self-pollinating nature, which makes it difficult to incorporate traits from other non-cultivated coffee species. Robusta coffee is a highly self-incompatible species with heterozygous seeds. Both species would greatly benefit from new technologies being developed at the cellular and molecular levels. This chapter deals with the development of cell biology methods and their application to coffee improvement and germplasm preservation. The fluctuation of purine alkaloids during leaf and fruit developmental stages is presented, suggesting a possible evolutionary defense mechanism for coffee species. The synthesis of caffeine and chlorogenic acid, and related regulatory factors are discussed for solid and liquid cell cultures. New advances on cell cultures methods based on embryogenic cell systems for propagation and genetic improvement will be presented. Much progress has been made in the mass production of somatic embryos in bioreactor vessels

and several test cases of scale-up programs are being reported. Finally, the opportunity of capturing in vitro variation for the development of new cultivars is exemplified in this chapter. The enhancement of our knowledge on organ differentiation and its metabolism, and the control of reliable methods for in vitro culture and plant regeneration, is essential for devising new processes for improving coffee plants and its beverage.

10.2 ORGAN DEVELOPMENT AND THE ALLOCATION OF DEFENSE COMPOUNDS 10.2.1 Introduction Studies on the development of coffee organs such as the root, fruit, leaf and flower are, if compared to other `crops', scarce and preferentially concentrate on arabica. Due to technical reasons these investigations were, and still are, largely performed under greenhouse or otherwise controlled conditions. However, despite the àrtificial' environment, the studies may render a good and reliable insight into the processes of both flower and fruit development including seed maturation, since the developmental program of these organs yielding finally the essential dispersal unit, the so-called diaspore (Van der Pijl, 1982), remains little or unaffected by external factors. Conversely, the growth of the vegetative plant parts, i.e. shoots, leaves and roots, is subjected to drastic alterations caused by all kind of biotic and abiotic factors. In a simplified manner one

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may consider generative development as an inert, but stringently controlled process orientated to dispersal and conservation of the entire species, whereas vegetative development is rather a dynamic and plastic event allied to protection and survival of the individual. This view has to be kept in mind when discussing below the organ-related allocation of chemical defense compounds such as purine alkaloids and chlorogenic acids.

10.2.2 The leaf The arabica leaf attains (under greenhouse or phytotron conditions) the full expansion and maximum dry weight after 30 to 35 days (MoÈsli Waldhauser et al., 1997) or 50 to 60 days (Frischknecht et al., 1982), respectively. This means that within 4 to 5 weeks after emergence, the surface growth of the still soft and glossy leaf blade is completed (see Fig. 10.1), but it takes another 2 to 3 weeks until the lamina has gained its final rigidity. Therefore, we can assign several stages or transitions to leaf development, which are characterized not only by means of morphological, but also physiological and phytochemical changes: (1) the quiescent bud (B1 in Fig. 10.2), (2) emergence from the bud (B2 to B4 in Fig. 10.2), (3) lamina expansion and mechanical strengthening, and (4) senescence.

Fig. 10.1 Leaf development of C. arabica. After 30±35 days (ca 5 weeks) leaf expansion is completed. During this period the fresh weight increases by a factor of 360.

(1)

In the quiescent bud (B1 in Fig. 10.2), the apical meristem together with the paired leaf primordia is covered by two firm stipules. Additionally, between the primordia and the stipules there is a resinous layer, of which remainders may still be attached to the leaf apices when emerged from the bud. With respect to purine alkaloids all structures, the primordial leaflets, the resin layer, and the stipules, exhibit a wide variation of

Fig. 10.2 Emergence of coffee (C. arabica) leaflets from the bud (taken from Frischknecht et al., 1986.

concentration from bud to bud (Fig. 10.3), indicating only moderate significance of chemical defense in favor of mechanical protection (Frischknecht et al., 1986). (2) Emergence from the bud is depicted in Fig. 10.2 (B2 to B4). The leaf pair develops to stage 4 within a few days, pushing apart the stipules, and with the leaves still tightly associated to each other at this stage. During this developmental process, the concentration of purine alkaloids markedly increases (up to 4% at stage B3) while their coefficient of variation decreases likewise, for example the latter is three times lower for caffeine at stage B3 than at stage B1. Clearly, caffeine biosynthesis is strongly accelerated during leaflet emergence and has been reported to reach a maximum rate of 17 000 mg dayÿ1 gÿ1 at stage B3 (Frischknecht et al., 1986). The considerably lower variation coefficient signifies that chemical defense by purine alkaloids has become a very important, stringent factor at the moment of emergence when the leaflets lack the mechanical protection by the stipules and have a very soft texture. Additionally, their dietary value for predators is very high at this stage. (3) During the next following expansion stage of the leaf blade, the rate of caffeine synthesis falls

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Fig. 10.3 Alkaloid content (theobromine and caffeine) of seven individual buds. Segments give the relative parts of stipules, resin layer and leaflets (after Frischknecht et al., 1986).

exponentially, reaching 16 mg dayÿ1 gÿ1 when the leaf is fully grown with respect to leaf area and photosynthetic capacity (Frischknecht et al., 1982). In parallel, the nutritional value per unit leaf area drops. The activity of the enzymes mediating the last steps of caffeine synthesis has been investigated during leaf expansion. The biosynthesis of caffeine in coffee most likely starts by the methylation of XMP (xanthosine monophosphate) at position N7 carried out by the first N-methyltransferase (NMT). After removal of the phosphoribose moiety, the resulting 7methylxanthine is further methylated via theobromine to caffeine by the second and third NMT respectively (Schulthess et al., 1996). For a comprehensive review on caffeine metabolism, see Ashihara & Crozier (1999). The activities of the second and third NMT are presented in dependence of leaf fresh weight in Fig. 10.4. As expected from the above-mentioned sharp increase of purine alkaloids, they both show a very high peak activity when the leaflets have completed their emergence from the protective bud. Thereafter, the activities decrease rapidly. The relative caffeine content drops as a consequence of

Fig. 10.4 Time course of NMT activities during leaf development in C. arabica. Leaves (5 to 70, depending on leaf size) of each stage were pooled and extracted. The youngest stage harvested had a fresh weight of ca 5 mg, the oldest of 1900 mg. The latter corresponded to the fully expanded, but still glossy and soft leaf, 30 to 35 days old, as shown in Fig. 10.1. (a) Activity per g leaf fresh weight; inset: purine alkaloid contents related to fresh weight (b) Total activity (pkat) per leaf; inset: absolute amounts of the purine alkaloids theobromine and caffeine per leaf. The data presented in the insets are from a separate experiment with 11 leaf classes and not from the leaf series that was used for the determination of the enzyme activities. (Taken from MoÈsli Waldhauser et al., 1997.)

`dilution by growth'. However, the absolute amount of caffeine increases steadily because of low enzyme activities persisting throughout the entire period of leaf expansion (MoÈsli Waldhauser et al., 1997). At the end of leaf expansion, the net photosynthesis (NPS) has attained a maximum rate which remains stable further on, while dark respiration gradually falls and thus the two events


(4)

in combination confer to an optimum dry matter production after 50 to 60 days of development. (Frischknecht et al., 1982. Note: erroneously, in this publication the dry weights listed in Table 1 are too small by a factor of ten!) To our knowledge, the related mechanical stabilization of the coffee leaf has not been investigated in depth. Certainly, the walls of the mesophyll cells will get thicker by the deposition of cellulose and the vascular system is fortified by phenolics. Moreover, the appearance of the upper surface of the lamina turns from glossy into dull, indicating a change preferably including a thickening of the waxy layer and/or cuticula. We should finally mention that during leaf development the chlorogenic acids are allocated in parallel to the purine alkaloids (Aerts & Baumann 1994). Similar to the NMTs catalyzing caffeine biosynthesis (see Fig. 10.4), the activity of the key enzyme of phenylpropane synthesis, phenylalanine ammonia lyase (PAL), is very high in the young leaflets and decreases during the further expansion (Aerts & Baumann, 1994). The concerted formation of both the alkaloids (mainly caffeine) and chlorogenic acids (mainly 5-caffeoylquinic acid; 5-CQA) has a physiological significance: caffeine, which easily permeates through all kind of biological barriers, is physico-chemically complexed by 5CQA and thus, compartmented in the cell vacuole in order to avoid autotoxicity (MoÈsli Waldhauser & Baumann, 1996). However, these processes eventually result in a coffee leaf in which these phytochemicals are not evenly distributed in the lamina. Preliminary investigations (Wenger & Baumann, unpublished data) revealed that both chlorogenic acids and purine alkaloids are considerably enriched at the leaf margin and sharply decrease in concentration towards the mid-vein. Conceivably, this `phytochemical leaf architecture' has an ecological significance: the leaf margin, a preferential site of insect attack, is particularly well furnished with these defense compounds. This insight could be of the upmost importance in modern breeding if phytochemical leaf architecture is genetically based. Senescence of the coffee leaf is not yet well investigated. The pioneering studies of Weevers (1907) may shed some light on the behavior of caffeine during aging: in his studies the adult leaf accumulated a maximum total amount which subsequently decreased by 30 to 50% in the old but still green leaf. He collected the leaves from

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plants growing in the `tropical greenhouse' in Amsterdam, and grouped them into `very young', `young', àdult' and òld'. Because of the long time span between the leaf classes àdult' and òld', their possibly divergent `life histories' (e.g. with respect to the light regime and other environmental factors), and merely degradation or export of caffeine may be responsible for the differences. Additionally, he tried to investigate the `caffeine status' in the leaf at shedding and recognized that only naturally aged leaves became caffeine-free, this in contrast to infected (e.g. Hemileia) leaves which still contained caffeine when shed. Despite these findings, it remains extremely difficult to tackle experimentally the problem of caffeine disappearance from the senescing arabica leaf. For instance, no metabolites were detected by Ashihara et al., (1996) after feeding ring-labeled caffeine to mature coffee leaves, except a trace of CO2 (0.03% of the applied radioactivity). Similarly, leaves still attached to the plant and fed with doubly labeled caffeine export only about 1% of the applied activity into the other leaves within one week (Baumann & Wanner, 1972). However, the recovered activity was about one fourth of the applied activity, indicating some catabolism in old leaves as it was found before in the classical investigations by Kalberer (1964, 1965). After feeding either [2-14 C], [8-14 C], [1-methyl-14 C], or [7-methyl-14 C] caffeine to aging arabica coffee leaves he always detected, besides allantoin and CO2 , the same three unknown degradation products which we speculatively classify as methylated ureides. The pathway from caffeine to these unknown compounds or to allantoin also remained obscure in more recent studies (Mazzafera et al., 1994; Ashihara et al., 1996). So far, neither a caffeine demethylase activity could be measured nor radioactivity in uric acid after feeding labeled caffeine could be detected in Arabica leaves (VitoÂria & Mazzafera 1999, and references cited therein). In conclusion, coffee leaf senescence and mobilization of related compounds certainly needs further investigation.

10.2.3 The fruit Fruit development in coffee, which covers the time between anthesis and full ripening, takes between 2 to 3 (e.g. C. racemosa) and 14 (e.g. C. liberica) months, depending on the species, genotype, climate and cultivation. The species of economic value, C. arabica

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and C. canephora, require 6 to 8 and 9 to 11 months for maturation, respectively (for related references see Guerreiro Filho, 1992). Almost 30 years ago, we performed a time-consuming investigation of fruit development in arabica coffee, whereas growth and alkaloid content were followed over a period of more than 7 months in both the pericarp and seed (Keller et al., 1971, 1972). Fruit development was divided into 11 stages (Fig. 10.5), as they are characterized in the legend, whereas at stage 1 (38 mg fresh wt) the separation into pericarp and seed tissue was not yet practicable. As depicted by the solid line, the dry wt of the pericarp exhibits a biphasic course due to the final ripening process (stages 8 to 11). The dry weight of the seeds increases gradually to reach a maximum value at stage 8 with an already tough endosperm texture. The time courses of the absolute amount of caffeine in the two fruit tissues is most remarkable. In the seed, it parallels more or less the dry weight curve, meaning that the relative caffeine content remains unchanged (> 1%) over the entire period of seed development.

Fig. 10.5 Absolute caffeine contents and dry weights during fruit development of C. arabica. (After Keller et al., 1972. Stages 1 to 11 (fresh wt in mg) are characterized as follows: 1 (38), separation into pericarp and seed tissue not possible, 1 to 2 weeks; 2 (240) green, 2 to 3 weeks; 3 (400) green, 3 weeks; 4 (800) green, 4 weeks; 5 (1200) green, 5 weeks; 6 (1180) green, endocarp hard, 2 to 3 months; 7 (1080) green, 4 months; 8 (1600) light-green/olive, mesocarp slightly fleshy, endosperm tough, 5 to 6 months; 9 (2180) exocarp partially reddish, mesocarp very fleshy, endosperm very tough, 5 to 6 months; 10 (2160) exocarp bright red, mesocarp very fleshy, endosperm very tough, 6 months; 11 (1800) exocarp dark red, mesocarp slightly dry, endosperm very tough, 7 to 8 months.


Unfortunately, we did not differentiate between the transient perisperm and the real endosperm as they are characteristic of coffee bean development (Carvalho et al., 1969). In the pericarp, however, the allocation of total caffeine stops early, that is already at stage 5, and hardly surpasses the total of 1 mg. Because the dry weight subsequently increases considerably, caffeine is diluted during fruit ripening to reach a final concentration of ca 0.24% (dry wt) in the fleshy pericarp. This is in contrast to stages 1 and 2 with a caffeine content of 2.2 and 1.7% respectively, distinctly higher than of a ripe arabica coffee bean. Interestingly, the cessation of further caffeine accumulation in the pericarp coincides with the formation of a hard endocarpic tissue (completed at stage 6), indicating a shift from chemical to mechanical defense. Clearly, the biochemical changes of the pericarpic tissues during ripening are directed to diaspore dispersal by animals (zoochory): the tough endocarp has to protect the seed from digesting enzyme activities in the gut of the frugivores such as birds or mammals, and the fleshy, sugar-containing (Urbaneja et al., 1996) mesocarp low in caffeine acts as a reward, while the vivid coloration, due to anthocyanins (Barboza & Ramirez-Martinez, 1991), of the exocarp is to attract the dispersing animal. By application of doubly-labeled caffeine to the epidermal layer of the pericarp of young coffee fruits attached to the tree, a considerable alkaloid transport into the endosperm tissue could be shown (Baumann & Wanner, 1972). The ability of the endosperm to form caffeine is indirectly testified by the fact that the very young, `liquid endosperm' of C. arabica served as an ideal source for the isolation of N-methyltransferases (Mazzafera et al., 1994; Gillies et al., 1995) catalyzing the last steps in caffeine biosynthesis. Moreover, endosperm tissue of over 6-month-old fruits, i.e. ca stage 8 in Fig. 10.5, exhibited distinct methyltransferase activity to synthesize theobromine and caffeine (Mazzafera et al., 1994). However, the ability of the coffee endosperm to form caffeine de novo has to our knowledge never been tested. There is one report dealing with radioactive precursor feeding to arabica endosperm, however, the authors (Keller et al., 1972) did not realize at that time that they actually fed the preceding, transient perisperm as one may conclude from the related fruit fresh weight of only 500 mg (about 25 days old). Nevertheless, the results were most intriguing: de novo caffeine synthesis in the perisperm is about 2.6 times higher than in the pericarp as determined by the incorporation of 14 CO2 in the presence of light. Conversely, methylation as estimated from the incorporation of radioactivity of [methyl-14 C]


methionine in the light is much higher (256) in the pericarp than in the perisperm. In the pericarp, light increases the methylation by a factor of 10 as compared to the dark condition. Unfortunately, the influence of light on caffeine formation (methylation) in the slightly greenish perisperm was not tested. (The background to light-dependent stimulation of caffeine biosynthesis will be discussed in the next section). It would be of the upmost importance to know the fate not only of the purine alkaloids but also of other secondary compounds such as chlorogenic acids allocated to the perisperm. Are they conserved and transported to the developing endosperm? Has the large fraction of dicaffeoylquinic acids present in the perisperm of arabica (Schulthess & Baumann, unpublished data) a significance similar to the occurrence of cyanogenic diglucosides in the seeds of many plant species (Selmar et al., 1988), namely to facilitate apoplastic transport by avoiding enzymatic degradation as required during seed development and/or germination? There are many other developmental key processes eventually leading to the lovely coffee bean that were completely neglected by science despite the high economic value of this product. However, very recently a remarkable investigation regarding the changes of various components during robusta and arabica seed development has been undertaken (Rogers et al., 1999). The authors followed various parameters such as sugars, polyols, organic acids and a few anorganic anions. Strikingly, the perisperm, which when fully developed fills the entire cavity later kept by the endosperm, is not only a `placeholder' but most likely also the full provider of sugars and organic acids for the endosperm. This view is essential for modern coffee breeding and implies that the maternal tissue (perisperm) controls not only the seed size but also the quantity and, to some extent, also the quality of the latter coffee bean (endosperm), this all reminding of the parental conflict theory (Grossniklaus et al., 1998).

10.3 PURINE ALKALOID FORMATION IN COFFEE CELL CULTURES 10.3.1 Introduction Even though the worldwide demand for caffeine in soft drinks has increased greatly in recent years, its production by plant tissue culture will not be economic because of the related costs. Nevertheless, caffeine formation by coffee cells deserves special attention, since this system has become a `standard of excellence'

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in designing and testing plant bioreactors for the production of secondary plant substances, as reviewed by Prenosil et al., (1987). The reasons are as follows: coffee cell cultures are easily established; they grow well on an artificial medium; they readily produce secondary compounds such as purine alkaloids and chlorogenic acids; the former diffuse into the culture medium allowing their direct determination, for example by HPLC, and thus a quick estimate of the alkaloid productivity and the product formation can be rapidly influenced by various factors, whereby light is experimentally the most prominent. This last reason points to an additional value of tissue culture exemplified below, namely its frequent use instead of difficult-to-handle plant organs and to facilitate many of the biochemical and physiological investigations. The coffee species used in these studies was preferentially arabica, though canephora is equally suitable or even superior with respect to alkaloid formation (Baumann & Frischknecht, 1982). However, the genetical homogeneity of arabica is an excellent prerequisite for such tissue culture investigations. So far, growth of purine alkaloid formation by cultured tissue of genera other than Coffea has been found, with the exception of Paullinia cupana (Baumann & Frischknecht, 1982), to be distinctly smaller, for example Camellia sinensis (Ogutuga & Northcote, 1970; Baumann & Frischknecht, 1982; Shervington et al., 1998) and Theobroma cacao (Baumann & Frischknecht, 1982; Gurney et al., 1992). Some of the older tissue culture work has been reviewed by Baumann & Frischknecht (1988a, b).

10.3.2 Callus culture Caffeine formation by callus cultures was first reported almost 30 years ago (Keller et al., 1971, 1972). The authors derived primary cultures from arabica coffee fruit transsects and revealed that after 4 to 5 weeks of cultivation 95 to 98% of the caffeine was present in the solid agar medium, most likely due to diffusion. Since the amount of caffeine detected in the whole culture increased by a factor of 6 and the biomass only by a factor of 2, it was hypothesized by Keller et al., (1972) that in vivo the caffeine synthesis is inhibited by product formation, and that in vitro the diffusion of caffeine into the medium diminished the inhibitory effect. Subsequently, the productivity of primary as well as of subcultures derived from arabica stem segments was investigated in detail by Frischknecht et al., (1977). Caffeine formation paralleled the increase of callus dry weight suggesting a metabolic connection of

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purine alkaloid synthesis to growth processes. Moreover, tissue caffeine concentrations of 900 to 1000 mg/ ml (ca 5 mM) and higher inhibited both callus growth and caffeine formation. The cultures grew in the dark which may explain why a caffeine concentration (5 mM) much lower than, for example, in a young coffee leaf (up to 50±60 mM) was inhibitory. In situ autotoxicity of caffeine is avoided by vacuolar complexation with chlorogenic acids mainly formed in the light (see below).

