WORLDWIDE DI S T I LLE D S P I R I TS CONFERENCE

PrProduction,oduction, TTecechnolohnologgyy andand InnoInnovvaationtion

EditedEdited byby JJ.H..H. BrBryyce,ce, JJ.R..R. PigPiggottgott andand GG.G.G.. StewStewarartt

Proceedings of the Worldwide Distilled Spirits Conference

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The Institute of Brewing & Distilling Scottish Section DISTILLED SPIRITS Production, Technology and Innovation

Distilled Spirits Production, Technology and Innovation

Edited by

J.H. Bryce1 J.R. Piggott2 G.G. Stewart1

1International Centre for Brewing and Distilling, Heriot-Watt University, Edinburgh, UK; 2Strathclyde Institute for Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK Nottingham University Press Manor Farm, Main Street, Thrumpton Nottingham, NG11 0AX, United Kingdom

NOTTINGHAM

First published 2008 © The several contributors names in the list of contents

All rights reserved. No part of this publication may be reproduced in any material form (including photocopying or storing in any medium by electronic means and whether or not transiently or incidentally to some other use of this publication) without the written permission of the copyright holder except in accordance with the provisions of the Copyright, Designs and Patents Act 1988. Applications for the copyright holder’s written permission to reproduce any part of this publication should be addressed to the publishers.

British Library Cataloguing in Publication Data Distilled Spirits - Production, Technology and Innovation I. Bryce, J.H., II. Piggott, J.R., III. Stewart, G.G.

ISBN 978-1-904761-64-8

Disclaimer

Every reasonable effort has been made to ensure that the material in this book is true, correct, complete and appropriate at the time of writing. Nevertheless, the publishers and authors do not accept responsibility for any omission or error, or for any injury, damage, loss or financial consequences arising from the use of the book.

Typeset by Nottingham University Press, Nottingham Printed and bound by The Cromwell Press, Trowbridge Foreword

The source material for this book originated in the second Worldwide Distilled Spirits Conference which took place in Edinburgh in the Autumn of 2005. At that event, organised by the Scottish Section of the Institute of Brewing and Distilling, a group of over 250 distillers from around the world gathered to share knowledge under the theme of Production, Technology and Innovation – Meeting the Challenges of Tomorrow. Tomorrow has become today and in the time since the conference those challenges have become more apparent, crystallising on the issue of global sustainability in its full environmental, social and economic sense. Application of new technology, understanding our supply chain and preserving our rich heritage will all play their part but most of all we must encourage the ability to think our way into a new tomorrow. I hope the information in this volume will make a contribution to thinking ahead in the distilled spirits industry.

It was a privilege to be the conference Chairman in 2005 and I would like to thank again the Organising Committee and the generous sponsors who supported the conference. A special thank you is due to the editors of this book for drawing together the many strands covered at the conference and making it into a lasting and coherent whole.

James M. Brosnan

Research Manager, Scotch Whisky Research Institute Chairman of the Institute of Brewing, Scottish Section 2005-2007 Chairman of the second Worldwide Distilled Spirits Conference Organising Committee

v

Contents

Preface xiii

1 Sustainability in the cereals supply chain 1 W. D. Rae The North British Distillery Company Limited, Wheatfield Road, Edinburgh, UK 2 New age spirit beverages – an Indian perspective 7 B. K. Maitin The UB Group, Spirits Division – Technical Centre, Bangalore, India 3 Orange press spirit: technical and economic aspects of a new distilled beverage 15 J.B. Faria, H. Roçafa Jr. and J.O. Ferreira UNESP, São Paulo State University, Departamento Alimentos e Nutrição, 14801-902, Araraquara, SP, Brazil 4 The influence of base wine composition and quality on the style and quality of South African brandy 21 C.L.C. Snyman1 and M.G. Lambrechts2 1Distell Group Ltd, PO Box 184, Stellenbosch 7599, South Africa; 2Institute for Wine Biotechnology, University of Stellenbosch, Stellenbosch, South Africa 5 New characterisation tools for whiskey raw materials 29 V. H. Beaumont Pernod Richard Research Center, 94015 Créteil Cedex, France 6 Mash rheology – Model studies to understand the distillery mashing process using the Rapid Visco Analyser 37 D. L. Goode1, V. H. Beaumont2, D. Quinn2 and E. K. Arendt1 1Department of Food and Nutritional Sciences, National University of Ireland, University College Cork, Ireland; 2 Irish Distillers Ltd, Midleton, Co. Cork, Ireland

7 The use of NIR spectroscopy to quantify various malted barley analytes 47 P. Lockyer and A. Wardlaw Diageo Global Supply, Brand Technical Centre, Menstrie, Clackmannanshire, UK 8 Wheat for Scotch whisky production: broadening the horizon 51 T.A. Bringhurst, R.C. Agu, J.M. Brosnan and A.L. Fotheringham The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK 9 Cereals for grain distilling: can wheat and other cereals ever achieve the potential alcohol production of maize? 59 R.C. Agu, T.A. Bringhurst and J.M. Brosnan The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK 10 The influence of nitrogen content and corn size on the quality of distilling wheat cultivars 67 R.C. Agu1, J.S. Swanston2, T.A. Bringhurst1, J.M. Brosnan1, F.R. Jack1 and P.L. Smith2 1The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK; 2Scottish Crop Research Institute, Invergowrie, Dundee, UK 11 A turbidity test for a genetic component of spirit yield in wheat 75 J. S. Swanston and P. L. Smith Scottish Crop Research Institute, Invergowrie, Dundee, UK 12 The regulation of limit dextrinase activity in malting, mashing and fermentation 81 H.R. Jenkinson, P.C. Morris and J. H. Bryce International Centre for Brewing and Distilling, Heriot-Watt University, Edinburgh, UK 13 The global crisis of energy and grain 87 B. J. Hoskins and M. P. Lyons Alltech, Inc., Nicholasville, Kentucky, USA and International Centre for Brewing and Distilling, Heriot-Watt University, Edinburgh, UK 14 Proteomic analysis of distillers’ yeast 93 R. Hansen1, A. M. Ferguson1, S.Y. Pearson2, J.M. Brosnan2, P. G. Meaden1, D. J. Jamieson1 1International Centre for Brewing and Distilling, Heriot-Watt University, Edinburgh, UK; 2The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK 15 Effect on new-make spirit character due to the performance of brewer’s yeast – (I) physiological changes of yeast during propagation and brewing 109 H. Yomo, Y. Noguchi and T. Yonezawa Suntory Ltd., Technical Development Center, Osaka, Japan 16 Effect on new-make spirit character due to the performance of brewer’s yeast – (II) various yeast strains containing commercial strains 117 Y. Noguchi, K. Urasaki, H. Yomo and T. Yonezawa Suntory Ltd., Technical Development Center, Osaka, Japan

17 The use of CO2 evolution monitoring as an indicator of yeast fermentation performance 123 S.Y. Pearson, J.W. Walker, T.A. Bringhurst and J.M. Brosnan The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK 18 Towards improved distilling yeast: effect of wort gravity and pitching rate on fermentation performance 127 J. W. Walker, S. Y. Pearson, T. A. Bringhurst and J. M. Brosnan The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK 19 Opportunities for improved yeast supplies in whisky and related spirits production 133 R. Munro Fermentis, Division of S.I. Lesaffre, 59703 Marcq-en-Baroeul Cedex, France 20 Application of novel yeast strains to the Scotch whisky fermentation process 139 H. B. de Amorim Neto1, S. Y. Pearson2, J. W. Walker2, G. M. Walker1 and J. M. Brosnan2 1School of Contemporary Sciences, University of Abertay, Dundee, UK; 2The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK 21 Speciality yeast and optimal yeast management practices for maximising fermentation efficiency in the fuel industry 145 C. L’Helgoualc’h Fermentis, Division of S.I. Lesaffre, 59703 Marcq-en-Baroeul Cedex, France 22 Spirit flavour release under mouth conditions 151 M. Maçatelli, A. Paterson and J.R. Piggott Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK 23 From sugar to rum - the technology of rum making 159 V. Persad-Doodnath Angostura Limited, Trinidad, West Indies 24 Discrimination between rum and cachaça 169 L. G. Andrade-Sobrinho1, J. R. Piggott2 and D. W. Franco1 1Departamento de Química e Física Molecular, Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil; 2Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK 25 Evaluation of Brazilian as an alternative to oak for cachaça aging in cask - their antioxidant ability 179 D. R. Cardoso1, A. M. Frederiksen2, A. A. da Silva1, D. W. Franco1 and L. H. Skibsted2 1Departamento de Química e Física Molecular, Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil; 2Department of Food Science, KVL, Rolighdsvej 30, Frederiksberg C, Denmark 26 Physical-chemical and sensory evaluation of Brazilian sugar cane distilled alcoholic beverage 189 P.H.A. Silva Departamento de Tecnologia de Alimentos, Universidade Federal de Viçosa, Viçosa-MG, Brazil 27 Characterising the volatile compounds in three fractions of distillates commonly recovered during sugar cane spirit processing 197 P.H.A. Silva Departamento de Tecnologia de Alimentos, Universidade Federal de Viçosa, Viçosa-MG, Brazil 28 Sensory implications of modifying practice in Scotch malt whisky production 205 F.R. Jack, J.M. Brosnan, K.A. Campbell, O. Fagnen, R.N. Fotheringham and I.C. Goodall The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK 29 Characterisation and differentiation of peat used in the preparation of malt for Scotch whisky production 213 B.M. Harrison1, 2, K.J.G. Reid1, G. M. Steele1 and F. G. Priest2 1The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK; 2International Centre for Brewing and Distilling, Heriot-Watt University, Edinburgh, UK 30 Understanding and enhancing cask performance 219 K.J.G. Reid, J.M. Conner, F. Jack, M. Patterson and J. Freeman The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK 31 Impact of brandy production processes on flavour 229 B. Colonna-Ceccaldi Pernod Ricard Research Centre, 94015 Créteil Cedex, France 32 Instrumentation of a pilot scale distillery with on line data recording to study the Cognac production process 237 G. Ferrari2, B. Galy2, A. Sommier1, C. Chipeaux1 and M. Decloux1 1ENSIA-UMR GENIA, Massy, France; 2Station Viticole du Bureau National Interprofessionnel du Cognac, Cognac, France 33 Esters – the most important group of flavour-active compounds in alcoholic beverages 243 G. G. Stewart International Centre for Brewing and Distilling, Heriot-Watt University, Edinburgh, UK 34 Environmental solutions and threats in today’s distilling industry 251 B. Higgs Diageo Plc, Elgin, UK 35 Wastewater treatment and energy recovery go hand in hand in the distilling industry 257 V. Groot Kormelinck Paques bv, 8560 AB Balk, Netherlands 36 Co-products from the distilling industry 265 R.W. Hall James & Son (Grain Merchants) Ltd, Wellingborough, Northants, UK 37 Packaging materials as a source of taints 269 J.M. Conner and K.J.G. Reid The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK 38 Proving that alcoholic beverage distillates are free from cereal, nut and milk derived allergens 277 I.C. Goodall, J.M. Brosnan, K.A. Campbell, C.D. Owen, G.M. Steele and J.W. Walker The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK 39 Authenticity indicators – enhancing consumer and brand protection 281 R. I. Aylott Diageo Plc, Brand Technical Centre, Menstrie, Clackmannanshire, UK 40 Meeting the challenges of tomorrow 289 G. Hewitt The Scotch Whisky Association, Atholl Crescent, Edinburgh, UK 41 Whisk(e)y production: raw materials, malting, cooking, mashing, fermentation and beyond 295 I.C. Goodall The Scotch Whisky Research Institute, The Roberton Trust Building, Riccarton, Edinburgh, UK 42 Gin and 299 R.I. Aylott Diageo Plc, Brand Technical Centre, Menstrie, Clackmannanshire, UK 43 Shochu 305 K. Hashimoto and S. Matsumoto Sanwa Shurui Co. Ltd., Usa-shi, Oita 879-0495, Japan 44 The production of Cognac 309 G. Ferrari, B. Galy, V. Dumot, and R. Cantagrel Station Viticole du Bureau National Interprofessionnel du Cognac, Cognac, France 45 Grappa: the Italian distillate 315 C. Da Porto1 and M. Longo2 1Dipartimento di Scienze degli Alimenti, Università di Udine, Udine, Italy; 2Distilleria Bonaventura Maschio S.r.L, Gaiarine (TV), Italy 46 Ouzo and raki 323 A. Varvagiannis, L. Margomenou and I. Zabetakis Laboratory of Food Chemistry, Department of Chemistry, University of Athens, Athens, Greece 47 Rum and Cachaça 327 J.B. Faria UNESP, São Paulo State University, Departamento Alimentos e Nutrição, 14801-902, Araraquara, SP, Brazil 48 Pálinka - Hungary’s national drink 335 L. Szabó Agárdi Distillery, Sreiner Tanya, Hungary 49 Tequila THIS CHAPTER NOT INCLUDED ON CD DUE TO COPYRIGHT ISSUES 339 M. G. López Centro de Investigación y de Estudios Avanzados del I.P.N., Campus Guanajuato, Irapuato, México 50 Education and training in the Scotch whisky industry 343 J. B. Eaton International Centre for Brewing and Distilling, Heriot--Watt University, Edinburgh, UK Index 349

Preface

The distilled spirits industry is changing rapidly molecular and biochemical techniques; one to meet the demands of globalisation. These chapter also reflects the development of yeast demands are being met by the use of completely for the fuel alcohol industry. novel raw materials and also by the continued Chapters 22 to 33 focus on the sensory development of raw materials that are currently implications of the raw materials used to in use. Novel spirits of high quality are being produce distilled spirits and changes in raw manufactured at a price and with flavours that material processing. Topics covered include meet the aspirations of consumers. These the effects of different peat sources on the products are being made at a time of ever flavour potential of peated malt, understanding increasing demands by legislatures to ensure that how distillation practice affects the flavour production is sustainable, with a minimal impact of whisky, and investigations into rum and on the environment. As always, the continued cachaça production. In chapters 34 to 40 there growth and success of well established brands is discussion of the environmental solutions and makes them a target for those who try to produce threats to the distilling industry with coverage counterfeit products and take advantage of the of the techniques for waste water treatment and established name of a product that has been the use of co-products produced by the distilling developed and perfected over many years. industry. These final chapters also deal with the Therefore, in 2008 there are many exciting importance of maintaining brand authenticity challenges facing the distilled spirits industry, and of ensuring that distilled spirits are free of but also a number of threats. potential taints and allergens. This book on distilled spirits contains Chapters 41-50 describe different means 50 chapters which arose out of the second of production of a wide variety of world wide Worldwide Distilled Spirits Conference organised distilled spirits, with the final chapter focussing on by the Institute of Brewing and Distilling, Scottish the development of education and examinations Section, in 2005. The opening 13 chapters to ensure that the quality of worldwide distilled reflect the changes that are taking place in the spirits is maintained and enhanced. production and use of raw materials. There is a It has taken time to gather together and edit particular emphasis on the methods of analysing the chapters in this book. However, we would the quality of cereals and the development of like to thank Dr Sandy MacGregor for help cereals to meet future demand. This is set against in editing a number of manuscripts, all the a global energy crisis where the distilled spirits authors for their excellent contributions and industry is competing with the fuel alcohol speedy response to our requests, and we would industry for raw materials. The focus of chapters also particularly like to thank Sarah Keeling of 14 to 21 is on the development of yeast for use Nottingham University Press for her dedication in the distilled spirits industry using the latest and expertise in this final production.

James H. Bryce1, John R Piggott2 and Graham G. Stewart1 1International Centre for Brewing and Distilling, Heriot-Watt University 2Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde

xiii xiv Sustainability in the cereals supply chain 1 Chapter 1 Sustainability in the cereals supply chain

W. D. Rae The North British Distillery Company Limited, Wheatfield Road, Edinburgh, UK

Introduction Whether it was maize from Rhodesia or Canada, or barley from Australia or northern Europe, a The word “sustainable” seems to be much used truly international supply chain was engaged. and often abused in business language today. Virtually all grain distilleries utilised maize as The definition of the verb to sustain in the Oxford their cereal of preference with high DP malt Dictionary is “to maintain, prolong or keep being secured from specialist maltsters. Good up the vitality of…” This will consider quality malting barley for malt distillation was whether the traditional cereal procurement only available from a limited number of sources. measures utilised by the Scotch Whisky industry However the process appeared to work and the do in fact encourage the long term engagement only issues of concern to the buyers, as they and commitment by the various participants in were then called, were weather patterns and the cereals supply chain. I would also add that political uncertainty in some of the more volatile the views expressed in this paper are entirely countries. personal and do not represent the official policy When the UK belatedly joined the EU, of the Scotch Whisky Association. They reflect commercial flexibility on cereal procurement rather the considerations of a career veteran in was greatly restricted as a consequence of the industry who has been exposed too long to the need for compliance with the Common the vagaries of the marketplace. However before Agricultural Policy (CAP) and its aims of we look forward it is often wise to look back protecting and nurturing EU grain production and evaluate the history of our procurement through a regime of direct subsidy to the grower. practices. Although opportunities to continue to import raw materials from other countries were still available through use of technical measures such as the Historical background dreaded Inward Processing Relief, by and large the importation of cereals from outwith the EU For those of us who have been in the industry ceased. This meant a significant increase in raw for a long time, we remember that the Scotch material costs that was compensated partly, by Whisky industry brought grain from all corners the award of EU financed Export Refunds. The of the globe to meet our processing needs. CAP, in short, created an artificial internal EU 2 W.D. Rae market for cereals that protected farmers and The breeders delivered the holy grail of high swallowed a huge share of the EU finances. spirit yields combined with excellent agronomic In response to these measures, companies performance, yet further strengthening the within the industry reviewed the process internal EU supply chain. of acquiring raw materials. Inevitably, the substantial cost hike in cereals resulted in a wholesale exodus by grain whisky producers Problems within the supply chain from procuring maize towards lower cost, domestically available, feed wheat. The high cost However all was not as rosy as might have been of malt was offset by the acceleration of selective first imagined. The EU subsidy and aid measures breeding programmes that delivered new high perpetuated an inefficient and expensive supply spirit yielding varieties of malting barley. Indeed chain. EU cereal prices remained consistently over the last two decades malt distillers have higher than in equivalent countries outside the witnessed their spirit yields increase by nearly EU, with the quality of crop often being poorer. 40 litres of alcohol per tonne. In addition, as the drinks industry accelerated Once these strategic moves had been into a programme of acquisition, consolidation completed, some kind of equilibrium was and globalisation it became clear that spirit established once again in the supply chain. manufacture could be located in centres of lower The farmer/grower, through the mechanism of raw material cost, other than within the EU. Only direct aid subsidies and the establishment of those drinks with geographic protection, such intervention pricing, was a willing supplier and as Scotch Whisky, were restricted in their point seller of cereals. He felt confident that provided of origin. However in most companies within he grew a reasonable quality of crop there would the drinks industry, products such as Scotch, be little problem in realising an economic value although vital to corporate performance, were for his efforts. Maltsters and merchants, often only components of a multi-faceted portfolio acting as middle men to the industry, were of alcoholic beverages. The protection on the able to apply strict quality parameters to their one hand, delivered significant constraints on procurement criteria often having the pick of the other. Input costs were substantially higher abundant supplies, provided no adverse weather for instance when comparing a mash bill for conditions had impacted on the crop. Distillers Scotch with that for Bourbon. For an industry that then negotiated with maltsters, setting key freely trades in over 200 countries throughout performance targets for the malt, through spirit the world such constraints make little or no yield performance. Some distillers who operated economic sense. their own maltings, compressed this supply However significant changes were on the chain but the same principles applied. way. The publication by the EU of Agenda 2000 Given the size of the direct subsidies available (European Commission, 1999) recognised the to the farmer/grower there appeared to be little difficulties that the existing support measures barrier to continuity of supply, even when had created and finally drove through the first crops were rejected for failing to meet quality stage of significant reform to the CAP in over parameters. Rejected material was simply two decades. The growth in the scale of the aid diverted to animal feeding. and subsidy, required to support the regime was In parallel to this, plant breeders saw lots of economically crippling the EU, and attracting opportunity to capitalise on a rapidly expanding significant criticism from the US and other domestic and EU market. Consequently, with countries at the WTO forum. The potential the direct encouragement of the supply chain, expansion of the EU to 18 and then 25 member varieties of cereal were developed that met the states further highlighted the looming funding climatic, agronomic and end user quality needs. crisis that faced the Brussels bureaucrats. Sustainability in the cereals supply chain 3

The advent of Agenda 2000 and its proposals to pressure to the cost of inputs to the farmer, with decouple support payments from units of farming fertiliser costs mirroring this price escalation. output was viewed as the only credible way Crops, therefore, that have high variable input forward for the EU. The payment of aid to farmers costs and low agronomic yield will become less was now to be targeted at maintaining land and attractive to farmers, unless these factors are agricultural practices that met environmental and reflected in their market value. quality standards with the level of aid gradually Wheat and barley will come under pressure being reduced over a fixed timeframe. It would from oilseed rape, peas and beans. In addition, have no reference to the level of output or type of farmers may well consider a move to fresh activity that the farmer engaged in. This updated produce crops, potatoes or sugar beet depending mechanism would, it was believed, improve the on ground conditions. competitiveness of European farmers in world Wheat and malting barley offer the highest markets. Following the implementation of these average yields at 3.75 tonnes per acre for wheat reforms, there has been a significant move in EU and 3.0 tonnes per acre for barley but input costs prices for cereals towards their world market remain the highest for all combinable crops. equivalent. However certain artificial barriers However these yields can vary by as much have been retained including intervention as 25%, depending on weather patterns and pricing for barley and maize. These mechanisms growing conditions. In addition, the farmer runs continue to distort the internal EU market, when the risk of not meeting the desired specification compared to world pricing levels. of his customers but this factor has been reduced, However, these decoupled support with the advent of dual purpose barley varieties mechanisms have for the first time created the such as Decanter, that attract customers over a opportunity for the farmer to consider how to range of nitrogen levels. obtain the highest economic return from the It is important at this point to reflect that land. In some extreme cases this may encompass farmers by nature remain conservative and will doing nothing other than land management. not radically change their behaviour or pattern Change of use for sporting and recreational of growing. However it is clear that with ex farm purposes seems to be a popular choice where prices for wheat at £65 per tonne and malting the proliferation of new golf courses and barley at £75 per tonne, these crops are unlikely leisure complexes is now almost at epidemic to achieve a long term commitment from the proportions. Those farmers that decide to stay grower. in their core business, are also closely reviewing Historically farmers have contracted with those crops that will deliver the maximum return their customers on an annual basis. This again from the market place. provides for no continuity or security to either Historically, combinable crop farmers’, party. In the past, these short term arrangements have taken little time to cost accurately their worked adequately, but where growers now production down to the level of £ per tonne have a required interest to maximise financial produced. This attitude has changed. Farmers returns, the ongoing availability of good quality will now assess from the range of combinable malting barley, in particular, could well be under crops a rotation that will deliver the maximum threat. Crops that maintain a poor profit margin potential return from their land. Fixed costs profile will become increasingly unpopular although important are not critical in this choices for farmers. decision making process. What is critical, are If that were not enough the growing threat the variable cost inputs and the average yield to of climate change and a weather pattern that in be achieved from a particular crop, in relation the UK has become increasingly unpredictable to its market value. The recent record levels of means that agronomic yields and the quality of oil and energy pricing have only added further crops at harvest will become more variable. 4 W.D. Rae

These factors will all combine to increase biodiesel from wheat, sugar beet, oilseed rape and short term volatility on supply availability and other feed streams will command a growing share ultimately market price. Indeed these conditions of available agricultural land. Whilst there is no have been mirrored in recent years where wheat short term threat, this alternative subsidised market and barley prices have fluctuated by as much as for combinable crops may encourage farmers to 40% from year to year. The only winner in such move away from their traditional markets. This circumstances is the market trader or speculator, is likely to pressurise further availability of good not the supplier or the customer. Increased quality cereals for distilling purposes. uncertainty on pricing of cereals is bad news for Today then, farmers are unhappy that their both farmer and distiller. Both seek the goal of traditional markets do not provide them with an a consistent and predictable cost and revenue adequate return on their investment. Farmers will profile, but their inability to work effectively increasingly seek markets that can provide them together means that neither party achieves this with a consistent and fair return. Plant breeders optimum goal. face difficulty in justifying future research into varietal development to meet our industry’s specialised needs. The weather is changing Potential shortage of high quality dramatically and industry has shown a distinct cereals lack of interest in attempting to manage and react to this change to the agricultural paradigm. Two other issues need to be borne in mind. After a flurry of varietal development from breeders in the 1980s and 1990s, the rate of The way forward delivery of improved varieties has significantly slowed. New varieties of wheat and barley to Is the spirits industry prepared to take its chances meet the specific needs of the distilling industry and let the market forces determine the solution? are now noticeable by their absence. Indeed the Is this an appropriate response from an industry industry today is almost exclusively reliant on that relies more than most on continuity of supply two barley varieties, Decanter and Optic and and the maintenance of quality raw materials to two wheat varieties, Consort and Claire. This is a underpin the development and growth of its worrying position, particularly given the lack of products and international brands? acceptable new varieties being brought through In my opinion it will be extremely foolhardy the Recommended and National Trial Lists. The of the industry to adopt such a laissez-faire rationalisation of breeding programmes, the attitude. Not only is this a recipe for ongoing increasing costs of varietal development and uncertainty and volatility but it increases the the lack of a substantive end market for these risk of there simply being insufficient providers varieties, will mean that distillers will have to rely of quality cereals prepared to commit to meet on those varieties that are developed for larger our specific needs as an industry. market sectors such as the brewing industry. Once What then is the solution? Can the industry again the risks to maintaining an available supply influence behaviour and protect itself and its of good quality cereals to meet our future needs relationship with its suppliers for the future? are greatly increased. There are obviously areas where little or no One new market that will become increasingly influence can be brought to bear. Control over available to the farmer in the UK and rest of Europe climate, the weather and growing conditions will be the biofuels industry. The EU Green Fuels remain, unfortunately, beyond even our grasp. initiative seeks to encourage member states to This remains one the largest factors affecting achieve a 5.75% inclusion rate of renewable agronomic yield. But even here, longer term energy in transport fuels by 2010. Bioethanol and solutions may well lie in developing varieties Sustainability in the cereals supply chain 5 and lines of crops that are more resistant to the However, until there is some openness on fluctuating weather patterns and more capable the costs of operating such a supply chain it is of withstanding heavy rainfall and strong winds. inevitable that each party will seek to protect Indeed the more entrepreneurial might even its own interest in preference to optimising the consider taking out hedge cover against weather overall effectiveness of the chain. At present conditions. there is no real value analysis undertaken that However there are areas where the industry tracks barley or wheat from seed through the can provide positive input to encourage the growing, harvesting, drying, processing and longer term commitment of suppliers. For any delivery activities to the end customer. There supply chain to work effectively it needs to is, therefore, no reliable benchmark to learn be well planned, responsive and sensitive to what the measure of fair value is, at any point customer needs. It also requires the involvement in the supply chain. As long as there remains of a limited number of participants, each of a perception by any one party in this chain that which is able to achieve a reasonable return it is not receiving its fair share of value, then from participating in the chain. Given their sustainability of the chain is under threat. Under commodity status, cereals can be traded both these circumstances it will be impossible to physically and on paper. However, it is clear build a longer term, secure trading relationship that any physical or paper transaction adds to from any participant’s perspective. What then the overall cost of supply. is required? It is essential, therefore, to consolidate Firstly it is essential that a value added the supply chain and remove the element of analysis is completed for the length of the speculative trading. How can this be achieved? supply chain, identifying economic margins that For any supply chain to work effectively, the require to be achieved by the limited number tighter and more direct the relationship between of participants. Secondly, a commitment should the supplier and the customer, the increased be given by all participants to invest in the likelihood there is of mutual commitment and relationship for a minimum period of three years. understanding to make the relationship work. Ideally the relationship should be for a longer In essence, the distilling industry needs to period, thereby enabling ongoing improvements get closer to its supply base. For many in the to the effectiveness of the supply chain as the industry, merchants, storekeepers and maltsters arrangement matures. Thirdly, transfer prices have become intermediate trading partners. should be established that meet the parameters For malting barley the use of maltsters has of the added value analysis, and crucially remove for many organisations been the sole point all participants from the volatility of the pricing of negotiation and trade. For wheat a limited in the market place. Fourthly, a commitment number of merchants and traders are utilised. should be given by all participants to achieve the This means that there are at least two separate required quality and environmental standards transactions for any trade in the chain from farm expected of the supply chain. In essence quality to processor. In practice there are many more! drivers underpin the agreed financial structure Is it effective that we hand over responsibility to established. Finally, a commitment should be these organisations to support our procurement given by all to encourage the development of processes? Does the farmer/grower receive a new crop varieties that will improve agronomic clear message from these organisations regarding and process yields. the strategic needs of the industry or are these Key concerns about this type of open organisations more intent on protecting their relationship generally gravitate to short term own profit margin than building relationships concerns by consumers or indeed growers that with growers? In reality the answer will vary open market conditions can be more favourable from relationship to relationship. than a structured arrangement. This is true but 6 W.D. Rae under these circumstances one party’s gain must Conclusions by definition be another party’s loss! In effect, open market trading tends to create winners and Given the many alternatives now available to losers within the supply chain! If a supply chain farmers/growers I believe we must as an industry is to truly succeed, and operate effectively, all review our current procurement strategies. Does participants must be winners. our present behaviour encourage sustainability? If the industry chooses to rely on a market It is my belief that it does not. We need to based procurement strategy it is inevitable consider whether we are all acting in the best that there will ongoing volatility of supply long term interests of the industry. The answer and pricing. Market forces will encourage lies in our own hands. entrants and leavers to the supply chain but With due deference to the Oxford Dictionary, what damage will be done in the interim? A I would therefore like to redefine sustainability sound, long term contractual basis of supply as follows:- that offers fair economic return will encourage ongoing commitment and participation and the Sustainability = Commitment + Sharing Value eradication of pricing volatility. Is this not what It is this formula that will truly deliver an effective we are truly seeking in an industry where we and long lasting cereals supply chain for our take on average eight years to bring our product industry. to market? Enhanced price predictability and assurance on the availability of quality raw materials must be key objectives for us all. Such Reference an environment, based on mutual benefit and stability, may well also encourage plant breeders European Commission (1999). Agenda 2000 – A to initiate re-investment in the development of an CAP for the future. http://www.ec.europa.eu/ enhanced range of suitable cereal varieties. agriculture/publi/review99/08_09_en.pdf (accessed 15 January 2008). New age spirit beverages – an indian perspective 7 Chapter 2 New age spirit beverages – an Indian perspective

B. K. Maitin The UB Group, Spirits Division – Technical Centre, Bangalore, India

Introduction The compatibilities of ingredients and formulations of the new age spirit beverages In recent years, there has been a pronounced are extremely challenging. It involves skilful growth in the trendy spirit beverages developed choice of flavours and speciality additives for specifically for the new generation of consumers. development of the product to meet with diverse This includes a wide variety of spirits and and varying consumer preferences. A brief liqueurs in exotic flavours and ready-to-drink account on these aspects along with the key form to suit the convenience and lifestyle of strategies of consumer driven innovative new young and women consumers. Incorporation product development is highlighted. of lifestyle and wellness or health related functions through herbs, spices, fruit juices, tea, coffee, and nutraceuticals has shown amazing New age spirit beverages - the global popularity and exponential growth among the soft drink segment worldwide. This trend has market trend considerably stimulated the Indian alcohol beverage industry to adopt similar concepts Ever changing consumer lifestyle has shown for developing innovative spirit beverages. significant impact upon demand for beverages, The consumer response on several prototype which are perceived as healthy and nutritious. products has been very encouraging. With rising concern about health among the This paper provides an insight into the consumers, the “Feel Good” factor is gaining prospects of unique youth and female oriented far more importance than the quantity of drink alcohol variants of several traditional Indian consumed. Other new generation spirit beverages beverages. The concepts, ingredients and show association with cocktail culture, image led preferred attributes are described in brief. designer drink and occasion driven choices such Additionally, an overview of the functionality as celebration, leisure, indulgence, business etc. of several well known Indian herbs and spices This in turn has provided market opportunity for acclaimed for prevention of diseases and the new age innovative beverages laced with longevity as per “Ayurveda”, the ancient Indian herbs, spices, juices and nutraceuticals which system of medicine, is discussed for potential appeal to different niches and satisfy a wide use in new age drinks. variety of thirsts. 8 B.K. Maitin

The current market trends of spirit beverages drinking style in India varies with different worldwide show an increased influx of new segments of consumers. The spirit is commonly and innovative flavoured and herbal or spicy mixed with carbonated soft drinks, soda or water spirit beverages, particularly vodka based drinks and consumed with or without ice. The drinks consumed by female and young consumers. are consumed mostly in the evening before food Flavoured vodka and rum have shown but with a sumptuous quantity of snacks. The phenomenal growth and escalating popularity drinking time is often compressed (within one leading to the proliferation of a rather wide range or two hours). of products by most leading manufacturers. While the growth of 5-8% abv RTD, FAB, Table 1. Consumtion of alcoholic beverages in LAB beverages has declined in the recent years, India.

15-20% abv shooter, shot and schnapps type Segment Volume products, such as “Slamma Shooters”, “Side (Million of 9 litre cases) Kick Shooter”, “Corky’s Vodka Shots”, “Bad Jelly Vodka”, “Kiss Me Tube Shooter”, “Baiki”, Whisky 67.20 “Cherry O” etc. have shown increased growth. Gin 3.30 Vodka 1.70 Likewise, 20-30% liqueurs and schnapps Rum 32.10 type products such as “Alize”, “Berentzen”, Brandy 19.60 “Archer’s Schnapps”, “ Amaretto”, “Bols Scotch 0.20 Blue Curacao”, are also gaining increased Beer 91.40 popularity. The preferred range of spirit variants RTD+Wines 0.97 include products with exotic flavours, prepared Total IMFL volume 124.00 cocktails and premixes, juice based spirits, herbs and spice associated drinks, low or no carbohydrate products with sugar substitutes, New age spirit beverages - current and frozen alcohol dessert. scenario and future potential

The trend and escalating popularity of new age Alcohol consumption in India beverages have stimulated the Indian alcohol beverage industry to adopt similar concepts for Alcohol drinking has a long history in many developing innovative spirit beverages. A range of the world’s cultures and prevails in most of exotic flavour variants of the mainstream societies. Mention of “Somaras” and “Sura” in vodka, gin, whisky, brandy and rum have shown the Vedas (Rig-Veda) evidences the consumption encouraging consumer acceptance. of invigorating beverages in ancient India. There In addition, many Indian traditional beverages is also evidence that in 2000-800 BC Aryans offer the potential for development of unique used fermentation and distillation to make their youth oriented alcohol variants. A few low drinks. alcohol ready-to-drink carbonated products The current consumption of alcoholic such as “Aampana” – a unique raw mango beverages in India is shown in the Table 1. flavour based mild spicy drink and “Kalakhatta” Besides IMFL (Indian Made Foreign Liquor), – a delightful sweet and sour beverage with beer and wine as well as the imported spirits and exotic fruit flavours were introduced into the wines, a huge quantity of more than 200 million marketplace. Prototypes of a few other drinks cases of country liquor is also consumed. such as “Thandai” and “Jaljeera” have also been Drinking practices vary substantially among tried out. The wide consumer acceptance of different countries basically due to differences these products has shown immense potential to in their ethnic and cultural characteristics. The grow and capitalise on tremendous Indian and New age spirit beverages – an indian perspective 9 global marketing opportunities. A few popular beverages. Some of the commonly used herbs Indian traditional drinks are listed in Table 2. and spices are listed in Table 3 along with their To propel this wave of popularity the needs reported health benefits. of a new generation consumers have been Some of these herbs and spices have great critically reviewed. Ever increasing efforts have potential for use in alcoholic beverages and been made to develop many new products on several such products are known internationally the basis of emerging insights using select herbs, and also in India. “Pastis”, “Pernod”, “Ozo spices, juices and health-promoting nutraceutical 12”, “Alize Bleu”, “Galliano”, “Benedictine”, ingredients. The consumer response on some “Kummel”, “After Shock - Hot & Cool”, “Kuya”, recent prototype products has shown good future “OP” and many other liqueurs, shots, shooters potential. Although, no health claim is permitted are known to incorporate herbs and spices. In in these products, there is immense scope to offer India, “Asha Liqueur”, an exotic heritage herbal unlimited range of products with exotic flavours drink incorporates more than 30 different herbs and “All Natural “benefits through herbs, spices and spices. A few variants of whisky with lime, and nutraceuticals . ginger and garcinia (kokum) were launched in India recently and have shown good consumer acceptance. Herbs and spices in alcoholic Like herbs and spices, association of tea and beverages coffee in spirit beverages is rather captivating and exciting. Many coffee based liqueurs such The association of herbs and spices with spirits as “Kahlua” and “Sambuca Caffe” are popular is known from ancient times. Historically India worldwide. A few tea based beverages are also is known for exotic herbs and spices. These known e.g. “Bacardi Rum Island Iced Tea”, are used for positive health benefits under “Tradeswind Gin Island Lemon Iced Tea” and “Ayurveda” – the knowledge or science of “Nikka Lemon Iced Tea & Vodka”. Tea offers life. “Ayurveda” was recorded in the “Veda”, opportunities for a large variety of products. world’s oldest known literature. Hundreds of Besides its unique taste, it provides proven Indian herbs and spices are known and they benefits of antioxidant and L-theanine. India is are invariably used in many exotic cuisines and well known for its quality tea. The introduction

Table 2. Traditional Indian drinks.

Drink Description Major ingredients

Aampanna Exotic cooling drink Raw mangoes, sugar, black salt, cumin , mint leaves Kalakhatta Refreshing sweet-sour Sharbet Indian blackberry, black salt, sea salt & pepper Jaljeera Classic Indian appetizer Cumin, mint leaves, jaggery, black salt, lemon juice, tamarind , chilli powder Thandhai Authentic nutritional Sugar, almond, dried seeds of muskmelon & water cooling drink melon, poppy seeds, aniseed, cardamom, rose petals, lotus stem seeds, cashew nut, pepper, saffron Rose Sharbet Heat fighter, fatigue buster Rose extract, sugar Shikanji Indian Lemonade - thirst quencher Lemon juice, sugar, salt, black salt Saffron Pista Sherbet Exotic healthy drink Sugar, pistachio, almond, rose water Lemony Ginger Sherbet Deliciously digestive drink Sugar, lemon, ginger, salt, black pepper & spices 10 B.K. Maitin

Table 3. Common herbs and spices. Common name Indian name Botanical name Potential health benefits

Winter Cherry Ashwagandha Withania somniferum Tonic, abortifacient, astringent, aeobstruent, mervine, aphrodisiac, sedative. Gives vitality and vigour. Cures rheumatism, leprosy and arthritis. Anise & Fennel Saunf Pimpinella anisum L . Anti-spasmodic, carminative expectorant, antacid, Foeniculum vulgare Anti microbial, memory tonic, skin conditioner, mother’s milk enhancer. Sacred Basil Tulsi Ocimum basilicum L. Antibacterial, antispasmodic, cures cough, colds fevers, arthritis, memory and nerve tissue strengthening, diaphoteric, anti periodic, stimulating, anti-catarrhal. Cumin Jeera Cuminum cyminum L. Stimulant, antispasmodic, carminative, respiratory health, stomach and intestinal health, kidney and liver function, circulatory and immune system support, Iron source. Cardamom Elaichi Elettaria cardamomum Appetizer, carminative, diaphoretic, digestive stimulant, expectorant, stomachic. Fenugreek Methi Trigonella foenum- Mucilaginous, emollient, febrifuge, restorative. graecum L. Useful for constipation, atherosclerosis, high cholesterol. Increases nursing mother’s milk. Ginger Adrak Zingiber officinale Antioxidant, analgesic, anti-emetic, aphrodisiac, Rosc. carminative, digestive, expectorant, useful in athero- sclerosis and high cholesterol, nervine, stimulant. Rose Gulab Rosa damascena Relieves stress, nervous tension, peptic ulcers, heart disease, positively influence digestion, womb disorders Quince Bael Aegle marmelos Antiscorbutic, carminative, alterative, nutritive, heart and brain tonic, digestive aid, useful in diahorrea, dysentery, laxative, stimulant and aphrodisiac. Carom Ajwain Carum copticum (L.) Useful in gastro intestinal disorders, respiratory disorders, migraine rheumatis. Mint Pudina Mentha arvensis Analgesic, anti-emetic, aromatic, aphrodisiac, carminative, diaphoretic, digestive, expectorant, nervine, sialagogue, stimulant. Liquorice Mulethi Glycyrrhiza glabra Tonic, diuretic, demulcent, expectorant and laxative. Used for allaying coughs and catarrhal infections. Coriander Dhania Coriandrum sativum Alterative, antibilious, antispasmodic, aphrodisiac, appetizer, aromatic, carminative, diaphoretic, diuretic, refrigerant, stimulant, stomachic, tonic, useful in dyspeptic complaints, loss of appetite. Tamarind Imli Tamarindus indica L. Mild laxative and digestive, useful in bronchial disorders, sore throat, antiseptic, treatment of ulcers, athartic, astringent, febrifuge, antiseptic, refrigerant. New age spirit beverages – an indian perspective 11

Table 3. Contd. Common name Indian name Botanical name Potential health benefits Cloves Lavang Eugenia caryophyllata Useful in digestive tract cancers, joint inflammation. Dentistry, toothache, anti-bacterial agent for sore throat, oral hygiene, antiseptic digestive aid. Nutmeg Jaiphal Myristica fragrans Aromatic, carminative, hallucinogenic, stimulant, Houtt expectorant, improves appetite and digestion. Myroblan Haradad Terminalia chebula Astringent, purgative, stomachic and laxative. Useful in asthma, piles and cough. Betel Leaf Paan Piper betle Treatment of stomach ailments, infections, and general tonic. It is often chewed in combination with the betel nut (Areca catechu), as a stimulatory. Has immune boosting & anti-cancer property. Aloe Vera Ghi Kunwar Aloe barbadensis Useful in X ray burns, dermatitis, skin disorders tonic used in jaundice, amenorrhoea, atonic seizure and piles. Heals wounds, ulcer and burns. Pepper Kali Mirch Piper nigrum Anthelmintic, antipyretic, antiperiodic, carminative, expectorant, stimulant, resolvent, rubefacient, stimulant. Cinnamon Dalchini Cinnamomum Aromatic, analgesic, antimicrobial, anti-rheumatic, zeylanicum antispasmodic, diaphoretic, digestive, diuretic, expectorant, stimulant, stomachic. Turmeric Haldi Lepidium sativum Alterative, anathematic, antibacterial, antibiotic stimulant. Amalaki Amla Phyllanthus amblica Rich in vitamin C, juice is tonic and is used in jaundice, amenorrhoea, atonic and piles, dermatitis. Saffron Kesar Crocus sativus Alterative, anodyne, antispasmodic, aphrodisiac, appetizer, carminative, expectorant, useful in fevers, melancholia, enlargement of the liver, and asthma, gout, whooping cough, snake bite. Long Pepper Pippali Piper longum Analgesic, anthelmintic, aphrodisiac, carminative, expectorant, useful in asthma, bronchitis, rheumatic pain, sciatica. Margosa Tree Neem Azadirachta indica Antiseptic, anti diabetic, anti bacterial tonic, useful in stomach, worms and ulcers, tonic, malaria, arthritis, eczema, skin disease. of a Rum Ice Tea with 5 % abv has received a Though no direct health claim is permitted, good consumer response. there is good potential for using them also in alcoholic beverages directly or as accessories. Development of such products, however, Nutraceuticals in alcoholic beverages requires in depth studies of the ingredients and skilful NPD efforts. Select nutraceuticals of Use of nutraceuticals in beverages has shown interests along with the possible health benefits exponential growth worldwide in recent years. are listed in Table 4. 12 B.K. Maitin

Table 4. Commonly used nutraceuticals.

Ingredients Potential health benefits Major manufacturers

Carotenoids (carotenes, lycopene, Antioxidants multiple protective Cognis, DSM, AIDP Inc., BASF, lutein, zeaxanthin & ,astaxanthin effects. Protection against Polyphenolics xanthophylls, vitamin A beta-carotene), developing certain, cancers, macular isoflavones, polyphenols, vitamin C degeneration, cataracts, protect & E, ellagic acid, flavanoids the heart from free radicals (Quercetin), etc. coenzyme Q-10 EGCG (Epigallocatechin gallate) Antioxidant, improves cardio- DSM vascular conditions and prevents cancer L-Theamine Provide relaxation, alleviates PMS Plantextrakt , AIDP Inc. L-Carnitine Energy nutrient, maintains Lonza AG heart health Branched Chain Amino Acid (BCAA) Decreases muscle damage, Amino Vital improves endurance, heightens mental focus and concentration Phytosterols and Phytostanols Lowers bad cholesterol, improves Reducol, Cargill, Cognis heart health Citicoline Protects brain-aging and head trauma Cognizin Soy Proteins and isoflavones Reducing the risk of cardiovascular Solae LLC,Cargill disease and certain cancers, prevents osteoporosis Conjugated Linoleic Acid - CLA Anti-Obesity Agent, lowers Cognis cholesterol and triglycerides Enhances immune system Omega 3 DHA Prevents cardiovascular conditions. BASF, Martek,DSM Reduces hypertension, aids in cancer prevention Glucosamine & Chondroitin Sulfate Joint pain relief, anti-inflammatory Cargill, NutraSense actions Creatine Muscle energy & repair, improves NutraSense, ProLab performance, fatigue & recovery Hyaluronic Acid & Collagen Joint health & skin nourishment, HyaMax,BioCell Collagen, Prevents premature aging AIDP,Inc Soluble Dietary Fibre Improves gut microflora Danisco

New product development – key Besides sensory profiling at every stage, it strategies requires comprehensive studies to optimise the levels of flavours and other additives used. Product development is an important aspect In fact, creating, selecting and using various of new age spirit beverages. The formulation additives in spirit beverages requires a rational of alcohol containing products with herbs and approach to balance various technical, legal and spices and/or nutraceuticals is rather complex. consumer issues. New age spirit beverages – an indian perspective 13

Intensive efforts are needed across a broad It saves time and money and avoids frequent spectrum of technical and marketing aspects re-visits to NPD drawing boards. for NPD (New Product Development) to meet Product Dimension Analysis is yet another the consumer’s perceived needs and delight. approach often used to accomplish consumer Flexible and responsive production is equally driven innovative product development. PDA crucial to meet the consumer demands and involves segmenting the market on the basis speedy launches of the products to market. of consumer preferences and consumption The traditional NPD practices need radical behaviours to capture the consumer needs changes in terms of consumer driven NPD of various segments. Consumer insights thus approaches. It requires structured NPD strategies obtained are then translated into quantitative involving applications of techniques such as and qualitative product attributes. PDA data Quality Function Deployment (QFD) and Product provides vital support to define the dynamic Dimension Analysis (PDA) to capture consumer marketing strategies for speedy introduction insight and their preferred attributes. The other of new products. A typical NPD process is important aspects of NPD include taste and illustrated in Figure 1. aroma profiling, analytical and sensory mapping, The availability of a wide range of innovative safety evaluation, regulatory compliance, colours, flavours and additives has enhanced the ingredients compatibility in terms of sensory versatility of a new generation of spirit beverages. quality and aesthetic appeal, as well as product These ingredients impart subtle character, stability. Optimal use of domain knowledge and aesthetic appeal, superb mouth feel, taste competency, production facilities and marketing and texture and better stability to the various networks of the mainstream spirit beverages products. Some of these are listed in Table 5. proves beneficial in accomplishing product success and achieving customer delight. Acknowledgements Quality Function Deployment (QFD) is a structured approach to define customer needs The author wishes to acknowledge his sincere or requirements and translate them into specific thanks to all whose information has been referred plans to produce products to meet those needs. in the listed bibliography.

Idea Idea Generation Screening Evaluation Development Commercialization

• QFD • Internal brainstorming • PDA • Consumers insight • Develop model • SWOT Analysis • Competitor products • Test marketing • Fit with objectives • Revise plans as • Market trends needed

• Finaliseproduct launch • “Roll Out”in select markets • Concept Testing • Market Research • Estimate cost, sales and profits Figure 1. Product development process. 14 B.K. Maitin

Table 5. Food additives. Drinks and Beverages. http://www. indiaforvisitors.com/food/drinks.htm Additives Perceived benefits Functional Foods and Nutraceuticals. http:// www.mednd.com/html/Healthy_Eating/ Food colours – Aesthetic appeal – natural Functional_Foods_And_Nutraceuticals. natural & artificial & artificial htm#Bibliography Flavours – natural Character enhancement – Grieve, M. (1931). A Modern Herbal. http:// & nature Identical masking undesirable botanical.com/botanical/mgmh/comindx. aftertaste html Sweeteners :sugars Sugar & non – sugar Herbal Medicine. http://www.holistic-online. (CHOs) & non-sugars (low or no carbohydrate) com/Herbal-Med/Hol_Herb_Directory_ (eg. Sucralose-600X, products Index.htm Stavia 30X, Thaumatin Jyotsna Kamat, “Drinking in Ancient India/ 2000X) Karnataka”. http://www.kamat.com/ Acidulants : Malic Mouthfeel, stability & pH kalranga/ancient/drinking/inkar.htm acid, Citric acid, Katzer, G., Spice Pages. http://www.uni-graz. pHase, etc. at/~katzer/engl/generic_frame.html?Rosa_ Preservatives – Microbial protection dam.html Benzoates, Sorbates Nimhans Report (1991). http://www.nimhans. kar.nic.in/deaddiction/lit/UNDOC_Review. Emulsifier & stabiliser Texture, appeal & stability pdf, pp. 6 -17 gums, pectin etc. – gum acacia , SIRC, Social and Cultural Aspects of Drinking. nanosove transparent http://www.sirc.org/publik/drinking_origins. emulsion html Swamy, S. S. , Ayurveda - Theory and Practice. http://www.dlshq.org/messages/ayurveda. Bibliography htm#importance Spirit Beverages. http://www.drinkfinder. co.uk/ Ayurveda Marketplace, Indian Herbs. http:// default.asp www.ayurveda-herbal-remedy.com/indian- Spirit Beverages. http://www.thedrinkshop. herbs/index.html com/ Cold Beverages. http://www.indianfoodsco.com/ Wright R. (2005). Neutraceutical World, 8, 3, Recipes/bevearge_images/ColdBeverages. 46 htm Wright R.( 2005). Nutraceutical World, 8, 9, Company Capabilities (2005). Nutraceutical pp. 32-48 World, 8,2, pp. 4-122 Supplements. http://www.wholehealthmd.com/ Cooper, R. (1982). Spirits and Liqueurs, HP refshelf/items_index/1,1538,HS,00.html Books, USA, pp. 80–107 The world’s healthiest foods. http://www. Costa, Ana I de A. (2003). New Insights into whfoods.com/nutrientstoc.php consumer-oriented food product design, Tarla Dalal Receipes. http://www.tarladalal.com/ Ph.D. Thesis, Wageningen University, The ViewContributedRecipe.asp?Id=6. Netherlands, pp. 28-55 Orange press liquor spirit 15 Chapter 3 Orange press liquor spirit: technical and economic aspects of a new distilled beverage

J.B. Faria, H. Roçafa Jr. and J.O. Ferreira UNESP, São Paulo State University, Departamento Alimentos e Nutrição, 14801-902, Araraquara, SP, Brazil

Introduction The sensory characteristics of the orange liquor spirit The orange juice industry produces, at the end of the process, a by-product called orange press The object of this study was to examine whether liquor. It has a high soluble solids content and it was possible to obtain a distilled spirit from is usually concentrated and added to citrus orange press liquor using a process similar pulp pellet (CPP) processing (Tetra Pak, 1988; to that used in the production of cachaça. Kimball, 1999). Considering the large volume of Samples of orange press liquor and sugar cane liquor produced by Brazilian orange juice plants juice were fermented, double distilled, aged in and for technical and economic reasons, the use small casks and compared using sensory of orange press liquor as a raw material for a new analysis techniques. The results of sensory product was evaluated. This work was carried acceptance tests for colour, aroma, flavour and out to investigate whether cachaça technology overall impression, obtained from the orange (Faria, Loyola, Lopez and Dufour, 2003) could liquor spirit and cachaça samples (see Figure be used to produce a new distilled beverage, 1), showed similar scores, indicating that this having typical characteristics and good flavour beverage, obtained under the same conditions and aroma.