10.3.3 Suspension culture Suspension-cultured coffee cells were first established by Townsley's group (Townsley, 1974; Van de Voort & Townsley 1974, 1975; Buckland & Townsley 1975), which recognized the potential of these cultures to maintain the biosynthetic capacity for compounds unique to the parent plant. Besides purine alkaloids the formation of sterols, fatty acids, chlorogenic acids and coffee aromatics was reported. Subsequently, the growth of coffee cells in suspension was optimized and alkaloid production thoroughly investigated (Frischknecht & Baumann, 1980). The latter was shown to be highest towards the end of the growth phase when the cells stop dividing and start to expand. This is in accordance with maximum alkaloid formation in the young coffee leaflets (see Section 10.2.2), when the cells expand rapidly. Additionally, the biotransformation capacity was tested using radioactively labeled theobromine (Frischknecht & Baumann, 1980). Finally, the related N-methyltransferase activities were measured during culture growth and, most interestingly, were found to be highest during the (mitotic) growth phase. At this stage the cells produce a short supply of purine rings. Later on, when the primary metabolism is decreasing, a surplus of purine metabolites meets with comparably low methyltransferase activities (Baumann et al., 1983). In short, purine alkaloid metabolism in suspension-cultured cells is per se not well coordinated, but can be optimized by several means as outlined below. It was found that productivity correlated with the selected cell aggregate type: the culture of the small aggregate type forms less purine alkaloid than the large aggregate type (Frischknecht & Baumann, 1980). This phenomenon led to the idea to entrap and immobilize coffee cells in alginate beads to form artificial aggregates under controlled conditions (Haldimann & Brodelius, 1987). Indeed, the immobilization resulted in a considerable increase in alkaloid production. It is difficult to explain why the formation of large cell


aggregates or immobilization should stimulate caffeine biosynthesis. One explanation is based on the stress concept, which postulates a highly modulating effect of external factors on secondary plant metabolism considered to have an ecological significance so as to improve adaptation to unfavorable conditions. Therefore, environmental stress conditions such as high temperature, UV radiation, low water potential and wounding (phytophagy) are expected to enhance accumulation of qualitative defense substances such as alkaloids, cardenolides, glucosinolates, and others. Similarly, the above-mentioned large cell aggregates may, in contrast to single cells or small aggregates, have suffered from a nutrient stress since the transport of the medium components into the core of the aggregate was impeded. Stress was first introduced into tissue culture in 1985 by Frischknecht and Baumann. They used suspension-cultured cells of arabica coffee which were exposed to either high light intensity and/or high salt (NaCl). The former was most effective and induced a 100-fold stimulation of caffeine synthesis, i.e. 450 mg/ l, in the small aggregate type culture. The formation of purine alkaloids was also enhanced by the application of ethephon (Cho et al., 1988; Schulthess & Baumann, 1995) or adenine (Schulthess & Baumann, 1995). The combination of both applied to dark-grown suspension cultures resulted in an 11-fold increase (Schulthess & Baumann, 1995). In a photoperiod, as compared to the control culture in the dark, caffeine formation was stimulated by a factor of 21, which was not additionally increased by the above-mentioned stimuli. Conversely, the combination of photoperiod and ethephon led to a drastic reduction of ca 50 to 60% in the formation of both caffeine and chlorogenic acid. It was concluded that caffeine formation is dependent on chlorogenic acid accumulation. If the latter is impaired, deficient caffeine complexation results in the inhibition of the purine alkaloid biosynthesis. Baumann & RoÈhrig (1989) visualized the vacuolar localization of the chlorogenic acids in arabica suspension-cultured cells. They found that due to complex formation, caffeine is intracellularly accumulated to a certain extent, which depended on the chlorogenic acid (5-CQA) concentration in the cells. The physico-chemical and metabolic interdependence between purine alkaloids and chlorogenic acids, which is valid also for the living plant, was investigated in detail by MoÈsli Waldhauser and Baumann (1996) using suspension-cultured cells of C. arabica. By means of various conditions such as the addition of a photoperiod or methyljasmonate (both stimulating the


synthesis of caffeine and chlorogenic acids), the application of a potent inhibitor of PAL, the key enzyme in the phenolic pathway eventually leading to chlorogenic acids, and of exogenous caffeine, they created metabolic situations shedding light on the above-mentioned interdependence: compartmentation of caffeine (and also that of theobromine) is highly correlated to the concentration of chlorogenic acids and relies exclusively on the physical chemistry of the complex; moreover, there is a regulatory connection between the complex partners, possibly guided by the cytoplasmic caffeine concentration. Since experimental inhibition of the chlorogenic acid synthesis drastically inhibited caffeine biosynthesis, the authors came to the conclusion that lowering the bean caffeine content by means of genetic engineering could be achieved by changing the expression not only of the caffeine, but also of the chlorogenic acid pathway.

10.4 NEW ADVANCES IN CELL AND ORGAN CULTURE 10.4.1 Brief review of the literature Detailed literature reviews of pioneer work on coffee tissue culture have been already published by Sondahl et al., (1984), Sondahl & Loh (1988), Dublin (1991) and Sondahl & Lauritis (1992). The ability to induce large quantities of somatic embryos, subsequent germination of these non-sexual embryos and recovery of normal coffee plants are techniques of paramount importance for multiple applications in coffee improvement programs. Some key reports that led to this development include the pioneer work with robusta shoot cultures (Staritsky, 1970), high-frequency embryogenesis from mature leaf explants of arabica (Sondahl & Sharp, 1977), production of somatic embryos from leaves of arabusta hybrids in auxin-free medium (Dublin, 1981), and somatic embryogenesis from young leaves of arabica (Yasuda et al., 1985). All the above protocols were based on in vitro solid cultures developed during a 20-year period, but in the early 1990s, progress was made with embryogenesis in liquid cultures. A liquid culture protocol for a highly synchronized somatic embryo production, based on a modified version of the two-step somatic embryogenesis method of Sondahl and Sharp (1977), was published by Neuenschwander and Baumann (1992). Large numbers of robusta somatic embryos were produced in 3-liter bioreactor cultures by Zamarripa et al.,

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(1991a) soon after, followed by the work of Noriega and Sondahl (1993) with arabica embryos using a 5-liter bioreactor system. Using a unique apparatus for a temporary immersion culture, a protocol for the development of coffee plantlets was reported by Berthouly et al., (1995a). These solid and liquid medium protocols for coffee somatic embryogenesis have provided the key for a series of applications in coffee improvement programs such as micropropagation, gene transfer, in vitro mutagenesis and selection, germplasm preservation (cryopreservation) and biochemical studies. Small clumps of cells can be indefinitely maintained in liquid suspension medium through periodic subcultures (3-day intervals). Cultures of such undifferentiated cells are useful for metabolic studies of aromaproducing compounds (Townsley, 1974), lipid synthesis (Van de Voort & Townsley, 1975), purine alkaloids (Neuenschwander & Baumann 1991) and other studies. Liquid cultures of specialized cell lines, like embryogenic tissue, are used either for protoplast isolation, gene transfer, or for mass propagation. Protoplasts are single cells without a cell wall, maintained in a high-osmoticum medium. Initial work with coffee leaf protoplasts led to production of microcolonies (Sondahl et al., 1980, Orozco & Schieder 1982) but later, somatic embryos were reported from protoplasts isolated from in vitro robusta embryos by Schoepke et al., (1987). More efficient protocols for coffee protoplast isolation and regeneration were reported when protoplasts were isolated from embryogenic cells growing in liquid media (Acuna & Pena, 1991; Spiral & Petiard, 1991). Protoplast culture is an ideal system for synthesis of somatic hybrids between distant related species and for gene transfer via DNA uptake by these naked cells.

10.4.2 New advances (a) Somatic embryogenesis Acuna (1993) presented new data for the production of embryogenic tissue (ET) in two selected genotypes and two culture media. The F5.305 line in combination with NAR 12 medium resulted in the induction of ET in 93% of the explants after 2 months in culture without any subculture. The most suitable explant was soft young leaves from new suckers after pruning. The NAR 14 medium consisted of 14 strength of Murashighe & Skoog (1962) MS macro salts, 12 strength MS micro salts, B-5 organic constituents; sucrose (30 g/l) and 2ip (1 mg/l).

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Scanning electron microscopy (SEM) studies on coffee embryogenic tissues and early stages of embryo differentiation have been previously reported by Sondahl et al., (1979) and Nakamura et al., (1992). More recently, an interesting SEM study was made by Tahara et al., (1995) using three types of coffee calli (C. arabica), one embryogenic callus (EC) and two nonembryogenic calli (yellow callus, NYC; white callus, NWC), maintained on MS medium with 2,4-D (10 mM). EC was composed of yellow, spherical cytoplasm-rich cells, uniform in size; NWC displayed elongated or swollen translucent cells; NYC had cells similar in appearance to EC cells, but more dispersed. It was observed that EC, in the presence of 2,4-D, was incapable of regenerating somatic embryos, but after transfer to a 2,4-D-free medium, globular stage embryos arose after 2 to 3 weeks. The authors reported that EC preserved its embryogenic potential when maintained in 10 mM 2,4-D medium for 6 years. These data demonstrate that 2,4-D is able to induce the proliferation of embryogenic-competent cells, but its presence inhibits the regeneration process leading to somatic embryo production. The effect of asparagine on coffee somatic embryo induction has been demonstrated by Nishibata et al., (1995). Embryogenic cell lines of C. arabica were maintained on 10 mM 2,4-D medium (growth medium) and were regularly transferred to a 5 mM 2-iP medium (regeneration medium) for the production of somatic embryos. The addition of asparagine (10 mM) to the regeneration medium promoted embryogenesis, while the addition of glutamine, glutamate or aspartate strongly inhibited somatic embryogenesis. Moreover, the addition of asparagine (10 mM) to 2,4-D growth medium was able to induce somatic embryos and inhibit further cell proliferation. The effects of plant growth regulators on somatic embryogenesis of leaf cultures of C. canephora were reported by Hatanaka et al., (1995). It was demonstrated that cytokinin (5 mM) was essential for the formation of somatic embryos in robusta leaf cultures and that 2-iP was the most effective cytokinin source. It was described that when only half of the leaf discs were immersed vertically into the solid medium, embryos developed only on the cut edges of the discs that were in contact with the medium. On the other hand, each auxin tested (IAA, IBA, NAA, 4-FA, 2,4-D) inhibited somatic embryogenesis in proportion to its concentration. The authors also evaluated the effect of ethylene and found that at a concentration of 12 ml/l somatic embryogenesis was promoted, but at 6 ml/l ethylene had no effect and at 24 ml/l ethylene had an inhibitory effect.


Culture conditions for induction of somatic embryogenesis in arabica and robusta tissues have been reported by Yasuda et al., (1995). Using young leaf explants, both species produced somatic embryos on A3 cytokinin-only medium (5 mM 2-iP or BA), but there was a different reaction in culture according to the genotype. In robusta cultures, somatic embryos arose immediately on the cut edges of young leaf explants in contact with the medium; if auxin was added, embryo formation was inhibited. In arabica cultures, embryogenic calli were induced only after a long time (16 weeks). If the embryogenic tissue was transferred to 10 mM 2,4-D medium, white nonembryogenic calli and yellow embryogenic calli were produced. The yellow calli could be maintained in continuous proliferation, retaining their ability to form somatic embryos for more than 4 years, upon transfer to a cytokinin or auxin-free medium. The induction of somatic embryogenesis was tested with ten F1 hybrids made between commercial arabica cultivars and wild genotypes from Ethiopia using young leaf explants (Etienne et al., 1997). Embryogenic cells were produced after 6 months on solid cultures, multiplied in Petri dishes, and transferred to 125 ml Erlenmeyers to establish embryogenic cell suspensions at 100 rpm and 278C, with subculture intervals of 10 weeks. Young somatic embryos were transferred to RITA1 vessels under periodic immersion technique for embryo germination and plantlet development. Plantlets with one pair of leaves and a tap root were obtained after 3 to 4 months of cultivation in RITA vessels. A genotypic differential response to somatic embryogenesis was observed among the F1 hybrids. In case of a high-embryogenic material (Family 1/hybrid 1), up to 9000 plantlets were obtained per RITA vessel, but in the case of a low-embryogenic hybrid, only 750 to 1000 plantlets were obtained per vessel. The authors emphasized the suitability of this method for largescale propagation of F1 hybrids. A critical study on `direct or low' somatic embryogenesis induction from arabica leaf explants was presented by Loyola-Vargas et al., (1999). Using soft leaves from in vitro plantlets on Yasuda et al.'s (1985) medium, somatic embryos were observed directly from mesophyll cells of the explants after 21 days. No embryogenic tissue (friable calli, embryogenic calli) were observed in these cultures. Single isolated embryos were transferred to germination conditions and more than 700 plants were produced under greenhouse conditions. No morphological differences were observed among regenerated plants, suggesting that there is no visible somaclonal variation in this


coffee cloned population. The authors studied the effect of nitrogen on coffee somatic embryogenesis and suggested that total levels of 4 to 9 mM give a maximum response. The optimum ratio of nitrogen sources should be 1 NO3 :2 NH4 for a maximum response.

(b) Cryopreservation of embryos Coffee germplasm has to be maintained under field conditions due to the short life of viable seeds and the difficulty of applying long-term conservation techniques to coffee seeds. Cryopreservation of somatic embryos under liquid nitrogen (71968C) may offer one alternative for back up preservation of valuable germplasm. The ability of arabica zygotic embryos and robusta somatic embryos to withstand freezing into liquid nitrogen was evaluated by several freezing methods by Florin et al., (1993). Zygotic embryos could be cryopreserved after a controlled drying under 43% relative humidity (RH) at 188C; after thawing, normal development of zygotic embryos was observed. With the same method, or with a simple method based on a sucrose pretreatment, followed by prefreezing at 208C, regrowth of somatic embryos was observed via a secondary embryogenesis process. Normal plants were obtained from C. arabica (cultivars Catuai and Caturra) and from C. canephora and arabusta hybrid. In subsequent work, Florin et al., (1995) evaluated three preservation techniques for robusta somatic embryos. It was found that hydrated embryos can be preserved at 208C for 1 to 2 months. Partially dehydrated embryos could be stored in liquid nitrogen for an indefinite period of time, and such frozen embryos were able to develop into plantlets similar to controls. It was also reported that coffee embryos could be dehydrated and stored at 15 to 248C under 43% RH for at least 1 month. The survival rates of alginate-coated robusta somatic embryos before and after freezing in liquid nitrogen were reported by Hatanaka et al., (1995). It was found that the critical dehydration was 13% and that below this level the embryos suffered desiccation injury. Unfrozen embryos had 77% recovery and frozen embryos a maximum of 66% survival rate. It was reported that more than half of the revived somatic embryos after cryopreservation developed shoots and roots directly (no callus or secondary embryos) within 50 days of thawing, and similar results were observed after 8 months of cryopreservation. To avoid the long process of inducing somatic embryos for cryopreservation purposes, Dussert et al.,

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(1997) proposed freezing the entire seeds and then excised the zygotic embryos after thawing. Seeds must be dehydrated from the original 0.5 down to 0.2 g H2 O gÿ1 dry weight (dw), surface sterilized, and slowly precooled to 7508C prior to immersion in liquid nitrogen. After thawing, 70% of the excised embryos were viable and could develop into normal plantlets (only 30% for germination of intact seeds).

(c) Protoplast culture Successful reports on coffee protoplast isolation and plantlet regeneration have been achieved by the use of embryogenic cells (Acuna & Pena 1991; Spiral & Petiard 1991). A simple protoplast protocol using only one medium for cell wall regeneration, microcolony formation and regeneration has been described by Yasuda et al., (1995). Protoplasts were isolated from embryogenic tissues of arabica coffee and cultivated in an A3 medium supplemented with 10% coconut water, mannitol (0.3 M) and 5 mM BA. The first cell division was observed after 3 weeks, when liquid A3 medium without mannitol was added. After 2 months of culture, somatic embryos were present and they developed into normal plantlets after subsequent subculture.

(d) Transformation and regeneration To perform a gene transfer to modify the genetic makeup of a plant species, one must successfully incorporate a gene cassette (desirable gene plus introns, promotor and terminating sequences) in a plant cell and subsequently recover a modified plant from such a single modified cell. Transformation deals with the techniques of `gene insertion' and regeneration refers to in vitro processes leading to the recovery of viable, positive transformed plants. The success rate of gene transfer varies with the in vitro cell culture system and the method utilized for gene insertion. Usually, the target cells are active proliferating embryogenic cells (embryogenic tissue) which would have the cell wall removed if the gene transfer method requires protoplast cultures. Basically, there are three main methods for gene transfer: (a) DNA uptake by protoplasts; (b) accelerated particles coated with DNA; and (c) cocultivation with Agrobacterium tumefaciens (or rhizogenes). The first two methods are physical methods and the third one is a biological method, mediated by the bacterial vector, which has the natural ability to penetrate plant cells and transfer DNA segments to the nucleus. Positive transgenic events (cells carrying foreign DNA) are usually at the 1 to 5% rate and from

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these cells, normal plants must be recovered. Transformation/regeneration protocols rely heavily on the in vitro regeneration protocol to recover a few positive transgenic cells. In practical terms, a gene transfer project must recover about 100 positive transgenic plants in order to make the selection of the best individuals. Positive transgenic plants may carry the new DNA make-up but it is not expressed, or it is poorly expressed. Sometimes, positive transgenic plants are in vitro or somatic mutated individuals and so they must be screened out at the plant level. Detailed information on molecular biology and gene transfer studies on coffee is covered in Chapter 11 in this book. Here, we will briefly review the in vitro methods of transformation/regeneration using coffee cultures. Two gene transfer methods have been used with coffee solid cultures. Barton et al., (1991) electroporated coffee protoplasts in an attempt to transfer a genetic marker (NPT II) for protocol development. The authors reported the subsequent recovery of callus and embryos from these experiments, but confirmation of the presence of an NPT II marker incorporated at the plant level has not been provided. Spiral & Petiard (1993) reported transformation/ regeneration in three genotypes (robusta, arabica (cv Red Catuai) and arabusta #1307) using Agrobacterium rhizogenes charged with GUS and NPT II gene markers and 35-S and NOS promotors. Coffee somatic embryos were co-cultivated with A. rhizogenes for 1 hour and transferred to an embryogenic medium for 11 days. After this period, embryos were subcultured to a medium containing 200 ml of cefaloridine to kill the bacteria. After 3 weeks, callus, hairy roots and embryos were observed. The authors reported a high frequency of positive transformation (+10%). Roots with a positive GUS reaction were subcultured on embryogenic medium and after 4 weeks, fresh somatic embryos were produced. After 2 months of culturing these rootderived embryos, coffee plantlets were recovered which allowed for the positive evaluation of gene integration (GUS and NP II). The integration of several copies of the same gene at the plantlet level was reported. The stability of integrated DNA and the presence of multi-gene copies would be traced to subsequent generations. Sugiyama et al., (1995) reported positive transformation/regeneration in arabica tissues using the wild type of Agrobacterium rhizogenes strain IF 14554. Cotyledon fragments produced callus (48%) and hairy roots (39%). Hypocotyl tissues formed only callus (95%) and leaf explants produced small numbers of hairy roots and callus. Callus tissues were non-


embryogenic, but small number of somatic embryos were recovered from hairy roots after 6 months in culture in a 2 mM 2-iP medium. After these rootderived embryos were transferred to hormone-free medium, plantlets were obtained. Positive transgenic plantlets were cloned by nodal culture and the resulting plantlets continued to express the same transformed phenotype of the donor tissues.

(e) In vitro selection studies In vitro selection using either solid or liquid cultures in the presence of phytotoxins offers the possibility of recovering cell lines and plantlets resistant to pathogen, if there is a correlation between toxin resistance and in vitro resistance. This in vitro selection system explores the natural variability existing in somatic cells and/or variability induced during the in vitro culture conditions. In several cases, a positive correlation between toxin resistance in vitro and in vivo pathogen resistance has been demonstrated (Hartman et al., 1984; Hammerschlag, 1990). Coffee berry disease (CBD) is caused by the fungus Colletotrichum coffeanum and constitutes a very serious limitation for coffee production in many African countries with losses of up to 50% if not controlled by frequent fungicide spraying (Griffiths et al., 1971) (see also chapter 9). Nyange et al., (1993) reported that partially-purified culture filtrates from C. coffeanum were used against crushed calli in liquid and with cell suspension of arabica materials (N 39 and Timor Hybrid). Growth and viability of susceptible cultures were significantly reduced by the presence of C. coffeanum filtrate and several somatic embryos and plantlets were recovered from the selected calli. The resulting plants will be tested in vivo to confirm (or not) the resistance reaction to CBD.

10.5 COFFEE SCALE-UP BY MICROPROPAGATION Seed is the most common form of plant propagation and it should be used when the species is autogamous (homozygous), or when reliable production of hybrid seeds can be made in large quantities. Vegetative propagation applies when the plant species does not produce seeds, or hybrid seeds cannot be commercially utilized. Rooting and grafting are the most common methods of vegetative propagation. Micropropagation is utilized when (a) it is difficult to apply traditional


propagation methods; (b) it is important to start from `disease-free' planting materials; or (c) there is a need to produce very large numbers of plants in a short period of time. Micropropagation can be achieved by different in vitro multiplication methods: (a) growth of preexisting axillary buds; (b) production of shoots via organogenesis; or (c) plantlets production from somatic embryogenesis. The great majority of commercial micropropagation protocols are based on axillary bud multiplication in solid medium (Kurtz et al., 1991). Micropropagation via organogenesis has been of limited use (Litz & Gray, 1992), either because of the lack of specific protocols or because high rates of somatic variation are suspected. Somatic embryogenesis is currently receiving a great deal of attention, due to its enormous multiplication rates (reduction of unit costs) and the relative genetic stability of the resulting plants under greenhouse and field evaluations (Jones & Hughes, 1989; Sondahl et al., 1999; Ducos et al., 1999). Indeed, somatic embryogenesis is a highly attractive propagation method for perennial species and also for tropical plants that carry elevated levels of phenolic compounds, which inhibit rooting and grafting. Arabica coffee is a self-pollinated species and so 98% of homozygous individuals can be obtained after six consecutive generations (ca 24 years of selfing and selection). Robusta is an out-crossing species and so fields propagated by seeds are composed of segregant individuals. Yield and other desirable characteristics would improve drastically in robusta plantations established from propagated elite individuals. One can easily evaluate the commercial advantages of cloning elite individual plants so bypassing the long time required for selfing and selecting homozygous seed donors. Multiple clone lines would be used at one time and would thus help preserve heterozygosity and plasticity in coffee plantations. Besides the establishment of commercial fields, coffee micropropagation could set up `seed orchard areas' for the production of multi-line bulked seeds, or to multiply segregating parental lines for the synthesis of intervarietal hybrid seeds. Coffee can be propagated by grafting, axillary bud development (nodal cultures) or by the `direct' embryogenesis pathway. All these methods are suitable for a limited cloned population due to its low multiplication rate. In this section we will focus on the use of `high-frequency (or indirect)' somatic embryogenesis for large-scale propagation of elite plants to establish commercial plantations with a population size of 100 000 to 1 000 000 plants per year.