9 Cachaça Orange liquor spirit 8 7 6 5 4

Hedonic scale 3 2 1 0 ColourAromaFlavour Overall impression Figure 1. Acceptance tests related to colour, aroma, flavour and overall impression of cachaça and orange spirit samples. 16 J.B. Faria et al. as cachaça, could be acceptable in sensory terms Optimizing the production process (Roçafa, Padovan and Faria, 2005). In order to optimize the orange liquor spirit process, the following options were tested and The orange juice industry and the compared (Ferreira, 2005). proposed new beverage 1. The effects of changing the pH value and Apart from the technical viability of using orange the soluble solid concentration of the orange liquor to produce a new beverage, another press liquor, before fermentation; important point to be considered was the interest 2. The kind of yeast used to ferment the liquor of the orange juice industry in incorporating this (bakers’ or re-used cachaça yeasts); new beverage in its production. 3. The maximum fermentation time (24 and 48 The Brazilian orange industry is a very well hours); established business, but lately it has been 4. The alembic material used to distill the spirits faced with important challenges, the main one (copper or stainless steel); being the international orange juice market. 5. The wood casks used for aging the spirit, oak The appearance of exotic new fruit juices in the or castanheira, and; market, and the establishement of new orange 6. The possibility of adding sugar to the final juice producers in Central America and South product (5.5 g/L or no sugar added). Africa, must be taken into account. The growing tarif barriers and orange diseases, as well as Based on the results obtained in the trials related the market dificulties mentioned above, are to fermentation, the following conditions were highlighting the necessity to increase efficiency adopted: and optimise investments, thus adding value to existing products as well as developing new • dilution of the liquor with water (1:1); ones. • pH correction from 5.4 to 4.4; So, based on the above reasons and taking • re-use of cachaça yeasts; into account the great volume of liquor generated • 24 h as maximum delay for fermentation. by the orange industry, as well as the growing international market for new distilled spirits, the Some characteristics of the press liquor before next steps of this study were: to optimize the and after fermentation and of the distillation orange liquor spirit process; and to evaluate the residue are presented in Table 1. economic aspects of its production.

Table 1. Characteristics of press liquor, wine and residue after distillation. Analysis Unit Crude press Diluted press Fermented Residue Residue Liquor Liquor press liquor (stainless steel) (copper) TSS (°Brix) 33.1±0.1 16.6±0.0 12.3±0.0 14.1±0.0 14.8±0.0 Sucrose (g/L) 86.19±1.58 43.10±1.22 0.01±0.00 1.98±0.24 1.87±0.24 (g/L) 54.84±1.62 27.42±0.98 1.37±0.10 2.06±0.32 1.98±0.28 Fructose (g/L) 67.63±1.06 33.82±1.00 7.95±0.31 11.70±0.23 10.88±0.25 Protein (g/L) 30.88±2.82 15.44±1.32 13.15±0.97 15.20±0.86 16.01±1.20 Ethanol (g/L) 2.80±0.00 1.40±0.00 69.80±0.92 3.58±0.02 0.17±0.00 pH - 5.4±0.0 4.4±0.0 4.6±0.1 4.6±0.0 4.6±0.0 COD (g/L) 181±18 90±11 114±8 150±16 152±19 BOD (g/L) 139±25 70±16 87±15 115±16 117±20 Copper (mg/L) 2.74±0.02 1.37±0.01 0.59±0.00 0.84±0.01 102.21±2.03 D-Limonene (%) 0.035±0.005 0.018±0.002 0.016±0.003 0.007±0.001 0.008±0.001 Orange press liquor spirit 17

The wine obtained, according to the above In fact the yield of both alembics as well as the fermentation conditions, was distilled in copper copper and dimethyl sulfide contents and the and in stainless steel alembics and aged for six absence of ethyl carbamate, indicated that the months in two miniature oak (Quercus spp) and distillation of the orange liquor wine, whether castanheira (Brazil nut, Bertholletia excelsa) copper or stainless steel alembics were used, casks with a capacity of 5 litres. The samples produced similar results. The results presented were then analysed and the results compared. in Table 3 about aging in oak and castanheira The distillation experiments (see Table 2) showed casks also showed similar results regarding the that in both cases the tests gave similar results. main components. Table 2. Characteristics of distilled liquor spirit sample. Dimethyl Ethyl Analysis pH Ethanol Copper COD BOD D-Limonene sulfide carbamate - (% v/v) (mg/L) (g/L) (g/L) (%) (µg/L) (mg/kg) Spirit - 1st distillation (stainless) 29.1±0.2 0.0±0.0 n.d. Spirit - 1st distillation (copper) 34.3±0.3 21.2±0.1 n.d. Spirit - 2nd distillation (stainless) 55.7±0.3 0.0±0.0 8.1±0.6 n.d. Spirit - 2nd distillation (copper) 54.8±0.2 3.6±0.0 4.7±0.2 n.d. Head - stainless steel 66.7±0.3 0.0±0.0 n.d. Head - copper 67.5±0.4 2.7±0.0 n.d. Tail - stainless steel 13.6±0.0 0.0±0.0 n.d. Tail - copper 7.2±0.0 19.8±0.2 n.d. Water - stainless steel 3.4±0.1 0.1±0.0 0.0±0.0 1.5±0.4 1.1±0.4 0.006±0.001 n.d. Water - copper 3.7±0.1 0.1±0.0 40.9±0.3 3.5±0.6 1.8±0.5 0.008±0.001 n.d. Detection limit for ethyl carbamate = 0.01mg/kg

Table 3. Aged orange liquor spirit sample characteristics. Analysis Unit Oak Castanheira Copper Stainless Copper Stainless Ethanol (after aged) % v/v 55.8±0.1 57.0±0.0 56.0±0.1 56.8±0.1 Ethanol (corrected with water) % v/v 40.0±0.0 40.0±0.0 40.0±0.0 40.0±0.0 Total sugar (g/100mL) <0.1 <0.1 <0.1 <0.1 Volatile acids as mg/100mL alcohol 53.1±2.3 52.0±1.0 52.9±2.1 52.2±2.0 acetic acid Alcohols mg/100mL alcohol 235.0±5.2 257.6±6.5 232.2±4.8 259.1±6.0 Furfural mg/100mL alcohol 7.8±0.5 5.1±0.4 7.2±0.6 5.3±0.5 Aldehydes as mg/100mL alcohol 0.027±0.002 0.013±0.001 0.015±0.001 0.021±0.002 acetic adehyde Esters as ethyl acetate mg/100mL alcohol 48.4±0.5 26.0±0.2 44.7±0.8 27.4±0.3 Secondary compounds mg/100mL alcohol 344.3±8.5 340.7±8.1 337.0±8.3 344.0±8.8 mg/100mL alcohol 26.0±0.5 25.0±0.6 25.6±0.5 25.8±0.7 Copper mg/L 1.6±0.1 0.1±0.0 2.6±0.1 0.1±0.0 Organoleptic analysis - OK OK OK OK 18 J.B. Faria et al.

Finally the sensory acceptance test results, medium size Brazilian plant (25 – 30 million comparing (copper and stainless steel) distilled boxes of oranges processed a year), operating and (oak and castanheira) aged samples (with 5.5 during 4 months a year, 26 days each month and g/L of added sugar) shown in Figure 2, as well as 23 hours a day. The proposed process, including the distilling and aging experiment results, lead the traditional CPP production and the orange to the conclusion that copper or stainless steel liquor spirit is presented in Figure 3. alembics as well as oak or castanheira casks may Based on the laboratory trials , and taking be used to produce the orange liquor spirit. into account the losses in these conditions, the estimated yield was 11% higher than that obtained by Braddock (1999) for orange Some economic aspects related to alcohol production (88.5 L/m3). The main orange liquor spirit production operational costs related to the orange liquor spirit production are shown in Table 4. For the economic evaluation of the spirit The steam used for the distillation process production, we considered only a small part represented 42% of the operational costs, i.e. (1/15) of the raw orange liquor generated in a approximately 18% of the final product overall costs. The required investments are mainly

7.0 6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 Copper/Castanheir a Stainles s Copper/Oa k Stainless Sell/Oat k Steel/Castanheir a Aroma Flavou r Overall Impression

Figure 2. Acceptance tests of aroma, flavour and overall impression of the orange liquor spirit samples.

Table 4. Operational costs per litre of orange liquor spirit produced. Operation Item Parameter R$/ano R$/L US$/L Labor 3 operators 52,650.00 0.022 0.007 Maintenance – 100,000.00 0.042 0.014 Electric power 70kwh 30,306.64 0.013 0.004 Steam 5t/h 577,428.80 0.241 0.080 Utilities – 150,000.00 0.063 0.021 CIP NaOH@3% 4,000.00 0.002 0.001 Ingredients (sacarose) 5.5g/L 23,230.72 0.010 0.003 Acid 2.26mL/L 5,640.65 0.002 0.001 CPP loss -f ueld oil econ. – 192,637.69 0.080 0.027 Logistics (FOB Santos) 500km 208,186.12 0.087 0.029 Others – 30,000.00 0.013 0.004 Total – 1,374,080.62 0.573 0.191 Orange press liquor spirit 19

27.5 million orange box (40.82kg) 563,750 t bagasses (peel, seed, pulp etc.) 80.5% moisture Bagasses Buffer Tank 563,750 t bagasses 80.5% moisture Mill 1,824 t lime 565,574 t bagasses

From Oil Recovery System Reaction Tank 94,079 t oil water 811,626 t bagasses5.0 ºBrix 79.7% moisture ST 454,765 t liquor 1 Press Buffer Tank 13.7ºBrix 356,861 t bagasses 548,844 t press liquor 71.4% moisture12.2 ºBrix 198,652 t liquor 202,632 t liquor Conveyor Waste Heat 33.0ºBrix 33.1 ºBrix 555,513 t bagasses 69.8% moisture 246,053 ND 346,213 t water + terpene 2 Press t liquor 22.0ºBrix 309,461 t bagasses 191,350 t liquor 63.3% moisture 33.1 ºBrix 12,860 181,114 t steam Dryer t fuel oil (waste heat) 128,346 dry peel 11.5% moisture 11,282 t liquor 33.1 ºBrix Pellet Machine Orange Spirit 721 Cooler Production t water 7,302 t liquor 127,625 t CPP31.0 ºBrix 11.0% moisture Storage 2,400,000 L Orange Spirit

Figure 3. Proposed lay-out for the orange industry, including liquor spirit production. related to the costs of the casks used for the Conclusions ageing process (68.2%) and of the building to store them (17%), following by the fermentation The production of orange liquor spirit (Faria, and distillation apparatus (10,7%), stainless Roçafa and Ferreira, 2005) represents an steel tanks and other small acessories, totalling interesting process both technically and approximately US$ 4,114,666.00. To evaluate economically. The double distillation and the investment costs the following methods were aging process achieved a quality for the orange proposed: Pay back of investiments (PB); Present liquor spirit comparable with cachaça and other value (PV) and Return investment tax (RIT), as similar products. The return of investment is described by Gitman (1997). The results obtained assured even using only 5.7% of the orange (Table 5) show that, even allowing for some liquor generated by a medium size Brazilian problems, orange liquor spirit production by the plant, indicating the great potential for further orange industry seems to be very interesting from growth. the economic point of view. 20 J.B. Faria et al.

Table 5. Investments analysis required for orange liquor spirit production.

Payback (PB) = 2.02 years: (+) Sales = 2,400,000L x 1.04US$ x 3.00R$ = R$7,488,000.00 (-) Operational Costs = R$1,374,080.62 (=) Cash Flow = R$6,113,919.38 Investments = R$12,344,121.15 Present Value (PV) = R$377,675.24 Investments = R$12,344,121.15 and Interest = 16.00% (per year)

Year 1 2 3 4 5 Total

Receipts 0.00 7,488,000.00 7,488,000.00 7,488,000.00 7,488,000.00 29,952,000.00 Costs (1,374,080.62) (1,511,488.68) (1,662,637.55) (1,828,901.30) (2,011,791.43) (8,388,899.58) Cash generated (1,374,080.62) (5,976,511.32) 5,825,362.45 5,659,098.70 5,476,208.57 21,563,100.42 Present Value (1,184,552.26) (4,441,521.49) 3,732,063.16 3,125,469.83 2,607,294.17 12,721,796.40

Return Investment = 17.01% with Spread = 1.01% Investments = R$12,344,121.15 and Interest = 16.00% (per year)

Year 0 1 2 3 4 5 Total

Receipts 0.00 0.00 7,488,000.00 7,488,000.00 7,488,000.00 7,488,000.00 29,952,000.00 Costs 0.00 (1,374,080.62) (1,511,488.68) (1,662,637.55) (1,828,901.30) (2,011,791.43) (8,388,899.58) Investment (12,344,121.15) 0.00 0.00 0.00 0.00 0.00 9,218,979.26

References Técnico-Econômica da Produção Industrial da Aguardente do “licor” de Laranja. Braddock, R.J. (1999). Handbook of Citrus Dissertação (Mestrado em Alimentos By-Products and Processing Technology. e Nutrição), Faculdade de Ciências University of Florida, John Wiley & Sons, Farmacêuticas, UNESP, Araraquara, 96p. Lake Alfred-USA, 247p. Gitman, L.J. (1997). Princípios de Administração Faria, J.B., Loyola, E., Lopez, M.G. and Dufour, Financeira. Editora Harbra, São Paulo, pp. J.P. (2003). Cachaça, pisco and Tequila. In: 100-378. Fermented Beverage Production. Edited Kimball, D.A. (1999). Citrus Processing: by Lea, A.G. and Piggott, J.R., Kluwer A Complete Guide. Aspen Publishers, Academic/Plenum Pub., New York, pp. Gaithersburg-USA, 450p. 335-363. Roçafa Jr, H., Padovan, F.C. and Faria, J.B. (2005). Faria, J.B., Roçafa H. and Ferreira, J.O. (2005). Obtenção de uma bebida fermento-destilada Processo de obtenção de uma bebida a partir do licor de laranja. Alimentos e fermento destilada a partir do “licor” de Nutrição 16: 321-325. laranja. Pedido de Patente (Brasil) 17 de Tetra Pak (1998). The Orange Book. Tetra novembro de 2005. Pak processing System AB, Lund-Sweden, Ferreira, J.O. (2005). Estudo de Viabilidade 206p. Influence of base wine composition and quality on style and quality of South African brandy 21 Chapter 4 The influence of base wine composition and quality on the style and quality of South African brandy

C.L.C. Snyman1 and M.G. Lambrechts2 1Distell Group Ltd, PO Box 184, Stellenbosch 7599, South Africa; 2Institute for Wine Biotechnology, University of Stellenbosch, Stellenbosch, South Africa

Introduction This study thus aimed to determine the influence of brandy base wine composition Brandy production is a multi-step process that on the quality of the respective unaged potstill involves harvesting of grapes, fermentation distillates as well as the ultimate quality and of the brandy base wine, distillation, wood character of the three-year old wood matured maturation and blending to produce a final potstill distillates. commercial product. Within each of these steps of the production process there are a number of factors that can influence the composition Experimental and resultant quality of the base wine, unaged and wood matured distillate. These factors The pipelines feeding to four potstills of 2000 include geographic and climatic features of litre capacity each within a large commercial the origin of grapes used, viticultural practices, distillery belonging to Distell in Worcester, grape maturity, grape variety, vintage variation, were adapted to be filled with brandy base vinification techniques, storage of the base wine wine from six 40 000 litre charger tanks. In this prior to distillation, distillation technique, age manner it was possible to separately receive, and origin of oak wood used for maturation and store and distill approximately 35 000 litres of barrel toasting levels. each brandy base wine selected from different In commercial brandy production, standard regions and producers. Thirty-three wines were operating procedures do not allow for the separate distilled in 1999 and twenty-five wines were distillation of different brandy base wines due to distilled in 2000. Wines were selected based the size of the wine receiver tanks as well as the on an initial chemical analysis and sensory continuity of the distilling process (24 hours a day, evaluation to ensure that all of the wines 7 days a week). Thus mixing of base wines occurs used in the experiment met the South African once they are delivered to the distillery. This legal specifications for brandy base wine. The makes it difficult to determine the exact influence specifications state: residual sugar < 4 g/L; total of base wine composition and quality on final SO2 less than 20 mg/L; volatile acidity less than brandy quality. However, a considerable amount 0.7 g/L; alcohol content = 10-12% v/v. Each of time goes into the analysis and approval of wine was treated separately (i.e. separate storage brandy base wines prior to purchasing. and distillation) and the resultant distillates were 22 C.L.C. Snyman and M.G. Lambrechts also stored separately. Wine samples were drawn their good extraction reproducibility (variation as soon as they had been offloaded from the coefficient < 20%). road tanker into the storage tank after thorough It is critical to bear in mind that, due to the mixing. scale of this experiment, and the ultimate aim Minimising the storage time of wine prior to of the study, the experimental outlay reflects distillation is a practical issue and depends on commercial practice. Distillation took place the volume of tank space available, tank cooling over a period of almost three months and ran facilities and the daily distillation capacity of concurrently with the season’s commercial a commercial distillery. In this study, no tank at the distillery. The distribution cooling facilities were available at the distillery of wines distilled per region and producer also and all wines were therefore stored for no longer closely reflects that of commercial practice than three days prior to distillation in order to during the course of the potstill distillation minimize the possibility of microbial infection season. in the base wines. Commercial scale double The brandy base wines and distillates were distillation took place in four identical copper evaluated by a panel of expert brandy judges potstills with a capacity of 2000 litres each. (with many years of experience in the South Samples of the low wine (first distillate) and African brandy industry) to assess the sensory of the heads (every 5 minutes until the liquid quality of these products. All samples were temperature reached 82°C), heart (well-mixed evaluated blind, in a random order and the sample from product receiving tank) and tails distillates were evaluated at 20% v/v through fraction (one sample every hour for the 8 hours dilution with distilled water. The base wines of the tails distillation) were taken for analysis. and unaged distillates were scored using a The heart fraction of each of these wines was 5 point scale, were 0 = unacceptable and 5 then filled into six 340-litre French oak barrels = outstanding. The matured distillates were to mature for three years, in compliance with the evaluated using a 10 point line scale, where 0 South African law on brandy. = unacceptable and 10 = outstanding. Base wines were analysed using the method The distillate sensory evaluation was then described in Steger and Lambrechts (2000). used to select 12 distillates (6 from 1999 and 6 Twenty-eight volatile compounds were identified from 2000) for aroma profiling using the South in the base wines. Among these 28 compounds, African brandy aroma wheel (Jolly and Hattingh, 21 were selected for statistical analysis because 2001) and sensory descriptive analysis, using of their good extraction reproducibility (variation a panel of trained tasters at the ARC Infruitec- coefficient < 20%). Nietvoorbij (The Fruit, Vine and Wine Institute The distillate analysis was performed using of the Agricultural Research Council). In each column gas chromatography. Five ml of the year two poor quality, two average and two potstill distillate samples were mixed with 0.25 ml good quality distillates were selected for the of a 2000 mg/L solution of 4-methyl-2-pentanol, full descriptive analysis (SDA). The SDA was which served as an internal standard. Samples performed by a panel of eleven judges. The were eluted using the method developed by brandy aroma wheel was developed using Blom (2000) and were run on a Hewlett Packard standardised descriptive aroma terminology HP5890 gas chromatograph, coupled to an (descriptors) for South African brandy aroma. The HP7673 auto sampler and injector, and an HP terminology was developed to be applicable at all 3396A integrator. The same operating conditions stages of brandy production and, consequently, were used as described in Steger and Lambrechts only small sections of the listed descriptors are (2000). Injection volume: 1 µl. Twenty-seven likely to be used at any one stage. Negative volatile compounds were identified in the descriptors are also incorporated to describe distillates. Among these 26 compounds, 21 faults that may occur during the production were selected for statistical analysis because of process. The positive descriptors have been Influence of base wine composition and quality on style and quality of South African brandy 23 arranged in a progression from aromas that all of the base wines, unaged distillates and occur most frequently in young distillates three-year old potstills over two successive (‘herbaceous’, ‘fruity’, ‘smooth associated’) to vintages in order to determine whether there more mature aromas (‘sweet associated’, ‘nutty’ was any significant correlation between these and ‘spicy’). The fruity aromas are arguably the scores. A correlation is significant at a p-value most important in brandy and make it uniquely of less than 0.05. As is evident from Table 1, different and distinguishable from other distilled a significant correlation was found in five of products such as whisky and rum (Jolly and the six relationships, the exception being the Hattingh, 2001). The ‘muscat’ and ‘floral’ notes correlation between the 2000 base wine and are particularly prominent in brandies made from three-year old potstill scores. The five significant aromatic Muscat type varieties, not applicable correlations were all found to be positive. in this study. ‘Woody’ and ‘toasted’ notes are those derived from maturation in oak wood Table 1. Spearman rank order correlations on (Jolly and Hattingh, 2001). The eleven judges sensory data. were trained on nosing standards for six sessions prior to evaluating the distillates. The samples Pair of variables Valid Spear- t(N-2) p-Value N man R were presented blind. Each judge was asked to mark the aroma intensity of the following 1999 wine score and 33 0.563 overall descriptors: fruity, herbaceous, smooth 3.7944 <0.001 1999 distillate score 1999 wine score and 33 0.375 2.2543 associated, floral, sweet associated and spicy 0.031 1999 3 yr score as well as negative aromas (heads, tails, musty, 1999 distillate score 33 0.439 2.7245 0.01 solvent/chemical), using a 10 cm line scale, and 1999 3 yr score where 0 indicated “not detectable” and 10 2000 wine score and 25 0.412 2.171 0.04 indicated “very prominent“. An average score 2000 distillate score was then calculated from the scores of the eleven 2000 wine score and 25 0.214 1.052 0.3 judges and was plotted graphically. 2000 3 yr score Analysis of variance, principal component 2000 distillate score 25 0.51 2.844 analysis, regression, Pearson correlation, 0.009 and 2000 3 yr score Spearman rank order correlation analysis and multivariate adaptive regression splines (MARS) were used to study the relationship between Regression analysis using sensory score as sensory quality of base wines and distillates and dependent variable on routine base wine composition of these base wines and distillates analyses using Statistica version 6.0. The significance level used as a guideline for accepting or rejecting It is commercial practice to do a so-called hypotheses was 5% (0.05). Classification and routine analysis on each brandy base wine that regression tree analysis was performed using the is presented to the distillery for purchasing. CART program from Salford Systems. This analysis has its origin with the South African liquor law and the dates back to the time when the South African Brandy Board Discussion had legislative power to control the quality of wines used for brandy distillation. Although Correlating wine to unaged distillate to three the activities of the South African Brandy Board year old distillate quality have been disbanded, the industry continues to adhere to these technical specifications as a A non-parametric Spearman rank order correlation guideline in the selection of base wines to be test was performed using the sensory scores of purchased. It is important to bear in mind that 24 C.L.C. Snyman and M.G. Lambrechts a brandy base wine is not merely purchased Table 2. Relationship between individual routine based on the results of this routine analysis, base wine analysis and average wine score. rather the final purchasing decision is based on its passing of a sensory evaluation. Thus, a base Independent variable R2 wine presented to the distillery for purchasing p-level may pass the technical requirements and may Alcohol 0.001 0.850 not be purchased if its sensory character is Residual sugar 0.090 0.102 displeasing. Although a base wine that fails the Volatile acidity 0.046 0.245 analytical criteria will not be purchased even if it Total acidity 0.110 0.069 is sensorially acceptable. The following analyses Total SO2 0.003 0.771 form part of this routine analysis: residual sugar, Free SO2 0.090 0.108 volatile acidity in mg/L, alcohol in %v/v, total pH 0.048 0.234 Polyphenols 0.187 0.015 SO2 in mg/L, and total polyphenol content less than 250 mg/L. The maximum allowable values for base wine are mentioned in the introduction of this paper. all six of the routine analyses and wine quality As these routine analyses already form showed that no significant inter-relationships existed part of the analytical infrastructure for brandy (data not shown). Wine quality tended to decrease production, it was decided to investigate whether as volatile acidity levels increased and wine quality any one of the above-mentioned analyses might tended to increase as wine pH decreased. yield an indication as to the expected quality From Table 3 it is evident that the only of the base wine, unaged distillate and/or the significant relationship found in the unaged three-year old matured potstill distillate. All base distillates was that of base wine alcohol content wines used for this study fell within the above- and distillate quality, where quality tended to mentioned specified parameters. It was hoped to increase as the concentration of alcohol in the find an indication of sensory quality within these base wine increased. Polyphenol concentrations analytical parameters using single and multiple exhibited the same trend as discussed for the base regression analysis. wines. Base wine pH also tended to decrease as distillate quality increased. Table 2 summarizes the R2 and p-level values for a single regression analysis for each of the six analyses that comprise the routine analysis. Table 3. Relationship between individual routine base wine analysis and unaged distillate score. The dependant variable used as an indication of quality was the average score of the base wines. As Independent variable R2 is evident from Table 2, only the total polyphenol p-level content showed any significant relationship to base wine quality. It is evident that, even though Alcohol 0.160 0.026 all of the base wines had a polyphenol content Residual sugar 0.037 0.302 less than or just equal to 250 mg/L (the maximum Volatile acidity 0.066 0.164 value of this specification), there is a tendency Total acidity 0.017 0.481 that as polyphenol concentrations increase, the Total SO2 0.001 0.844 quality of the base wines decrease. Although not Free SO2 0.004 0.746 pH 0.032 0.338 deemed significant, total acidity and wine score Polyphenols 0.087 0.107 yielded the same trend i.e. as the total acidity increases, so too did the average wine score (data not shown). A multiple regression analysis, which As was noted in the unaged distillates, there tries to establish the inter-relationship between was a significant relationship between the Influence of base wine composition and quality on style and quality of South African brandy 25 three-year old potstill quality scores and the An analysis of variance was performed on the base wine alcohol concentration (data not volatile compound data of each of the twelve shown). Although there were no other significant composite distillates profiled using sensory relationships between three year old distillate descriptive analysis. This was done in order quality and the remaining 5 analyses, there was to determine significant differences in the a tendency for the score of the three-year old concentration of these compounds between the distillate to increase as the value for base wine good, average and poor quality distillates after pH and volatile acidity decreased. The multiple one, two and three years of wood maturation. regression analysis using the three-year old This paper only focuses on the results of 6 potstill score yielded no significant results (data distillates in order to illustrate the results obtained not shown). in the study. Additionally, only the results after the third year of maturation are discussed. The results discussed in this paper reflect the results Volatile compound concentration differences obtained in the complete study. between distillates profiled using sensory Table 5 lists the concentrations of selected descriptive analysis volatile compounds present in six base wine samples. Table 6 lists the concentrations of Twelve distillates (6 from 1999 and 6 from 2000) selected volatile compounds present in the same, were identified from the sensory evaluations for respective six matured distillate samples (3 years a further sensory descriptive analysis. For each old). Please note that Tables 5 and 6 focus only on of these two years, 2 were selected on the basis those compounds where differences did occur. of their evaluation as good quality distillates, 2 Other volatile compounds were quantified (as average quality and two poor quality distillates. discussed in the experimental design) but are Although they were selected based on their not listed or discussed in this paper. quality as unaged distillates, it was proven in From table 5, it is evident that the concentration the first part of this discussion, that distillates of acetic acid and diethyl succinate is significantly retained their sensory quality rating after three higher in the base wines judged to be of poor years of wood maturation when they were quality. On the other hand, the concentration of organoleptically evaluated again. As the sensory ethyl caprylate was higher in the good quality evaluation scale was a 10 cm line scale, scores base wine samples and in one sample of the below 4 were regarded as being poor quality, base wine judged to be of average quality. between 4 and 7 as average quality and above From table 6 it is evident that the concentration 7 as good quality. Table 4 lists the respective of acetaldehyde, ethyl lactate, hexanol and sensory scores for the six samples. diethyl succinate is significantly higher and the concentration of iso-amyl acetate is significantly Table 4. Sensory scores for six distillate samples lower in the two poor quality 3 year old using for sensory descriptive analysis. distillates. The concentration of hexyl acetate Sample No. Wine Distillate 3 yr old is significantly higher in the average and good score score distillate quality three year old distillates when compared Score (5-point) (5-point) to those of poor quality. The concentration of (10-point) 2-phenethyl acetate was significantly higher Sample 1 0.5 0.5 0.85 in one sample of the good quality, matured Sample 2 1 1.3 3 distillates. The volatile compound composition Sample 3 2.2 2.7 5.15 of the distillates was also found to correlate Sample 4 2.5 2.3 5.4 to the sensory quality of the three year old Sample 5 3 3 7.25 distillates using a Pearson correlation analysis Sample 6 4 4 9 with p<0.05. In this analysis it was found that 26 C.L.C. Snyman and M.G. Lambrechts

Table 5. Volatile compound concentrations in the six base wine samples (mg/L).

Compound Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 (mg/L) (poor) (poor) (average) (average) (good) (good)

Ethyl caprylate 0.86 0.79 0.84 1.98 1.37 1.31 Acetic acid 343.12 748.29 114.26 295 200.47 202.51 Ethyl caproate 3.76 1.64 1.01 1.22 1.3 1.55 Diethyl succinate 1.43 1.37 0.29 0.7 0.4 0.54 2-phenethyl acetate 0.17 0.14 0 0.36 0.77 0.9

Table 6. Volatile compound concentrations in the six 3 year old distillate samples (mg/L).

Compound Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 (mg/L) (poor) (poor) (average) (average) (good) (good)

Acetaldehyde 1900 264 85 185 74 117 i-Amyl acetate 4 4 13 16 18 64 Hexyl acetate 0 0 0.291 2.262 1.539 4.504 Ethyl lactate 64 121 11 10 18 10 Hexanol 25 26 16 18 20 16 Ethyl caprate 11 14 25 28 29 27 Diethyl succinate 3.56 3.01 0.98 1.37 2.50 1.22 2-Phenethyl acetate 0.00 0.31 0.00 0.83 0.93 2.79

iso-amyl acetate, hexyl acetate, ethyl caproate, between the good and average quality distillates ethyl caprylate, n-butanol, octanoic acid, ethyl profiled over three years of wood maturation, caprate and decanoic acid showed some positive there were distinct differences between the correlation to the quality of the three year old good quality and poor quality aroma profiles distillates. Isobutanol, ethyl lactate, acetic throughout the course of maturation. The acid, acetaldehyde and ethyl acetate showed a same observation was made on the unaged significant negative correlation to three year old distillates. distillate quality (data not shown). Figure 1 and Figure 2 illustrate the aroma profiles obtained for two three year old distillates of good and poor quality respectively. Where Sensory descriptive analysis (SDA) on the woody aromas were prominent at one and selected distillates two years of wood maturation in the good and average quality distillates, the woody aroma A sensory descriptive analysis was performed was found to become less prominent and more on the 12 distillates mentioned in the previous integrated into the aroma profile after three section of this discussion in order to gain a better years of wood maturation. This was however not idea of the impact of the volatile compound the case for poor quality distillates. After three concentration differences on the sensory aroma years of wood maturation, the aroma profile characteristics of these distillates throughout of poor quality distillates can be characterised the three-year maturation period. Although few by prominent herbaceous and woody aromas, distinctive differences in aroma profiles existed which are more intense than the fruity aromas. Influence of base wine composition and quality on style and quality of South African brandy 27

Herbaceous Good Quality 1 Herbaceous Poor Quality 1 Good Quality 2 6 6 Poor Quality 2 Negative aromas 5 Fruity Negative aromas 5 Fruity 4 4 3 3 Nutty 2 Floral Nutty 2 Floral 1 1 0 0

Spicy Toasted Spicy Toasted

Smooth associated Woody Smooth associated Woody Sweet associated Sweet associated

Figure 1. SDA on three year old distillates Figure 2. SDA on three year old distillates of good quality. of poor quality.

The distinct differences in the aroma profile of the base wine, unaged distillate and three-year old poor quality distillates, when compared to those wood maturated distillates. Thus, three years of of good and average quality after all three years wood maturation do not significantly alter the of oak maturation indicate that wood maturation perceived sensory quality of potstill distillates as does not significantly alter the perceived originally determined in their unaged distillates aroma profile of a poor quality distillate. The and in the case of the 1999 distillates also as undesirable characteristics originally present originally determined in their base wines. are still present, even if at lower intensity than The routine analyses performed on the brandy in the unaged product. This implies that a poor base wines, whether viewed individually or as quality, unmatured distillate will not significantly a group, showed little statistical correlation to improve in quality with three years of wood the sensory quality of the base wines, unaged maturation. Similarly, a good quality unmatured and three-year old distillates. The exceptions distillate, on condition that it is not matured in were total polyphenol concentration and base a faulty barrel or under incorrect conditions, wine quality, wine alcohol content and unaged will retain its quality throughout maturation, distillate quality as well as three-year old distillate although it is difficult to distinguish between a quality. Thus, when already within the required distillate of good and average quality after three specification, these analyses cannot provide a years of wood maturation. This correlation was clear guideline as to the expected quality of the also largely confirmed in a Spearman rank order base wines, unaged and three-year old distillates. correlation analysis performed during the study Some interesting tendencies were however (data not shown). noted in terms of the relationship between quality, total and volatile acidity and pH even though these were not statistically significant. Summary The volatile compound composition was also found to correlate to the sensory quality of the In summary, it was found that, with the three year old distillates. exception of the 2000 base wine score and the It is important to bear in mind that this study 2000 three-year old distillate score, there is a has focussed on relatively few compounds and significant correlation between the score of the only volatile compounds in determining their 28 C.L.C. Snyman and M.G. Lambrechts relationship to wood matured potstill distillate References quality. Those compounds derived from the wood maturation process may also play a role Blom, M. (2000). Determination of flavour in this relationship. In future studies, these compounds in wines, rebate spirits and should also be taken into account. Steger and brandies. Distell Chemical Analysis Method, Lambrechts (2000) showed that three large Distell, South Africa brandy producing companies within South Jolly, N.P. and Hattingh, S. (2001). A brandy Africa differed in their sensory evaluation of aroma wheel for South African brandy. brandy base wines and distillates, due to the South African Journal of Enology and inherent brandy style differences between the Viticulture 22:16-21 two companies. Thus, the findings of this study Steger, C.L.C. and Lambrechts, M.G. (2000). The are closely related to Distell’s perception of base selection of yeast strains for the production wine and distillate quality in the style of brandies of premium quality South African base that are produced by the company. Thus findings products. Journal of Industrial Microbiology of this nature may be different for other brandy 24:431-440. producing companies, depending on the style of brandy desired. New characterisation tools for whiskey raw materials 29 Chapter 5 New characterisation tools for whiskey raw materials

V. H. Beaumont Pernod Richard Research Center, 94015 Créteil Cedex, France

Introduction The old Midleton distillery used to handle barley from harvest to malting, including In the Irish distilling industry both malted and choosing, screening, drying and storing the unmalted barley are being used for the production grain. Nowadays, the distillery buys malted and of whiskey. The quality of unmalted grain supply unmalted barley from merchants and no longer to the industry can be variable. Despite an controls these -harvest steps. One result of this extensive list of grain specifications, traditional situation is that various batches are delivered to methods of analysis do not accurately predict the distillery and the processing ability of these industrial brewing performance. This can result in batches is variable. This is why we tried to define reduced extraction, poor lautering performance more precise specifications for our unmalted and reduced alcohol yields. Recognition of barley supplies. this fact has led to the development of novel Raw materials’ specifications should be based alternative methods based on mash rheology and on the plant requirements, i.e. generally, yield Near Infrared Reflectance (NIR) spectroscopy. and throughput. Potential Spirit Yield (PSY), a The Midleton distillery produces the standard lab-scale brewing and fermentation component spirits for all the Irish Distillers Group protocol (European Brewing Convention, 1998), whiskey brands, including the most famous Irish predicts alcohol yields of malts. Many other whiskey in the world: Jameson. The complex malt analyses were developed to evaluate the was opened in 1975. Its activities include all level of modification of the malt: friability, steps of whiskey production, from brewing up 0.2mm/0.7mm extract difference (Bathgate and to maturation. Two separate processes generate Cook, 1989). However, none of these analyses two main products: a grain whiskey and a are a direct measurement of the malt processing barley/malt pot still whiskey. In the barley/malt ability. For barley, specifications are based on process, barley and malt are mashed together in moisture, hectolitre-weight and total nitrogen. a mashing tank before their transfer to a lauter Again, no specification is directly correlated to the tun. The wort obtained is fermented for four days processing ability of the grain. As a result, there is before its distillation in pot stills. A number of pot a need to characterise both malted and unmalted still whiskey types are produced, all involving barley in order to predict yield in the plant and triple distillation. behaviour during the process. This report gives 30 V.H. Beaumont considerations and preliminary results for new analysed. For the reference method, estimates of analytical systems. precision (repeatability, reproducibility if many labs are involved) are needed, against which the calibration will be compared. There are individual NIR spectra spectral peaks for protein, water and other chemical components, but they are not normally Near Infrared Reflectance (NIR) or Transmittance used specifically for analysis because the whole (NIT) have been subjects of increasing number NIR spectrum is influenced by the overall sample of publications in various areas for the last few composition. This is why multivariate analysis is years (Norris, 1992). Near infrared spectra are required to establish a model taking into account nowadays easy to collect: the entire spectrum (Bertrand, 2000). Once the calibration is set, one needs to • No intensive sample preparation is needed evaluate its qualities: accuracy, precision and since near infrared wavelengths (800-1100 robustness. Quality criteria are computed from nm) can go through thick material; a second set of samples, the validation set. • New generation spectrometers (Fourrier- Accuracy is generally not a problem for NIR Transformed NIR) allow an excellent since the calibration is set so that there is no bias reproducibility; between prediction and reference analyses. If any, • Spectra are available within seconds; the bias might be an indicator of a shift between calibration samples and validation samples. The For all these reasons, NIR is the method of choice precision is estimated by the standard error of for grain analysis at delivery. The problem, prediction. The standard error of prediction will however, is to link the spectra obtained to never be smaller than the standard error of the some reference analysis. A calibration phase is reference method. Robustness can be described required. as the quality of the calibration to provide a Calibration includes three steps of equal good prediction although the sample analysed is importance: creation of a library of calibration different from the calibration samples. Robustness samples, analysis of these samples with reference is the most difficult quality parameter to evaluate methods and spectra collection, and setting up of since statisticians have difficulties to giving a good a prediction model using multivariate statistical estimate of it. methods. The choice of calibration samples is Table 1 presents some of the calibrations essential for a good calibration: they must represent currently under development by Irish Distillers the range of the future unknown samples to be Ltd. The wash calibration is the most advanced

Table 1. Precision of some calibrations under development by Irish Distillers Ltd. Material Samples in Parameter Range Samples in Standard error calibration validation of prediction Lab and 265 Original Gravity 65 to 100 145 1.70 Plant Alcohol strength 8.9 to 15% 145 0.16 Wash Final gravity (-10) to 2 145 0.56

Barley 67 Total Nitrogen 1.5 to 2.1% 10 0.03 Moisture 12 to 16% 10 0.06 Screenings 80 to 97 % 10 1.08 Malt 170 Hot water extract 297 to 307 17 1.82 Soluble Extract 76.5 to 80 17 0.45 Fermentability 85.4 to 88.5 17 0.45 New characterisation tools for whiskey raw materials 31 one. It gives satisfactory results and is currently maximise the fermentability, while decreasing used by the quality control department. The the soluble extract. Surprisingly, supplier 2 is barley calibration, although in need of more having difficulties both in term of soluble extract samples, is very encouraging. and fermentability. Figure 2 also shows that there However, malt calibration developed for the is more heterogeneity in the malts coming from components of PSY (Potential Spirit Yield, i.e. supplier 2 than from the two others. These same soluble extract and fermentability) gave poor samples were scanned with Near Infrared and a results in term of precision. To understand the Principal Component Analysis (PCA) was used source of inaccuracy, we compared the PSY to analyse the data (Figure 3). The PCA indicates values obtained by two independent laboratories that supplier 3 is well discriminated from the (Figure 1). The figure clearly shows that PSY others. Differences between suppliers 1 and 2 analysis has three inconveniences: are less obvious but could be maximised through the use of a discriminant analysis. • the range is narrow: most samples gave values We also tried to develop a Near Infrared tool between 405 and 420 LPA/t; that would detect non-standard supplies among • the precision is poor: all results were given our unmalted barley deliveries. As a first step, we at +1 LPA/t; collected samples during the barley harvest in • the reproducibility is limited; 2004, to obtain a representation of our deliveries to come. The library contained 67 samples In conclusion, it will probably be very difficult sourced from 3 suppliers, presented as spring or to create a satisfactory NIR calibration for PSY, winter barley. Four outliers were removed from because the method used to determine PSY is the library (the distance between their individual not precise and reproducible enough. spectrum and the spectral center of the library, The components of PSY, soluble extract and was higher than 10). A discriminant analysis fermentability, are more informative than PSY model was then developed to classify samples alone. As an example, Figure 2 represents the based on their origin (Table 2: calibration set). soluble extract and fermentability for 170 malt Winter samples could clearly be segregated samples. The strategies used by the suppliers are from the spring barley. Supplier 3 was found easy to understand: supplier 1 tries to optimise to provide spring barley distinct from the two the soluble extract of its malt, supplier 3 goes other suppliers. The model was then validated toward more modification of the barley to with 183 deliveries over the period 2004-2005

420 418 416 414 412 410 408 406

Supplier PSY (LPA/t) 404 402 400 400 402 404 406 408 410 412 414 416 418 420 QC PSY (LPA/t)

Figure 1. A comparison of PSY values obtained in two independent laboratories (106 samples), LPA (litres pure alcohol). 32 V.H. Beaumont

88.5

88

87.5

% FER 87

76.5 77 77.5 7886.5 78.5 79 79.5 80

86

85.5

85 % SE

Figure 2. Relation between Soluble Extract (SE) and Fermentability (FER) for malt samples coming from 3 suppliers (supplier 1 , supplier 2 , supplier 3 ).

PC2 Scores 0.025

0.020

0.015

0.010

0.005

0

-0.005

-0.010

-0.015

-0.020

-0.025

-0.030 PC1 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4

Figure 3. Principal component analysis of the NIR scan from malt samples. Labels differentiate the suppliers. New characterisation tools for whiskey raw materials 33

Table 2. Source of unmalted barley and corresponding NIR prediction.

Calibration Set ↓ Prediction / Actual→ Supplier 1 Supplier 2 Supplier 3 Winter Total Supplier 1 13 1 14 Supplier 2 1 13 14 Supplier 3 14 14 Winter 21 21 Total 14 14 14 21 63

Validation Set ↓ Prediction / Actual→ Supplier 1 Supplier 2 Supplier 3 Winter Total Supplier 1 52 23 3 78 Supplier 2 7 36 4 47 Supplier 3 1 46 47 Winter 10 1 11 Total 69 60 54 183

(Table 2: validation set). The classification programs (13 and 33 min) were applied to the obtained fitted what was expected i.e. samples 67 samples of the library and 10 parameters from supplier 3 could generally be identified were recorded, including initial viscosity, while samples from supplier 1 and 2 were closer peak viscosity, time to peak viscosity and final to each other. Surprisingly, 11 samples from 2 viscosity. A PCA was run to analyse the data. suppliers were sorted as winter barley. Further Unfortunately, these rheological parameters analysis of these samples is currently ongoing. could not discriminate among the suppliers, or between spring and winter barley. As a second trial, viscosity during the longest mashing RVA Data program was recorded every 30 seconds. The curves obtained were normalised before being Many authors have previously described the use used in a second PCA (Figure 4). The analysis of of various viscometric measurements to evaluate this second set of data could clearly discriminate barley and malt quality (Glennie Holmes 1995). the winter from the spring barleys. It is also The RVA (Rapid Visco-Analyser) apparatus interesting to note the heterogeneous behaviour consists of a viscometer (stirring device applying of barley coming from supplier 1. Three spring constant shear rate and measuring resultant barley samples were sorted as winter barley torque) coupled to a temperature controlled with the RVA analysis: further analysis (DNA water bath. The system allows the study of fingerprinting) showed that these three samples the evolution of viscosity in water/cereal grist actually contained one winter barley variety in suspensions under various simulated mashing various proportions. conditions. Parameters generally recorded A closer look at the PCA results explains include initial viscosity, maximum viscosity, where the information comes from. The first two time to and/or temperature at peak viscosity principal components defined by the PCA explain (Glennie Holmes 1995). 95% of the variability, so a projection on these two In order to evaluate our library of barley axes gives a good representation of the samples harvest 2004 described above, we developed (Figure 4). The loading scores of the principal mashing programs with various duration and components explain the origin of information temperature profiles. Two different mashing provided by the RVA data (Figure 5): 34 V.H. Beaumont

PC2 Scores 1.2

1.0

0.8

0.6

0.4

0.2

0

-0.2

-0.4

-0.6

-0.8 PC1 -2.0 -1.5 -1.0 -0.5 0 0.5 1.0 1.5 2.0 Figure 4. Principal component analysis of the RVA curve from unmalted barley samples. 1 to 3: 3 different suppliers; W: winter barley.

0.30

0.25

0.20

0.15

0.10

0.05

0

-0.05

-0.10

-0.15

Source 2.5 5 7.5 10 12.5 15 17.5 20 22.5 25 27.5 30 32.5 Figure 5. Loading scores of the first two principal components based on the RVA viscosity measurement. These two axes explain 95 % of the total variability. New characterisation tools for whiskey raw materials 35

• the first axis describes the peak of viscosity (at References around 10 minutes) and the final viscosity; • the second axis takes into account the initial Bathgate, G.N., Cook R. (1989). Malting of viscosity and the rate of breakdown (between barley for Scotch whiskies. In: Science and 10 and 20 minutes). Technology of Whiskies, Edited by Piggott J.R., Sharp R., Duncan R.E.B., Longman Altogether, the PCA analysis demonstrates Scientific and Technical, Essex, pp. 19-63 that the whole RVA curve, not only the peak Bertrand, D. (2000). Les methodes d’analyses viscosity, provides valuable information. rapide dans les industries agroalimentaires. In: La spectroscopie infrarouge et ses applications analytiques, Edited by Bertrand Conclusions D.and Dufour E., Tec and Doc, Paris, pp. 3-27 For rheological studies as well as routine European Brewing Convention (1998). Analytica analysis, the RVA has many advantages over a EBC, 5th Edition, Verlag Hans Carl, Nurnberg, classic system: it requires a small sample, it is Germany fully automated, it uses disposable vessels, data Glennie Holmes, M.R. (1995). Studies on barley can be stored and processed through a computer and malt with the rapid viscoanalyser. and reproducibility is excellent. The use of Journal of the Institute of Brewing 101: multivariate analysis together with RVA may 29-32 open new areas for grain and malt evaluation. Norris, K.H. (1992). Early history of near infrared First results obtained with NIR are very for agricultural applications. NIR News 3: encouraging. NIR has shown its ability to 12-13. quantify various parameters like alcohol or original gravity on wash, nitrogen and moisture of grain. Poor precision and reproducibility of standard methods are probably inherent to PSY, so it will probably be difficult to establish a precise NIR calibration for PSY. However, the results obtained show that Near Infrared could be useful in classifying malts based on the supplier. For barley also, there is probably a potential to detect deliveries that deviate from the expected grain profile. Future research will try to assess the ability of NIR and RVA to classify grains based on profile recognition. These profiles may help to 1) allow acceptance/rejection of grain at in-take, 2) predict downstream brewing performance of grain supplies and 3) develop specific profile- based processing procedures to cope with the quality of supply. 36 V.H. Beaumont Mash rheology – model studies to understand the distillery mashing process using the rapid visco analyser 37 Chapter 6 Mash rheology – Model studies to understand the distillery mashing process using the Rapid Visco Analyser

D. L. Goode1, V. H. Beaumont2, D. Quinn2 and E. K. Arendt1 1Department of Food and Nutritional Sciences, National University of Ireland, University College Cork, Ireland; 2Irish Distillers Ltd, Midleton, Co. Cork, Ireland

Introduction The distillery mashing process is a temperature- and time-dependent enzymatic process. Mash In the Irish distilling industry a high proportion viscosity is influenced by macromolecules of unmalted barley (up to 70%) is currently such as β-glucan, arabinoxylan, protein, starch, being used for the production of whiskey. The and dextrins. Monitoring of such degradation quality of grain supply to the industry is diverse. processes by rheological means could provide Barley grain supplies can differ greatly in terms of a more in-depth understanding of raw material source, varietal content, chemical and physical quality and distillery mashing processes. compositions, and their pre-distillery handling This could provide the distiller with a more histories. Routine methods of analysis do not efficient quality and process control tool aimed always predict industrial brewing performance. at characterising brew-house raw materials In the distillery this can result in extraction and thus minimising downstream processing problems, poor lautering performance, and difficulties. reduced alcohol yields. Therefore, there has The Rapid Visco Analyser (RVA) was been much interest in the development of developed for measuring the pasting properties alternative methods aimed at characterising of starch and was initially used to determine raw materials. One such technique, which is of pre-harvest damage of cereals. It is widely used interest to the brewing and distilling industries, as an instrument to measure grain quality in is rheology. By scientific definition, rheology the cereal industry. The RVA has been used is the study of the deformation and flow of for a wide range of applications such as (a) matter under the influence of an external force establishing the effects of processing on the (Rao, 1999; Mezger, 2000, 2002). In practice, pasting characteristics of maize tortilla chips it is already a successful and proven research-, (Gomez, Lee, McDonough, Waniska and and quality-control- tool in many industries, Rooney, 1992); (b) the evaluation of sprout including the food industry and in particular the damage in barley (Bason, Ronalds, Wrigley and cereal baking and starch sectors (Dobraszczyk Hubbard,1993); (c) the noodle quality of wheat and Morgenstern, 2003; Tabilo-Muinzaga and (Panozzo and McCormick, 1993); and (d) the Barbosa-Canovas, 2005). starch pasting characteristics of wheat, potato, and barley (Kim and Walker, 1992; Deffenbaugh 38 D.L. Goode et al. and Walker,1989b; Song and Jane, 2000). In The temperature-time programme used for the brewing industry the RVA has been used the analysis was based on industrial mashing as a tool for monitoring the overall quality of procedures. The effect of a range of substrates malted barley (Glennie Holmes, 1995a-e; Dunn, and substrate combination (starch, beta-glucan) Trinh, Bonnici and Stuart,1997; Stuart, Dunn and on the viscosity of the mashes was evaluated. Allan,1997; Broadhead, Brosnan and Bringhurst, The impact of enzymes such as amylase, beta 2004; Zhou and Mendham, 2005) and wheat glucanase and mash pH on viscosity of the mash adjunct additions (Broadhead et al., 2004). model systems were tested. The overall objectives of this study were 1) to develop novel rheological methods with the capability of accurately detecting Methodology sensitive viscosity changes, that occur during the distillery mashing process; 2) to use the Rapid Visco Analyser (RVA) mash rheological rheological methods for developing user-friendly method applications for the purpose of raw material characterisation in the distilling industry; and 3) The RVA Super3 (Newport Scientific, to use the newly developed method applications Warriewood, Australia) is a controlled shear to further characterise and understand the rate instrument, in that it applies a constant biochemical interactions that occur during the shear rate (rpm) and then measures the resultant distillery mashing process due to the influence torque (force, shear stress). Data can be produced of raw material quality. in both tabular and graphical format. The RVA In this study model systems were developed system (Figure 1) consists of a one-way plastic based on purified starch and unmalted barley. stirrer and aluminium sample canister. Further

1. Sample Preparation Disc Mill: 0.20 mm Grind Liquor:Grist: 2.57:1 Grain: 7.840 g ±0.001 g Water: 20.160 g ±0.001 g

2. Rheological Analysis Initial Mixing: 960 RPM for 10s Constant Shear Rate: 160 RPM Mashing: 50°C×30min, 62°C×40min, 72°C×20min, 78°C×5min. Plastic Paddle & RVA Super 3 Sample Cannister Measurements taken every 0.5 s.