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10.5.1 Mass production of somatic embryos Many protocols are available for production of coffee somatic embryos but all of them are experimental in nature and not suitable for large-scale propagation. Embryogenic tissues from somatic cells of elite plants should be produced on solid cultures and maintained viable through a periodic subculture regime. Mass production of somatic embryos must be set up in liquid embryogenic suspension cultures growing in Erlenmeyer or bioreactor vessels.

(a) Erlenmeyer cultures Zamarripa et al., (1991a) made a very detailed report on the conditions to establish and maintain embryogenic suspensions of coffee cells. Embryogenesis was induced in solid media from leaf explants cultivated in Dublin (1984) medium in the dark and later, transferred to light on Yasuda (1985) medium containing 1.0 mg/l BA. Yellow, friable, embryogenic tissues were transferred to 50 ml Erlenmeyer flasks containing 20 ml of a modified Yasuda medium (Zamarripa et al., 1991a). After initial growth, suspensions are transferred to 100 ml and later to 250 ml Erlenmeyer flasks, under 100 rpm at 238C. The density of the initial inoculum is important in this liquid suspension establishment and it should be at least equal to 10 g fw/ liter. The total time to establish stable embryogenic suspension is about 8 months. Liquid cultures are maintained through a 21-day subculture regime. At each subculture, the biomass is collected in a nylon filter (mesh 50 mm) and transferred to a fresh 250 ml Erlenmeyer flask containing 100 ml of liquid medium to keep a final density of 10 g fw per liter. These coffee suspension cultures consist of cellular aggregates of 430 mm diameter in Catuai, 630 mm in arabusta and 760 to 940 mm in robusta. Once the embryogenic suspensions were well established (Zamarripa et al., 1991a), somatic embryos were induced by transferring cellular aggregates to a fresh Dublin (1984) medium. In this phase, the dilution of the initial density to 0.1±0.2 g fw/liter is critical. The lower the density, the better the development of the somatic embryos. After 6 weeks, the embryo concentration reaches a plateau of 240 000 embryos/liter. After 6 weeks, about 90 000 torpedo shape embryos can be found. This behavior is similar to all coffee genotypes studied. However, the embryo formation is not synchronized since one can observe somatic embryos at all stages of differentiation (globular, heart and torpedo

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shape). The authors noted that this embryo production may vary with the culture conditions like inoculum density, periodicity of fresh medium addition, rate of agitation, and light/dark conditions. Weekly addition of fresh medium is better than biweekly for torpedo embryo production. After 8 weeks of embryo differentiation in liquid medium, embryos were transferred to solid medium (Zamarripa et al., 1991a) containing 0.225 mg/l BA. Following another 4 weeks, embryos were subcultured to a cytokinin-free medium and the first pair of leaves were observed within 8 additional weeks. The conversion rate of embryos to plantlets was 50 to 70% after 12 weeks in BA-free medium. Plantlets with 2 to 3 pairs of leaves were transferred to a greenhouse for hardening and subsequent growth.

(b) Bioreactor cultures Robusta embryogenic suspension cells (clone R2) were charged at the rate of 1.0 mg fw/liter to a 3-liter liquid medium, inside a stirred Setric SGI bioreactor apparatus, running at 60 rpm, with aeration at 0.04 air volume/medium volume per minute at 268C (Zamarripa et al., 1991b). Under this system, embryogenic tissue proliferates up to a yield of 200 000 embryos per liter on day 49 of culture. From this total embryo population, about 20% (40 000/liter) were torpedoshaped embryos. The conversion rates of somatic embryos derived from the bioreactor were similar to the ones produced in Erlenmeyer flasks (50 to 70%) which translates into 60 000 plantlets/bioreactor every 2 months. The success rate during hardening in the greenhouse was described at 80 to 95% after 5 months. This means that a 3-liter bioreactor culture of robusta embryogenic cells can yield ca 48 000 cloned plants every 2 months which is equivalent to 19 ha of a coffee plantation at a planting density of 2500 plants/ha. The potential of this process for scaling up elite robusta plants can be realized if the bioreactor is charged with higher working volumes and multiple bioreactor units run simultaneously. An additional study on the critical parameters for bioreactor mass production of robusta (and arabusta) coffee was presented by Ducos et al., (1993). A Setric SGI model SET4CV stirrer bioreactor was charged with an embryogenic cell suspension at rate of 0.5 g fw/ liter, operating at a 3-liter working volume, renewed every week. Agitation was kept at the lowest level of 50 rpm until day 21 and then, increased slowly to 100 rpm. Air flow was maintained at the lowest level just to maintain dissolved oxygen (DO2 ) above the


critical level. Specific oxygen uptake and the specific production rates of CO2 and ethylene decreased as a function of culture time. The increases in CO2 and ethylene were linked with the increase in the aeration rate, and this finding is in contrast with Erlenmeyer flask cultures. Production of embryos began on day 21 and it was completed on day 58. Ducos et al., (1993) reported a maximum yield of 180 000 embryos/liter for robusta cultures. The embryo±plantlet conversion rate observed in robusta was 47%, similar to control Erlenmeyer flasks. For arabusta, a population of 160 000 embryos/liter was achieved, and a conversion rate of 37% was observed for bioreactor-derived embryos. This rate was higher than the Erlenmeyer flask rate (20%). Bioreactor mass production of arabica somatic embryos has been reported by Noriega and Sondahl (1993). Friable embryogenic tissue (FET) was obtained according to Sondahl's protocols (Sondahl & Sharp 1977; Sondahl et al., 1984), using mature leaf explants of C. arabica cv Red Catuai, cultured on conditioning medium (MSI) for 6 weeks and then transferred to the induction medium (MSII). Friable embryogenic tissue colonies were isolated after 4 to 6 months of secondary culture, and this tissue was maintained on solid medium by periodic subcultures for ca 3 years. In a preliminary study, FET cultures were inoculated at low density in a bioreactor vessel where a 20-fold proliferation increase was observed (Sondahl & Noriega, 1992). After this multiplication phase, torpedo-stage embryos were observed at low frequency and the bioreactor was kept running without medium exchange for 2 months. At the end of this period, the suspension differentiated entirely into embryos, which were then cultivated into a liquid `maturation medium' for 4 weeks. Mature embryos were plated onto solid germination medium producing normal plantlets. This experiment revealed a yield of 12 500 embryos per 1.0 g fw inoculum of FET cells. In a subsequent study of arabica mass production of embryos in bioreactors, Noriega & Sondahl (1993) used embryogenic cell suspension cultures of Red Catuai, initiated in MSII medium using 125 or 250 Erlenmeyer flasks in a rotary shaker at 100 rpm at 258C in the dark. Fresh MSII medium was added twice a week. The suspension cultures were established after a period of 3 months and after that, cultures were split every 3 weeks, maintaining the packed cell volume (PCV) of about 5 to 10 ml/100 ml. These suspensions contained only FET cells with cluster of 0.5 to 1.0 mm in diameter. These FET suspension cultures were used for


bioreactor inoculation (Noriega & Sondahl, 1993). A 5-liter magnetic stirring bioreactor vessel was used, running at 70 to 120 rpm and kept at 258C in the dark. Clusters of FET were charged to liquid MSII, which was replaced every week for the next 5 weeks. Cell density was maintained at 1 to 5 PCV/liter inside the bioreactor by removing the excess of tissue at the time of medium exchange. At the end of the fifth week, no more fresh medium was added to force the culture to enter into a rest period (to stop the multiplication phase). After another 4 weeks, fresh `developing medium' (DM) was added. After an additional 5 weeks, somatic embryos were harvested from the bioreactor and plated on solid agar medium for germination. Summarizing this protocol, FET cells were allowed a multiplication phase of 5 weeks, a resting phase of 4 weeks (embryo differentiation) and an additional 5 weeks on developing medium. At the end of production (3.5 months), a population of 45 000 embryos/5 liter bioreactor was estimated. The total embryo population was reduced by the periodic FET cell removal during the multiplication phase. Samples of the somatic embryo population produced by this bioreactor method revealed about 25% of torpedo, 45% of heart and 30% of globular embryos. Debris of FET cells could still be seen inside the bioreactor, which is an indication that the bioreactor differentiation process was not fully completed at the time of opening. Plated embryos began chlorophyll development after 1 to 2 weeks under light and fully germinated embryos were observed at the tenth week on solid medium (Noriega & Sondahl, 1993).

(c) Periodic immersion cultures An autoclavable filtration unit (500 ml capacity) was modified to facilitate periodic immersion flushes of liquid medium and it has been tested for coffee propagation via axillary buds and somatic embryogenesis (Berthouly et al., 1995a). A glass tube is installed to connect the upper and lower compartments. A fine screen is placed in the bottom of the upper part to hold cultivating tissues. The system is charged with explant in the top part and fresh medium in the bottom section, which is connected to a small air pump, controlled by an electric clock. When the pump is turned on, the air passes through a 0.22 mm filter to maintain sterility and enters the lower section; as the pressure builds, the liquid medium is suspended to the upper section. When the pump turns off, the liquid returns to the lower section by gravity. Using coffee orthotropic nodal segments in the

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upper section, the authors were able to induce six to seven axillary buds to develop within 5 to 6 weeks, in contrast with solid medium cultures that take about 12 weeks. It was reported that time and frequency of periodic immersion were critical factors for optimizing the final results. Again, the protocol must be adjusted according to the genotype being cultivated. For arabica nodal cultures, the best conditions were four pulses of 15 minutes per 24 hours in the presence of 1.0 mg/ 1 BA. Prolonged immersion times would lead to vitrification. For robusta nodal cultures, four pulses of 1 minute per 24 hour period should be used in the presence of 0.1 mg/l BA. Under these conditions, an average of 6.8 shoots and 7.2 shoots were recorded for arabica and robusta cultures, respectively. The same periodic immersion system (called RITA) was charged with FET and after 40 days it produced the same amount of fresh weight as control cultures in the Erlenmeyer flasks. Immersion time and frequency were also critical for coffee embryogenic cultures. The optimum conditions for somatic cell proliferation and embryo regeneration employed four pulses of 15 minutes per 24 hours.

10.5.2 Applications Case 1: Bourbon LC cloned field To evaluate the stability of coffee somatic embryos produced via solid and liquid media, an experiment field was established using C. arabica cv Bourbon LC line B. Young plants derived from in vitro cultures were shipped from the New Jersey laboratory to Brazil and seedlings of the same line were produced as control plants at the local nursery (Sondahl et al., 1999). Friable embryogenic tissues were produced from leaf explants of adult plants according to the two-stage method of Sondahl & Sharp (1977). Liquid suspension cultures of FET were established and then propagated into bioreactor vessels as described by Noriega & Sondahl (1993). Two bioreactor models were tested: a 5-liter, magnetic-stirring Ono model and a 7-liter, blade-stirring Aplikon model. Somatic embryos from bioreactor cultures and from solid agar cultures were allowed to germinate and the resulting plantlets were transferred to 35 6 145 mm tubettes and placed in a greenhouse for hardening. Stage 4 plantlets were introduced in Brazil with the assistance of the Quarantine Service (Cenargen), and later transferred to a local coffee farm. Seeds of the same Bourbon LC line B were germinated in sand beds and later transferred to 35 6 145 mm tubettes to complete their development.

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Planting was carried out in randomized plots in a coffee farm at 2.0 6 1.0 m spacing, during the period of 2 to 4 March 1996. This experimental plot consisted of the following materials: 220 bioreactor-derived plants, 230 solid medium plants and 500 seed-derived plants. Morphological evaluations were made after the first harvest (1998) and second harvest (1999) which can be summarized as follows: Bioreactor plants = 01 Solid medium plants = 02 Seed-derived plants = 02 01

variegated (01/220 = 0.4%) broad leaf type (02/230 = 0.9%) murta type angustifolia (03/500 = 1.0%)

After 4 years under field conditions and at the second crop, very few differences among coffee plants (as shown above) could be seen. This field test plot demonstrates that coffee plants derived from somatic embryos, produced either by solid or liquid bioreactor cultures, can be used for micropropagation. For practical purposes, all micropropagated plants evaluated can be considered to be similar to each other. Since the plants show a very low rate of variation (less than 1%), it can be concluded that the process is safe for largescale multiplication for the coffee variety tested. The frequency of in vitro variability is highly dependent on the genotype, and so each elite plant or hybrid selected for micropropagation must be field tested before large plantation areas are established.

Case 2: NestleÂ fields of Robusta clones Robusta is a self-incompatible species, and so vegetative propagation must be performed in order to maintain the genetic potential of selected plants. Before entering into a full scale planting program, several aspects have to be evaluated: (a) regeneration capacity of embryogenic cell lines for each selected genotype; (b) logistics of production and distribution of cloned plants; (c) cost of production of plants produced by somatic embryogenesis; and (d) true-to-type status of regenerated plants (Ducos et al., 1999). Five elite robusta plants were selected on the basis of their agronomic traits for this micropropagation evaluation. Embryogenic cell lines were isolated from young leaf explants after 8 months on solid cultures. Liquid embryogenic suspensions were established after 2 months and subcultured every 2 weeks at a density of 10 g fw/liter. Somatic embryos were induced by decreasing the inoculum density from 10 to 1.0 g fw/l and by transfer to a full strength MS medium. After 2 to 3 months, embryos were plated onto germination solid medium. The authors observed that the

embryogenic suspension cell lines decrease their embryo-producing capacity after 6 months of continuous culture. So it has been recommended to maintain the embryogenic cell lines under solid medium and only transfer to liquid suspension according to the mass production schedule (3-monthold suspensions are ideal). Based on these assumptions, the following calculations were made by Ducos et al., (1999): (a) 500 explants produce 1.0 g FET; (b) 60 g FET are generated after 3 months of liquid multiplication; (c) 1.0 g fw of 3-month-old FET liquid culture produces 56 000 plantlets. In conclusion, about 3.0 million plantlets can be produced from an initial inoculation of 500 leaf segments, that is sufficient for planting ca 1800 ha of robusta fields. NestleÂ laboratory sent in vitro torpedo shaped somatic embryos to a collaborating facility in the Philippines (Department of Plant Industry) from which about 70 000 plants were recovered. The conversion of embryo to plantlets in the recipient country was four to five times lower than in France, and so it was recommended to ship ex vitro acclimatized plantlets to local nursery facilities. The average recovery of ex vitro plantlets is currently 37%. This group made initial cost calculations and derived production costs of US $0.169 per somatic-embryoderived plant versus a cost of US $0.158 for a robusta cutting. Based on 1600 plants/ha plantations, this difference is only US $18.4 per hectare, which is not significant considering the several benefits offered by mass production of cloning operations. These five robusta clones are being field tested in five coffee-producing countries (4000 plants/location): Philippines, Thailand, Mexico, Nigeria and Brazil. Based on visual inspection of 8000 plants under field conditions in the Philippines, all micropropagated robusta plants have normal vegetative aspects and are developing normal flowers and fruits 2 years after planting. The first test field in the Philippines produced 1 t green coffee at its second harvest. These field tests will continue to evaluate long-term growth and production capacity in addition to comparing in vitro-derived plants with plants originated by other vegetative methods such as cuttings and microcuttings.

Case 3: Cloned fields of F1 Arabica hybrids A cooperative program between coffee-producing countries of Central America (Promecafe), the CATIE research center in Costa Rica, and a French Cooperation Consortium (Cirad, Orstom, Mae) has embarked on an interesting program for synthesis of F1 hybrids


between high-yielding arabica varieties (Caturra, Catuai, Catimor, Sharchimor) and wild arabica genotypes from Ethiopia and Sudan (Etienne et al., 1997). The goal is to combine superior cup quality with yield and disease resistance. This hybridization program was initiated in 1992 and now F1 hybrids are being evaluated under field conditions and final selections should be made by year 2003 (Etienne et al., 1999). A pilot scale micropropagation of 10 F1 hybrids is already under way, using the RITA periodic immersion technique described above (Berthouly et al., 1995a). Differences in the embryogenesis capacity among different F1 hybrids have been reported, but the amount of FET recovered from any F1 hybrid is sufficient for multiplication in liquid for large-scale clone production of any single hybrid (Etienne et al., 1997). The current data reveal a production of 7500 to 15 000 embryoderived plants per gram of embryogenic suspension culture. The authors have successfully tested `direct sowing', that is transfer of early stages of cotyledonary somatic embryos to trays containing artificial soil under controlled greenhouse conditions. A total of 20 000 plants from these F1 hybrids was produced and these plants are being used to establish test field plots in four Central American countries (Etienne et al., 1999). The objective is to evaluate the performance of the embryo-derived plants under distinct farming conditions. It is reported that among 4000 vitroplants under field and nursery conditions so far evaluated, no somaclonal variation has been observed (Etienne et al., 1999).

Case 4: Uganda Robusta cloning program The Uganda Ministry of Agriculture through the Farming System Support Programme sponsored by the European Union has launched a project for largescale propagation and distribution to farmers of six selected robusta clones. Propagation will be accomplished by the cutting process and by in vitro methods (Berthouly et al., 1995a). A local cloning facility of 182 m2 was constructed in Uganda, equipped with two temperature controlled culture rooms and a nearby greenhouse. The micropropagation effort will utilize the periodic immersion technique (Berthouly et al., 1995a) for cloning via axillary bud development (microcuttings) and via somatic embryogenesis. A total of 2000 RITA vessels are being installed and the expectation is to produce 600 000 plants/year from microcuttings and 2.0 to 2.5 million plants/year via somatic embryogenesis (Berthouly et al., 1995b).

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10.6 SOMACLONAL VARIATION AND NEW BREEDING LINES 10.6.1 Definitions and examples Variation among plants regenerated from in vitro cultures was first described for plants from tobacco callus cultures by Butenko et al., (1967). However, this in vitro variability was not clearly recognized and defined until the review made by Larkin and Scowcroft (1981). Somaclonal variants can appear when an explant (any plant part) is subjected to a tissue culture cycle. This cycle includes establishment of a dedifferentiated cell or tissue culture under defined conditions and the subsequent regeneration of plants (Hammerschlag, 1992). This phenomenon was further defined to include in vitro variability from cultivated haploid cells and named `gametoclonal variation' (Evans et al., 1984). Somaclonal variation is the expression of the naturally occurring variability of plant cells, or the result of in vitro induced variability of cells following plant regeneration (Larkin & Scowcroft, 1981; Evans & Sharp, 1986). Most of this spontaneous variability from in vitro plants is associated with chromosome alterations such as breakage, translocation, deletions, aneuploidy, polyploidy and somatic crossing-over. In addition, somaclonal variation can also have a single gene origin, for example a point mutation, alteration in gene copy number, activation of transposon elements and variation in DNA methylation (Karp et al., 1982; McCoy et al., 1982; Orton, 1983; Phillips et al., 1990). Somaclonal variation is an excellent method for shortening breeding programs, since it can provide access to genetic variability within existing cultivars (Evans & Sharp, 1986). Somaclones carry few genetic alterations and so the overall genetic integrity of the original commercial cultivar is preserved. In the case of coffee, no variability has been observed beyond the diversity already known for the arabica species (Sondahl & Bragin, 1991). Somaclonal variation has contributed to the release of improved varieties of sexually (tobacco, tomato, rapeseed, corn, blackberry, celery, coffee) and non-sexually propagated species (potato, sweet potato, sugarcane) (Evans, 1988; Hammerschlag, 1992; Sondahl & Lauritis, 1992).

10.6.2 Coffee somaclonal variation program Several agronomically important coffee genotypes were used for a somaclone variation study (Sondahl & Bragin, 1991; Sondahl & Lauritis, 1992). Since

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somaclonal variation frequency varies with genotype and culture procedure, a wide array of genotypes were used in this program, including tall stature varieties (Yellow Bourbon, Mundo Novo, Icatu and Aramosa) and short stature varieties (Red and Yellow Catuai, Caturra, Catimor, Laurina and other genotypes). Tissue culture was initiated from mature leaf explants following the Sondahl and Sharp (1977) protocol and donor plants were maintained in a greenhouse collection. Plantlets were recovered from both the `lowfrequency pathway (LFSE)' and the `high-frequency pathway (HFSE)'. Plantlets were hardened in a greenhouse and then shipped to a coffee nursery in Brazil, with the assistance of local quarantine service. In vitro-derived plants were transferred to the field as they reached transplanting size, and different experimental fields were made according to the donor variety. Seed-derived control plants for each coffee variety being studied were planted in each experimental field. Planting was in a coffee farm in Cajuru SP, Brazil at 218 latitude south, at 1040 m altitude, with a spacing of 3.5 6 2.0 m. Normal coffee fertilization and disease control practices were used in this field. A total of 14 948 in vitro-derived plants were established in the field, representing nine different coffee genotypes. Screening was done at the Ro generation during the first and second crops. The most interesting variant forms were studied in the next generation by establishing progeny fields. The overall variability found in this in vitro-derived coffee population was 10%, but variability was highly genotype-dependent, for instance: 30.6% for Yellow Bourbon and only 3.3% for Red Catuai. The most common mutation was for fruit color (42.35% yellow to red) followed by change in plant stature (3.8% tall to short). Based on 7772 in vitro plants evaluated, the frequency of variability was similar for plants originated from HFSE (or indirect embryogenesis; 12%) and LFSE (or direct embryogenesis; 10.4%). These data demonstrate that both HFSE and LFSE could be used for micropropagation programs, since there is no enhanced variability among plants from the HFSE pathway, as initially suspected. More detailed information about this program can be found in Sondahl and Bragin (1991) and Sondahl and Lauritis (1992). Many interesting variants have been selected from this program and their progenies are being studied in subsequent generations. The most interesting mutations are being carried on by standard breeding methods aiming for the release of new varieties in the future. Emphasis is being made in selecting superior cup qualities associated with desirable agronomic traits.