3. Data Processing / Interpretation

16 85 14 80

y 75 it 12 os 70 e

10 ur sc

65 at Vi 8 60 ve ve mper

ti 6

55 Te la 4 Re 50 2 45 0 40 9 7 5 3 .6 .2 .9 .7 .4 .4 .7 .1 .8 .7 26 54 77 7. 10 11 12

0. 15 23 30 38 46 61 69 84 92 99 40% Malt Time (min) Temperature

Figure 1. Basic design principles of the RVA mash rheological profiling methods. Mash rheology – model studies to understand the distillery mashing process using the rapid visco analyser 39 details on the operation of this method are values of the repeated experiments together outlined in Goode and Arendt, 2005; and with standard deviations. The graphical results Goode, Wiltschko, Ulmer and Arendt, 2005. represent one individual analysis. All viscosity data is given as cP.

Temperature profile Results and discussion For all rheological experiments the temperature profile during the viscosity-measuring phase Interpretation of the rheological profile of a was kept constant. The temperature profile was mash? as follows: 50°C × 30 min, 62°C × 40 min, 72°C × 20 min and 78°C × 5 min. The heating The first step is mashing at 50˚C. During this step, rate applied between stands was 1˚C/min. This the viscosity was observed to initially increase temperature profile was chosen so as to simulate from the mash-in viscosity at 50˚C to the peak an upward infusion-mashing programme as is viscosity at 50˚C (Figure 1). This peak maximum typically used during a mashing programme. at 50˚C is primarily associated with solubilisation of high molecular weight β-glucans and mixing of the grain with mashing liquor, resulting in Experimental design hydration and swelling of the grain components. The viscosity was then observed to decrease from Five different parameters which are known the peak 50˚C viscosity to the viscosity recorded to influence viscosity during mashing were after 30 min mashing at 50˚C. This decrease simulated in these experiments. These simulated (happening at a constant rate) is most likely conditions were then rheologically assessed due to the combined polymer degrading effects using the RVA mash rheological method: of glucanolytic, proteolytic and xylanolytic enzymes which, have optimum temperature (1) The effect of grist of varying degrees activities at approximately 50˚C (Senge, of quality imparted by the inclusion of Schwarzlos, Blochwitz and Annemüller,1996b). unmalted barley at varying proportions With an increase in mash temperature from together with malted barley. 50˚C to 62˚C the viscosity was seen to increase (2) The effects of amylolytic enzyme levels on rapidly and reach a viscosity peak (Figure 1). mash viscosity, when mashing with raw This temperature of viscosity increase is noted unmalted barley. as being the point of gelatinisation of the barley (3) The effects of amylolytic enzyme levels on starch. Amylolytic enzymes (α- and β-amylase mash viscosity, when mashing with a pure and debranching enzyme) can therefore act barley starch substrate model system (pH on this accessible starch substrate, thereby 5.8). decreasing the volume fraction of the granules in the suspension. Heating from 50 to 62°C causes (4) The effect of pH on mash viscosity when deactivation of the proteases and β-glucanases using a pure barley starch substrate model (Narziss, 1992). The mash was held at 62˚C. system (buffered at pH 5.8 and 4.8). Further enzymatic breakdown of the gelatinised (5) The effects of glucanolytic enzymes when starch is occurring due to the combined action using a pure barley glucan substrate. of the α- and β-amylases. Due to the action of β-amylase, maltose production is continuing. Experimental procedure The viscosity of the mash was thereby seen to reduce from the viscosity recorded at the start Each experiment was repeated at least three of the 62˚C hold to the viscosity recorded at the times. The tabular results quoted are the mean end of the 62˚C hold. This viscosity decrease 40 D.L. Goode et al. can be attributed mainly to the endo-hydrolysing of barley adjunct on the rheological curve, when actions of the α-amylase and to a lesser extent the proportion of malted barley was increased to the exo-hydrolysing actions of β-amylase. The in the mashing system. As the % malt was mash temperature was then raised to 72˚C. At increased, the observed trend was a decrease this temperature, it is reported that β-amylase is in viscosities recorded at 50˚C. The higher data less active (Narziss, 1992). Enzyme inactivation values are most likely related to the higher levels is an interaction between time and temperature. of undegraded macromolecular substances With an increase in time at temperatures ≥ 72˚C, (i.e. β-glucan, arabinoxylans and proteins) β-amylase is inactivated. The α-amylases further and lower amounts of endogenous enzymes breakdown the gelatinised starch and high at the higher levels of barley adjunct. Clear molecular weight dextrins, into low molecular exponential correlations were found between, weight dextrins and glucose. The viscosity of the the % malt from 5% to 100%, with regard to mash was observed to reduce from the viscosity the peak viscosity at 50˚C (y = 1122.2e-0.0182x, recorded at the start of the 72˚C stand to the R2 = 0.9931) and the viscosity breakdown rate -0.0415x 2 viscosity recorded at the end of the 72˚C stand. at 50˚C (y = 31.159e , R = 0.9522). Mash The temperature was then raised to 78˚C where viscosity at 50˚C was therefore dependent on enzyme inactivation is reported to occur and the the level of added adjunct/under-modified grain final viscosity at 78˚C was attained. (P<0.001). This can mainly be attributed to the higher levels of water-holding capacity of under degraded macromolecular substances (i.e. Grist quality and its affect on the mash β-glucan, arabinoxylan(s) and proteins). rheological profile With regard to starch gelatinisation and amylolytic breakdown, as the % malt was In order to simulate the effects of different levels increased, the trends observed were a significant of grist quality, the RVA rheological method (P<0.001) decrease in the rheological was applied to mashing systems consisting of parameters recorded immediately prior to unmalted barley together with malted barley at primary gelatinisation and the parameters various proportions. Figure 2 shows the effects recorded after gelatinisation (Figure 3). The

Figure 2. Rheological profiles of grist compositions representing various ratios of malt:barley adjunct over the course of the 50˚C × 30 min mash stand. Mash rheology – model studies to understand the distillery mashing process using the rapid visco analyser 41

Figure 3. Rheological profiles of grist compositions representing various ratios of malt:barley adjunct over the course of the gelatinisation/liquefaction mashing step. primary gelatinisation temperature was found was applied to mashing systems consisting of to be significantly dependent on the level unmalted barley together with different levels of barley adjunct (P<0.05). It tended to rise of an amylolytic enzyme preparation. as the percentage of malt in the grist was The level of amylase was not found to have a increased (mean value of 58.8˚C over the malt significant affect on the gelatinisation temperature range of 0-20%; 59.7˚C at 100% malt). Clear and the pre-gelatinisation viscosity. As the level exponential correlations were found regarding of amylase was increased, the trends observed the % malt from 5% to 100%, with regard to (Figure 4) were a decrease in the peak viscosity the viscosity peak recorded after gelatinisation recorded after gelatinisation, the time recorded (y = 18994x-0.7342, R2 = 0.9988) and the area at this peak and the area recorded under the recorded under the peak (y = 83438x-0.6475, R2 = peak. The level of added amylase was found to 0.9928). With regard to the temperature stands significantly affect these rheological data points at 62˚C and 72˚C, as the % malt was increased, (P<0.001). When best-fit curves were applied the trends observed were a decrease in recorded to the data versus the level of amylase, inverse rheological parameters. These rheological data power correlations were found between the points were found to be significantly dependent level of malt amylase and the peak viscosity (y = on the level of unmalted barley (P<0.001). 4869.3e-0.1957x, R2 = 0.9444) and the peak area Overall the study showed that the mash (y = 25067e-0.2686x, R2 = 0.9522). The results rheological profiles were directly related to the show, that the level of amylolytic breakdown quality of the grist. following starch gelatinisation has an influence

in determining the peak viscosity. Therefore, the higher the level of amylase the faster the Mash α-amylase and its affect on the mash liquefaction processes and the lower the peak rheological profile viscosity. The breakdown of this viscosity was found to be not only dependent on the level In order to simulate the effects of different levels of amylase, but also on the level of available of mash α-amylase, the RVA rheological method under-hydrolysed substrate. The rate of any 42 D.L. Goode et al.

Figure 4. Rheological profiles of mashes consisting of 100% unmalted barley together with increasing levels of an amylase preparation. enzyme-catalyzed reaction is increased (up to their viscous effects. It has been reported that, if a maximum) by raising the concentration of the these smaller starch granules are not degraded substrate. Therefore, with a higher peak viscosity, during mashing, they can then impede wort more under-hydrolysed substrate was available filtration by cross-linking with other polymers and therefore the rate of viscosity breakdown (Barrett, Clapperton, Divers and Rennie, 1973). was found to increase. With close observation Therefore, this highlights the importance of to Figure 4, secondary increases in viscosity adequate amylolytic enzyme levels as being not were recorded after 90 min of mashing. These only necessary for the production of fermentable peaks represent secondary starch gelatinisation worts, but also in the reduction of these points of the smaller barley starch granules. secondary viscous complexes thus reducing the The temperature of gelatinisation of these potential of poor wort run-off. secondary peaks was independent of the level of amylase (70.96 ± 0.38˚C). Starch composition, gelatinisation and pasting properties, enzyme Effect of amylase level on the breakdown of susceptibility, crystallinity, swelling and purified barley starch solubility are all affected by the starch granule size (Lindeboom, Chang and Tyler, 2004). In this In this trial a purified barley starch substrate study, it was observed that increasing amylase (buffered at pH 5.8) was used, in order to levels resulted in corresponding decreases see the rheological effect that malt amylases in the rheological data points of the viscosity have on barley starch in the absence of other recorded prior to secondary gelatinisation and grain components such as protein, β-glucans, the peak in viscosity recorded after secondary arabinoxylans, lipid etc (Figure 5). gelatinisation. However, no clear correlation was When mashing at 50˚C for 30 min, no found between the level of amylase and these significant differences in the rheological data secondary gelatinisation rheological data points. points recorded at 50˚C due to amylase content As they undergo the gelatinisation process, were observed. Therefore, no degradation there are sufficient amylase levels to reduce of the ungelatinised starch due to amylase Mash rheology – model studies to understand the distillery mashing process using the rapid visco analyser 43

Figure 5. Rheological profiles of buffered mashes (pH 5.8) containing purified barley starch together with increasing levels of an amylase preparation. content could be seen using this procedure. ~ 67˚C. No deviation from this value was This is expected since amylases are reported found to occur due to amylase addition. The to have a very limited ability of degrading viscosity increase at this point was found to be ungelatinised starch. Only the surface of an dependent on the level of amylase (P<0.001). intact, ungelatinised starch granule is available Clear correlations could be established between for enzymatic attack. Although the purified the secondary starch gelatinisation rheological starch substrate is unrelated to the raw barley data points and the level of added amylase (R2 grain, it is seen that the starch fraction contributes = 0.9576). only ~ 40-100 mPa.s of a total viscosity of ~ 1000 mPa.s prior to gelatinisation. Therefore, it can be concluded, that the contribution of Mash pH and its affect on the rheological starch, to the viscosity of a raw barley system, amylolytic induced breakdown of starch? prior to gelatinisation, is quite low. The inherent viscosity must therefore be largely Simulated mashing systems consisting of a attributed to the remaining barley components purified barley starch substrate were buffered such as β-glucans, arabinoxylans, proteins etc. to pH 5.8 and 4.8. PH was found to have no The primary gelatinisation temperature of the significant influence on the primary temperature purified starch (~61-62˚C) was not found to be of gelatinisation of the starch granules or the significantly influenced by the amylase content. viscosities recorded prior to gelatinisation. The peak area recorded under the gelatinisation/ The remaining rheological data points (post liquefaction curve was found to correlate quite gelatinisation) were all observed to be higher, closely with the level of mash amylase (R2 = when the starch solution was buffered at pH 0.9576). Therefore, as previously concluded in 4.8 in comparison to pH 5.8 (Table 1). The malt the barley amylase trial, the level of amylolytic amylase preparation used in this study has a pH enzyme determines the viscosity peak after optimum ranging from 5.0 to 6.0. It is reported gelatinisation and also the resultant breakdown to undergo rapid irreversible inactivation below of this viscosity. The secondary gelatinisation pH 5.0. Therefore, this accounts for the higher point of this purified starch was found to be viscosities which were recorded when mashing 44 D.L. Goode et al.

Table 1. Rheological data pertaining to mashes containing purified unmalted barley starch (buffered at pH 5.8 and pH 4.8) together with increasing levels of a malt amylase.

Amylase (g) pH PVG a (cP) SPVG b (cP) SPGA c VE78 d (cP)

0.025 5.8 6041 ± 582 9229 ± 910 302812 ± 35281 8268 ± 1025 4.8 7787 ± 146 9063 ± 326 261624 ± 10385 5890 ± 261 0.050 5.8 2270 ± 123 2258 ± 113 62517 ± 3761 1646 ± 94 4.8 4155 ± 111 3936 ± 130 103505 ± 4212 2219 ± 108 0.075 5.8 942 ± 33 873 ± 53 23037 ± 1767 592 ± 60 4.8 2468 ± 186 2291 ± 180 56235 ± 5155 1181 ± 118 0.100 5.8 391 ± 38 458 ± 61 12238 ± 1956 320 ± 53 4.8 1247 ± 22 1444 ± 53 34040 ± 1533 705 ± 42 0.125 5.8 246 ± 8 312 ± 16 7984 ± 219 203 ± 8 4.8 740 ± 10 936 ± 30 21537 ± 809 431 ± 20 0.150 5.8 177 ± 33 257 ± 20 6498 ± 1013 170 ± 32 4.8 496 ± 36 697 ± 29 16379 ± 935 319 ± 26 0.175 5.8 162 ± 10 249 ± 13 7045 ± 451 191 ± 13 4.8 331 ± 18 553 ± 18 13753 ± 528 272 ± 8 0.560 5.8 76 ± 2 100 ± 6 2934 ± 174 82 ± 1 4.8 78 ± 15 269 ± 8 7159 ± 507 148 ± 14 1.000 5.8 63 ± 9 132 ± 25 5331 ±1014 189 ± 39 4.8 57 ± 14 356 ± 22 4930 ± 265 79 ± 9 a PVG = peak viscosity recorded after primary gelatinisation. b SPVG = peak viscosity recorded after secondary gelatinisation. c SPGA = area recorded under the gelatinisation/liquefaction viscosity curve. d VE78 = viscosity recorded at the end of the mashing period. at a buffered pH of 4.8. The rheological data end of the 78˚C mash stand (y = 30.506x-0.193, R2 clearly show the important influence of pH on = 0.9516). These results give an indication of the the gelatinisation viscosity and the subsequent ability of the enzyme preparation to breakdown amylolytic breakdown of the gelatinised the barley beta-glucan. starch. Table 2. Rheological data of mashes containing a purified beta-glucan substrate together with Mash beta-glucanase and its affect on the increasing levels of a beta-glucanase preparation. rheological hydrolysis of barley beta-glucan Beta- End viscosity Area under In this trial, a purified beta-glucan substrate Glucanasea (mPa.s) curve (average MW 321,000) was buffered at pH 5.8. The objective was to determine the 0 U 137 ± 4 18331 ± 1682 0.000673 U 112 ± 2 15647 ± 1436 impact of an endo-beta-glucanase preparation 0.00673 U 86 ± 17 11253 ± 3164 (ability of hydrolysing beta-1,4 bonds next to a 0.0673 U 57 ± 16 6335 ± 1663 beta-1,3 linkage) (Table 2). Overall, correlations 0.673 U 33 ± 0 4719 ± 439 were found between the added levels of the 6.73 U 26 ± 15 2876 ± 1469 commercial beta-glucanase and the total area 10.095 U 15 ± 20 2477 ± 971 beneath the viscosity curves (y = 4060.2x-0.19, R2 = 0.9926) and the end viscosity recorded at the a Total enzyme activity units added. Mash rheology – model studies to understand the distillery mashing process using the rapid visco analyser 45

Overall discussion and conclusions that the RVA mash rheological profiling method can be used to provide the distiller with more The determination of viscosity of wort solutions functional information regarding raw materials gives an indication of the quality of the raw and distillery mashing conditions. materials from which the wort was produced. However, it gives little precise information regarding the specific degradation processes Acknowledgements that occur during the mashing process. The rheological methods developed in this paper The authors acknowledge that this research was provide a good indication of the degradation partly funded by Enterprise Ireland and the Food processes, taking place during mashing. In Industry Research Measure (FIRM) under the comparison to the rheological studies of National Development Plan, 2000-2006. We thank other research groups, this study utilises the Dirk Simons, Eric Wiltschko and Helge Ulmer temperature/time conditions of a full commercial (Department of Food and Nutritional Sciences, type mashing programme. Other studies UCC) for their contributions to this work. focussed on isothermal processing, with much shorter analysis times, and were therefore not in a position to determine the enzymatic References processes which are taking place during the mashing process. The method developed was Barrett, J., Clapperton, J. F., Divers, D. M. able to detect the major viscosity changes which and Rennie, H. (1973). Factors affecting occur during starch gelatinisation/liquefaction, wort separation. Journal of the Institute of as well as the minor viscosity changes, which Brewing 79:407-413 were found to occur during the proteolytic and Bason, M. L., Ronalds, J. E., Wrigley, C. W. saccharification steps. The reproducibility of the and Hubbard, L. J. (1993). Testing for replicated trials was found to be very satisfactory. sprout damage in malting barley using the The simplicity of the rheological procedure Rapid Visco Analyser. Cereal Chemistry would suggest, that it could not only be used 70:269-272 as a method in a research laboratory but may Broadhead, A. L., Brosnan, J. M. and Bringhurst, also be suitable as a quality control tool in an T. A. (2004). The role of the Rapid Visco operator laboratory. Analyser (RVA) in minimizing distillery The results from these experiments have processing problems. In: Distilled Spirits: demonstrated the potential of the RVA mash Tradition and Innovation, Edited by Bryce J. rheological methods for many applications for the H. and Stewart G. G., Nottingham University distilling and brewing industry. Clear correlations Press, Nottingham, pp. 89-94 were found between the controlled amounts of Deffenbaugh, L. B. and Walker, C. E. (1989). Use exogenous amylase additions and the recorded of the Rapid Visco Analyser to measure starch rheological data points. This demonstrates pasting properties. Starch 41:461-467 the ability of the rheological method to give Dobraszczyk, B. J. and Morgenstern, M. P. (2003). a qualitative assessment of enzyme activity. Rheology and the breadmaking process. The test also has the ability to identify viscosity Journal of Cereal Science 38:229-245 changes due to added enzymes. Likewise, Dunn, C. A., Trinh, M. L. T., Bonnici, M. J. and mash rheological profiling can be used in the Stuart, I. M. (1997). The effect of viscometric assessment of commercial enzyme preparations and gelatinization properties of barley when brewing with high adjunct ratios as well as on malt quality. Proceedings of the 47th in the assessment of barley and malt quality for Australian Cereal Chemistry Conference, distilling purposes. Overall, it can be concluded pp. 189-194 46 D.L. Goode et al.

Glennie Holmes, M. R. (1995a). Studies on physicochemical aspects of starch granule barley and malt with the rapid viscoanalyser: size, with emphasis on small granule (I) The effects of the variation in physical and starches: a review. Starch 56:89-99 chemical parameters. Journal of Institute of Mezger, T. G. (2000). Handbuch der Rheologie: Brewing 101:15-18 für Anweder von Rotations - und Oszillations Glennie Holmes, M. R. (1995b). Studies on Rheometern, Edited by Zoll, U. and Curt, R., barley and malt with the rapid viscoanalyser: Vincentz Verlag, Hannover, Germany (II) The effects of modification on viscograms. Mezger, T. G. (2002). The rheology handbook: Journal of Institute of Brewing 101:19-28 for users of rotational and oscillatory Glennie Holmes, M. R. (1995c). Studies on rheometers. Vincentz Verlag, Hannover, barley and malt with the rapid viscoanalyser: Germany (III) The prediction of malting potential Narziss, L. (1992). Die Technologie der from viscograms. Journal of the Institute of Wurzebereitung, 7th edition. Ferdinand Brewing 101:29-32 Enke Verlag, Stuttgart, Germany Glennie Holmes, M. R. (1995d). Studies on Panozzo, J. F. and McCormick, K. M. J. (1993). barley and malt with the rapid viscoanalyser: The rapid visco-analyzer as a method for (IV) Amyloclasis, amylolysis and cytoclasis testing for noodle quality in a wheat breeding of malt and malting quality. Journal of the programme. Cereal Science 17:25-32 Institute of Brewing 101:33-38 Rao, M. A. (1999). Rheology of fluid and semisolid Glennie Holmes, M. R. (1995e). Uses for the foods – principles and applications. Aspen rapid viscoanalyser in a brewery. MBAA Publishers, Maryland, USA Technical Quarterly 32:72-75 Senge, B., Schwarzlos, B., Blochwitz, R. Gomez, M.H., Lee, J.K., McDonough, C.M., and Annemuller, G. (1996). Rheological Waniska, R.D. and Rooney, L.W. (1992). examination of mashing in the brewery Corn starch changes during tortilla chip process. Applied Rheology 6:11-18 processing. Cereal Chemistry, 69:275-279. Song, Y. and Jane, J. (2000). Characterization of Goode, D. L. and Arendt, E. K. (2006). Model barley starches of waxy, normal, and high studies characterizing the rheological amylose varieties. Carbohydrate Polymers behavior of simulated mashing conditions 41:365-377 using the Rapid Visco Analyser. Journal of Stuart, I. M., Dunn, C. A. and Allan, G. R. the American Society of Brewing Chemists (1997). The prediction of malt quality using 64:100-110 the Rapid ViscoAnalyser: a new technique Goode, D. L., Wiltschko, E. A., Ulmer, H. M. and for the evaluation of sprout damage. Arendt, E. K. (2005). Application of the Rapid In: Proceedings of the 6th Scientific and Visco Analyser as a rheological tool for the Technical Convention of the Institute of characterisation of mash viscosity as affected Brewing, Central and Southern African by the level of barley adjunct. Journal of the Section, Johannesburg, South Africa, pp. Institute of Brewing 111:165-175 170-174 Kim, C. S. and Walker, C. E. (1992). Changes Tabilo-Munizaga, G. and Barbosa-Canovas, in starch pasting properties due to sugars G. (2005). Rheology for the food industry. and emulsifiers as determined by viscosity Journal of Food Engineering 67:147-156 measurements. Journal of Food Science Zhou, M. X. and Mendham, N. J. (2005). 57:1009-1013 Predicting barley malt extract with a Rapid Lindeboom, N., Chang, P. R. and Tyler, R. Viscoanalyser. Journal of Cereal Science T. (2004). Analytical, biochemical and 41:31-36. The use of NIR spectroscopy to quantify various malted barley analytes 47 Chapter 7 The use of NIR spectroscopy to quantify various malted barley analytes

P. Lockyer and A. Wardlaw Diageo Global Supply, Brand Technical Centre, Menstrie, Clackmannanshire, UK

Introduction thought that this technology might be amenable for the analysis of malted barley. This paper Malted barley is one of the most important raw explains how NIR has been investigated as a tool materials used in the Scotch whisky industry as it for the rapid quantitative assessment of malted forms 100% of the carbohydrate source for malt barley and that it has been shown to be accurate whisky and approximately 10% of the source for and reliable. grain whisky. Moreover, the real advantage of the NIR is Key analytical parameters that may be used in the ease of use of the equipment and the for the quality assessment include PSY (potential transferability of the calibrations, allowing spirit yield), HWE (hot water extract), moisture, the potential for malted barley analysis to be friability and homogeneity. Many of these performed at the maltings allowing more timely traditional techniques are time consuming and interaction with the process. very operator dependent, which has led to much of the industry performing the tests off-site at a central laboratory. This will inevitably lead to a Sample selection delay in the assessment of how well the barley has been modified. 200 samples of malted barley were selected from The use of NIR (Near Infrared) has previously 4 different commercial malting sites within the been implemented at many sites for the routine Diageo group. These covered a sufficient range analysis of unmalted barley (mainly nitrogen to construct a working NIR model. The range of and moisture) for a number of years and it was reference results are shown in Table 1: Table 1. The range of analytical values of 5 parameters for 200 malt samples used to construct a working NIR model. Moisture Hot Water Extract PSY (Litres alcohol Friability Homogeneity (%) (%) per tonne of malt) (%) (%) Minimum 3.9 73.6 391 78.3 85.3 Maximum 5.9 79.3 430 98.5 99.3 Mean 5.1 77.0 411 89.3 97.3 Standard Deviation 0.3 0.8 8.2 3.7 1.7 48 P. Lockyer and A. Wardlaw

Equation development • Friability • Homogeneity Reference analysis was performed for the five analytes and samples spectra obtained using All five equations were then put together to the FOSS Infratec 1241 Grain Analyser as soon create an Application Model using ODIN as possible thereafter (to minimize any ageing software. of the samples). This was then validated and demonstrated to PLS calibration development was performed have the following capability: using the whole spectrum obtained (850 nm to 1050 nm) using Win ISI II software, without Accuracy any mathematical pre-treatment. A repeatability Over 50 samples analysed and investigated file was also used to take into account sample Overall accuracy : PSY ± 5 litres alcohol/tonne repeatability, sample temperature (+5 to +35oC) and instrument variability (3 instruments). A plot Reproducibility (and ease of use) of predicted versus reference results for PSY is Analysed “AQC” sample twice daily for one shown in Figure 1. month with 13 different operators (non- This process was repeated for all five skilled) analytes : Overall reproducibility : PSY ± 2 litres alcohol/tonne • Predicted Spirit Yield • Hot Water Extract Transferability • Moisture Model transferred to three different Infratec

Figure 1. “XY-Plot” of NIR predicted results vs reference results for potential spirit yield. The use of NIR spectroscopy to quantify various malted barley analytes 49

1241 instruments and analysed over 50 A single (multi-analyte) model can be transferred samples on each to multiple instruments. Overall transferability : PSY ± 3 litres alcohol/ tonne At the time of writing, the authors were looking for other interested partners to develop an “Industry Standard” model with a view to Conclusions and next steps generating a networked calibration that could be used for the basis of trading (as is already the case It has been demonstrated that NIR of malted for non-malted barley and wheat). Examples of barley can give “real time” information at the the equipment and spectra are given in Figures point of malt manufacture to allow timely 2 and 3. interaction to improve process efficiency.

The technique can be used by non-skilled operators.

Sta

Standard Transmision Spectra for Malted Barley 850 – 1050nm

Figure 2. NIR equipment and spectra. 50 P. Lockyer and A. Wardlaw

Overlay of all calibration sample spectra (SNV & Detrend to normalise)

Figure 3. Overlay of all sample calibration spectra. Wheat for Scotch whisky production: broadening the horizon 51 Chapter 8 Wheat for Scotch whisky production: broadening the horizon

T.A. Bringhurst, R.C. Agu, J.M. Brosnan and A.L. Fotheringham The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK

Introduction Bringhurst, Fotheringham and Brosnan, 2003). These include soft endosperm texture, high Although traditionally Scotch whisky grain starch and low nitrogen (protein) levels, which distillers used maize for the production of grain come together to provide good alcohol yields spirit, the Scotch whisky industry has generally and low processing problems (Brown 1990; been using winter wheat as their main cereal raw Riffkin, Bringhurst, McDonald and Hands, 1990; material since the middle 1980s. Brosnan et al., 1999; Broadhead et al, 2004). This Scottish winter wheat currently comprises less knowledge has been used to support agronomic than 6 percent of a total UK wheat production data to assist in the selection of suitable distilling of 15.5 million tonnes (DEFRA, 2005). While wheat for growing in Scotland and the UK. Scotch whisky distillers’ total requirements Perhaps fortuitously, the change over from for unmalted cereals represent a significant maize to wheat was shortly followed by the percentage (around 60 percent) of the market emergence of the soft wheat cultivar Riband (first for Scottish wheat, this is a very small proportion listed in 1989) as the dominant variety grown (in the region of 3.5 percent) of the total wheat in the UK. During this period, Riband found produced in the UK. Hence, one of the problems universal acceptance with Scotch whisky distillers facing distillers is that they are dependent on as the ideal wheat variety for the production of the availability of suitable distilling varieties, grain spirit, and has since, consistently given low which make up a very small part of the UK nitrogen grain with good starch accessibility, wheat market, which is largely dominated by the resulting in good performance in the distillery requirements of the milling and bread making both in terms of production efficiency and industries. processing characteristics (Brosnan et al., It is now well established that wheat has very 1999), as well as giving reliable agronomic different processing characteristics to maize performance. (Palmer, 1986; Walker, 1986; Broadhead, Although Riband is no longer a major UK Brosnan and Bringhurst, 2004), and since wheat variety, as a result of a strong decline in its the introduction of wheat, there has been a agronomic viability in recent years, it is still sown large increase in our understanding of the in significant quantities in Scotland. However, parameters defining high quality distilling wheat Riband is now being replaced by newer varieties (Brosnan, Makari, Paterson and Cochrane, 1999; such as Consort, Claire and Robigus, which are 52 T.A. Bringhurst et al. now dominant in Scotland (National Institute of Table 1. SWRI quality ratings for distilling wheat. Agricultural Botany, 2005). However, none of these is considered to be as good for distillers Rating Definition as Riband (Brosnan, 2001), and efforts are on- going to find more suitable alternative distilling Good Established soft wheat variety producing consistently good laboratory distilling wheat cultivars. yield as well as giving good distillery Official UK wheat trials are now conducted performance by Crop Evaluation Ltd (CEL), a wholly owned subsidiary of the Home Grown Cereals Authority Medium/ Newer soft wheat variety producing Good consistent, good, laboratory results or (HGCA), based on samples grown on a range of established variety giving satisfactory sites throughout the UK, including sites in the laboratory and distillery performance North and Central Scotland. The Scottish sites operated by the Scottish Agricultural College Medium Soft wheat with average laboratory (SAC) and Scottish Agronomy, are of particular results or distillery performance interest to distillers who prefer a convenient and Medium/ Soft wheat with significantly below inexpensive supply of locally grown material. Poor average laboratory or distillery The CEL Cereals Committee, which now also performance includes a representative from the distilling industry, Poor All hard wheat or soft wheat carrying as well as from the HGCA, Scottish Agronomy, the 1b1r rye gene translocation the British Society of Plant Breeders (BSPB)) and the National Association of British and Irish Millers (NABIM), discuss these trials and make recommendations regarding which of the new Hard wheat, or soft wheat varieties containing wheat varieties will progress through the system the 1b1r rye gene translocation, are unsuitable from National List (NL1) trials, through NL2 to final for distilling, since they are associated with approval for growing in the UK (RLT). serious processing problems arising from high Recommendations are primarily decided on levels of viscosity. the basis of agronomic data, but during the last The purpose of this paper is to review some 5 years, the Scotch Whisky Research Institute of the data generated at the Scotch Whisky (SWRI), have been actively participating in this Research Institute during the last 5 - 6 years and system by providing additional alcohol yield illustrate how it has been used in the selection data on soft winter wheat varieties specifically of good quality distilling wheat. aimed at the distilling market. The Institute also maintains contacts with all the major wheat breeders to ensure that distillers’ requirements are Materials and methods considered when developing new varieties. Currently the quality ratings used by SWRI for Wheat samples were obtained over a period of distilling wheat are defined as shown in Table 1. years from official UK trial sites operated by the Wheat which is suitable for distilling into Scottish Agricultural College (SAC), and Crop Scotch whisky, has different characteristics to Evaluation Ltd (CEL). These were analysed at bread making and other milling wheat, and SWRI using the standard methodology described is generally characterised by soft endosperm below. texture, high levels of starch and low nitrogen levels (<<2 percent (dwb) (<<11.4 percent Alcohol yield analysis protein)). This is in contrast with bread making wheat for which high protein (gluten) levels are The method used was based on that of Brosnan et considered an essential requirement. al. (1999), and simulates the production process Wheat for Scotch whisky production: broadening the horizon 53 conditions in a ‘typical’ Scotch whisky grain Results and discussion distillery. Wheat flour (30 g) was milled into a fine grist on a Buhler Miag disc mill (0.2 mm), Figure 1 summarises alcohol yield and total and slurried with water (81 ml), and Termamyl nitrogen data obtained over the years from 1999 120L (25 µl) (fungal α-amylase), and gradually to 2004. These data represent the overall site heated up to 85°C, in a stainless steel mashing averages of the growing sites, which were used beaker, with frequent stirring, before pressure in each year. These results give an indication of cooking at 142°C for 15 minutes in an autoclave. the large amount of annual variation. The cooked slurry was cooled to 85°C and The figure also illustrates the well established given a second treatment with Termamyl (25 inverse relationship between alcohol yield µl) to prevent starch retrogradation. After further and total grain nitrogen (Taylor and Roscrow, cooling to mashing temperature, the slurry 1990). Generally, the correlation between total was then mashed at 63°C with an added high nitrogen and alcohol yield is very significant and enzyme malt for 1 hour. The malt inclusion rate is normally in the region of -0.70 to -0.86, but was equivalent to 20 percent (dry weight basis) this can vary from year to year, and in some cases of the total grain bill. The mash was then cooled the correlation can be much lower, particularly to room temperature and pitched with Quest if the overall nitrogen range is small, or under (Kerry) ‘M’ yeast (or equivalent) (0.4 percent poor or uneven harvest conditions where site to w/v) and fermented at 30°C for 68 hours, prior site variations may be abnormally high. to distillation to collect the alcohol. The alcohol Historically, the number of sites has increased yield was determined from the alcohol strength from just 3 Scottish sites in 1999 to 6 - 8 sites by of the distillate, which was measured using a Paar 2003 – 2004. These additional trial sites are in a 5000 density meter. The results were quoted on broader range of locations within the UK (from a dry weight basis. the North of Scotland to Northern/East England) and allow a more representative assessment of quality than was previously possible. Total nitrogen analysis The variations which were observed between each year were probably the result of several Nitrogen analysis was determined using a factors including prevailing weather and Kjeldahl method similar to that for barley environmental conditions, nitrogen treatments, described in Method 1.8 of the Institute of soil type, field topography, exposure and other Brewing Recommended Methods of Analysis conditions at the different growing locations (Institute of Brewing, 1997). (Orth and Shellenberger, 1988; Agu and Palmer, 2003).

455 1.95 5 Sites Alcohol yield 450 Total N 1.90 445 4 Sites 3 Sites 6 Sites 1.85 440 1.80 435 8 Sites 1.75 430 5 Sites 1.70 Alcohol Yield (LA/tonne) 425 Total N (% dry weight) 420 1.65 415 1.60 1999 2000 2001 2002 2003 2004

Figure 1. Overall averages for alcohol yield and total nitrogen 1999 – 2004. 54 T.A. Bringhurst et al.

Performance of individual wheat cultivars the standard for new distilling wheat, even though the agronomics of growing this variety Table 2 gives an indication of the wheat are becoming increasingly difficult. varieties which have been going through the Figure 3 gives a good illustration of the Recommended Level Trials system since 1999. contrast between Buchan and Madrigal, which A number of these varieties have been on the were not considered to be good distilling HGCA Recommended lists for growing in the wheats, and Eclipse, which was potentially an UK, but only a few have been recommended exceptional distilling variety. for distilling. Between 1999 and 2001, Buchan and Figure 2 shows the performance of Claire and Madrigal both performed relatively poorly, Consort (current Riband replacements), over the compared with Riband, with the exception of period 1999-2004, and confirms that although 2000, where the alcohol yields improved for their alcohol yields were generally comparable Buchan and Madrigal, but were still slightly with Riband, this variety generally continues to lower than Riband. Poor performance by Riband have a slight edge on the others. The exceptions during 2002 reflects the overall harvest quality for were during the relatively poor harvest years of that year. Overall, neither Buchan nor Madrigal 2002 and 2003, where overall performance was was able to sustain good distilling performance poor for all varieties. in the longer term. Both of these varieties carry Although Riband appears to falter from time the 1b1r rye gene and under current selection to time, particularly in a poor harvest year, in criteria would not be considered suitable for the long term this cultivar is far from being distilling due to viscosity issues impacting on outclassed as a distilling variety and still sets processability.

Table 2. List of new wheat varieties appearing in RLT from 1999 – 2004 (the year corresponds to the year of entry into RLT; wheat with distilling recommendations are shown in bold).

Control varieties 1999 2000 2001 2002 2003 2004

Claire Buchan Arlington CPBT W64 Arran Dart Ambrosia Consort Eclipse Deben Savannah Brunel Dickson Atlanta Riband Harrier Windsor Storm Goodwood Gladiator Glasgow Madrigal X119 Robigus Istabraq Wizard Nijinsky Quest Steadfast

460 Riband 450 Consort Claire 440

430

420 Alcohol yield (LA/tonne dry ) 410

400 1999 2000 2001 2002 2003 2004

Figure 2. Long term performance comparisons of Riband, Consort and Claire over 6 harvest years (1999-2004). Wheat for Scotch whisky production: broadening the horizon 55

455 450 445 440 435 430 425 420 415 1999 Alcohol Yield (LA/Tonne dry ) 2000 410 2001 Riband 2002 Eclipse Buchan Madrigal Figure 3. Comparison of Buchan and Madrigal and Eclipse with Riband Control.

In contrast to Buchan and Madrigal, Eclipse seed production (HGCA, 2005). However, in was consistently ahead of Riband in all of the that year, in terms of distilling performance, trials in which it was entered, but gave problems both Robigus and Nijinsky were outclassed by with its agronomic characteristics, and so was Istabraq and Glasgow, although they still remain not acceptable to growers and thus was not on the list of Recommended distilling varieties. included on the Recommended List. The robustness of the assessment process is Figure 4 shows more recent data from the illustrated by Table 3 which shows the ranking 2004 harvest. The 2004 harvest was assessed of the distilling performance of each variety on by considering 6 individual sites, in North the individual sites. This confirms that although Scotland (NS1, NS2) South/Central Scotland there were some minor differences in the ranking (CS1, CS2) and England (East Anglia (EA), North on each site, the overall ranking of the top Yorkshire (NY)). Early data for the cultivars performers was a reasonably reliable indication Robigus, Nijinsky, Dickson and Istabraq had of their relative overall performance. originally suggested that all of these varieties Thus, of the top 2 performers, Glasgow gave might have promising distilling potential, and the highest alcohol yield on three of the six sites, in 2004 Robigus was the dominant UK wheat and came second on two sites, while Istabraq variety, accounting for 17 percent of certified

450

445

440

435

430 Alcohol Yield (LA/tonne dry )

425

n w kso lanta Claire ic Deben At Riband Consort Istabraq Nijinsky Robigus D Glasgo

Figure 4. Distilling performance of current distilling wheat varieties 2004 (Riband, Consort and Claire are control varieties). 56 T.A. Bringhurst et al.

Table 3. Overall ranking of distilling performance Table 4. Current SWRI quality rating of distilling of wheat varieties compared with individual sites wheat from the 2004 winter wheat harvest. (2004 Harvest). (EA – East Anglia, NY – North Yorkshire, NS – North Scotland, CS – Central/South Good Medium/Good Medium Medium/Poor Scotland). Riband Consort Atlanta Deben Overall EA NY NS1 NS2 CS1 CS2 Istabraq Claire ranking Nijinsky Robigus Glasgow Dickson Glasgow 1 2 1 6 1 2 1 Istabraq 2 1 2 1 2 3 4 Ambrosia 3 4 3 5 8 1 3 promising new distilling wheat varieties, such as Atlanta 4 3 4 8 6 4 2 Glasgow, Istabraq and even Robigus, Riband is Robigus 5 6 6 3 3 8 6 still the aspirational standard for distilling wheat. Dickson 6 5 8 4 5 5 7 Continuing efforts will be on-going to find viable Nijinsky 7 7 7 2 7 7 5 replacements for this variety. Deben 8 8 5 7 4 6 8 The assessment of new distilling wheat has developed from very limited studies of wheat gave the best performance on two of the sites, but samples grown on a few localised sites, to came second on three sites. Although Glasgow much larger scale trials, at a range of locations was the best overall performer, it came sixth on throughout the UK and is now capable of giving one of the North Scotland sites. Agronomically, a more robust assessment of distilling wheat this variety is not considered to be ideally suited varieties. for growing in Scotland (despite its name!). During the last 5 – 10 years, work at SWRI Ambrosia, which came third in the overall has contributed to major advances in our ranking, gave a more varied performance on the understanding of what makes good distilling different sites. This variety, although capable of wheat, and the input of distilling quality giving good alcohol yields, carries the 1b1r rye parameters by the Institute is now used in gene translocation, and would not be acceptable the UK Recommendation process for wheat. for distilling, due to concerns about high Importantly, distillers’ requirements are taken viscosity, leading to potentially serious distillery into account when new wheat varieties are processing problems. assessed for growing in the UK, particularly in Robigus, Dickson and Nijinsky all performed Scotland, where the demand for distilling wheat relatively poorly on individual sites, while is greatest. In this context SWRI are continuing to Deben consistently gave the poorest ranking, maintain contacts with plant breeders to ensure indicating that some varieties appearing on the continuing availability of a sustainable the Recommended List, on the basis of their supply of wheat for distilling end users. As a agronomic performance, would not necessarily result, plant breeders are now beginning to be the best ones for distilling use. respond positively by looking for wheat varieties which are targeted specifically at distillers.

Conclusions Summary On the basis of the SWRI distilling quality ratings outlined in Table 1, the current grading for winter Winter wheat has been well established as the distilling wheat varieties are shown in Table 4. main cereal for the production of Scotch grain While the UK Recommendation process whisky for the last twenty years, and during has been successful in identifying several this period there have been significant changes Wheat for Scotch whisky production: broadening the horizon 57 in the way that wheat has been selected for T.A. (2004). The Role of the Rapid Visco distilling. Work carried out at SWRI on the Analyser (RVA) in Minimising Distillery potential alcohol yield of these varieties, has Processing Problems. In: Distilled Spirits: an important role in identifying suitable wheat Tradition and Innovation. Edited by Bryce, cultivars for distilling, and is an essential part of J.H. and Stewart, G.G. Nottingham University the official recommendation process for selecting Press, Nottingham, UK, pp. 89-94 new wheat for the UK. As a result of this work Brosnan, J.M. (2001), There’s something about we have a much better understanding of the Riband, Arable Farming, 26 May 2001, p. characteristics which define distilling wheat, 29 which we can now use to give guidance to plant Brosnan, J.M, Makari, S., Paterson, I. and breeders in the search for potential new distilling Cochrane, M.P. (1999). What Makes a Good varieties. Grain Distilling Wheat?. In: Proceedings of Scotch whisky distillers have traditionally the Fifth Aviemore Conference on Malting purchased wheat as a commodity, and for many Brewing and Distilling, 1998. Edited by years the main wheat cultivar grown in Scotland, Campbell, I., Institute of Brewing, London, Riband, was regarded as the ‘ideal’ distilling pp. 225-228 wheat. However this variety became much less Brown, J.H. (1990). Assessment of Wheat for economic to cultivate successfully, and has now Grain Distilling. In: Proceedings of the Third declined significantly, being replaced by Consort Aviemore Conference on Malting Brewing and Claire, which now provide the yardstick and Distilling, 1990, Edited by Campbell, I., against which new varieties are compared. Institute of Brewing, London, pp. 34-47 However, neither of these provides the same DEFRA (2005). 2004 Harvest: Final Estimates level of distilling performance as Riband. It is of Cereals Production, United Kingdom, thus essential to continue searching for new (Stats 03/05), Joint Announcement by the improved distilling varieties. Agricultural Departments of the United Contacts between distillers (SWRI) and Kingdom, Department for Environment plant breeders have stimulated an increase Farming and Rural Affairs, York/London in the numbers of new potential distilling HGCA (2005). HGCA Recommended Lists wheat cultivars entering trials. Some of these 2005-2006, for Cereals and Oilseeds, Home are now showing great promise for the future Grown Cereals Authority, London development and sustainability of wheat as a raw Institute of Brewing (1997). Method 1.8, Total material for Scotch whisky grain distilling. Nitrogen in Barley: Kjeldahl Method. Recommended Methods of Analysis, Institute of Brewing, London References National Association of British and Irish Millers (2005). Nabim Wheat Guide 2005, Agu, R.C. and Palmer, G.H. (2003). Pattern London of nitrogen distribution in barley grains National Institute of Agricultural Botany (2005). grown in the field. Journal of the Institute Wheat Certifications, NIAB, 2004 of Brewing 109: 110-113 Orth, R.A. and Shellenberger, J.A. (1988). Origin Bringhurst, T.A., Fotheringham, A.L., and Production and Utilisation of Wheat, In: Brosnan J. (2003). Grain Whisky: Raw Wheat: Chemistry and Technology, Third Materials and Processing. In: Whisky: Edition. Edited by Pomeranz, Y., American Technology, Production and Marketing. Society of Cereal Chemists, St Paul, USA, Edited by Russell, I., Academic Press, pp. 1-14 London, pp. 77-115 Palmer, G.H. (1986). Adjuncts in Brewing and Broadhead, A.L., Brosnan, J.M. and Bringhurst, Distilling. In: Proceedings of the Second 58 T.A. Bringhurst et al.

Aviemore Conference on Malting, Brewing affecting the qualtiy of wheat grain for and Distilling, 1986. Edited by Campbell, distilling in Northern Scotland. Aspects of I. and Priest, F.G., Institute of Brewing, Applied Biology, 25: Cereal Quality II, pp. London, pp. 24-45 183-191 Riffkin, H.L., Bringhurst, T.A., McDonald, A.M.L. Walker E.W. (1986). Grain Spirit – Which and Hands, E. (1990). Quality requirements Cereal. In: Proceedings of the Second of wheat for distilling. Aspects of Applied Aviemore Conference on Malting, Brewing Biology 25: Cereal Quality II, pp. 29-40 and Distilling, 1986. Edited by Campbell, Taylor, B.R. and Roscrow, J.C. (1990). Factors I. and Priest, F.G., Institute of Brewing, London, pp. 375-380. Cereals for grain distilling 59 Chapter 9 Cereals for grain distilling: can wheat and other cereals ever achieve the potential alcohol production of maize?

R.C. Agu, T.A. Bringhurst and J.M. Brosnan The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK

Introduction downtime, leading to substantial production losses. Traditionally maize (corn) was the cereal of Long standing experience with maize by the choice used by Scotch whisky grain distillers distilling industry, has shown that these problems until the general adoption of wheat during the are reduced when maize is used and although 1980’s (Walker, 1986; Brown, 1990). While the use of wheat is now almost ubiquitous in wheat is now well established as the main raw the production of Scotch grain whisky, the material for Scotch grain whisky production, processability of maize is considered to be better mainly as a result of its local availability suited to the optimum operation of most grain and favourable economics, it still has some distilleries. disadvantages compared with maize. The However, recent advances in the development yield of alcohol from wheat is generally much of potential new wheat cultivars which are lower than maize, in some cases by as much suitable for grain distilling are now beginning as 10-20 litres of alcohol per tonne. Wheat to yield examples which could potentially give also contains significant amounts of pentosans higher alcohol yield levels, similar to those of (such as arabinoxylans) (Palmer, 1989) which maize. The work reported here investigates some can survive through the distillery process and of the physiological and processing properties can cause processing problems (Preece and of such wheat varieties and contrasts them with MacDougall, 1958; Preece and Garg, 1961; maize and some other cereals. Bathgate and Palmer, 1972; Coote and Kirsop, 1976; Palmer, Gernah, McKernan and Nimmo, 1985; Ahluwalia and Fry, 1986). The presence of Materials and methods pentosans in the distillation residues (spent wash) can give potentially serious problems due to the Cereals samples, wheat (low and high nitrogen), high viscosity of the post-distillation evaporator maize, sorghum and millet were obtained feed stream, reducing the effectiveness of the from various sources, including plant breeding evaporators used to recover co-products. These companies, distillers and other sources (including problems can result in significant process Heriot Watt University). 60 R.C. Agu et al.