Three main breeding populations have been derived from this tissue culture program: Laurina somaclones, Icatu somaclones and Aramosa somaclones. Some characteristics of segregating individuals of the Icatu and Aramosa populations were reported by Sondahl et al., (1997). Other small populations are also being studied, such as short-stature mutants of Mundo Novo and Yellow Bourbon. Another interesting population is based in one Maragogype mutant plant that was derived from Yellow Catuai leaf cultures. Second and third generations of this somaclone are showing segregation for the typical Maragogype phenotype, normal Catuai and an intermediate phenotype with short stature but very large beans.

10.6.3 Commercialization of new varieties Laurina is a natural mutation from Red Bourbon plants found in Reunion Island by the mid-1800s. These mutated plants had small leaves, thin lateral branches, short stature and elongated fruits and beans, and it has been referred to in the literature as `Laurina', `Leroy', `Bourbon Pointu' and `Smyrna' coffee (Raoul, 1897; Boutilly, 1900; Coste, 1955). This Laurina sport was immediately introduced into commercial plantations in Africa (and transferred to South America) because of its drought resistance and superior beverage properties (Raoul, 1897; Krug et al., 1954). It was not until much later (the mid-1950s) that studies of coffee collections reported that Laurina plants had a natural 50% reduced caffeine concentration (Lopes, 1973). More recently, Baumann et al., (1998) explained that the reduction in caffeine content in Laurina is due to a reduced synthetic activity. Among more than 800 in vitro-derived plants of Laurina, 15 elite plants were selected at the R0 generation in June 1991. These selected plants were clearly more vigorous than sister plants and donor controls, as demonstrated by greater leaf area, lateral branches, plant height, plant diameter, and superior yield. Seeds of these selected plants were taken to establish a separate experimental field (4 ha in size) to evaluate the performance of each somaclone line. A total of 360 plants per line was established in a coffee farm in Brazil, in March 1992, in nine random replicated blocks plus control plants (seed-derived donor Laurina) and border lines, at a spacing of 3.5 6 1.5 m, with two plants per hill. Growth pattern, yield and caffeine content were monitored for each of these 15 lines during the first 5 years under field conditions (three successive crops). It was observed


that the caffeine content was stable and equal to the donor plants, the growth pattern was stable for all lines (no segregation) and the yield from the top five lines was twice as high as for the control plants. Yield evaluation continued up to the sixth harvest (1999 crop) in the experimental field, thus confirming the initial selection of the top five lines for superior yield and reduced biannual cycle. A third generation of selected lines was established in a semi-commercial plot design of 25 ha in size. Seeds from the top five highyielding lines are being scaled under the name of `Bourbon'. At the time the first round of selection of elite Laurina somaclones was completed, filings for patent protection were made on this discovery. A utility patent was awarded in the USA under Patent No 5 436 395 on 25 July 1995 (Sondahl et al., 1995). The Bourbon LC is the first example of a patent awarded for a coffee variety and it is also the first case of the release of a coffee variety derived from natural variability, isolated from somatic embryo cultures. Bourbon LC is the only naturally reduced (50%) caffeine variety being produced in commercial quantities. It is a product that should capture the interest of `coffee lovers' since it enables the consumer to drink twice as many cups per day before reaching his/her body caffeine threshold level.

10.7 SUMMARY The ontogeny of leaf and fruit formation in arabica coffee plants and the importance of the purine alkaloid tissue fluctuation as a defense mechanism for survival in tropical and sub-tropical environments have been discussed. The most recent advances in cell and organ culture have been reviewed. Enormous progress has been made in the induction and regeneration of somatic embryos from both arabica and robusta species, as well as in the maintenance of embryogenic-competent cell lines. These achievements are key for future progress in protoplast work and gene transfer programs. Greater understanding of the control of the somatic embryogenesis process, coupled with the development of bioreactor and periodic immersion culture techniques, has led to the development of reliable methods for mass coffee propagation. Vegetative propagation of a perennial species like coffee can bring flexibility for introducing new genotypes into production. The ability to shift planting material rapidly will enable farmers to enjoy the

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agronomic advantages of elite plants, but it will also bring the opportunity for the coffee industry to work more closely with the green coffee production sector to address industry and marketing needs. Green coffee quality is controlled by genetics, environment, and farm processing and mass propagation offers the opportunity to control the first of these variables. Vegetative propagation of heterozygous elite plants and the use of multiple clone lines at one time will assure the preservation of heterozygosity and plasticity to environmental changes within coffee plantations. Micropropagation via somatic embryogenesis is the technology that has the scale to satisfy commercial plantations and to be competitive in cost with other propagation methods. Current field evaluation of somatic embryo-derived coffee plants, for both arabica and robusta, provides confidence that clonal fidelity is very high, with minimum or no somatic variation on the genotypes tested so far. First practical applications of natural variability at the cellular level are being released for production. New and improved varieties will be developed through cell biology techniques to address production constraints at the farm level, and at the same time to permit the adoption of more sustainable coffee production. These techniques are based on spontaneous somatic mutations. Plant regeneration techniques are just the tool to uncap such variability at the cellular level, and so the genetic make-up of the new mutant type selected is very similar to the original one. This similarity with the original genetic make-up of donor plants helps to hasten the release of new varieties, since the other characteristics are kept constant. Progress is still needed in the area of anther or microspore culture leading to the recovery of double homozygous plants. This technique has already been mastered with other plant species, and thus it could assist the introgression of genes from wild coffee species to cultivated species, speeding up coffee breeding programs. Another area that deserves more attention is germplasm preservation. Man is altering the natural habitats where wild coffee species have naturally evolved, and in consequence much valuable germplasm is in danger of destruction. Live coffee germplasm collections must continue to be supported, but efforts should be made to improve the techniques of long-term coffee seed and embryo preservation.

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ABREVIATIONS BA or 6BA 2,4-D 2-iP KIN NAA PAL

6-Benzylaminopurine 2,4-Dichlorophenoxyacetic acid 2-Isopentenyladenine Kinetin 1-Naphthaleneacetic acid Phenylalanine ammonia lyase

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Chapter 11

Agronomy III: Molecular Biology John I. Stiles Integrated Coffee Technologies, Inc. Honolulu, Hawaii USA 11.1 INTRODUCTION Despite the importance of coffee as a commodity in world trade and as a major component of foreign exchange for many producing countries, the application of molecular biology and biotechnology to coffee has lagged behind many other crops. The first coffee gene sequences were not entered into GeneBank until 1994 and still only a handful of complete coding sequences are known. Even today, considering its economic importance, only a few laboratories are working on coffee molecular biology and biotechnology. Undoubtedly, this is due in part to the development of these techniques in research centers located predominantly in northern temperate zones, where coffee is not commercially grown, and in part to the perennial nature of coffee which makes it less attractive as a model organism and less attractive to life science companies that rely on the sale of seeds of high volume annuals, such as corn, soybeans and cotton, for revenue. As will be discussed below, many of the techniques that are standard with temperate crops are still difficult to apply to coffee. However, with the continued development and widespread dissemination of molecular techniques and biotechnology, the application of this technology to coffee will continue to increase. Coffee farmers face many challenges in producing an abundant and high quality crop. Biotic stresses, especially from insects and fungal pathogens, are particular problems. There appears to be no naturally occurring resistance gene for certain important insect pathogens such as coffee berry borer (Hypothenemus hampei). Although biological control mechanisms are being developed, coffee berry borer is still considered the most economically important insect pathogen of coffee and effective resistance introduced through biotechnology would have a major effect on the lives of coffee farmers in many areas of the world.

While there are known resistance genes to some fungal diseases of coffee, the long-term nature of the breeding cycle and the need for durable resistance, due to the perennial nature of coffee, make biotechnology an important potential tool for sustainable coffee production. The development of resistant cultivars is particularly difficult due to the long breeding time (25 to 30 years) and the need for not only agronomic characteristics, but also cup quality for acceptance of new varieties. The ability of biotechnology to move natural coffee resistance genes and non-coffee resistance genes into established cultivars and to pyramid resistance genes will save considerable time and provide sustainable and high quality coffee production. Abiotic stresses, such as freezing and drought, also represent significant problems in the coffee industry. For example, the periodic freezes experienced in certain production areas of Brazil not only cause severe economic loss to the farmers involved, but also disrupt commodity markets affecting importers, roasters, and consumers. The introduction of even a minimal increase in frost tolerance through molecular techniques would be of great importance in alleviating the disruption that a freeze can cause the coffee industry. Finally, cup quality is increasingly important as a result of the growth of the specialty coffee industry. Biotechnology will undoubtedly play a progressively more important role in assuring cup quality by reducing defects resulting from contamination by microorganisms and insects. However, it may also play a more direct role by modifying the chemicals present in the green bean. For example, coffee grown without caffeine would negate the need for chemical decaffeination and the resulting decrease in coffee quality. Although the application of molecular biology and biotechnology is still in its infancy, the current progress will be reviewed and prospects for the coffee industry discussed.

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Agronomy III: Molecular Biology

11.2 COFFEE GENES The complete sequences of only a few coffee genes have been reported. And, even fewer genes have been characterized to any extent. Most of the nucleic acid sequences that have been reported are partial sequences, used only for phylogenetic studies. However, this is now changing rapidly. The first coffee gene to be isolated was a-galactosidase from coffee seeds (beans). Coffee bean a-galactosidase can cleave the terminal a1,3-linked galactose residues from the surface of the B-type blood group, converting it to O-group serology and permitting it to be used as a ùniversal donor' for transfusion therapy (Goldstein et al., 1982; Goldstein, 1989). The idea was to clone a-galactosidase from coffee seed and then produce it in high quantities in a microbial or cell culture system to obtain enough enzyme for commercial use. Zhu and Goldstein (1994) purified agalactosidase from dried green coffee beans and obtained a partial amino acid sequence using Nterminal sequencing and sequencing of CNBr fragments. Part of the cDNA was obtained by polymerase chain reaction (PCR) amplification of a segment of the gene using cDNA made from total seed mRNA and primers constructed using the partial amino acid sequence. The 50 and 30 ends of the cDNA were obtained using the RACE technique (Zhu & Goldstein, 1994). Coffee a-galactosidase shows about 80% similarity to guar (Cyamopsis tetragonoloba) a-galactosidase, previously isolated by Overbeeke et al., (1989), even though the guar enzyme principally cleaves a 1,6 glycoside linkages (Guiseppin et al., 1993), whereas the coffee enzyme cleaves mainly a 1,3 and a 1,4 linkages. The coffee a-galactosidase shows more than 50% homology to a number of other a-galactosidases from diverse organisms including human, yeast and Aspergillus niger, although there is little homology to agalactosidases from prokaryotic organisms (Zhu & Goldstein, 1994). The identity of the coffee seed agalactosidase was proven by insertion of the coffee cDNA into a baculovirus expression system and identification of a-galactosidase activity in transformed but not non-transformed insect cells. Perhaps the best-characterized coffee genes are those encoding the 11S seed storage protein. AcunÄa et al., (1999) and Rogers et al., (1999) have recently published detailed investigations on the structure and sequence of coffee 11S proteins and cDNAs. Marraccini et al., (1999) have cloned a complete 11S seed storage protein gene and carried out promoter analysis in transgenic tobacco plants. The 11S seed storage

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protein is the most abundant protein in coffee seeds and is found principally in the storage vacuole of endosperm cells. The coffee seed 11S storage protein is similar to other legumin-like seed storage proteins. In the absence of reducing agents, the 11S protein has an apparent molecular mass of about 55 kDa and can be converted by reducing agents into two polypeptides of about 32 to 33 kDa (designated a-subunit) and 20 to 24 kDa (designated b-subunit) (Rogers et al., 1999; AcunÄa et al., 1999). Figure 11.1 shows an SDS polyacrylamide gel separation of seed proteins under reduced and non-reduced conditions. Microsequencing of seed proteins separated by two-dimensional gel electrophoresis indicates that there is heterogeneity among both the a subunit and the b subunit of the 11S proteins (Rogers et al., 1999). This heterogeneity was found in C. arabica, a tetraploid, and C. canephora, a diploid, that is thought to be one of the parents of C. arabica (Lashermes et al., 1999). Furthermore, comparison of the deduced amino acid sequences from three cDNA sequences revealed a number of amino

Fig. 11.1 SDS polyacrylamide gel electrophoresis of coffee seed (bean) proteins under reducing and nonreducing conditions. P indicates the major species of the mature 11S seed storage protein under nonreducing conditions where the a chain and b chains are attached by a disulfide bridge. a and b indicate the a chain and b chains separated by reduction of the disulfide bridge. Reproduced from Rogers et al. (1999) with permission.

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acid substitutions as well as deletions/insertions, indicating that the 11S seed storage proteins are probably members of a gene family, as is the case with seed storage proteins in other plants, although the existence of a multigene family cannot, at this time, be confirmed by Southern blotting (Rogers et al., 1999; AcunÄa et al., 1999). As with other seed storage proteins, regulation of expression is at the transcriptional level. Rogers et al., (1999) found a large accumulation of 11S mRNA concomitant with the increase of 11S protein in developing coffee seeds. The 11S mRNA is absent in developing seeds until about 15 weeks after flowering. The message accumulates rapidly and remains high between 18 and 27 weeks after flowering, the time that 11S protein accumulation is at its peak (Fig. 11.2). Message levels decrease after week 27 after flowering, as does further accumulation of the 11S protein.

Fig. 11.2 Accumulation of 11S seed storage protein and mRNA during seed development. Reproduced from Rogers et al. (1999) with permission.

Synthesis and processing of the coffee 11S seed storage protein are similar to that of other legumin-like seed storage proteins. Marraccini et al., (1999) cloned a genomic copy of the 11S protein using the inverse polymerase chain reaction. The DNA sequence of this gene is consistent with it being the gene encoding the csp1 cDNA that they had previously isolated (Rogers et al., 1999). The gene contains three introns, two of 111 base pairs, and one of 79 base pairs. The introns are located at exactly the same positions as those in the other known legumin genes. The presumed full-length csp1 cDNA predicts an mRNA with a 32 base 50 untranslated leader region, a 1476 base open reading frame encoding a 492 amino acid protein (55 kDa), and


a 30 untranslated region of 195 bases. Comparison of the predicted protein sequence in the cDNA and the N-terminal sequences determined from the isolated protein predicts a signal sequence of 26 amino acids at the N-terminus. The predicted protein encoded by the cDNA contains a sequence NGLEET. This is identical to a highly conserved cleavage site found in 11S storage proteins from other plants. Cleavage occurs between the N and the G. The functionality of this site in coffee 11S proteins is confirmed by the occurrence of the GLEET sequence at the N-terminus of two different b-chains purified by Rogers et al., (1999), using twodimensional gel electrophoresis, and also by AcunÄa et al., (1999) using SDS polyacrylamide gel electrophoresis and N-terminal sequencing. Figure 11.3 shows the sequence of synthesis and processing of the 11S protein. The 55 kDa preproprotein is the initial product. Cleavage of the Nterminus at the sequence E/QPRL 26 or 27 amino acids from the ATG translational initiation codon, depending on the gene family member, was determined by N-terminal sequencing of the a-peptide (AcunÄa et al., 1999; Rogers et al., 1999). This is consistent with other 11S storage proteins. N-terminal sequencing indicates that the conserved NGLEET sequence directs the cleavage of the pre-protein into an acidic peptide (designated a chain by Rogers et al., 1999, and used in this chapter) and a basic peptide (designated b chain by Rogers et al., 1999, and used in this chapter). In other 11S storage proteins the two chains are held together by a disulfide bridge between two conserved cysteines. AcunÄa et al., (1999) predict, based on the analogy to other legumins, that the disulfide bridge involves C112 and C307. They also predict an internal a chain disulfide bridge between C36 and C69 based on analogy to other legumins. Overall, the coffee 11S seed storage protein appears to be a fairly typical legumintype seed storage protein. Marraccini et al., (1999) cloned a coffee 11S storage genomic gene using the inverse polymerase chain reaction (IPCR). They obtained about 1 kb of the promoter region and about 0.9 kb of the 30 region in addition to the coding region. Sequence analysis of the promoter region indicated several motifs that occur in other seed storage protein genes that are responsible for both temporal and spatial regulation. The sequence TGTAAAG appears 757 bp and 181 bp upstream of the ATG translational initiation site. This sequence is similar to the endosperm motif TGTAAAGT found in barley and wheat glutenins, pea legumin, maize zein and barley hordein promoters (see Marraccini et al., 1999 for references). Marraccini et al., (1999) also


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Fig. 11.3 Structure of the 11S seed storage protein gene and processing steps to give the mature 11S seed storage protein. The position and identity of conserved motifs in the promoter are shown: . endosperm-like motif; ^ GCN4-like motif; $ TGAC-like motif; $ soybean box; ^ E-box motif; * RY repreats. See text for full description. Pre-mRNA is shown with position of three introns indicated. Pre-proprotein is shown with signal sequence cleaved at the PQPRL site. a and b chains are produced by cleavage at the NGLEET consensus cleavage site. The arrows indicate the exact point of cleavage.

reported several other potential motifs including a `GCN4-like motif' similar to that found in the barley C-hordein promoter at positions 7742 and 7181 (with respect to the translational initiation site). The coffee sequences are TGAGTC and TGAGT, respectively, and the GCN4 motif is ATGA(C/ G)TCAT. There is also a TGAC-like motif at position 7326. This motif has been shown to be essential for pea lectin seed-specific expression (de Pater et al., 1993). There are two `soybean boxes' at positions 7248 and 742. This sequence has been found to be essential for proper expression of several soybean genes (Goldberg et al., 1989). Marraccini et al., (1999) reported the existence of sequences similar to a number of other motifs including the E-box of phaseolin, the RY repeat regions of the legumin box, and the AT-rich enhancer motif of soybean b-conglycinin. However, transcriptional regulation consensus sequences by their nature, are not exact and are generally fairly short. Thus, similar sequences can often be found in DNA sequences of reasonable length and confirmation by other means, such as promoter deletion analysis, is required to adequately address their significance. Marraccini et al., (1999) fused the upstream sequences of the coffee 11S promoter to the uidA gene (GUS gene) and transformed tobacco to address this question. GUS expression was measured in seed and leaf tissue of the transformed tobacco to assess the level and specificity of expression. Four different promoter constructs were analyzed, along with a control with no promoter, and a positive control with constitutive expression driven by the 35S promoter. Fusion of the 11S promoter to the GUS coding

sequence was immediately after the region coding for the fifth amino acid of the coffee 11S protein in all cases. The four promoter constructs used contained, with respect to the ATG translational initiation codon, 945 bp, 695 bp, 445 bp, and 245 bp of the promoter region. The level of expression in tobacco seeds was relatively high and not significantly different between the 945 bp and 695 bp promoters. There was a decrease when the promoter was shortened to 445 bp and a further decrease when shortened to 245 bp. None of the constructs showed detectable levels of expression in leaves, and all constructs, including the 245 bp promoter that showed the lowest level of activity, were significantly higher than the 35S promoter. Since the 945 bp and 695 bp promoters were not different in either strength or specificity it can be concluded that no essential sequences lie in the 250 bp between positions 7695 and 7945. There are several potential consensus sequences in this 250 bp region, but additional copies of all of these also occur within the 7695 region. Although it is difficult to show in a statistically rigorous manner, there appears to be a drop of about 50% in promoter strength by deletion of the region between 7445 and 7695. This region is relatively devoid of consensus sequences except for the only occurrence of the AT-rich enhancer motif and an E-box sequence. It is tempting to speculate that the drop of 50% in promoter strength is due to the removal of the putative AT-rich enhancer. However, a more detailed analysis is necessary to confirm the importance of this motif. Promoter strength, but not specificity, is further reduced by removal of the sequences between positions

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7245 and 7454. This region contains two putative RY-motifs, an endosperm motif, and a soybean box that overlaps the 7254 site. From the initial analysis presented by Marraccini et al., (1999) it is not possible to assign specific roles to specific motifs; however, their analysis does point the way for future work to uncover the exact details of the coffee 11S promoter. A common problem with promoter analysis by transformation, and present in the data of Marraccini et al., (1999), is the relatively large deviation from one transformant to another. This is most likely a result of `position effects' resulting from the random integration that occurs with current plant transformation technology. However, until technology for site-specific transformation has been perfected this is a technical limitation that one must live with. The seed specificity of the 11S storage protein promoter may make it a useful tool for coffee biotechnology. By varying the length of the promoter it should be possible to impart the desired level of expression in a tissue-specific manner to coffee seeds (beans). This technology might be used for expression of disease-resistant genes specifically in the seed at the desired amount. It could also be used to express genes that affect quality factors involved in cup quality or soluble solid, important to the soluble coffee market. Neupane et al., (1999) have cloned two genes involved in ethylene biosynthesis in ripening coffee fruit. Fruit-expressed 1-amino-cyclopropane-1carboxylic acid (ACC) synthase and ACC oxidase were isolated from a cDNA library constructed using mRNA from ripening coffee fruits. Degenerate deoxyoligonucleotide primers were synthesized based on the amino acid sequence of highly conserved regions of known ACC syntheses and ACC oxidases. These primers were used in reverse transcriptase polymerase chain reactions (RPCR) to synthesize a portion of the ACC synthase and ACC oxidase genes using cDNA synthesized from ripening fruit mRNA as the template. The RPCR products were then used as probes to screen a cDNA library constructed from mRNA isolated from ripening coffee fruits to obtain full (or near full) length cDNAs of both genes. The largest ACC synthase cDNA isolated was 2040 bp in length. It contained an open reading frame of 488 amino acids. The deduced amino acid sequence of this cDNA is between 51% and 68% identical to other ACC syntheses and also contains all of the highly conserved regions. The largest ACC oxidase cDNA is 1320 bp with a 318 amino acid open reading frame that is between 50% and 83% identical to other ACC oxidases.