Alcohol yield Results and discussion

The method used was based on that of Brosnan, Potential distilling performance of cereals Makari, Paterson and Cochrane (1999), and is described in full in Bringhurst, Agu, Brosnan and The results obtained for the total nitrogen (TN) Fotheringham (2006) (in this volume). Alcohol spirit yield and the residue viscosity analysis yield was reported as litres of alcohol per tonne of samples of low and high nitrogen wheat (of (LA/tonne) on a dry weight basis. the same variety), maize, sorghum and millet are shown in Table 1. The low nitrogen wheat, which had a similar nitrogen content to the Residue viscosity maize sample, gave a high level of alcohol yield (472 LA per tonne dwb), and was similar to the Residues from the alcohol distillations were other cereals. This was considerably higher than adjusted to a fixed volume (250 ml) and would normally be expected for typical distilling centrifuged to remove the grain solids and wheat (about 435-455 LA per tonne) (Bringhurst spent yeast. The viscosity of the supernatant was et al., 2008). measured at 20°C using an Ostwald viscometer in a similar fashion to that described for the Table 1. Distilling potential of wheat, sorghum, viscosity of beer, in Institute of Brewing Method maize and millet samples. 9.2 (Institute of Brewing, 1997a). Residue viscosity is a measure of the viscosity Cereal variety/ TN PSY PSY Residue of the unfermentable material remaining in the type % LA/tonne LA/tonne viscosity (dwb) (dwb) (as is) (mPas) spent wash, prior to converting into co-products at the end of the process, and is an indication Wheat (Low N) 1.45 472 414 1.60 of the potential for processing problems. This Wheat (High N) 1.93 451 401 1.74 is quite distinct from the ‘front end’ process Maize (Yellow) 1.44 475 417 1.15 characteristics which are determined by RVA. Sorghum (White) 1.48 472 415 1.15 Sorghum (Red) 1.53 475 415 1.16 Millet 1.60 462 422 1.16 Total nitrogen

Total nitrogen analysis was determined on Buchi The sorghum samples also gave high alcohol Kjeldahl apparatus using a method similar to yields, comparable with maize, with red that for barley described in Institute of Brewing sorghum giving a marginal improvement on Method 1.8 (Institute of Brewing, 1997b). the white variety. Since the high yielding, red sorghum had a slightly higher level of nitrogen, this suggests that this cereal is less sensitive to RVA characteristics the effect of nitrogen levels than wheat, for which the inverse relationship between alcohol yield Processing characteristics of wheat, maize, and and nitrogen has been well established (Brosnan other cereals were studied on a rapid visco et al., 1999). Millet gave a slightly lower alcohol analyser (RVA) (Newport Scientific, supplied by yield than the other cereals. Calibre Control Ltd) (Newport Scientific, 1998), The result for the low nitrogen wheat was using a standard program for unmalted cereals. about 20 litres of alcohol per tonne higher than This method measures the starch properties the sample of high nitrogen wheat of the same which influence cereal handling during the ‘front variety, which was grown on the same site. This end’ cooking cycle of the process. gave an alcohol yield of 450 LA/tonne, which Cereals for grain distilling 61 was also higher than expected for a distilling peak than the high nitrogen wheat. Maize shows wheat sample, at this nitrogen level (1.9 percent a very different profile from wheat, giving a much (dwb)). The high alcohol yields, for these ‘elite’ higher, but less well defined peak viscosity. wheat samples, reflect careful management of While both types of sorghum had similar RVA nitrogen application on the growing sites, but characteristics to maize, millet, which gave the indicate that it is now possible to produce wheat second highest peak viscosity, had a profile which which can produce high levels of alcohol yield in some ways was more like that of wheat. All of similar to maize and other cereals. these cereals gave higher levels of RVA viscosity The data shown in Table 1 also contrast than wheat, suggesting significant physiological the residual viscosities of the spent wash from similarities between maize and sorghum as well the laboratory distillations, measured using an as strong differences between maize and wheat Ostwald viscometer, and show that while the (and to some extent, millet). It is well established other cereals, maize, sorghum and millet, all that maize, sorghum and millet have similar gave similar, low, viscosity values, those for chemical and physical properties and have much the wheat samples were much higher, with the higher gelatinization temperatures than wheat high nitrogen wheat giving the highest residual (Haln, 1966; Palmer, 1989), possibly reflecting viscosity. These results, which were typical for the warm climactic habitats associated with these wheat, suggest that there would still be a potential cereal grains (Agu and Palmer, 1998) in contrast risk of downstream processing problems with to wheat, which is a temperate crop. These might wheat, even at very high alcohol yields. explain some of the observed differences in the processing characteristics of these cereals. Numerical data generated by the RVA RVA profiles for wheat, maize, sorghum and instrument software are shown in Table 2. These millet confirm that the lower nitrogen wheat gave approximately double the peak viscosity of the Figure 1 contrasts the RVA profiles of low and higher nitrogen wheat sample. high nitrogen wheat samples, with other cereals However, the full implications of the RVA (maize, red and white sorghum and millet). This results for the cereals studied are not clear at shows that both low and high nitrogen wheat present, but they are probably related to the samples gave similar profiles, but that the low relative amounts of starch which are available nitrogen wheat gave a much higher gelatinisation for hydrolysis, and thus alcohol production,

Red sorghum 1600 Yellow maize

1200 White sorghum Millet 800 Low N wheat Viscosity (cP) 400

High N wheat 0 036912 151 Time (min) Figure 1. RVA profiles for wheat, maize, red and white sorghum and millet (from Agu et al., 2006, Figure 2). 62 R.C. Agu et al.

Table 2. RVA data for wheat (low/high N), maize, sorghum (red/white) and millet (modified from Agu et al., 2006, Table III). Sample Pasting Peak Peak Peak Final Temp (oC) Time (min) Temp (oC) Viscosity (cP) Viscosity (cP)

Sorghum (Red) 72.60 5.93 95.00 795 1652 Maize (Yellow) 67.65 6.60 94.90 712 1390 Sorghum (White) 76.65 6.93 95.00 495 1145 Millet 70.05 4.87 94.90 722 917 Low N wheat 60.25 5.00 94.80 440 636 High N wheat 66.10 4.67 94.40 169 120

during the processing of these cereals. In the This hypothesis was tested, by looking at a context of the Scotch whisky grain distilling range of low and high nitrogen wheat samples process, the cooking phase for wheat was and seeing if these followed similar trends. originally developed to handle maize, and on the The alcohol yield, total nitrogen content, and basis of these results should be able to efficiently RVA profiles for 4 varieties grown under low process the other cereals studied. and high nitrogen conditions are shown in Figures 2, 3, 4 and 5 and these indicate that the observed relationship appears to be valid. RVA RVA characteristics of low and high nitrogen data generated by these samples are shown in wheat samples Table 3. These results confirm that there is a pattern, Overall, the experiments suggest that the and support the hypothesis that for an individual potential alcohol yield of wheat might be related wheat variety, high peak and final viscosity will to RVA peak and final viscosities, since the RVA be associated with higher alcohol yields and that profile is strongly influenced by the relative this is strongly influenced by the level of nitrogen amounts of gelatinised polysaccharide substrate accumulated in the grain. (such as starch) so that low nitrogen wheat While it is not clear whether these relationships which produces more alcohol yield will give will hold for all wheat varieties, the correlations higher peak and final viscosities in comparison generated by these sets of samples are very high, to higher nitrogen wheat samples (of the same and show that there is a potential to influence variety), which are associated with lower alcohol the alcohol yield and processing characteristics yields. by careful manipulation of the nitrogen levels

Low N wheat A 900 PSY = 464 LA/tonne (dwb) TN = 1.62% (dwb) (Residue viscosity = 1.63 mPa)

600

Viscosity (cP) 300 High N wheat A PSY = 450 LA/tonne (dwb) TN = 1.85% (dwb) (Residue viscosity = 1.82 mPa) 0 0136912 5 Time (min) Figure 2. RVA curves for Low N and High N wheat A. Cereals for grain distilling 63

Low N wheat B PSY = 462 LA/tonne (dwb) TN = 1.51% (dwb) 600 (Residue Viscosity = 1.63 mPa)

High N wheat B 400 PSY = 446 LA/tonne (dwb) TN = 1.82% (dwb) (Residue Viscosity = 1.78 mPa) Viscosity (cP) 200

0 036912 15 Time (min) Figure 3. RVA curves for Low N and High N wheat B.

Low N wheat C PSY = 457 LA/tonne (dwb) 450 TN = 1.59% (dwb) (Residue viscosity = 1.72 mPa)

300 Viscosity (cP) 150 High N wheat C PSY = 450 LA/t (dwb) TN = 1.84% (dwb) (Residue viscosity = 1.83 mPa) 0 0136912 5

Time (min) Figure 4. RVA curves for Low and High N Wheat C.

Low N wheat D 450 PSY = 455 LA/tonne (dwb) TN = 1.62% (dwb) (Residue viscosity = 1.71 mPa)

300 High N wheat D PSY = 445 LA/t (dwb) TN = 1.83% (dwb) (Residue viscosity = 1.79 mPa)

Viscosity (cP) 150

0 0136912 5 Time (min)

Figure 5. RVA curves for Low and High N Wheat D. 64 R.C. Agu et al.

Table 3. RVA data for low and high nitrogen wheat samples.

Sample Pasting Peak Peak Peak Final Temp (oC) Time (min) Temp (oC) viscosity (cP) viscosity (cP)

Low N Wheat A 74.25 5.13 94.90 566 873 High N Wheat A 66.80 5.93 93.60 101 70 Low N Wheat B 72.60 5.00 94.80 430 727 High N Wheat B 76.60 5.00 94.85 304 458 Low N Wheat C 66.15 4.93 94.75 385 462 High N Wheat C 67.00 4.67 94.40 100 69 Low N Wheat D 69.35 4.93 94.75 327 447 High N Wheat D 72.50 5.00 94.80 190 243

during growth, since the level of nitrogen Further physiological differences between uptake by wheat in the field during growth is wheat and maize were also highlighted, and influenced by the growing conditions (Orth and wheat produced spent wash residues with much Shellenberger, 1988; Agu and Palmer, 2003). higher viscosity than maize, sorghum and millet. Swanston, Newton, Brosnan, Fotheringham This is probably due to the presence of high and Glasgow (2005) suggest that different levels of arabinoxylans in wheat (Ahluwalia and wheat varieties will uptake nitrogen from the Fry, 1986; Palmer, 1989). Low nitrogen wheat field at different rates. As well as supporting samples, however, produced lower spent wash this hypothesis, the work reported here also viscosity residues than higher nitrogen wheat. suggests that the degree of nitrogen uptake will This confirms the importance of selecting have differing effects on both the alcohol yield low nitrogen wheat for distilling to minimise and processing characteristics of different wheat processing problems associated with wheat. varieties. The RVA characteristics of wheat were also The reasons for the variable and in some very different from maize and are related to cases, very low, viscosity profiles of some of the differences in the relative amounts and structures high nitrogen wheat samples are uncertain, but of carbohydrate polymers, such as starch, in could possibly indicate that some of the high these cereals, which have a strong influence on nitrogen wheat samples might contain trace gelatinisation temperatures, and will have an levels of a-amylase, however further work would important impact on ‘front end’ processes such be required to confirm this. as cooking, mashing and wort handling. The similarities between the RVA pasting curves for maize, sorghum and millet are Conclusions interesting, particularly if the use of sorghum is ever considered by grain distillers in the future. The work presented in this report confirms that Although currently Scotch whisky distillers do it is now possible to produce wheat varieties not use sorghum or millet, the alcohol yield capable of yielding alcohol at similar levels to and processing data which have been obtained those obtainable from maize, which was the show that these cereals could conceivably be grain traditionally used by Scotch whisky grain used for the production of whisky, provided distillers. It is also clear that the nitrogen content that the economics were favourable, and that of the wheat is a key parameter and must be any handling problems resulting from using very carefully controlled during the growing season small grains could be overcome. to achieve high alcohol yields. Cereals for grain distilling 65

Low nitrogen wheat gave much higher RVA These experiments show that it is now possible peak and final viscosities than high nitrogen to obtain wheat which is capable of producing wheat, indicating that low nitrogen wheat is similar alcohol yields to maize, which was the associated with greater amounts and better cereal, traditionally used by Scotch whisky grain accessibility of starch, and suggests that there distillers, until the general adoption of wheat could be a relationship between RVA viscosity during the 1980’s. Provided that viscosity issues parameters and potential alcohol yield. This can be managed to give acceptable processing might potentially be of use when developing a performance, this work confirms that there is model to predict the alcohol yield of wheat. continuing scope for developing increasingly While it is now possible for wheat varieties high alcohol producing wheat varieties. to produce comparable alcohol yields to maize, the processing difficulties arising from wheat viscosity will still remain, although it should be References possible to minimise these problems in future by selecting for appropriate distilling wheat quality, Agu, R. C. and Palmer, G. H. (1998). A as we understand more about the processing reassessment of sorghum for lager-beer aspects of wheat. brewing. Bioresource Technology 66: 253-261 Agu, R. C. and Palmer, G. H. (2003). Pattern Summary of nitrogen distribution in barley grains grown in the field. Journal of the Institute The distilling potential of samples of wheat, of Brewing 109: 110-113 sorghum and maize, at comparable nitrogen Agu, R.C., Bringhurst, T.A. and Brosnan, J.M. levels, was studied with regards to alcohol yield (2006). Production of grain whisky and and residue viscosity. In these experiments we ethanol from wheat, maize and other cereals. compared wheat samples (high/low nitrogen, Journal of the Institute of Brewing 112: same variety), with maize and sorghum (red/ 314-333 white). The results showed that the low nitrogen Ahluwalia, B. and Fry, S. C. (1986). Barley wheat, gave an alcohol yield which was 20 endosperm cell walls contain a feruloylated litres of alcohol per tonne higher than the high arabinoxylan and a non-feruloylated ß-glucan. nitrogen sample. This was comparable to levels Journal of Cereal Science 4: 287-295 associated with maize. The sorghum samples Bathgate, G. N. and Palmer, G. H. (1972). A also gave high alcohol yields which were reassessment of the chemical structure of comparable with maize, with the red sorghum barley and wheat starch granules. Die Starke giving a marginal improvement on the white 24: 336-341 variety. Since the red sorghum had a higher Bringhurst, T.A., Agu, R.C, Brosnan, J.M. level of nitrogen, this suggests that this cereal and Fotheringham, A. (2008). Wheat for is less sensitive to the effect of nitrogen levels Scotch Whisky production:- Broadening the than wheat. horizon, In: Proceedings of the 2005 World Comparisons of the viscosity characteristics Wide Distilled Spirits Conference held in of wheat, maize and sorghum, showed that the Edinburgh, Edited by Bryce, J.H., Piggot, J.R. low nitrogen sample of wheat gave a much lower and Stewart, G.G, Nottingham University viscosity than at high nitrogen levels. However Press, Nottingham, UK, pp. 51-58 wheat gave a higher residue viscosity than either Brosnan, J. M., Makari, S., Paterson, L. and maize or sorghum. There appeared to be no Cochrane, M. P. (1999). What makes a good substantial difference between the processing distilling wheat, In: Proceedings of the Fifth characteristics of the maize and either of the two Aviemore Conference on Malting, Brewing sorghum samples. and Distilling 1998. Edited by Campbell, 66 R.C. Agu et al.

I., Institute of Brewing, London, UK, pp. Science and Technology (ed. Palmer, G.H.) 225-228. Aberdeen University Press, Aberdeen, pp. Brown, J.H. (1990). Assessment of wheat for 80-86 grain distilling, In: Proceedings of the Palmer, G. H., Gernah, D. I., McKernan, G. and Third Aviemore Conference on Malting Nimmo, D. H. (1985) Influence of enzyme Brewing and Distilling, 1990, Edited by distribution on endosperm breakdown Campbell, I., Institute of Brewing, London, (modification) during malting. American UK, pp.34-47 Society of Brewing Chemists 42: 17-28 Coote, N. and Kirsop, B. H. (1976). A haze Preece, I, A. and MacDougall, M. (1958) Enzymic constituting largely of pentosan. Journal of degradation of cereal II. the Institute of Brewing 82: 34 Pattern of pentosan degradation. Journal of Haln, R. R. (1966). Sorghum as a brewing the Institute of Brewing 58: 353-362 adjunct. Brewer’s Digest. 49: 70-74 Preece, I, A. and Garg, N. K. J. (1961) Enzymic Institute of Brewing (1997), Recommended degradation of cereal hemicelluloses IV. Methods of Analysis, Institute of Brewing, Quantitative aspects of â-glucan degradation. London, UK Journal of the Institute of Brewing 67: Newport Scientific (1998). Application Manual 267-273 for the Rapid Visco Analyser. Newport Swanston, J. S., Newton, A. C., Brosnan, J. M., Scientific Pty. Ltd Warriewood, Australia, Fotheringham, A., and Glasgow, E. (2005), pp. 19-26 Determining the spirit yield of wheat Orth, R. A. and Shellenberger, J. A. (1988). varieties and variety mixtures, Journal of Origin, production and utilisation of wheat. Cereal Science 42: 127-134 In: Wheat: Chemistry and Technology, 3rd Walker E.W. (1986), Grain Spirit – Which edition, Volume 1, Edited by Pomeranz, Y., Cereal, In: Proceedings of the Second American Association of Cereal Chemists, St Aviemore Conference on Malting, Brewing Paul, USA, pp. 1-14 and Distilling, edited by Campbell, I. and Palmer, G. H. (1989). Properties of â-glucan Priest, F.G., Institute of Brewing, London, extracts of endosperm cell walls. In: Cereal pp. 375-380. The influence of nitrogen content and corn size on the quality of distilling wheat cultivars 67 Chapter 10 The influence of nitrogen content and corn size on the quality of distilling wheat cultivars

R.C. Agu1, J.S. Swanston2, T.A. Bringhurst1, J.M. Brosnan1, F.R. Jack1 and P.L. Smith2 1The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK; 2Scottish Crop Research Institute, Invergowrie, Dundee, UK

Introduction An important aspect of this will be the development and application of rapid screening The work described below was carried out techniques, which, along with the identification as part of the initial stages of a collaborative of genetic markers, will facilitate selection project entitled ‘Genetic Reduction of Energy for suitable attributes in wheat cultivars. Use and Emission of Nitrogen through Cereal However before commercial breeding can Production (GREEN Grain)’ which is sponsored take place, it is essential that we have a clearer by the Department of Environment, Food and understanding of the influence of grain total Rural Affairs (DEFRA) and the Scottish Executive nitrogen on the attributes which we would like to Environment and Rural Affairs Department develop, particularly on the potential for ethanol (SEERAD), through the UK Sustainable Arable production for end users, such as Scotch whisky LINK Programme. The Project is supported by the distillers. Home Grown Cereals Authority (HGCA) and the Currently, winter wheat is still the major following Research and Industrial partners; ADAS raw material for the production of Scotch grain UK Ltd, Foss UK Ltd, Grampian Country Food whisky, and the quantity (yield) of alcohol Group Ltd, the Scottish Crop Research Institute produced by a distillery is very much dependent (SCRI), the Scotch Whisky Research Institute on the quality of the wheat used in the process. (SWRI), Syngenta Seeds and Wessex Grain Ltd. Although there is a substantial body of work The main aims of this project are to improve published on wheat quality, much of this is the value of UK wheat for ethanol production and focussed on processes such as bread making or non-ruminant feeding, and to reduce the costs of feed production (e.g. Pomeranz, 1988), which growing wheat for these and other applications require different wheat quality from that desired (Sylvester-Bradley, 2004). In practical terms for distilling. Distilling wheat is characterised this means developing new wheat varieties by soft endosperm texture, high levels of starch with high energy content (starch) and low input and low total nitrogen (protein) content (Brown, requirements (particularly in terms of nitrogen 1990; Riffkin, Bringhurst, McDonald and Hands, fertiliser), which are suitable for these markets 1990; Brosnan, Makari, Paterson and Cochrane, as well as providing guidance for wheat growers 1999; Bringhurst, Fotheringham and Brosnan, in determining optimal, more environmentally 2003; Broadhead, Brosnan and Bringhurst, friendly, agronomic practices for wheat. 2004). 68 R.C. Agu et al.

For many years, research studies into the data are available this can be used to formulate a quality characteristics of wheat have been in model for assessing the distilling quality of wheat progress at SWRI to assess the distilling potential based on simple, rapid techniques. This would of new and up and coming distilling wheat give substantial benefits by facilitating rapid varieties harvested each year. These studies intake testing of wheat for grain distilleries. complement the annual agronomic assessments carried out by other organisations such as the Scottish Agricultural College (SAC) and Crop Materials and methods Evaluation Ltd (CEL), and are now included in the official Recommendation process for new wheat Samples varieties in the UK. However, greater in-depth knowledge of the physiology and composition A range of winter wheat samples were obtained of distilling wheat is necessary to facilitate the from trial sites grown at various locations in the production of increasingly high quality wheat, UK during 2004 (including Northern/Central capable of delivering substantial improvements Scotland and the North and East of England). in alcohol yield. In addition, while it is the aim Additional individual wheat varieties were of the plant breeders to produce new wheat grown on trial sites run by the Scottish Crop varieties with improved genetic potential, the Research Institute (SCRI). major interest of growers and end users is in the environmental and agronomic factors that determine whether quality potential is achieved Alcohol yield of wheat in known varieties. Aside from alcohol yield, the most important The alcohol yield of wheat samples was parameter influencing the quality of wheat determined using standard methodology used for distilling is the total nitrogen content described in Bringhurst, Agu, Brosnan and of the grain, and it is well established that there Fotheringham, (2006 (in this volume)). Prior to is a significant inverse relationship between the determination of alcohol yield samples were grain nitrogen and alcohol yield (Brosnan et al, analysed for moisture content, and the results 1999). However this does not explain all of the were expressed in litres of alcohol per tonne (LA/ differences which have been observed between tonne) on a dry weight basis (dwb). different wheat samples. This paper describes efforts to look at the relationship between these parameters in more detail. Total nitrogen of wheat A further variable, corn size, was also studied. At present it is not known to what extent corn size The total nitrogen of wheat samples was will affect the distilling potential of wheat. Corn determined using a Kjeldahl method similar to size is known to influence the physical properties that described for barley in the Recommended of other cereals such as barley, as well as the Methods of the Institute of Brewing (Method efficient recovery of extract during the brewing 1.8) (Institute of Brewing, 1997a). Results were process (Home, Wilhelmson, Tammisola and reported as a percentage on a dry weight basis Husman, 1997; Agu and Palmer, 1998). Hence (dwb). it is suspected that corn size might also be an important determinant of the distilling quality of wheat, and we have now further explored the Measurement of corn size relationship between the corn size distribution (percentages of small corns), total nitrogen and Corn size was determined using a screening alcohol yield. It is hoped that once sufficient box, originally designed for barley, essentially The influence of nitrogen content and corn size on the quality of distilling wheat cultivars 69 as described in the Recommended Methods of around 30 litres of alcohol per tonne), and this the Institute of Brewing (Method 1.13) (Institute disparity suggests that the relationship with grain of Brewing, 1997b), but with manual shaking. nitrogen is more complex than was previously In this method 100 g wheat grains were placed considered, and that other factors may also have in the screening box, and shaken for 2 minutes. important effects. The mesh sizes were 2.8 mm, 2.5 mm and The observed variation is partly a result 2.2 mm. The proportions of small corns were of differences between growing sites, where determined as the percentage (weight/weight) wheat can uptake nitrogen at different rates of the corns which passed through the 2.5 mm depending on the growing conditions which are screen together with those which passed through experienced (soil fertility, fertiliser application, the 2.2 mm screen (i.e. all the corns < 2.5 mm). field topography and weather) (Orth and The proportion of large corns was defined as the Shellenberger, 1988, Agu and Palmer, 2003). percentage of the sample which was retained The complexity of the relationship between by the 2.8 mm screen. The corns which lay grain nitrogen and alcohol yield is confirmed between 2.5 and 2.8 mm were designated as an by a separate study carried out by the Scottish intermediate fraction. Crop Research Institute, on a series of individual wheat varieties, grown over a range of sites in two seasons. The relationships between total Results and discussion nitrogen and alcohol yield for a selection of individual varieties (Istabraq, Consort and Influence of nitrogen on alcohol yield Deben) are shown in Figure 2. These indicate that the relationship between these parameters Wheat cultivars (10), grown on 6 trial sites at may not necessarily be linear, and that the link various locations throughout the UK, were between total nitrogen and alcohol yield might analysed for alcohol yield potential (PSY), approximate more closely to a polynomial and total nitrogen, as part of the normal SWRI (quadratic) relationship. assessment of the 2004 wheat harvest. The Additionally, the shape of the curve is quite correlation between alcohol yield and total different for each wheat variety, indicating that nitrogen levels, shown in Figure 1, accounted they are subject to different interactions between for 52.2 percent of the variability in the alcohol alcohol yield and total nitrogen. While in this yield data. case, the significance may be questionable, The range of alcohol yields for a given due to the small number of data points, the nitrogen content can be very large (up to relationships are supported by previous reports

470

460

450

440

430

PSY (LA/Tonne) dry 420 Correlation = -0.72226 2 R = 0.5217 410

400 1.51.7 1.92.1 2.32.5 TN (%) dry

Figure 1. Correlation between alcohol yield (PSY) and the grain total nitrogen content of wheat samples grown during 2004 season on a range of trial sites in the UK. 70 R.C. Agu et al.

Istabraq Consort 460 460 455 y = 54.191x2 - 245.16x + 705.41 450 450 440 445 2 430 440 R = 0.7821 420 Spirit Yield 435 Spirit Yield y = 5.593x 2 - 76.903x + 558.35 410 430 R2 = 0.8231 425 400 1.51.7 1.9 2.1 2.3 1.5 1.7 1.9 2.1 2.3

% Nitrogen % Nitrogen

Deben 450 y = -2- 1.508x2 + 13.058x + 477.3 440

430 R2 = 0.7923

420 Spirit Yield 410

400 1.5 1.7 1.9 2.1 2.3

% Nitrogen Figure 2. Relationship between total nitrogen and alcohol yield for samples of individual wheat cultivars for 2003 and 2004 seasons (SCRI data).

(Swanston, Newton, Brosnan, Fotheringham and and genetic factors play an important role in Glasgow, 2005). the factors defining the distilling potential of Together, these observations imply that there wheat. is an additional degree of complexity in the relationships between these two parameters which affects the ability of different wheat Influence of corn size varieties to maintain their alcohol potential with increasing nitrogen levels. One factor which was considered to be This is most apparent with Deben, which potentially important in influencing the potential is not considered to be a good distilling wheat alcohol yield is corn size. This was quantified variety. The ‘convex’ shape of the curve for this by determining the proportion of small corns in variety indicates that the alcohol yield potential the wheat samples. falls off rapidly as the nitrogen level increases Figure 3 shows that there is a correlation above 2 percent. This is in contrast to the data between the percentage of small corns (<2.5mm), for Istabraq, which gives a more ‘concave’ and the PSY, which accounts for 31.7 percent of profile, showing a more gradual fall off in the variation in the alcohol yield data. alcohol yield as the nitrogen level increases. These results confirm that together with total Consort approximates a more linear decline in nitrogen, corn size may also be an important alcohol yield with increasing nitrogen levels. determinant of alcohol yield and that in general, These results suggest that both environmental wheat samples with a high percentage of small The influence of nitrogen content and corn size on the quality of distilling wheat cultivars 71

470

460 Correlation = - 0.563419 R2 = 0.3174 450

440

430

PSY (LA/Tonne) dry 420

410

400 0510 15 20 25 30 35 Percentage of small corns (< 2.5mm)

Figure 3. Correlation between percentage of small corns (<2.5mm) and alcohol yield (PSY) of wheat samples grown during 2004 season on a range of trial sites in the UK. corns will give relatively low alcohol yields, influence on alcohol yield, but that individually, while conversely, those with a high proportion neither can fully explain the variation in the data. of large corns will tend to give higher alcohol To explore this further, a multiple regression yields. model was constructed and used to determine As the proportion of small corns increases, the whether an improved prediction of alcohol total nitrogen level of the wheat also increases, yield could be obtained by taking account of suggesting that the relative proportions of starch the combined influence of these two parameters. and protein may differ in corns of different sizes. Figure 5 shows the resulting model of predicted However, the correlation between total nitrogen versus actual alcohol yield. and the proportion of small corns (R2 = 0.2662), This model gave an improvement in the indicates that these two parameters are related, prediction of alcohol yield, accounting for 57.1 but that they are to some degree independent of percent of the variation in the data, compared each other (Figure 4). with the total nitrogen and the corn size individually (52.2 percent and 31.7 percent respectively). Prediction of alcohol yield Although the multiple regression analysis was able to account for a sizeable proportion The data summarised above show that both of the variation in the model, it is clear that a grain nitrogen and corn size have a fundamental significant component of the alcohol yield is not

35 Correlation = 0.51595 30 R2 = 0.2662 25

20

15

10

Percentage of small corns 5

0 1.5 1.7 1.9 2.1 2.3 2.5 TN (%) dry

Figure 4. Correlation between the percentage of small corns (<2.5mm) and the total nitrogen content of wheat. 72 R.C. Agu et al.