Although it has been known for some time that coffee fruit past a certain stage of development will ripen in response to ethylene, it has yet to be demonstrated that coffee fruit exhibits a climacteric. The data of Neupane et al., (1999) indicate that coffee is a climacteric fruit based on the accumulation pattern of both ACC synthase and ACC oxidase mRNA during fruit ripening. Figure 11.4 is a northern blot showing the accumulation of ACC synthase and ACC oxidase mRNA in coffee fruit at various stages of development. Messenger RNA was isolated from immature green, mature green, approximately 25% red, 50% red, 75% red and 100% red coffee fruits, separated by gel electrophoresis, blotted to a membrane and simultaneously probed with ACC synthase and ACC oxidase cDNAs. Both ACC synthase and ACC oxidase mRNAs accumulate during fruit ripening in a manner consistent with that of a climacteric fruit (Neupane et al., 1999). Conclusive proof that coffee is a climacteric fruit will come from observing the effect of inhibition of ethylene biosynthesis. These experiments are in progress.

Fig. 11.4 Northern blot of seed (bean) total RNA isolated at the developmental stages indicated. The blot was simultaneously probed with radioactively labeled cDNA for fruit-expressed ACC synthase and ACC oxidase.

An area of considerable interest in coffee molecular biology and biotechnology is caffeine biosynthesis. Figure 11.5 shows a consensus caffeine biosynthetic pathway that has developed over the years from radiolabeled feeding experiments and a limited amount of biochemical studies (see Crozier et al., 1997). The first step unique to the caffeine biosynthetic pathway is the methylation of xanthosine at the N7 position by xanthosine-N7 -methyl transferase. It has also been reported that XMP can serve as the initial substrate (Schulthess et al., 1996). After cleavage of the ribose to


Fig. 11.5 Caffeine biosynthetic pathway. Initial substrate is xanthosine or perhaps xanthosine monophosphate. After methylation at the N7 position, the ribose is cleaved to form 7-methylxanthine. Two subsequent methylation reactions produce caffeine (trimethylxanthine).

form 7-methylxanthine, two additional methylations occur to form caffeine. The only caffeine biosynthetic pathway gene isolated to date is the gene encoding xanthosine-N7 -methyl transferase from coffee (Moisyadi et al., 1998, 1999). The gene was cloned using the `classical' biotechnology approach of purifying the enzyme, obtaining a partial amino acid sequence and back translating this amino acid sequence to obtain a degenerate oligonucleotide sequence. Degenerate PCR primers were synthesized based on the amino acid sequences obtained and a portion of the gene was synthesized using PCR. This partial gene sequence was then used to obtain the entire coding region by screening a cDNA library constructed from young leaf tissue mRNA. The most pure XMT preparations, when analyzed by two-dimensional gel electrophoresis, contain four peptides that separate into groups of two peptides of about 41 kDa and two peptides of about 40 kDa. One peptide of each size class has a charge very similar to a peptide of the other size class (Fig. 11.6) (Moisyadi et al., 1998, 1999). There are also two higher molecular weight proteins present in the most purified preparations. Partial amino acid sequencing of peptide fragments from these proteins identifies them as known `housekeeping' enzymes (Moisyadi & Stiles, unpublished data). Two cDNA clones have been completely sequenced. The largest of these contains an open reading frame encoding a protein of 41 kDa (Fig. 11.7). There is moderate similarity to an Arabidopsis cDNA that

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Fig. 11.6 Two-dimensional gel showing the most purified fraction of xanthosine-N7-methyltransferase. The most purified xanthosine-N7-methyltransferase preparation has four major peptides that have slightly different isoelectric points and are slightly different in size.

contains an open reading frame encoding a protein of unknown function and to some putative Arabidopsis GDSL-motif lipase/hydrolases. The coffee XMT also contains sequences similar to motifs determined to be involved in adenosine and s-adenosylmethionine binding sites in certain prokaryotic modification enzymes that methylate the N6 position of adenine residues in restriction/modification sites in DNA. One

Fig. 11.7 Amino acid sequence of xanthosine-N7methyltransferase from coffee. The sequence YPPY (light shading) is similar to a conserved motif (D,S,N)PPY found in most N6-adenine methyltransferases and is most likely involved in the active site of the enzyme. The dark shaded sequence is similar to those found in N6-adenine methyltransferases and has been identified as the s-adenosylmethionine binding site.

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such conserved motif, (D,S,N)PPY, appears in most N6 methyltransferases (Timinskas et al., 1995). Mutation of the D or the Y of the DPPY sequence found in the EcoRV adenine-N6 -methyltransferase greatly decreased enzymatic activity without affecting s-adenosylmethionine binding. A related sequence, YPPY, is found starting at position 60 in the coffee XMT sequence. A second sequence starting at position 250 in XMT shows some similarity to the s-adenosylmethionine binding site described by Roth et al., (1998). However, these consensus sites and the match between them and the XMT gene and the differences in the structures of adenine and xanthine make conclusions based on these similarities difficult. In an attempt to prove the identity of the coffee XMT gene, transgenic tobacco plants were generated that expressed the XMT cDNA sequence under control of the 35S promoter. Transgenic tobacco plants expressing the coffee XMT gene were selected by northern blot analysis. Proteins were extracted from young leaves of these plants and assayed directly or partially purified and assayed. Figure 11.8 shows that transgenic tobacco expressing the coffee XMT cDNA has detectable levels of XMT activity and that the specific activity increases with purification. Coffee plants with the XMT cDNA in antisense are currently being characterized and it is anticipated that caffeine-free arabica coffee, at least to the extent of

Fig. 11.8 Expression of the coffee xanthosine-N7methyltransferase in tobacco. The coffee xanthosineN7-methyltransferase cDNA was expressed in tobacco under control of the CaMV 35S promoter. Total proteins were extracted from young leaves of normal tobacco plants or from those expressing the xanthosine-N7-methyltransferase mRNA and assayed directly or purified by hydrophobic interaction chromatography (HIC) or both HIC and Cibacron blue F3GA (BioRad) (Affi-blue).


currently available decaffeination process, will be produced.

11.3 TRANSFORMATION SYSTEMS FOR COFFEE A number of different transformation systems have been reported for arabica and robusta coffee, although there are only a few reports of recovery of whole plants with stable integration of DNA. Transformation has been reported using the biolistic method (gene gun) (Van Boxtel et al., 1995), DNA electroporation using protoplasts (Barton et al., 1991), and various Agrobacterium systems (Spiral & Petiard 1991; Ocampo & Manzanera, 1991; GreÁzes et al., 1993; Spiral et al., 1993; Leroy et al., 1997), however, there are few reports of stable transformation of whole plants. The first report of stable transformation of coffee plants used a protoplast system and electroporation (Barton et al., 1991). Barton and co-workers reported the recovery of plantlets after selection on kanamycin for transformed protoplasts and regenerating tissue. Unfortunately, the root systems of the regenerated transgenic plants were not well developed and plants capable of flowering were not produced. This work was done on a single not well-defined arabica and it is not known if this technology will transfer to all or most other varieties. Protoplast regeneration is known to be quite genotype-dependent. Although many different systems have been investigated, most of the current work has utilized the standard Agrobacterium tumefaciens system, despite the relatively low rate of transgenic plant production obtained to date. The most advanced transformation work is that describing the production of coffee expressing the Bacillus thuringiensis CryIA(c) gene. The CryIA(c) gene product is an insecticidal protein that is toxic to certain insects including the coffee leaf miner, Perileucoptera coffeela (Guerreiro Filho et al., 1998). Transgenic coffee plants containing the CryIA(c) gene were produced using both the Agrobacterium rhizogenes and Agrobacterium tumefaciens systems (Leroy et al., 1997). Although higher rates of transformation were initially found using the A. rhizogenes system, the `hairy root' phenotype could not be suppressed and led to plants with unacceptable agronomic traits including lack of flowering. These problems resulted in further work using the A. tumefaciens system. Spiral and co-workers found that the NPTII kanamycin resistance gene was not effective as a selection system when coffee somatic embryos were


transformed. However, they reported that the gene for resistance to the herbicide chlorsulfuron was effective (Spiral et al., 1999). The initial frequency of transgenic plants produced is considerably below that of many other plant transformation systems. Using the A. tumefaciens system only, about 0.4% of the somatic embryos infected gave transgenic plantlets (Leroy et al., 1997). However, improvements in the selection system have recently been reported. Leroy and coworkers found between 30 and 80% of embryogenic calli transformed with the chlorsulfuron resistance gene and the GUS gene were GUS positive after 10 to 12 months of selection on chlorsulfuron (Leroy et al., 2000). As yet, only limited DNA analysis has been presented; nonetheless, this represents a potentially significant advance in transformation efficiency. However, the results are still quite variable and show a genotype dependence (see below) (Leroy et al., 2000). The work of Leroy and co-workers is also the first, and thus far only, report of stable transformation and regeneration of coffee plants with a useful agronomic trait, insect resistance. As previously mentioned, the cryIA(c) gene was inserted into robusta (C. canephora) and two different arabica (C. arabica) coffee varieties, a Catimor (8661±4) and an F1 hybrid (Et29 6 Ca5). The Catimor and robusta varieties transformed at approximately the same efficiency, but the F1 hybrid produced for fewer transgenic plants. Of the 23 transgenic plants examined for the cryIA(c) protein using an antibody, 18 plants had detectable levels. Preliminary bioassay data indicate that at least some of the transgenic plants that express cryIA(c) also have significant decreases in their overall bioassay score and in the number of P. coffeela pupae detected on the leaves. These plants are intended for field trials starting in 2000.

11.4 PROSPECTS Despite the current controversy in some parts of the world, molecular biology and biotechnology will play an increasingly important role in the improvement of coffee cultivars. These technologies will have benefits for both farmers and consumers. Coffee is a perennial crop grown in a tropical environment. Like other tropical crops, coffee is under severe pressure from fungal and insect pathogens. Biotechnology can have a significant impact on the ability of farmers to produce high quality coffee, perhaps with unique properties, while preserving the ecosystem by reducing chemical use.

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A potential application of biotechnology is resistance to the coffee berry borer (Hypothenemus hampei). There is no known resistance in coffee to H. hampei and, because a significant portion of its life cycle is in the berry, insecticides are often not highly effective. Reported losses can range up to 96% (Nyambo & Masaba, 1997). A biotechnology-based control would be effective, relatively inexpensive compared to chemical treatments, and would reduce chemical contamination of the ecosystem. This situation is analogous to the use of Bt-maize in the United States to control European corn borer. Under significant disease pressure, yields increased by as much as 14%, while pesticide usage decreased (Gianessi, 1999). Broad-spectrum fungal resistance such as that imparted by certain hydrolytic enzymes, either alone or in combination, could play an important role in coffee disease resistance. Pathogenic fungi result in significant losses and include diseases such as coffee berry diseases (Colletotrichum kahawae), coffee leaf rust (Hemileia vastatrix), and a number of other diseases such as those caused by Fusarium and Micena. Although there are naturally occurring resistance genes for many of the fungal diseases, additional mechanisms of resistance would be of great value. Since coffee is a perennial plant with a long breeding cycle, breakdown of an existing resistance gene is especially significant as the development of new resistant lines takes many years. In coffee this is a particular problem in that cultivars must not only carry an effective resistance gene, but also have acceptable cup quality and even have the cup characteristics expected for a coffee from the country of origin. This makes breeding of new lines especially complex and time consuming. A biotechnology approach that could `pyramid' additional resistance genes into existing cultivars that are accepted by the industry would save considerable time and avoid potential acceptance problems. A second problem with fungal contamination of coffee beans is the production of fungal toxins such as ochratoxin. Ochratoxin is now a serious concern, especially in the European community. Vega and Mercadier (1999) have shown that insects such as H. hampei can act as a vector for fungi such as Aspergillus flavus and A. ochraceus that can produce aflatoxins in infected coffee beans. The introduction of resistance genes to H. hampei and/or the introduction of broadbased fungal resistance genes should decrease the instances of contaminated coffee beans. Biotechnology can also decrease susceptibility to environmental stresses. Abnormal weather, especially freezes, can cause severe disruptions in coffee markets,

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affecting both farmers and roasters. Since coffee is grown in areas where freezing weather is infrequent and generally of short duration, coffee may be an ideal crop for engineered freeze protection. There are multiple changes that occur in plants during cold acclimation to stabilize membranes, thought to be the initial site of cold/freezing damage (Tomashow, 1999). These changes include alteration in lipid composition, accumulation of sucrose and other sugars and synthesis of certain proteins that help to stabilize membranes such as the late embryogenesis abundant proteins (LEA proteins) and highly hydrophilic proteins such as the COR156 protein of Arabidopsis. Intervention using biotechnology to produce one or more of these protective factors could give coffee the ability to withstand most low temperature stresses that are likely to be encountered in the present growing range. The use of genetic transformation technology to manipulate quality and flavor traits is still problematic. Most quality traits are not yet understood in detail great enough to identify specific genetic changes that will result in specific flavor or quality characteristics, although it may be possible to remove some defects. One potential defect found in most robusta coffees at varying levels is methylisoborneol (MIB). Although it is not yet known whether MIB is synthesized in the plant or an associated microbe, biotechnology could be used to eliminate MIB production, increasing the quality of robusta coffees. A considerable amount of criticism of genetically engineered plants has resulted from the use of foreign genes used as selectable markers, especially markers that are antibiotic resistance genes. At this time most genetically engineered plants carry a foreign gene that imparts a selection that is used during the transformation process. This is needed since the DNA uptake process is not efficient and many untransformed cells remain mixed with the transformed cells. The gene most commonly used for selectable markers is the neomycin phosphotransferase type II (NptII) gene. This gene gives transformed cells the ability to grow in the presence of kanamycin and related antibiotics, whereas growth of normal plant cells is inhibited. Genes that give resistance to herbicides such as chlorsulfuron have also been used. Although a considerable body of scientific evidence indicates that these genes are not a danger to the environment or to consumers, they have nonetheless met with considerable resistance in certain countries. New technologies based on different selection markers or even the elimination of such genes are under development and promise alternatives to the present situation.


One type of new selection system is based on giving the transformed cells the ability to grow on carbon sources that normal plant cells cannot utilize. One such system uses the phosphate-6-mannose isomerase gene to give plant cells the ability to grow on mannose as the sole carbon source (Joersbo et al., 1998). This system is reportedly up to 5-fold more efficient as a selection system than existing markers such as NptII. Selection systems such as this do not impart a trait that would give the plants a selective advantage in the field (as would a herbicide resistance gene) and do not involve antibiotic resistance genes, so most of the objections to current selection genes are met. New more efficient transformation systems such as the use of pollen may alleviate the need for selectable markers. Smith et al., (1994) have reported a pollen transformation system that uses electroporation to introduce DNA into pollen to give transgenic plants at a rate of up to 44% when this pollen was used to pollinate tobacco flowers. With transformation rates this high, selectable markers would not be needed. Technologies that can impart site-specific genetic changes may also alleviate the need for the introduction of foreign DNA. These technologies could be-used to inactivate an existing gene to turn off production of some product such as caffeine or MIB, or perhaps even alter the gene product to impart some new property such as alternation of an existing disease resistance gene to give resistance to a new pathogen or a broader spectrum of pathogens. Two different approaches show promise for site-specific genetic changes. Two groups have recently demonstrated the use of chimeric RNA/DNA oligonucleotides to selectively and specifically mutate plant genes, at least in model systems. Zhu et al., (1999) were able to use chimeric oligonucleotides to mutate the maize acetohydroxyacid synthase gene to confer resistance to either imidazolinone or sulfonylurea herbicides. They were also able to restore activity to a mutant green fluorescent protein gene. The chimeric oligonucleotides were designed to change a single base in the plant DNA that changed a single amino acid in the acetohydroxyacid synthase protein and resulted in herbicide resistance. In one experiment, 34 out of 40 events characterized had the expected base change. The conversion frequency was generally in the range of about 1 6 10ÿ4 . However, there were also a number of unexpected changes in the target gene. This system appears to work by inducing mismatch repair and it may be that correction of the mismatch required to fix the mutation is not exact. Beetham et al., (1999) reported similar results in mutating the tobacco acetohydroxyacid synthase gene


using essentially the same system. Although the frequency of the events is moderate, as compared to site-specific homologous recombination, it would be difficult to use this system at present to make changes that did not impart some type of selection. An additional limitation is that the mutations produced have been limited to single base changes in single-copy genes. To use this technology to change the characteristics of a gene might require several rounds of mutation if multiple changes in a single gene were required. Also, plant genes often exist as multi-gene families. Mutation of all members of a family would require multiple rounds of mutation. Nonetheless, the demonstration of the potential of chimeric oligonucleotides to mutate plant genes in a site-specific manner is an important advance. A second approach to site-specific genetic changes is enhanced homologous recombination. Homologous recombination has been used in yeast and some animal systems to make specific genetic changes in specific genes. However, except for some model systems, the efficiency of homologous recombination in plants has been too low for practical use. The use of proteins, such as the E. coli RecA protein, involved in the recombination process to enhance the rate of sitespecific recombination, has been reported (Pati et al., 1997). An advantage of this system, if adaptable to plants, is that it should allow more extensive changes than those reported using chimeric oligonucleotides. However, changes to all members of a gene family or duplicated genes in a tetraploid, such as C. arabica, would still require multiple rounds of mutagenesis. The prospects are excellent for applying molecular biology and biotechnology to the improvement of agronomic and quality traits in coffee. The field trials now being initiated to test coffee transformed with the Bt protein to give resistance to leaf miner should demonstrate the potential of biotechnology to alleviate significant agronomic problems. Transgenic caffeinefree coffee now undergoing the final stages of development should demonstrate the potential for quality improvement using biotechnology. Coffee, like other perennial tropical crops, is under significant disease pressure that often requires considerable input of chemicals to obtain satisfactory yields. Many coffee farmers do not have the cash needed for effective chemical control. Furthermore, large-scale use of chemicals leads to environmental damage. Biotechnology can play an important role in improving yields while decreasing environmental damage and in adding traits of value to coffee. This should stabilize and increase the profitability of coffee farming and

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improve the standard of living of many people involved in coffee production.

REFERENCES AcunÄa, R., BassuÈner, R., Beilinson, V. et al. (1999) Coffee seeds contain 11S storage proteins. Physiol. Plant., 105, 122±31. Barton, C.R., Adams, T.L. & Zarowitz, M.A. (1991) Stable transformation of foreign DNA into Coffea arabica plants. In: Proceedings of the 14th ASIC Colloquium (San Francisco), pp. 460±64. ASIC, Paris, France. Beetham, P.R., Kipp, P.B., Sawycky, X.L., Arntzen, C.J. & May, G.D. (1999) A tool for functional plant genomics: chimeric RNA/DNA oligonucleotides cause in vitro gene-specific mutations. Proc. Natl. Acad. Sci. USA, 96, 9774±9778. Crozier, A., Baumann, T.W., Ashihara, H., Suzuki, T. & Waller, G.R. (1997) Pathways involved in the biosynthesis and catabolism of caffeine in Coffea and Camellia. ASIC, Paris, France. In: Proceedings of the 17th ASIC Colloquium (Nairobi), pp. 106± 13. Gianessi, L. (1999) Agricultural Biotechnology: Insect Control Benefits. National Center for Food and Agricultural Policy, Washington, DC. Goldberg, R.B., Baker, S.J. & Perez-Grau, L. (1989) Regulation of gene expression during plant embryogenesis. Cell, 56, 149± 60. Goldstein, J. (1989) Conversion of ABO blood groups. Transfusion Med. Rev., 3, 206±12. Goldstein, J., Siviglia, G., Hurst, R., Lenny, L. & Reich, L. (1982) Group B erythrocytes enzymatically converted to group O survive normally in A, B and O individuals. Science, 215, 168±70. GreÁzes, J., Thomasset, B. & Thomas, D. (1993) Coffea arabica protoplast culture: transformation assays. In: Proceedings of the 15th ASIC Colloquium (Montpellier), pp. 745±7. ASIC, Paris, France. Guerreiro Filho, O., Denolf, P., Peforoen, M, Decazy, B., Eskes, A.B. & Frutos, R. (1998) Susceptibility of coffee leaf miner (Perileucoptera spp) to Bacillus thuringiensis d-endotoxins: a model for transgenic perennial crops resistant to endocarpic insects. Curr. Microbiol., 36, 175±9. Guiseppin, M.L., Almkerk, J.W., Heistek, J.C. & Verrips, C.T. (1993) Comparative study on the production of guar a-galactosidase by Saccharomyces cerevisiae SU50B and Hansenula polymorpha 8/2 in continuous cultures. Appl. Environ. Microbiol., 59, 52±9. Joersbo, M., Donaldson, I., Kreiberg, J., Petersin, S.G., Brunsted, J. & Okkels, F.T. (1998) Analysis of mannose selection used for transformation of sugar beet. Mol. Breed., 4, 111±17. Lashermes, P., Combes, M.-C., Robert, J. et al. (1999) Molecular characterisation and origin of the Coffea arabica L. genome. Mol. Gen. Genet. 261, 259±66. Leroy, T., Henry, A.-M., Royer, M. et al. (2000) Genetically modified coffee plants expressing the Bacillus thuringiensis cry 1Ac gene for resistance to leaf miner. Plant Cell Rep. (in press). Leroy, T., Paillard, M., Royer, M. et al. (1997). Introduction de

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geÁnes d'inteÂreÃt agronomique dans I'espeÁce Coffea canephora Pierre par transformation avec Agrobacterium sp. In: Proceedings of the 17th ASIC Colloquium (Nairobi), pp. 439±46. ASIC, Paris, France. Marraccini, P., Deshayes, A., PeÂtiard, V. & Rogers, W.J. (1999) Molecular cloning of the complete 11S seed storage protein gene of Coffea arabica and promoter analysis in transgenic tobacco plants. Plant Physiol. Biochem., 37, 273±82. Moisyadi, S., Neupane, K.R. & Stiles, J.I. (1998) Cloning and characterization of a cDNA encoding xanthosine-N7 -methyltransferase from coffee (Coffea arabica). Acta Hort., 461, 367± 77. Moisyadi, S., Neupane, K.R. & Stiles, J.I. (1999). Cloning and characterization of xanthosine-N 7 -methyltransferase, the first enzyme of the caffeine biosynthetic pathway. In: Proceedings of the 18th ASIC Colloquium (Helsinki), pp. 327±31. ASIC, Paris, France. Neupane, K.R., Moisyadi, S. & Stiles, J.I. (1999) Cloning and characterization of fruit-expressed ACC synthase and ACC oxidase from coffee. In: Proceedings of the 18th ASIC Colloquium (Helsinki), pp. 322±6. ASIC, Paris, France. Nyambo, B.T. & Masaba, D.M. (1997) Integrated pest management in coffee: needs, limitations and opportunities. In: Proceedings of the 17th ASIC Colloquium (Nairobi), pp. 629±38. ASIC, Paris, France. Ocampo, C.A. & Manzanera, L.M. (1991) Advances in genetic manipulation of the coffee plant. In: Proceedings of the 14th ASIC Colloquium (San Francisco), pp. 378±82. ASIC, Paris, France. Overbeeke, N., Fellinger, A.J., Toonen, M.Y., Van Wassenaar, D. & Verrips, C.T. (1989) Cloning and nucleotide sequence of the a-galactosidase cDNA from Cyamopsis tetragonoloba (guar). Plant Mol. Biol., 13, 541±50. de Pater, S., Pham, K., Chua, N.H., Memelink, J. & Kijne, J. (1993) A 22-bp fragment of the pea lectin promoter containing essential TGAC-like motifs confers seed-specific gene expression. Plant Cell, 5, 877±86. Pati, S., Mirkin, S., Feuerstein, B. & Zarling, D. (1997) Sequence-specific DNA targeting. Encyclopedia of Cancer, Vol. III, pp. 1601±25. Rogers, W.J., BeÂard, Deshayes, A., Meyer, I., PeÂtiard, V. & Marraccini, P. (1999) Biochemical and molecular characterization and expression of the 11S-type storage protein from Coffea arabica endosperm. Plant Physiol. Biochem., 37, 261±72.


Roth, M., Helm-Kruse, S., Friedrich, T. & Jeltsch, A. (1998) Functional roles of conserved amino acid residues in DNA methyltransferases investigated by site-directed mutagenesis of the EcoRV adenine-N6 -methyltransferase. J. Biol. Chem., 273, 17333±42. Schulthess, B.H., Morath, P. & Baumann, T.W. (1996) Caffeine biosynthesis starts with the metabolically channelled formation of 7-methyl-XMP ± a new hypothesis. Phytochemistry, 41, 169± 75. Smith, C.R., Saunders, J.A., Van Wert, S., Cheng, J. & Matthews, B.F. (1994) Expression of GUS and CAT activities using electrotransformed pollen. Plant Sci., 104, 49±58. Spiral, J., Leroy, T., Paillard, M. & PeÂtiard, V. (1999) Transgenic coffee (Coffea species). Biotechnol. Agric. Forestry, 44, 55±76. Spiral, J. & PeÂtiard, V. (1991) Protoplast culture and regeneration in Coffea species. In: Proceedings of the 14th ASIC Colloquium (San Francisco), pp. 383±91. ASIC, Paris, France. Spiral, J., Thierry, C., Paillard, M., PeÂtiard, V. (1993) Obtention de plantules de Coffea canephora Pierre transformeÂse par Agrobacterium rhizogenes. C. R. Acad. Sci. Paris, t316, SeÂrie III, 1±6. Timinskas, A., Butkus, V. & Janulaitis, A. (1995) Sequence motifs characteristic for DNA [cytosine-N4] and DNA [adenine-N6 ] methyltransferases. Classification of all DNA methyltransferases. Gene, 157, 3±11. Tomashow, M.F. (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Ann. Rev. Plant Physiol. Plant Mol. Biol., 50, 571±99. Van Boxtel, J., Berthouly, M., Carasco, C., Dufour, M. & Eskes, A. (1995) Transient expression of b-glucuronidase following biolistic delivery of foreign DNA into coffee tissues. Plant Cell Rep., 14, 748±52. Vega, F.E. & Mercadier, G. (1999) The coffee berry borer and associated fungi. Presented at the 18th ASIC Colloquium (Helsinki). Zhu, A. & Goldstein, J. (1994) Cloning and functional expression of a cDNA encoding coffee bean a-galactosidase. Gene, 140, 227±31. Zhu, T., Peterson, D.J., Tagliani, L., St Clair, G., Baszczynski, C.L. & Bowen, B. (1999) Targeted manipulation of maize genes in vivo using chimeric RNA/DNA oligonucleotides. Proc. Natl. Acad. Sci. USA, 96, 8768±73.

Appendix 1

International Standards Organization (ISO) R.J. Clarke Formerly Chairman, ISO/TC34/SC15 The International Standards Organization, through its sub-committees SC15 of ISO/TC34, has continued to be concerned with the initiation and development of standards for coffee and coffee products. Since its inception in 1963, some 24 different standards have been issued, which can be categorised under the following headings, with dates of the most recent versions.

1.1 GLOSSARY RELATING TO COFFEE AND ITS PRODUCTS ISO 3509±1989, 3rd edn (1st edn 1977) BS 5456±1989

1.3 INSTANT COFFEE (SAMPLING PROCEDURES) Method of sampling from cases with liners ISO 6670±1983 = BS 6379±1984 under revision.

1.4 METHODS OF TEST (CHEMICAL OR PHYSICAL) Moisture content Green coffee Determination of moisture content Reference method: ISO 1446±1978 2nd edn = BS 5752±1979 Part 1 Routine method: ISO 1447±1983 2nd edn = BS 5752±1984 Part 2

1.2 GREEN COFFEE (GUIDES AND SAMPLING PROCEDURES) Guide to storage and transport ISO 8455±1986 = BS 6827±1987

Determination of loss in mass at 1058C Routine method: ISO 6673±1983 = BS 5752±1984 Part 7

Guide to specifying ISO 9166±1992 = BS 7601±1992

Guide to defects ISO 10470±1993 = BS 7683±1993

Roasted and ground coffee Determination of moisture content by Karl Fischer method

Method of sampling

Reference method: ISO 11187±1994 = BS 5752±1995 Part 13

ISO 4072±1982 = BS 6379±1983 Part 1

Specification of coffee tryer ISO 6666±1983 = BS 6379±1984 Part 3

Determination of moisture content (loss in mass at 1038C) Routine method: ISO 11294±1994 = BS 5752±1995 Part 14

Method of preparation for use in sensory analysis ISO 6608±1991 = BS 6379±1991 Part 4 235

236


Instant coffee

Green and roasted coffee

Determination of loss in mass at 708C (under reduced pressure)

Determination of a free flow bulk density of whole beans, by a free-flow method

ISO 3726±1983 = BS 5752±1984 Part 4

ISO 6669±1995 = BS 5752±1996 Part 16

Caffeine content

Instant coffee

Green coffee (also roasted and instant)

Particle size analysis

Determination of caffeine content (Levene method) Reference method: ISO 4052±1983 = BS 5752±1984. Part 3

Determination of caffeine content (HPLC method) Routine method: ISO 10095±1992 = BS 5752±1992 Part 12.

Other chemical content Instant coffee Determination of free and total carbohydrate contents by high performance anion exchange chromatography (HPAE) ISO 11292±1997 (corrected edition) = BS 5752±1995 Part 15

Visual and physical characteristics Green coffee Visual, olfactory examination and determination of foreign matter and defects ISO 4149±1980 = BS 5752±1980 Part 4

Size analysis (manual sieving) ISO 4150±1991 (2nd edn) = BS 5752±1991 Part 5

Determination of proportion of insect damaged beans ISO 6667±1985 = BS 5752±1986 Part 8

ISO 7532±1985 = BS 5752±1986 Part 10

Determination of free flow and compacted bulk densities ISO 8460±1987 = BS 5752±1987 Part 11

1.5 GENERAL COMMENTS All these standards (analytical methods, guides, or glossaries) are available in both English and French versions from the ISO in Geneva; while most will also have been published, practically verbatim, as National Standards in the national language by the respective National Standards bodies. Of particular interest are those most recently issued, which seek to use the most modern analytical methodology, for example Karl Fischer's methods for the measurement of true moisture content, ISO 11817 (1994), and also planned for green coffee. Similarly, high performance liquid chromatography is recommended now for caffeine determination (ISO 10095± 1992); and the even more sophisticated HPAE chromatography for carbohydrate determination (ISO 11292±1997). While many coffee standards for green coffee are for field use, close to harvesting and processing, it is also felt that so-called developing countries do now also have access to modern analytical laboratory techniques. A particular feature of the guides available is the comprehensive standard for defects in green coffee, issued 1993, ISO 10470, which should attract continued attention It is designed to develop full agreement and as much accuracy as possible in definitions, causes and effects of all the different kinds of defective beans that can be encountered in wet processed and dry processed arabica coffee, and dry processed robusta. It was decided that only qualitative statements can be made about the influence of particular defective beans, on brew flavour (after roasting), on account of the differing national perceptions, as reflected in marketing specifications.

Appendix 1

Increased harmonisation in this area is desirable, thus, for example, use of weighing methods rather than counting. So far SC15 has decided that it should not be involved in setting actual specifications for any aspect of green, roasted or instant coffee, but rather provide glossary guides and methods of test (by ring testing, where possible) for all chemical and physical characteristics of concern to the coffee trade. A description of the activities of ISO/TC34/SC15 was given in the ISO Bulletin, 1995, pages 13±15, by the former Chairman, R.J. Clarke, and of the ISO generally, in food standardisation, pages 8±12. A meeting of SC15, under the new Chairman, Dr R. Viani, was held in Paris, December 1999, to develop future strategy and activity. A question is often asked about the respective roles of the ISO and of Codex

237

Alimentarius (of the FAO/WHO) in food standardisation in general, and coffee in particular. Their activities can be said to be in parallel, and the two organizations in liaision. The scope of the latter, however, is primarily regulatory (for example in health/nutritional issues), as befits its governmental basis; whereas the former is non-governmental, dealing with the standardization of those issues of concern to international trade (for example, test methods, terminology, sampling, etc.), which it has done since 1947. Codex Alimentarius decided, some time ago, not to involve itself in coffee or instant coffee standardization; although the regulatory aspects in Europe are covered by European Directives in force from the relevant Council of Ministers.

Appendix 2

International Coffee Organization (ICO) C.P.R. Dubois Head of Operations, ICO 2.1 THE INTERNATIONAL COFFEE AGREEMENT 1994 2.1.1 Background The International Coffee Agreement 1994 was the fifth long-term agreement since 1962. The process of negotiation was long-drawn out since the initial aim had been to secure an Agreement with mechanisms for price stabilization, as in the past. This proved politically impossible and the Agreement which was eventually negotiated and entered into force on 1 October 1994 had no regulatory economic clauses, concentrating instead on maintaining the International Coffee Organization (ICO) and promoting international cooperation on coffee by other means. Mr Celsius A. Lodder, a Brazilian national, was appointed Executive Director. Mr. Lodder had worked in a variety of senior positions in the Brazilian civil service, including in coffee, and was also a professor of economics.

2.1.2 Priorities In line with the general objectives of the 1994 Agreement, the International Coffee Council approved a programme of action in May 1995 which identified four priority areas: expanding membership; reviewing statistical services and developing new analytical documents and information services on the coffee market; developing the Organization's capacity to sponsor projects for financing by the Common Fund for Commodities (CFC); and undertaking studies and surveys.

2.1.3 Coffee development projects As the designated International Commodity Body for coffee at the Common Fund for Commodities (CFC), the Organization is able to assist producing countries with projects to improve production and combat pests. Six major projects valued at over US $31 million were approved between 1995 and 1999. Funding was prin-

cipally secured from the Common Fund for Commodities, but significant co-funding from other bodies, such as the European Union and bilateral donor agencies, has been achieved. Funds were mainly invested in coffee-producing countries and were provided in the form of grants rather than loans. Areas covered included improvement of quality to secure added value, combat of pests and diseases, and improvement of marketing structures. The innovative aspect of this programme has been to get away from the usual one-country project approach to address issues of concern to coffee in a number of regions and environments, and to establish new techniques and methodologies relevant to a number of other countries.

2.1.4 Promotional activity With limited funds and modest budgets, using resources remaining from the Promotion Fund established under the 1987 and 1983 Agreements, the ICO has sought to enhance the image of coffee drinking and to increase the consumption of coffee in two of the world's largest markets, China and Russia. ICO generic promotion was only one of several factors that influenced consumption, but was widely perceived in China and Russia to have been beneficial. Activities included high profile Vanessa Mae concert promotions; production and dissemination of educational materials, including a new `Coffee story' booklet to create greater awareness of coffee; developing annual coffee festivals; and a programme of media briefings, supported by tastings and demonstrations, in order to educate journalists about the benefits of coffee. The development of strong and positive relationships with leading coffee companies, and the increasing extent of private sector participation in ICO promotions, were among the most important achievements. The educational elements of the promotion such as the `Coffee story' booklets, the programme of coffee tasting and the media briefings should continue to bear fruit in terms of creating a favourable image of `coffee culture'

238

Appendix 2

with a positive impact on consumption over the long term.

2.1.5 Involvement of the private sector

239

indirectly to several thousand man-years of research project work. This should greatly enhance the effectiveness of future coffee research expenditure, increase income from coffee exports, and improve productivity.

The creation in 1997 of the Coffee Industry and Trade Associations Forum (CITAF) established a consultative mechanism which allowed private sector concerns (such as statistics, legislation and the environment) to be addressed, albeit informally, at regular meetings during the Board and Council sessions. The work of the private sector was subsequently strengthened through two important new initiatives: a new Private Sector Consultative Board (PSCB) to advise the Council and Executive Board on ICA matters, established in July 1999 and composed of eight representatives of the private sector from exporting countries and eight from importing countries, and a regular World Coffee Conference to bring government and private sector representatives together to discuss matters of common concern to the world coffee industry. The first world coffee conference held by the ICO will take place in May 2001, and will be chaired by Mr Jorge CaÂrdenas GutieÂrrez, General Manager of the FederacioÂn Nacional de Cafeteros de Colombia.

2.1.8 Economic studies and publications

2.1.6 Statistics and information

In July 1999 the International Coffee Council, at its 78th (Special) Session, adopted Resolution number 384 providing for the extension for 2 years from 1 October 1999 of the International Coffee Agreement 1994. The Resolution also indicated that the Council would take measures as soon as possible to strengthen the involvement of the private coffee sector in the work of the Organization, to promote consumption of coffee, and to improve the Organization's system of statistics. In addition, a Negotiating Group would be established to draft the text of a new International Coffee Agreement by 30 September 2000, thus giving a full year for such a text to be ratified by Member countries. A new Negotiating Group, chaired by Mr Arnoldo LoÂpez Echandi, President, Instituto Costarricense del CafeÂ, Costa Rica, began discussions at the end of 1999 with a remit of drafting the text of a new International Coffee Agreement by 30 September 2000, which could enter into force on 1 October 2001.

A new Statistics Committee was established by the Council in 1999, open to all Members of the Organization, to representatives of the private sector and to experts in the area of coffee statistics. Its remit is to ensure that the Organization's statistical services continue to be strengthened and adapted to changing conditions. The 1994 Agreement also saw the launch of iCoffee (www.iCoffee.com), a joint ICO/Dow Jones initiative, the world's first major subscription Internet service totally dedicated to the global coffee industry, and the establishment of the Organization's Web site (www.ico.org), visited on average by 650 users throughout the world each day.

2.1.7 Global research network on coffee A global research network was implemented in 2000, following preliminary research and a feasibility study. ICO Members currently invest around 1500 man-years every year in coffee-related research projects. However, dissemination of research results is often slow and incomplete. The global research network should, within a few years, provide a database linking Members

Since 1994, studies have been undertaken on key economic and environmental aspects of the coffee market by members of the Organization's technical staff and published as documents. They include regular reviews of the market situation; four studies on price determination and volatility; a study on organic coffee; a study on the impact of Èl NinÄo' on coffee production; and a study on sustainability and its relevance for the coffee sector. Other studies are currently in progress or planned on the economics of speciality coffees, forecasting models, coffee and biodiversity, and coffee, trade and the environment. In addition, 13 country coffee profiles had been published by the end of 1999, with nine more planned for 2000. They contain an upto-date and comprehensive account of the coffee sector in the countries concerned.

2.1.9 Towards a new Agreement in 2001

2.2 CONCLUSIONS At the end of the coffee year 1998/99 the Organization had 63 Member countries, 45 exporting and 18 importing Members. However, there is little doubt that the increasing involvement of the private sector in the

240


work of the Organization reflects the shift in emphasis of government involvement in world coffee trade from marketing to establishing regulatory frameworks related to protection of consumers and the environment, and to facilitate the development of market-oriented trading environments. The new PSCB had, by January 2000, already identified three priority areas: the need to disseminate positive news about coffee, the need to promote sustainable development and the need to

ensure that regulations to protect public safety are appropriate to the specific conditions of coffee. The ICO, as an international forum for all stakeholders in the world coffee economy, is uniquely placed to address these challenges. We have attempted in this narrative to tell the latest history of the Organization in words (also see References). For those interested in statistics, we have added two tables (Tables A.I and A.2, Section 2.3).

2.3 STATISTICAL INFORMATION Table A.1

Volume, value and unit value of exports to all destinations, calendar years 1964 to 1998.

Year

Exports to all destinations Volume (million bags)

1964 1965 1966 1967 1968 1969 1970 1971 1972 1973 1974 1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998

46.2 43.2 49.4 50.7 54.1 54.9 52.8 53.8 58.1 62.8 55.0 58.6 60.0 48.2 57.3 64.3 60.2 60.5 64.5 66.3 68.6 71.4 64.5 72.0 65.8 75.9 80.6 75.8 78.2 75.0 70.5 67.6 77.5 79.8 79.1

Value in Unit value current terms in current (million terms (US US $) cents/lb) 2306 2114 2309 2191 2368 2404 3018 2688 3221 4294 4200 4254 8395 12524 11235 12411 11778 8087 9014 9243 10680 10831 14309 9589 9437 8683 6866 6501 5326 5689 10125 11611 9993 12871 11321

37.7 37.0 35.3 32.7 33.1 33.1 43.2 37.8 41.9 51.7 57.7 54.9 105.7 196.6 148.2 145.9 147.8 101.0 105.6 105.5 117.7 114.8 167.7 100.8 108.4 86.5 64.4 64.9 51.5 57.4 108.6 129.9 97.5 122.0 108.3

Values in constant 1990 terms1

Index 1964 = 100

Value of exports (million US $)

Unit value (US cents/ lb)

Volume

Value in constant terms

10480 9609 10037 9528 10295 10017 12074 9954 11106 12630 10244 9249 17861 24557 19371 18805 15916 11720 13256 14219 16953 17192 18827 11281 10258 9542 6866 6501 5171 5865 10227 10555 9428 13134 11917

171.4 168.0 153.5 142.2 143.8 138.0 172.9 140.0 144.5 152.0 140.8 119.3 225.0 385.4 255.5 221.0 199.7 146.4 155.3 162.2 186.8 182.1 220.6 118.5 117.8 95.0 64.4 64.9 50.0 59.1 109.7 118.1 92.0 124.5 113.9

100 94 107 110 117 119 114 116 126 136 119 127 130 104 124 139 130 131 140 143 148 154 140 156 142 164 174 164 169 162 152 146 168 173 171

100 92 96 91 98 96 115 95 106 121 98 88 170 234 185 179 152 112 126 136 162 164 180 108 98 91 66 62 49 56 98 101 90 125 114

Unit value UN in constant index terms (1990 = 100) 100 98 90 83 84 81 101 82 84 89 82 70 131 225 149 129 117 85 91 95 109 106 129 69 69 55 38 38 29 35 64 69 54 73 66

22 22 23 23 23 24 25 27 29 34 41 46 47 51 58 66 74 69 68 65 63 63 76 85 92 91 100 100 103 97 99 110 106 98 95

Source: ICO database. 1 Value in current terms deflated by the UN index of unit values of exports of manufactured goods from developed market economies.

Appendix 2

Table A.2

241

Prices of coffee, 1965 to 1999 (US cents/lb).

Year

Other mild arabicas

Robustas

Composite indicator price

Colombian mild (New York)

Brazilian naturals (New York)

1965 1966 1967 1968 1969 1970 1971 1972 1973 1974 1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999

45.08 42.12 39.20 39.33 39.78 52.01 44.99 50.33 62.30 65.84 65.41 142.75 234.67 162.82 173.53 154.20 128.23 140.05 132.05 144.64 146.05 194.69 113.62 137.60 108.25 89.46 84.98 64.04 70.76 150.04 151.15 122.21 189.06 135.23 103.90

31.07 33.53 33.52 33.86 33.11 41.44 42.27 45.19 49.88 58.68 61.05 127.62 223.76 147.48 165.47 147.15 102.61 109.94 123.90 137.75 120.14 147.16 101.99 94.31 75.09 53.60 48.62 42.66 52.50 118.87 125.68 81.92 78.75 82.67 67.53

40.37 39.61 37.22 37.36 38.71 50.52 44.66 50.41 62.16 67.95 71.73 141.96 229.21 155.15 169.50 150.67 115.42 125.00 127.98 141.19 133.10 170.93 107.81 115.96 91.67 71.53 66.80 53.35 61.63 134.45 138.42 102.07 133.91 108.95 85.72

48.00 47.35 41.61 42.42 44.44 56.66 49.01 56.70 72.52 77.81 81.31 157.72 240.21 185.20 183.41 178.82 145.33 148.60 141.61 147.33 155.87 220.04 123.45

43.58 40.56 37.72 37.36 40.90 55.80 44.71 52.52 69.20 73.34 82.57 149.48 308.04 165.29 178.47 208.79 179.55 143.68 142.75 149.65 151.76 231.19 106.37 121.84 98.76 82.97 72.91 56.49 66.58 143.24 145.95 119.77 166.80 121.81 88.84

REFERENCE International Standards Organization (1988) In Coffee Volume 6: Commercial and Technical-Legal Aspects (eds R.J. Clarke and R. Macrae), pp. 29±54. Elsevier Applied Science, London.

107.14 96.53 89.76 67.97 75.79 157.27 158.33 131.23 198.92 142.83 116.45

Appendix 3

Units and Numerals 3.1 UNITS 3.1.1 SI base units Quantity Length Mass Time Thermodynamic temperature Amount of substance Electric current Luminous intensity

Unit name metre kilogram second kelvin mole ampere candela

Unit symbol m kg s

Dimensions [L] [M] [T]

K mol A cd

[y] [N] [I] [Iv]

3.1.2 Some SI derived units used in engineering (a) Units with special names Quantity Frequency Energy, work, quantity of heat Force Pressure Power

Name

Unit symbol

hertz

Hz

Symbol expressed in base units s±1

joule newton pascal (newtons per square metre) watt (joules per second)

J N Pa

kg m2 s±2 kg m s±2 kg m±1 s±2

W

kg m2 s±3

(b) Examples without special names Physical quantity Density Heat capacity Heat transfer coefficient Thermal conductivity Velocity Viscosity (dynamic) (kinematic)

SI unit kilograms per cubic metre joules per kilogram per kelvin watts per square metre kelvin watts per metre kelvin metres per second

Unit symbol kg m±3 J kg±1 K±1 W m±2 K±1 W m±1 K±1 m s±1

pascal second square metres per second

Pa s m2 s±1

NB These unit symbols may alternatively be expressed using a solidus, e.g. W m±1 K±1 = W/m K.

242

Appendix 3

243

3.1.3 Some prefixes for SI units Multiplication factor 1012 109 106 103 102 10 10±1 10±2 10±3 10±6 10±9

Prefix tera giga mega kilo hecto deka deci centi milli micro nano

Symbol T G M k h da d c m m n

NB The use of prefixes hecto-, deka-, deci-, and centi- is not recommended except for SI unit multiples for area and volume. The litre is an acceptable derived SI unit (equalling 1 6 10±3 cubic metres, or 1 cubic decimetre, or 1 dm3), to which the above prefixes are commonly applied; thus, the millilitre (1/1000 or 10±3 litre), the centilitre (1/1000 or 10±2 litre) and the decilitre (1/10 or 10±1 litre). The millilitre is equivalent in older use to the cubic centimetre (cm3 or cc). In weight and mass measurement, these prefixes are used in relation to the gramme, e.g. the microgramme (mg) is 10±6 g. The common use of the millimetre, etc. in linear measurement is entirely consistent with basic SI units. The micron (often cited as m, but more correctly as mm) is 1 6 10±6 m. The aÊngstroÈm is equal to 10±10 m; it is preferable to replace this unit with the nanometre (i.e. 1 AÊ = 0.1 nm).

3.1.4 Some conversions of SI and non-SI units Type of unit Linear Area Volume

Mass Density

Flow rate

from inch (in) foot (ft) miles square foot (ft2) acre cubic foot (ft3) litre gallon (US) gallon (British) pound (lb) gallons (British) ounce (oz) pound per cubic foot

cubic foot per minute (cfm) gallon (British or Imperial) per hour (igph)

To convert

to metre (m) metre (m) kilometres square metre (m2) hectare cubic metre (m3) cubic metre gallon (Imperial or British) cubic metre kilogram (kg) per mile litres per kilometre kilogram kilograms per cubic metre (or grams per litre) grams per cubic centimetre (or per millilitre) cubic metre per second cubic metre per second

Multiply by 0.025 4 0.304 80 1.6211 0.092 90 0.404 69 0.028 32 10±3 0.833 4.546 6 10±3 0.453 6 2.80 28.35 6 10±3 16.02 0.01602 4.72 6 10±4 1.263 6 10±6

244

Type of unit Heat and power


from Btu (BThU) calories (cal) thermochemical international table watt (W) horsepower (hp) (550 ft lbf per second Btu per pound

Heat transfer coefficient

Btu per square foot per hour per 8F calorie per square metre per second per 8C

Pressure

Pound-force per square inch (psig, gauge; psig, absolute pressure) psig inHg (328F) mmHg (08C) mmHg absolute bar

Thermal conductivity

Viscosity (dynamic)

atmosphere (= 760 mmHg or &30 inHg &14.7 psi abs. = 0.00 psig Btu per hour square foot per 8F/in Btu per hour per square foot per 8F/ft calorie per second per square centimetre per 8C/cm (= calorie per second per centimetre per 8C)

}

gram per centimetre per second (or dyne second per square centimetre) poise centipoise gram per centimetre per second centipoise

To convert calories joule (J)

to

joule per second watt (W) per kilogram {calorie kilocalorie per kilogram

calorie per square metre per second per 8C watt per square metre per kelvin kilogram per square centimetre pascal (Pa) kilopascal (kPa) megapascal (MPa) bar pascal pascal torr pascal megapascal pascal megapascal

{

calorie per second per square centimetre per 8C/cm watt per square centimetre per kelvin/cm (= watt per centimetre per kelvin) watt per metre per kelvin poise (P)

Multiply by 252 4.184 4.187 equivalent 7.457 6 102 555 0.555 1.356 4.187 0.070 31 6.895 6.895 0.006 895 0.06895 3.386 6 103 1.333 6 102 equivalent 1.0 6 105 0.1 1.013 6 105 0.101 3

}

3.447 6 10±4 4.13 6 10±3 4.187

4.187 6 102 equivalent

centipoise (cP) pound (mass) per foot per hour pascal second

102 2.42

millipascal second (m Pa s)

equivalent

0.1

*

Appendix 3

Type of unit Viscosity (kinematic) Diffusivity (diffusion coefficient) Force Mass transfer flux

245

To convert

from square centimetre per second stokes square centimetre per second

stokes

dyne pound-force gram per square centimetre per second

Multiply by

to

equivalent

square metre per second square metre per second

1.0 6 10±4 1 6 10±4

newton (N) newton (N) gram per square metre per second

1.0 6 10±5 4.448 1 6 104

References Kirk-Othmer, Concise Encyclopaedia of Chemical Technology, John Wiley, New York, 1985.

3.2 NUMERALS – CARDINAL Greeka

Latinb

eis, mia, en duo treis, tria tessares, tessara pente hex hepta

unus, una, unum; I duo, duae, duo; II tres, tria; III quattuor; IV quinque; V sex; VI septem; VII

okte ennea deka eicosi pentekonta hekaton

octo; VIII novem; IX decem; X viginti, xx quinquaginta; L centum; C

pentakosioi kilioi

quingenti; D mille; M

Direct English, and Prefixc,d one [uni-, L.] two [duo-, L. and Gk] three [tri, L. and Gk] four [quadri-, L.] five [quinqu(e)(i)-, L.; pent(a), Gk] six [hex(a)-, Gk; sex(i)-, L.] seven [hepta-, Gk; sept(em)(i)-, L.] eight [oct(a)(o)-, L. and Gk] nine [nan-, Gk] ten [dec(a)-, Gk; deci- (tenth), L.] twenty [eicosa-Ck] fifty [Ð] hundred [hecto-, Gk; centi(hundredth), L.] five hundred thousand [kilo-, Gk; milli(thousandth), L.]

a Greek nouns in nominative singular form, together with the indefinite form, where this is different, Latinised characters used, with e (long = Z; e (short) = e; o (short) = o; o (long) = o; kh = w; ph = f. Greek u = u (upsilon) is the Latin y. b Latin nouns given in nominative singular form with their m., f. or n. genders; where applicable. c Greek prefixes are only usable in combination with Greek-based words, and Latin prefixes with Latin words. d In general, Greek prefixes are used as multiples (e.g. kilo-) and Latin prefixes for fractions (e.g. milli-).

Reproduced with permission from Clarke & Macrae (1987, 1988) by Kluwer Academic Publishers.

Index Note: page references in italic are to figures and tables. abortion, spontaneous 176±7 acetic acid brews 154 caffeine recovery processes 120 formation mechanisms 23 green coffee 19 roast coffee 19±20, 21, 22 storage effects 26, 56 acidity 27±30, 55±6, 154, 158 acids bean swelling 94±5 brews 55±6, 154 caffeine recovery processes 120 effects of roasting 22±5, 29±30, 54±6, 59, 158 formation mechanisms 13, 23±6 green coffee 18±19, 23, 25, 26, 54±5, 59 roast coffee 19±23 sensory characteristics 27±30, 55, 147, 154, 158 solvent decaffeination 109 storage effects 26, 55±6 suspension-cultured cells 208±9 volatile 26±7, 28 activated carbon caffeine recovery 119±21 chlorinated water 156 activity coefficients, volatile compounds 135 adsorption processes, decaffeination 111, 112±13 adulterants see contaminants aflatoxins 168, 231 after-taste, brews 158±9 agronomy see breeding practices; cell biology; molecular biology air±water partition coefficients 133±4, 135 alcoholic cirrhosis 178 aldehydes aroma analysis methodology 70, 71 brews 81, 82 green coffee 73, 74 odorant formation 82, 83±4, 85 physical properties 133±4, 135, 136, 137 roast coffee 71, 76, 77, 80 storage effects 79 alkaloid formation 203, 205, 207±9 alkanes 44

alkylpyrazines aroma analysis methodology 70, 71, 72 brews 80, 81, 82 formation 82, 84 green coffee 73±4, 75 optimum roast detection 93 physical properties 134±5, 136, 137 roast coffee 75, 76, 77, 78, 79, 80 storage effects 79 amino acids 13±14, 50±1 11S seed storage proteins 225±6 carbohydrate breakdown products 13±14, 52 Maillard products 13, 14 odorant formation 82, 83, 84, 85 protein-bound 50, 51, 52±3 ripening coffee fruits 228 Strecker degradation 82, 83, 84 1-amino-cyclopropane-1-carboxylic acid (ACC) oxidase 228 1-amino-cyclopropane-1-carboxylic acid (ACC) synthase 228 aminohexose reductones 53±4 antibiotic resistance genes 232 antimutagenic effects, coffee 167±8 antioxidative compounds 57±8, 168 arabinogalactan coffee fibre 15 extract viscosity 14 green coffee 3±5 odorant formation 85 roast coffee 7, 9, 14 soluble coffee 13, 14 spent coffee grounds 132 arabinose green coffee 2, 5 roast coffee 7, 8, 9, 14 soluble coffee 8, 9, 12 aroma, brews 77±8, 80±2, 126, 159 aroma-active compounds see volatile compounds aroma analysis methodology 69±73 aroma extract dilution analysis (AEDA) 69, 70, 72, 73 aroma formation proteins 51, 53 roasting processes 79, 92 see also volatile compounds

246

Index

aroma retention, instant coffee 82, 126, 129, 131 aromatization, instant coffee 132 arrhythmias 170 artificial-flavour-reinforced coffees 141, 161±2 astringency, brews 160 atractylglycosides 37 green coffee 2±3, 4 roast coffee 6±7 bean defects, standards 236±7 bean development 205±7 bean mass 94, 96±100, 101 bean moisture international standards 235±6 and roasting 94±5, 96±7, 101 bean porosity 95, 99, 101 bean swelling mechanisms 92, 94±5 bean temperatures, roasting 92±5, 96, 97, 98±9, 101 bean volume 95, 101 beverage characterization acids 154 carbohydrates 154±5 chemical 153±7 density 152 dispersed phases 152 foam 151±2 lipids 154 minerals 155±6 nitrogenous compounds 155 organoleptic 157±60 physical 151±3 refractive index 153 surface tension 153 total solids 153±4 viscosity 152±3 water 151, 156±7 beverage preparation 140±3 decoction 143±4 and health 143, 171±2 infusion 144±5 pressure 145±51 bioreactor cultures 214±16 biotechnology 185, 224 11S storage protein promoter 228 caffeine biosynthesis 228±30, 233 coffee transformation systems 230±1, 232±3 disease resistance 231 environmental stresses 231±2 flavour traits 232 fruit development 228 fungal toxins 231 pest resistance 231

247

prospects 231±3 quality traits 232 selectable markers 232 site-specific changes 232±3 birth weight, and maternal caffeine 175±6 bispyrrolidinohexose reductones 53±4 bitterness brews 158±9 protein 53±4 black rot 195 bladder cancer 166±7 blood pressure 170±1 body, brews 160 boiled coffee 143, 154, 155, 171±2 bone health 173±4 bottled coffee drinks 161 Bourbon LC variety 215±16, 219 breast cancer 166 breeding practices 184 before 1985 184±5 biotechnology 185 clonal propagation 196±7 for disease resistance 192±5 for drought tolerance 196 genetic resources 186±9 germplasm conservation 188±9 for insect resistance 195±6 methods 189, 190 for nematode resistance 195 new cultivar propagation 196±7 new developments 185±6 objectives 189 for productivity 189, 191 for quality 191±2 seed propagation 196, 212±13 species relationships 186±8 world germplasm collections 186 world production levels 184 see also cell biology; molecular biology brewing yield 142 brews acidity 55±6, 154 aromas 77±8, 80±2, 126, 159 in-home decaffeination 119 international standards 236±7 modified 141, 160±2 preparation methods 140±51, 171±2 see also beverage characterization bubbling beds 92 2,3-butanedione 70, 71, 76, 79 brews 80, 81 odorant formation 82±3, 84

248

cafestal 40, 41 cafestol 37, 38, 39±40, 41 and health 168, 172, 178, 179 cafetieÂre (plunger) coffee 145±6, 154, 155, 172 caffeic acid antioxidation 57±8, 168 decaffeination 109, 110, 112 zinc-chelating compounds 64±5 caffeine 108 antioxidative effects 57, 168 applications 123 biosynthesis 203±4, 206±9, 228±30 and bitterness 53 and blood pressure 170±1 and bone health 173±4 brews 154, 155 callus cultures 207±8 demand for 122±3 international standards 236 and leaf development 203±4, 205 molecular biology 228±30 and neuroactivity 177±8 and pregnancy 174±7 recaffeinated coffee 119 recovery processes 119±21 removal see decaffeination processes and roasting speed 92 safe consumption levels 179 caffeine-free coffee 230, 232, 233 caffeoyltryptophan 51, 57 calcium, bone health 173±4 callus cultures 207±8 caloric content, brews 155, 160 cancers 15, 166±9, 178 canned coffee drinks 141, 161 cappucino 150, 160±1 caramelization 13 heat caramelization equipment 106 caramel odours brews 81 roast coffee 75, 76, 77, 78, 79, 80 carbohydrates 1 brews 154±5 colour development 51±2 extract viscosity 14±15 functional properties 14±15 green coffee 1±6 high molecular weight green coffee 1, 3±6 roast coffee 7±8 soluble coffee 12±13 see also arabinogalactan; mannan

Index

low molecular weight green coffee 1±3 roast coffee 4, 6±7, 8, 13 soluble coffee 8±12 odorant formation 82±5 reactions on roasting 6±7, 9, 13±14, 23, 51±2 roast coffee 4, 6±8, 13±14 sedimentation 15 soluble coffee 8±13, 14±15, 127 spent coffee grounds 132 carbon, activated caffeine recovery 119±21 chlorinated water 156 carbon dioxide decaffeination 108±9, 113±18, 119, 122 carbon fibres, decaffeination 111 carbonic acid 5-hydroxytryptamides (C-5-HT) 45±6 carbonyl compounds, aromas 79, 82±3 carboxyatractylglycosides 2±3, 4 carcinogenicity, coffee 41, 168 cardiac arrhythmias 170 cardiovascular disease 169±72 cavities, roast coffee 95, 100 cell biology 202 alkaloid formation 203, 205, 207±9 embryo cryopreservation 211 gene regeneration 211, 212, 219 gene transfer 211±12, 219 in vitro selection studies 212 micropropagation 212±17, 219 organ development 202±7 protoplast culture 209, 211, 219 somaclonal variants 217±19 somatic embryogenesis 209±11, 213±17, 219 cellulose green coffee 3, 6 soluble coffee 13 CHARM analysis 69, 70, 72 chemoprotection, from coffee 178 chimeric oligonucleotides 232±3 chlorination, water 156 chlorogenic acids (CGA) antioxidative compounds 57±8 bean swelling 94±5 brews 154 coffee acidity 28±9, 55±6, 158 decaffeination 109, 112 effects of roasting 54±6, 59, 158 green coffee content 18 and health 168 and leaf development 205 as quinic acid precursor 23, 25 roast coffee content 19±20, 22, 24

Index

suspension-cultured cells 208±9 cholesterol 42, 43 boiled coffee 143, 171±2 coffee consumption levels 179 serum levels 41, 171±2 cigarette smoking and fertility 177 in pregnancy 176 cinnamic acid 56, 57 cirrhosis 178 citric acid caffeine recovery processes 120 coffee acidity 29, 56 formation mechanisms 25 green coffee content 18, 19 roast coffee content 19±20, 21, 22, 24 storage effects 26, 56 clonal propagation 196±7, 213±17, 219 coffeadiol 44 coffee berry borer 195, 224, 231 coffee berry disease (CBD) 185, 193±4, 212, 231 Coffee Industry and Trade Associations Forum (CITAF) 239 coffee leaf rust (CLR) 184, 185, 192±3, 231 coffee leaf scorch 195 coffee machines 141, 142±4, 145±6, 150±1, 156 coffee oil 33±4 see also lipids coffee white stem borer 195 Colletotrichum coffeanum (C. kahawae) 193, 212, 231 colloids, and after-taste 158±9 colorectal cancer 15, 167, 178 colour development instant coffee 129 protein reactivity 51±2 coloured macromolecular compounds polymers 58±62 melanoidin 13±14, 53, 58±9, 60, 62 zinc-chelating 62±5 Cona coffee 144 conductivity, thermal, beans 97, 100, 101 congenital malformations 174±5 contaminants causing off-flavour 75 coffee extraction process 10, 11±12 international standards 236 cooling gas roasters 104 cooling methods, roasters 104, 106 coronary heart disease 169±70 CROSSPY 14, 52 cryopreservation, germplasm 189, 211 cyclic peptides 53, 54

249

cyclohexanoic acid ethylester 74, 137 L-cysteine 53±4 û-damascenone aroma analysis methodology 70, 71 brews 80, 81 green coffee 73, 74, 75 physical properties 136 roast coffee 71, 75, 76, 77, 78, 80 decaffeinated coffee 108 bean behaviour 95±6 economics 122±3 production see decaffeination processes decaffeination processes 108±9 caffeine recovery 119±21 economics 122±3 fatty materials 118, 122 in-home 119 liquid CO2 118, 122 solvent 108, 109±10, 122 supercritical CO2 108±9, 113±17, 122 water 108, 110±13, 120±1, 122 decoction, brew preparation 143±4 degustation, brews 158±9 dehydrocafestol 39±40, 41 dehydrokahweol 39, 41 demineralization and bone health 173 water 157 density, brews 152 dephosphorylation 6 desorption processes, decaffeination 111±12, 113, 120, 121 developmental biology (plant) 202±7 developmental outcomes (human) 174±7 development projects 238 dichloromethane (DCM) 109, 110, 122 diketopiperazines 53 dimethyl sulphoxide 109 1,1,diphenyl-2-picryl hydazil (DPPH) 57 disaccharides see carbohydrates, low molecular weight disease resistance breeding for 192±5 molecular biology 224, 231, 232 dispersed phases, brews 152 diterpenes 33, 34, 36±41 boiled coffee 143 and health 41, 168, 172, 178, 179 see also atractylglycosides drip (filter) coffee 144±5, 154, 171, 172 drought tolerance, breeding for 196

250

Index

drum roasters 101, 104±5, 105

extracts, liquid coffee 132±3

earthy odours brews 81 roast coffee 75, 76, 77, 78, 79, 80 economic studies 239 embryo cryopreservation 189, 211 embryogenesis, somatic 209±11, 213±17, 219 environmental stresses biotechnology 231±2 drought tolerance 196 enzymes caffeine synthesis 204 chlorogenic acid synthesis 205 coffee extraction processes 14±15, 128 detoxifying 168 Erlenmeyer cultures 213±14 espresso coffee 146 acid content determination 23, 24 and cappucino 150, 160±1 chemical characteristics 154, 155 definitions 146, 148, 149 foam 15, 151±2, 157±8 and health 41, 172 as lifestyle 146±7 machines 150±1, 156 physical characteristics 151, 152, 153 pressure in preparation 147±9 quantitative definition 149 rapidity of extraction 149 sensory characteristics 147, 157±9, 160 water hardness 151, 156, 157 esters decaffeination processes 118 diterpene fatty acid 33, 34, 38±9, 40, 41 sterol 34, 41±2 ethyl 2-methylbutyrate 74, 75 ethyl 3-methylbutyrate 74, 75 ethyl acetate decaffeination 109, 122 physical properties 133±4, 135 ethylene biosynthesis, fruit ripening 228 evaporation, instant coffee processing 129±30 export values 240 export volumes 240 extraction methods, brew preparation 143±51 extraction processes carbohydrates 9, 10, 13, 14±15 chlorogenic acids 55 coffee fibre 15 instant coffee 127±8 spent coffee grounds 132

F1 arabica hybrids, clonal propagation 216±17 fast roasting processes 92, 95, 98, 104±6 fats see lipids fatty acids 34±6, 38±9, 40, 41, 45 decaffeination processes 118 fertility, and caffeine 177 fibre 15 Fick's law of diffusion 142 filter coffee 144±5, 154, 155, 158, 171, 172 flavour, brews 159, 236±7 flavoured coffee drinks 141, 161±2 fluidized bed roasting 91±2, 93 bean behaviour 93±4 bean volume 95 equipment 101±3, 105 foam 151±2 stability 15 visual importance 157±8 foreign matter see contaminants formic acid brews 154 caffeine recovery processes 120 coffee acidity 29, 56 formation mechanisms 23 green coffee content 19 roast coffee content 19±20, 21, 22 storage effects 26, 56 free fatty acids (FFA) 35±6 free radicals 14 freeze concentration 128 freeze drying 131±2 freezing weather 231±2 French press (plunger; cafetieÂre) coffee 145±6, 154, 155, 172 friable embryogenic tissue (FET) cultures 214±15, 216 fructose green coffee 2 roast coffee 6, 13 soluble coffee 9, 10, 12 fruit development 205±7, 228 fruity odours brews 81 roast coffee 75, 76, 80 fungal toxins 168, 231 fungi coffee berry disease 193, 212, 231 coloured polymer characterization 59±60, 61 resistance genes 224, 231 fungicides coffee berry disease 193±4

Index

contamination with 75 furanones aroma analysis methodology 71, 72 brews 80, 81 formation 82, 83±4 green coffee 75 physical properties 136 roast coffee 75, 76, 77, 78, 79, 80 furfural arabinogalactan scission 14 odorant formation 85 fusarium wilt disease 195, 231 Fusarium xylarioides 195, 231 galactomannan foam stability 15 roast coffee 7 use of term 6 see also mannans galactose green coffee content 3±4, 5, 6 roast coffee content 7, 8, 9, 14 soluble coffee content 8, 9, 12±13 a-galactosidase 225 gas chromatography-olfactometry (GCO) 69±72, 73, 74 gas circulation, roasters 104 gastric cancers 167 gastrointestinal tract cancers 15, 167, 178 reactions to wax 45 gene regeneration 211, 212, 219 gene sequences 225±30 genetic resources, for breeding 186±9 gene transfers 211±12, 219 gene transformation systems 230±1, 232±3 geosmin 75 germplasm collections 186, 219 germplasm conservation 188±9, 211, 219 global research network 239 glucose green coffee 2, 5 roast coffee 6, 13, 14 soluble coffee 9, 12 glycolic acid coffee acidity 29, 56 formation mechanisms 23 roast coffee content 20, 21, 22, 24 storage effects 26, 56 glycosides 37 green coffee 2±3, 4 roast coffee 6±7

251

Greek coffee 143 green coffee amino acids 50, 51 antioxidative compounds 57±8 and brew astringency 160 brewing 140±1 carbohydrates 1±6 contaminants 75 content of odorants 73±5 decaffeination 109, 111±17, 118 heat capacity 96±7, 98 for instant coffee 132, 133, 137 international standards 235, 236 lipids 33, 34, 35±6, 37, 43±4, 45±6 OAVs of odorants 73±5 organic acids effects of roasting 54±5, 59 formation mechanisms 23, 25, 26 quantitative data 18±19 physical values 91, 100±1 potent odorants 73 protein 51 volatile compounds 73±5, 137 zinc-chelating compound 64 grinding processes, instant coffee 127 ground coffee, standards 235, 236 guaiacols aroma analysis methodology 70, 71 brews 81, 82 green coffee 74, 75 physical properties 136 roast coffee 75, 76, 77, 78, 79, 80 health considerations 165 antioxidative compounds 57, 58, 168 benefits of coffee 177±8 birth defects 174±6 boiled coffee 143, 171±2 bones 173±4 cancers 15, 166±9, 178 cardiovascular disease 169±72 chemoprotection 178 coffee fibre 15 coffee wax 45 diterpenes 41, 168, 172, 178, 179 neuroactivity 177±8 neurodevelopment 175 preserved coffee drinks 141, 161 solvent decaffeination 109 zinc-chelating compounds 62 heat capacity, beans 96±7, 98 heat caramelization equipment 106

252

heat conductivity, beans 97, 100, 101 heat sterilization, and aroma 82 heat transfer, gas±bean 99±100 heat transfer coefficients 99±100 heat uptake, beans 97±8 Hemileia vastatrix 192, 231 hepatitis infections 178 4-heptenal 74 herbicide resistance genes 232 high performance anion-exchange chromatography (HPAEC) 10±11, 12 home decaffeination 119 homocysteine, serum levels 172 homologous recombination, genetic changes 233 husk adulteration 10, 12 hydrogen peroxide, formation 57±8 hydrolysis coffee extraction process 127 carbohydrates 9, 10, 13, 14±15, 127 chlorogenic acids 55 fibre 15 spent coffee grounds 132 hydrolyzing enzymes 14±15, 128 hypercholesterolaemia 171±2 hypertension 171 Hypothenemus hampei 195, 224, 231 ibrik 143 industrial roasting equipment 101±7 infusion, brew preparation 144±5 in-home decaffeination 119 inositol phosphates (IPs) coffee acidity 29, 154 green coffee content 2 phosphoric acid formation 25±6 roast coffee content 6 insect damage international standards 236 odour activity values 74 insect resistance 195±6, 224, 231 instant coffee 125±6 agglomeration 130±1 aroma of brews 82, 126 aromatization 132 brew preparation 140 carbohydrates 8±15, 127, 132 extraction processes 127±8 freeze concentration 128 freeze drying 131±2 green coffee for 132, 137 and health 41, 172 international standards 126, 235, 236

Index

legislation 126 liquid extracts 132±3 Loncin's role 125±6 organic acid determination 23, 24 reverse osmosis 130 sales 125 spent grounds disposal 132 spray drying 130±1 technology 125 thermal concentration 129±30 Thijssen's legacy 125 volatile compounds extraction 126±7 freeze drying 131±2 handling 130 physical properties 133±7 recovery 129±30 zinc-chelating compounds 62±4 integrated pest management (IPM) 195 International Coffee Agreement (1994) 238±9 International Coffee Organization (ICO) 238±41 International Standards Organization (ISO) 11, 126, 235±7 in vitro selection studies 212 ion exchange chromatography 1 iron-chelating compounds 62, 63 isotope dilution assays 72 Israeli `mud' coffee 143 kahweal 40 kahweol 37, 38, 39, 40, 41 and health 168, 172, 178, 179 Koleroga noxia 195 lactic acid brews 154 coffee acidity 29 formation mechanisms 23 roast coffee content 19, 21, 22, 24 storage effects 26 lactones, chlorogenic acid 55±6 Laurina somaclones 218±19 `leaching', beverage preparation 141 leaf development 203±5 leaf miners 195±6 Leucoptera spp. 195±6 lipids 33 alkanes 44 boiled coffee 143, 171±2 brews 154 brew viscosity 152±3 coffeadiol 44

Index

decaffeinated coffee 95±6 decaffeination processes 118, 122 determination methods 33±4 diterpenes 34, 36±41, 143, 168, 172, 178, 179 fatty acids 34±6, 38±9, 40, 41, 45 and health 41, 45, 168, 172, 178, 179 isolation for analysis 34 squalene 44 sterols 34, 41±2, 143, 171±2, 179 tocopherols 42±4 wax 33, 45±6 liquid CO2 decaffeination 118, 122 liquid coffee extracts 132±3 liver disease 178 Loncin, M. 125±6 low birth weight infants 175±6 lysine 51±2 Maillard reaction 13, 14, 58±9, 62, 64 malic acid brews 154 coffee acidity 29, 56 formation mechanisms 25 green coffee content 18, 19 roast coffee content 19±20, 21, 22, 24 storage effects 26, 56 mannans green coffee 3, 5±6 roast coffee 7±8, 9 soluble coffee 12±13, 14±15, 127±8 spent coffee grounds 132 mannitol roast coffee 6 soluble coffee 10, 12, 13 spent coffee grounds 132 mannose green coffee 2, 5 roast coffee 7, 8, 9, 14 soluble coffee 8, 9, 12±13 spent coffee grounds 132 mass transfer, brew preparation 142 mass transport, during roasting 96±100, 101 measurement units 242±5 melanoidin antioxidant activity 168 brews 155 characterization 58±9, 60, 62, 64 formation 13±14, 53, 58 Meloidogyne spp. 195 metal-chelating compounds 62±5 2-/3-methylbutanoic acid 27 2-/3-methylbutyric acid 27, 74

253

16-O-methylcafestol (16-OMC) 37±8, 40, 41 2-methylisoborneol (MIB) 75, 78, 137 16-O-methylkahweol 37 methylthio groups 52±3 microbiological characterization, coloured polymers 59±62 micropores, cell walls 95 microporous resins, decaffeination 111 micropropagation 212±17, 219 milk±coffee admixtures 160±1 mineral-chelating compounds 62±5 minerals bone health 173±4 brews 154, 155±6 miscarriage (spontaneous abortion) 176±7 modified coffee beverages 141, 160±2 moisture content international standards 235±6 roasting beans 94±5, 96±7, 101 Moka coffee 146, 154, 155 molecular biology 185, 224 11S storage protein promoter 226±8 caffeine biosynthesis 228±30, 233 coffee genes 225±30 coffee transformation systems 230±1, 232±3 disease resistance 231 environmental stresses 231±2 flavour traits 232 fruit development 228 fungal toxins 231 pest resistance 231 prospects 231±3 quality traits 232 selectable markers 232 site-specific changes 232±3 monosaccharides see carbohydrates, low molecular weight mouldy flavours 75, 78 mouthfeel, brews 159±60 multidimensional gas chromatography (MDGC) 72 mutagenic effects, coffee 167±8 myocardial infarction 169±70 Napoletana coffee 145, 154, 155 nematode resistance 195 Nepro Vortex Fluidat 103, 105 NestleÂ, clonal propagation 216 neuroactivity, and caffeine 177±8 neurodevelopment, and caffeine 175 nitrogenous compounds, brews 155 see also caffeine; melanoidin nitrous oxide, decaffeination 114

254

(E)-2-nonenal aroma analysis methodology 70 brews 82 green coffee 74, 75 roast coffee 75 non-volatile compounds acids 18±30, 54±6, 59 amino acids 13±14, 50±1, 52±3 antioxidative 57±8 carbohydrates 1±15 coloured macromolecular 58±65 lipids 33±46 protein 51±4 Nusselt equations 99 ochratoxin 231 odorants see volatile compounds odour, brews 77±8, 80±2, 126, 159 odour activity values (OAVs) 68±9 green coffee 73±5 oesophageal cancers 167 oil, coffee 33±4 see also lipids olfaction 159, 236 oligonucleotides, chimeric 232±3 oral cancers 167 organic acids bean swelling 94±5 brews 55±6, 154 caffeine recovery processes 120 effects of roasting 22±5, 29±30, 54±6, 59, 158 formation mechanisms 13, 23±5 green coffee 18±19, 23, 25, 26, 54±5, 59 roast coffee 19±23 roast kinetics 29±30 sensory characteristics 27±30, 55, 147, 154, 158 solvent decaffeination 109 and storage 26, 55±6 suspension-cultured cells 208±9 volatile 26±7, 28 organic solvents, decaffeination 109 organoleptic characteristics see sensory characteristics osmosis, reverse 130 osteoporosis 173±4 ovarian cancer 166 2-oxopropanal 82, 84 packed bed roasters 104, 105 pancreatic cancer 167 partition coefficients 133±4, 135 pectin, green coffee 6 percolation 144

Index

espresso coffee 148, 149, 157 and water hardness 157 percolator coffee 143±4, 154, 155 Perileucoptera coffeella 195 periodic immersion cultures 215 pest resistance 195±6, 224, 231 pharyngeal cancers 167 phases, brews 152 phenolic compounds antioxidant activity 168 melanoidin characterization 58, 59, 60, 64 metal binding 62, 64±5 phenolic odours 75, 76, 78, 80 brews 81 phenols formation 84 physical properties 136 roast coffee 78 see also guaiacols phenylindans 58, 168 phosphoric acid coffee acidity 29, 56, 154 formation mechanisms 25±6 green coffee content 18 roast coffee content 20, 21, 22 and storage 56 phosphorylation 6 phytic acid (IP6) coffee acidity 29, 154 phosphoric acid formation 25±6 plunger (cafetieÂre) coffee 145±6, 154, 155, 172 pollen transformation system 232 polyalcohols 2±3 see also atractylglycosides polysaccharides see carbohydrates, high molecular weight porosity, bean 95, 99, 101 potassium, brews 156 Pratylenchus spp. 195 predrying processes 92, 95 pregnancy 174±7 premature births 175±6 preserved coffee drinks 141, 161 pressure methods, brew preparation 145±51 see also espresso coffee prices, coffee 241 promotional activities 238±9 propagation new cultivars 196±7 using micropropagation 212±17, 219 prostate cancer 166 protein 51

Index

bitter tasting compounds 53±4 brews 154 carbohydrate breakdown products 13±14 Maillard products 13, 14 reactivity 51±3 seed storage genes 225±6 zinc-chelating compounds 64 protoplast culture 209, 211, 219 Pseudomonas syringae pv garcae 195 psicose 2, 6 purine alkaloid formation 207±9 pyrazines aroma analysis methodology 70, 71, 72 brews 80, 81, 82 formation 82, 84 green coffee 73±4, 75 optimum roast detection 93 physical properties 134±5, 136, 137 roast coffee 75, 76, 77, 78, 79, 80 storage effects 79 pyrrolidinohexose reductones 53±4 pyruvic acid, roast coffee content 19 quinic acid antioxidative compounds 57±8 brews 154 coffee acidity 29, 55, 56 effects of roasting 54±6, 59 formation mechanisms 23, 25 green coffee 18, 19 roast coffee 19, 20, 21, 22 storage effects 26, 56 quinides 54±5 radiotherapy 178 raw coffee see green coffee recaffeinated coffee 119 refractive index, brews 153 renal cancer 166 reproduction, coffee effects 174±7 research network 239 resins, decaffeination 111, 112±13 reverse osmosis 130 rhamnose green coffee 2, 6 roast coffee 14 soluble coffee 12 roast coffee antioxidative compounds 57±8 arabica aroma profiles 77±8 aroma changes in storage 79±80 brewing techniques 141±51

255

carbohydrates 4, 6±8, 13 cavities 95, 100 degree of roast 79 international standards 235, 236 lipids 33, 34 diterpenes 37, 39±40 fatty acids 34±5, 36, 40, 41 tocopherols 43 wax 46 liquid CO2 decaffeination 118 odorant concentration 75±7 odorant evaluation 77 organic acids 19±23 physical values 91, 100±1 robusta aroma profiles 77±8 supercritical CO2 decaffeination 117 volatile compounds 75±80, 136 zinc-chelating compounds 62±3 see also roasting processes roasters, industrial 101±7 roasting processes 90 amino acids 50, 51, 52, 53 antioxidative compounds 58 and aroma 79, 92 bean behaviour 93±6 beans' physical values 100±1 and bitter taste 53±4 cafestol/dehydrocafestol ratio 39±40 carbohydrate reactions 6±7, 9, 13±14, 23, 51±2 chlorogenic acids 54±6, 59, 158 colour development 51±2 conventional 91, 93±4 detecting optimum roast 79, 92±3 fast 92, 95, 98, 104±6 fluidized bed 91±2, 93±4, 95, 101±3, 105 heat transport in bean 96±100, 101 industrial equipment 101±7 for instant coffee 127 mass transport in bean 96±100, 101 organic acids 22±5, 29±30, 54±6, 59, 158 protein 51, 52, 53±4 zinc-chelating compounds 64 roasty odours brews 81 roast coffee 75, 76, 77, 78, 79, 80 root-knot nematodes 195 root-lesion nematodes 195 rotating bowl roasters 101, 105 rotating fluidized bed (RFB) roasters 101±3 safety, coffee consumption 165, 178±9 Secoffex decaffeination 111, 112

256

sedimentation, carbohydrate role 15 seed development 206 seed propagation 196, 212±13 seed storage protein gene 225±8 sensory characteristics 157±60 acids 27±30, 55, 147, 154, 158 espresso coffee 147, 148, 158 international standards 236 serum cholesterol 41, 171±2, 179 serum homocysteine 172 sexual organs, cancers of 166 silicas, decaffeination 112 SI units (appendix) 242±5 smoking and fertility 177 in pregnancy 176 smoky odours brews 81 roast coffee 75, 76, 77, 78, 79, 80 solid±liquid extraction, brewing 140, 141±51 solids, brew characteristics 153±4 soluble coffee see instant coffee solvent decaffeination 108, 109±10, 122 somaclonal variation 217±19 somatic embryogenesis 209±11, 213±17, 219 sourness 27±8 specific heat, coffee 96±7 spent coffee grounds, disposal 132 spicy odours brews 81 roast coffee 75, 76, 80 spinning cone columns (SCCs) 129±30 spontaneous abortion 176±7 spouting beds fast roasting 92 fluidized bed roasting 91±2 spray drying, instant coffee 130±1 squalene 44 stable isotope dilution assays 72 starch green coffee 6 soluble coffee 12 statistical services 239 steaming, instant coffee processing 130 steam roasting 106±7 sterols 33, 34, 41±2, 143, 171±2, 179 stomach cancers 167 storage of coffee 26, 79±80 Strecker degradation 82, 83, 84 succinic acid green coffee 19 roast coffee 20, 21, 22, 24

Index

storage effects 26 sucrose brews 155 green coffee 1±2 roast coffee 6, 13, 14, 23 sudden infant death syndrome 175 sulphur-containing amino acids 52±3 sulphurous odours brews 81 roast coffee 75, 76, 77, 79, 80 supercritical carbon dioxide decaffeination 108±9, 113±17, 119, 122 supercritical nitrous oxide decaffeination 114 supernatant foam 151±2 surface tension, brews 153 suspension-cultured coffee cells 208±9 sweetness, brews 158 sweet odours brews 81 roast coffee 75, 76, 77, 78, 79, 80 swelling mechanisms, beans 92, 94±5 taste acidity 27±8, 154, 158 bitterness 53±4, 158±9 brews 158±9 olfactory component 159 temperatures, bean roasting 92±5, 96, 97, 98±9, 101 temperature stresses, coffee plants 231±2 teratogenicity, caffeine 174±5 thermal concentration, instant coffee 129±30 thermal conductivity, beans 97, 100, 101 Thijssen, H.A.C. 125 thiols aroma analysis methodology 69, 70, 71, 72, 73 brews 80, 81±2 formation 84±5 heating effects 82 physical properties 136, 137 roast coffee 76, 77, 78, 79, 80 storage effects 79 tocopherols 42±4 tracheomycosis (fusarium wilt disease) 195, 231 transformation systems, coffee 230±1, 232±3 transgenic plants 230±1, 232±3 2,4,6-trichloroanisole (2,4,6-TCA) 75, 137 triglycerides 33, 34±5, 41 trigonelline 53, 54, 154, 155 Turkish coffee 143, 171, 172 Uganda robusta cloning program 217 units of measurement 242±5

Index

urinary tract cancers 166±7 vacuum coffee 144 viscosity, brews 14±15, 152±3 visual characteristics brews 157±8 international standards 236 volatile acids 26±7, 28 volatile compounds 68±9 aroma analysis methodology 69±73 brews 77±8, 80±2 green coffee 73±5 instant coffee processing 126±7, 129±30, 131±2, 133±7 odorant formation 82±5 olfaction 159 physical properties 133±7 roast coffee 75±80 Volatile Organic Compound (VOC) Directive 109 water brew characteristics 156±7 brew preparation 141, 143±6, 147±8, 149 chlorination 156 demineralizers 157

257

hardness 151, 156, 157 softening 151, 156±7 water content, roasting beans 94±5, 96±7, 101, 235±6 water decaffeination 108, 110±13, 120±1, 122 water shortages, drought tolerance 196 wax 33, 45±6 decaffeinated coffee 95±6 weather stresses 196, 231±2 World Coffee Conference 239 world coffee production 184 world germplasm collections 186 world research network 239 xanthosine-N7-methyl transferase (XMT) 228±30 Xylella fastidiosa 195 xylose green coffee 6 soluble coffee 9, 10, 12 Xylotrechus quadripes 195 zeolites decaffeination processes 111±12, 117 roasting processes 106 zinc-chelating compounds 62±5

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