450 De Cl De De G G De Cl Cl G GG 440 De Cl Cl

De 430

420 Cl G 2 R = 0.5712

410 PSY predicted from TN and % small corn s 400 410 420 430 440 450460 Actual PSY Figure 5. Predicted alcohol yield from TN and % small corns based on a multiple regression model (Outlying samples: Glasgow (G), Claire (C), Deben (D)). accounted for. This, together with the presence these relationships will change from season of a number of outliers, suggests that for some to season. Further work is necessary, before a wheat samples there are other parameters which reliable model can be developed. might also contribute to the alcohol yield. The corn size distribution of a wheat sample, together with its nitrogen content, is probably Conclusions influenced by the growing and environmental conditions within each trial plot. However, The work described above shows that the while the relationship between corn size and relationship between alcohol yield and grain alcohol yield appears to apply for individual total nitrogen is more complex than was varieties, it is known that certain wheat cultivars previously considered, and that wheat varieties such as Claire and Glasgow are associated with are influenced by nitrogen levels in different relatively small corns, but can still give good ways, with some having a better (or poorer) alcohol yields This is supported by Swanston ability to maintain their alcohol yield potential et al (2005) who noted that the relationship with increasing nitrogen levels. between potential alcohol yield and thousand In addition, other parameters such as corn corn weight appeared to break down when small size can also have a significant influence on grained samples such as Claire were included in the distilling potential of wheat samples, and the data. Both Claire and Glasgow gave outlying combining the effects of corn size and total values in the dataset shown in Figure 5. nitrogen gives an improved prediction of wheat The work which was reported was based on alcohol yield. However, the multiple regression a wide range of samples deriving from a number model which was developed did not completely of trial sites located around the UK, and as a explain all of the variation in the alcohol result the range of nitrogen levels in the sample yield data, indicating that there are still some set (1.6 - 2.5 percent nitrogen) is much higher significant factors to be considered. than would normally be expected for actual The results of the study suggest that the wheat deliveries to distilleries, reflecting a wide inclusion of corn size, together with total range of growing sites in the UK. It is possible nitrogen, at intake testing would give distillers that this wide range of nitrogen levels might a rapid and more accurate means of predicting have an impact on the relationship between wheat distilling quality. the parameters which are discussed here. In The GREEN Grain Project is a 5 year project addition the data were obtained over a single designed to closely study the impact of nitrogen harvest year, and it is likely that the detail of uptake and utilisation of wheat and its effects The influence of nitrogen content and corn size on the quality of distilling wheat cultivars 73 on grain quality for Scotch whisky production, such as soil fertility, fertiliser application, field as well as a wide range of other applications, topography and weather conditions during the such as bioethanol and non-ruminant feeds. growing season, all of which could influence During the course of the project, a large number the precise relationship between alcohol of samples will be generated and analysed yield and nitrogen content for different wheat over several growing seasons. This will add cultivars. Consequently, batches of wheat considerably to the database, and should grown at different locations can achieve similar facilitate the development of a much improved nitrogen levels despite having very different model to predict the alcohol yield potential of patterns of grain filling, and this could have a wheat. significant impact on potential alcohol yield for Study of the relationship between nitrogen grain distilleries. This paper studies some ways and corn size has resulted in an increased that alcohol yield can be influenced by other understanding of the factors which influence parameters. the potential alcohol yield of wheat, (and Close study of these parameters has resulted potentially, other cereals) and this will pave the in an increased understanding of the factors way for the development of new techniques to which influence the potential alcohol yield of measure the distilling quality of wheat, such wheat, and other cereals, and this will pave the as NIR. This should eventually enable us to way for the development of new techniques to predict the distilling quality of wheat (and other measure the distilling quality of wheat. This will cereals) in a similar way that Bishop’s equations not only facilitate grain distillers’ intake testing, have been used for malting barley (Briggs, but will also provide guidance for growers in Hough, Stevens and Young, 1981), and will determining optimal, more environmentally not only facilitate grain distillers’ intake testing, friendly, agronomic practices for wheat. but will also provide guidance for growers in determining optimal, more environmentally friendly, agronomic practices for wheat. References

Agu, R. C. and Palmer, G. H. (1998). Some Summary relationships between the protein nitrogen of barley and the production of amylolytic The work described was carried out as part of enzymes during mashing. Journal of the the initial stages of a DEFRA/HGCA funded LINK Institute of Brewing 104: 273 – 276 collaborative project entitled ‘Genetic Reduction Agu, R. C. and Palmer, G. H. (2003). Pattern of Energy use and Emission of Nitrogen through of nitrogen distribution in barley grains cereal production (GREEN Grain)’, funded under grown in the field. Journal of the Institute the UK Sustainable Arable LINK programme. of Brewing 109: 110-113 It has been well established, that the alcohol Briggs, D.E., Hough, J.S., Stevens, R. and yield from cereals such as wheat, for grain Young, T.W. (1981). Malting and Brewing distilling, is inversely related to the level of Science, Malt and Sweet Wort. Second nitrogen (protein) in the grain, and this is a major Edition, Chapman and Hall, London, pp. quality indicator for wheat for grain distilling. 113 – 115 However more recent work at the Scotch Bringhurst, T.A., Agu, R.C, Brosnan, J.M. Whisky Research Institute and at the Scottish and Fotheringham, A. (2008). Wheat for Crop Research Institute, has indicated that the Scotch Whisky Production: Broadening the relationship between these two parameters is Horizon. In: Distilled Spirits: Production, more complex than expected. The nitrogen Technology and Innovation. Edited by available to growing plants is affected by factors J.H. Bryce, J.R. Piggott and G.G. Stewart, 74 R.C. Agu et al.

Nottingham University Press, Nottingham, Institute of Brewing (1997a). Method 1.18, Total UK, pp. 51-58 Nitrogen in Barley, Recommended Methods Bringhurst, T.A., Fotheringham, A.L., and of Analysis, Institute of Brewing, London Brosnan J.M. (2003). Grain Whisky: Raw Institute of Brewing (1997b). Method 1.13, Materials and Processing. In: Whisky: Sieving Test for Barley, Recommended Technology, Production and Marketing. Methods of Analysis, Institute of Brewing, Edited by Russell, I., Academic Press, London London, pp. 77-115 Orth, R. A. and Shellenberger, J. A. (1988). Broadhead, A.L., Brosnan, J.M. and Bringhurst, Origin, production and utilisation of wheat. T.A. (2004). The Role of the Rapid Visco In: Wheat: Chemistry and Technology, 3rd Analyser (RVA) in Minimising Distillery Edition. Edited by Pomeranz Y., American Processing Problems. In: Distilled Spirits: Association of Cereal Chemists, St Paul, Tradition and Innovation. Edited by USA, pp. 1-14 Bryce, J.H. and Stewart, G.G., Nottingham Pomeranz, Y. (1988). Wheat: Chemistry and University Press, Nottingham, UK, pp. Technology, 3rd Edition, Edited by Pomeranz 89-94 Y., American Association of Cereal Chemists, Brosnan, J. M., Makari, S., Paterson, L. and St Paul, USA Cochrane, M. P. (1999). What makes a good Riffkin, H.L., Bringhurst, T.A., McDonald, A.M.L distilling wheat. In: Proceedings of the Fifth and Hands, E. (1990). Quality requirements Aviemore Conference on Malting, Brewing of wheat for distilling. Aspects of Applied and Distilling. Edited by Campbell, I., Biology 25, Cereal Quality II, pp. 29-40 Institute of Brewing, London, pp. 225-228 Swanston, J. S., Newton, A. C., Brosnan, J. M., Brown, J. H. (1990). Assessment of wheat for Fotheringham, A. and Glasgow, E. (2005). grain distilling. In: Proceedings of the Third Determining the spirit yield of wheat Aviemore Conference on Malting, Brewing varieties and variety mixtures. Journal of and Distilling. Edited by Campbell, I ., Cereal Science 42: 127-134 Institute of Brewing, London, pp 34-47 Sylvester-Bradley, R. (2004). Genetic Reduction Home, S., Wilhelmson, A., Tammisola, J. and of Energy use and Emissions of Nitrogen Husman, J. (1997). Natural variation among through cereal production (GREEN Grain), barley kernels. Journal of the American LINK Project Proposal to DEFRA/HGCA Society of Brewing Chemists 55: 47-51 March 2004. A turbidity test for a genetic component of spirit yield in wheat 75 Chapter 11 A turbidity test for a genetic component of spirit yield in wheat

J. S. Swanston and P. L. Smith Scottish Crop Research Institute, Invergowrie, Dundee, UK

Introduction it has been shown that the precise relationship between these factors differs between varieties In the production of grain whisky, the starch from (Swanston, Newton, Brosnan, Fotheringham, unmalted cereal grain (wheat or maize) is broken and Glasgow, 2005) and that spirit yield declines down by enzymes from the small proportion of more rapidly with increasing protein content, in malted barley that is added to the mash. The varieties with poorer distilling quality. Varieties sugars thus produced are then fermented into in which starch is more readily released from the alcohol. The best wheat varieties for distilling are protein may thus be inherently higher in spirit those that give the highest quantities of alcohol yield and a test to identify this characteristic after distillation, i.e. the best spirit yields, from could be a useful plant breeding tool. a given quantity of grain. In barley, Koliatsou and Palmer (2003) Although spirit yield is a starch-related measured the turbidity of finely milled and property, it is not simply a function of starch sieved flour suspended in ethanol. They showed content. Riffkin, Bringhurst, McDonald and that varieties with a mealy endosperm released Hands (1990) showed starch content to have starch more readily and therefore had higher a positive, but not particularly high correlation turbidity than those with steely endosperms. with spirit yield. They suggested that the relative Consequently, feed varieties had low turbidity, proportions of large and small starch granules while, among malting barleys, the variety Optic could be important, a view also expressed in had very high turbidity. It was therefore decided a later study (Brosnan, Makari, Paterson and to apply this technique to wheat, to ascertain Cochrane, 1998). However, it is also possible whether turbidity could be a useful predictor that endosperm structure and in particular, of distilling quality. The effects of site, season ease of access to the starch may influence spirit and nitrogen application on turbidity were also yield. studied. The starch in wheat occurs in the form of granules contained within a matrix of protein and there is a very strong negative association Materials and methods between grain protein (usually measured as nitrogen) content and spirit yield (Riffkin et al., The turbidity method was based on that of 1990; Taylor and Roscrow, 1990). More recently, Koliatsou and Palmer (2003), who noted that 76 J.S. Swanston and P.L. Smith consistency of milling was of major importance. Results Greatest repeatability and discrimination between samples of differing turbidity was thus obtained Initial comparison of varieties in our laboratory, by milling the wheat grain in a Retsch mill with a 0.5mm sieve size and shaking The varieties Claire, Consort, Deben, Riband and the resultant flour through a 250µm Endecotts Wizard were included in trials at SCRI harvested sieve (Endecotts Ltd., London). A portion of the in 2004. Figure 1 shows means and standard sieved flour (0.255g) of each sample was placed deviations for turbidity measured on fungicide in a test tube (150mm x 15mm diameter) and treated samples. Consort was significantly higher 8.5ml of 70% ethanol was added. The sample in turbidity than Claire, Deben and Wizard, but was mixed for 5 sec on a vortex mixer, inverted the difference between Consort and Riband twice, then allowed to settle for 2 min. A portion was not significant. Riband was not significantly (1.5ml) was then drawn off as described by higher than the other 3 varieties. Koliatsou and Palmer (2003), mixed with 4.25ml ethanol and the turbidity was determined using a Unigalvo Type 200 nephelometer (Corning Effect of seasons and treatments Ltd., Halstead, Essex). To optimise comparisons between samples, this was calibrated so that All the varieties (above) except Wizard had also distilled water gave a reading of 0 and the been in trial during the previous season, in which Perspex standard, supplied with the apparatus Deben had been shown to have significantly (refractive index 1.5), was set to 50. Results are lower spirit yield that Claire when untreated therefore recorded in arbitrary units. and to be significantly lower than all the other The samples measured were obtained either varieties when treated (Swanston et al., 2005). from trials grown at the Scottish Crop Research Turbidities were therefore measured on both Institute (SCRI), Dundee and harvested in 2003 treated and untreated samples from 2003, and and 2004, or were provided by the Scotch on untreated samples from 2004, to complete Whisky Research Institute from samples grown in the data collected on treated samples (above). those same seasons. In the SCRI trials, genotypes Analysis of variance (Table 1) showed significant were grown under two fungicide regimes, either effects of variety and of season, but no effect of a full treatment to eliminate foliar diseases (Trt) or fungicide treatment. Turbidities were higher in left untreated (Unt) and there were 3 replications 2004 compared to 2003 (Table 2) and Consort for each genotype in each regime. had the highest turbidity in both seasons. There

70

65

60 urbidity (arb. units)

T 55

50 Claire Consort Deben Riband Wizard

Figure 1. Mean turbidity of 5 wheat varieties grown in trial at SCRI in 2004. Vertical lines indicate standard deviations. A turbidity test for a genetic component of spirit yield in wheat 77 was no significant variety x season or variety x Effects of site and nitrogen content treatment interaction. Samples harvested in 2003 were provided by Table 1. Analysis of variance for turbidity in 4 SWRI, from a number of varieties, covering a wheat varieties grown over 2 seasons, with 2 range of sites, spirit yields and grain nitrogen treatments, i.e. with or without fungicide. contents. Turbidities were determined on 5 samples from each of the 3 varieties Consort, Between df MS Istabraq and Deben and plotted against grain Reps 2 3.13 nitrogen content (Figure 2). While there were Genotypes 3 91.99*** differences between sites for both nitrogen Treatments 1 1.06 content and turbidity in all three varieties, there Years 1 199.99*** was no consistent association between the two Geno.x Treat. 3 6.57 characters. Turbidity values for Consort were Geno.x Year 3 6.99 Treat.x Year 1 2.04 higher than those for the other 2 varieties and Geno.x Treat.x Year 3 5.39 Istabraq, which had higher spirit yield than Residual 30 13.38 Deben (data not shown) also, generally, had Total 47 higher turbidity.

*** - significant at the 0.1% level.

Table 2. Mean values for turbidity of 4 wheat Extension to further varieties varieties grown over 2 seasons, with 2 treatments, i.e. with (Trt) or without (Unt) fungicide. Means SWRI also provided samples from the 2004 followed by a different letter are significantly harvest that had been grown at 5 sites in the different at the 5% level. UK. These included samples from some recent varieties such as Ambrosia, Atlanta and Dickson. Year/Treatment Mean values across sites for turbidity of these Genotype 2003 Unt Trt 2004 Unt Trt varieties were compared with those of Consort Claire 54 c 54 c 57 bc 59 b and Deben (Figure 3) and Atlanta and Dickson, Consort 60 b 61 ab 64 a 63 a both of which have Consort in their pedigrees, Deben 52 c 53 c 57 bc 58 b had similarly high levels of turbidity. Ambrosia, bc bc b b Riband 57 55 59 59 which has distilling potential, although it is

80 Consort

75 Deben Istabraq 70

65

60 Turbidity (arb. units) 55

50 1.5 1.7 1.9 2.1 2.3 2.5 Grain N (%) Figure 2. Turbidity plotted against grain nitrogen content for 5 samples of each of 3 varieties, grown at a range of sites in 2003. 78 J.S. Swanston and P.L. Smith

80 75 70 65 60 55

Turbidity (arb. units ) 50 45 40 AmbrosiaAtlanta Consort Deben Dickson

Figure 3. Mean turbidities across 5 sites, for 5 wheat varieties grown in 2004. Vertical lines indicate standard deviations. classed as a Grade 4 feed variety (HGCA, 2005), breeding programme. Breeders need to identify had low turbidity, similar to that of Deben. the lines that give the best distilling quality at a given nitrogen level. The absence of any effects of nitrogen or fungicide application on turbidity, Discussion or any genotype x season interaction (Table 1), indicate that turbidity is largely a genetically Grain whisky distillers began to use wheat as determined character. It is thus a potentially the adjunct of choice during the 1980s (Riffkin useful procedure for wheat breeders. et al., 1990). However there has been little The turbidity test in barley (Koliatsou and plant breeding effort to develop wheat varieties Palmer, 2003) was of limited value in predicting specifically for distilling (Swanston et al., 2005), malting performance, but did indicate a due, in part, to limited understanding of the characteristic of Optic that had a positive effect underlying genetics and the lack of suitable early- on quality in that particular variety. Turbidity generation screening tests for large populations. in wheat also picks out certain varieties, and This resembles the situation that breeders of contributes to the distilling quality of Consort malting barley faced in the 1970s, when many and some of its progeny, although not Wizard screening systems were introduced, but could (Figure 1). Cultivars with good distilling quality, not be applied over a wide range of germplasm but without high turbidity, presumably achieve (Ellis, Swanston and Bruce, 1979). This was their quality through other mechanisms, so because good malting quality could result from future research will be aimed at identifying these several different mechanisms and, as screening mechanisms and deriving appropriate tests. By tests often relied on single characters, they were combining useful attributes from current distilling only applicable to certain crosses. However, wheat varieties, it should be possible to increase using a range of appropriate tests to select the spirit yields of future varieties, addressing the the progeny of parents with complementary previously disadvantageous position of wheat characteristics could be a means of identifying compared to maize (Walker, 1986). lines superior to either parent. Swanston et al. (2005) showed that the relationship between grain protein and spirit Acknowledgements yield varied between varieties. Thus, while low nitrogen level would be a useful means of Supply of wheat samples from the Scotch discriminating the best samples of known varieties Whisky Research Institute (SWRI) is gratefully at intake, it is unsuited to selecting lines in a acknowledged. The work described was carried A turbidity test for a genetic component of spirit yield in wheat 79 out during the initial stages of a project entitled HGCA (2005). Recommended lists 2005/06 for ‘Genetic Reduction of Energy Use and Emission cereals and oilseeds. HGCA, London of Nitrogen through Cereal Production (GREEN Koliatsou, M. and Palmer, G.H. (2003). A new Grain)’ and sponsored, under the UK sustainable method to assess mealiness and steeliness of agriculture LINK programme, by the Dept. of barley varieties and relationship of mealiness Environment, Food and Rural Affairs (Defra) with malting parameters. Journal of the and the Scottish Executive Environment and American Society of Brewing Chemists 61: Rural Affairs Deptartment (SEERAD). Partners 114-118 comprising HGCA, who provided additional Riffkin, H.L., Bringhurst, T.A., McDonald, A.M.L. funding, SWRI, ADAS, Syngenta Seeds, Foss UK, and Hands, E. (1990). Quality requirements Wessex Grain and Grampian Country Foods are of wheat for distilling. Aspects of Applied thanked for their support. Biology 25: 29-40 Swanston, J.S., Newton, A.C., Brosnan, J.M., Fotheringham, A. and Glasgow, E. (2005). References Determining the spirit yield of wheat varieties and variety mixtures. Journal of Brosnan, J.M., Makari, S., Paterson, L. and Cereal Science 42: 127-134 Cochrane, P. (1998). What makes a good Taylor, B.R. and Roscrow, J.C. (1990). Factors grain distillery wheat? In: Proceedings affecting the quality of wheat grain for of the Fifth Aviemore Conference on distilling in northern Scotland. Aspects of Malting, Brewing and Distilling, Edited by Applied Biology 25: 183-191 Campbell, I., Institute of Brewing, London, Walker, E.W. (1986). Grain spirit – which pp. 225-228 cereal? In: Proceedings of the Second Ellis, R.P., Swanston, J.S. and Bruce, F.M. (1979). Aviemore Conference on Malting, Brewing A comparison of some rapid screening tests and Distilling, Edited by Campbell, I. and for malting quality. Journal of the Institute Priest, F.G., Institute of Brewing, London, of Brewing 85: 282-285 pp. 375-380. 80 J.S. Swanston and P.L. Smith The regulation of limit dextrinase activity in malting, mashing and fermentation 81 Chapter 12 The regulation of limit dextrinase activity in malting, mashing and fermentation

H.R. Jenkinson, P.C. Morris and J. H. Bryce International Centre for Brewing and Distilling, Heriot-Watt University, Edin- burgh, UK

Introduction be inactivated by kilning, and that any surviving enzyme would be rapidly denatured during Limit dextrinase (EC 3.2.1.142) hydrolyses α-1,6 mashing. glucosidic linkages in pullulan, amylopectin, and The extensive research that has been done on branched dextrins derived from amylopectin limit dextrinase over the last 15 years portrays a (Bryce,2003; MacGregor, 2003). The renaissance very different scenario. It has been discovered of research on limit dextrinase began in the that the enzyme can survive both kilning and early 1990s following the development of mashing temperatures (Sissons, Taylor and assays based on hydrolysis of dyed pullulan Proudlove, 1995; MacGregor, Macri, Bazin (McCleary, 1992). Pullulan is a polysaccharide and Sadler, 1995; Stenholm and Home, 1999; consisting of maltotriose molecules linked by Walker, Bringhirst, Broadhead, Brosnan and α-1,6 glucosidic bonds. The hydrolysis of dyed Pearson, 2001; McCafferty, Jenkinson, Morris pullulan by limit dextrinase releases lower and Bryce, 2004). 'Total' limit dextrinase that molecular weight fragments from the pullulan can be extracted from malt (ie extraction with a chain. These dyed fragments are soluble and reducing agent) differs between barley varieties can thus be determined spectrophotometrically and between malts. There is good evidence with a release of solubilised dye being correlated that reducing agents activate proteases which with limit dextrinase activity. In the procedures then activate limit dextrinase (Longstaff and for measurement of malt limit dextrinase, the Bryce,1993). enzyme can either be extracted at pH 5.0-5.5 It is now known that in barley, there is a for 5 hours at 40°C with a reducing agent to proteinaceous inhibitor of limit dextrinase and obtain the 'total' enzyme's activity or without that this inhibitor is degraded during malting a reducing agent to obtain the 'free' enzyme (Macri, MacGregor, Schroeder and Bazin, 1993; (soluble and without activation due to a reducing MacGregor, Macri, Schroeder and Bazin, 1994; agent) (McCleary, 1992). MacGregor et al., 1995; MacGregor, 2004; In the late 1980s, malt limit dextrinase was Stahl, Alexander, Coates, Bryce, Jenkinson and regarded as an enzyme with low activity that Morris, 2007). Barley varieties however differ in was heat-sensitive and therefore it was thought their level of inhibitor and the rate at which it is that to a very large extent limit dextrinase would degraded (Ross, Sungurtas, Ducreux, Swanston, 82 H.R. Jenkinson et al.

Davies and McDougall, 2003). Transgenic Limit dextrinase extraction and assay barley has been produced using antisense technology to down-regulate inhibitor levels Limit dextrinase of malts was extracted and in barley grains. This barley has altered starch assayed in 100 mM sodium maleate buffer composition and has been used to investigate as specified by the Limit Dextrizyme method the function of the inhibitor (Stahl, Coates, Bryce (McCleary, 1992). Ground malt was sealed in and Morris, 2004). Furthermore, pH affects the tubes and allowed to shake in an incubator at binding of inhibitor to limit dextrinase in the 40 ºC for 5 h. The procedure was scaled down mash and fermentation, and thus the potential for individual grain analysis. ‘Free’ activity is the of limit dextrinase to degrade α-1,6-glucosidic activity measured after extraction for 5 h without bonds (McCafferty et al., 2004). DTT, and ‘total’ activity is measured after 5 h Malt enzymes are a major factor in determining extraction with DTT. the composition of wort. An increase in limit dextrinase activity during mashing and fermentation will allow greater breakdown of Results branched dextrins and enhance the fermentability of wort. Thus, increased limit dextrinase activity Optic was malted in the Seegar micromalting during the production of spirit will increase plant and freeze dried after either 5 or 6 days of alcohol production with ensuing economic germination. Analysis was then carried out to benefits to the distilling industries. This work determine the activity of limit dextrinase in 100 was carried out with the aim of investigating the individual grains. Extraction and assays were at variation in limit dextrinase activity of individual pH 5.5 for 5 h with and without DTT. Extraction grains from samples of malted barley. with DTT provides a measurement of ‘total’ activity and without DTT a measurement of ‘free’ activity. Figure 1 shows the pattern of activities Materials and methods found in 100 individual grains after 5 days. The highest proportion of grains had activities of 2 Chemicals and biological material mU/grain or less. Since the average weight of grains was 0.037 mg, this equates to an activity of Barley Hordeum vulgare (variety Optic) was 54 mU/g or less, an activity typical of ‘free’ limit supplied by a local maltings. Chemicals were dextrinase activities in malt. However, a number obtained from the Sigma Chemical Company, of grains showed activities up to 11 mU/grain Poole, Dorset, England or BHD, Leicestershire, or 297 mU/g. Overall, the distribution of ‘free’ England and were of analytical or the highest activity in individual grains was heavily skewed grade available. Limit Dextrizyme Kits were to the lower end of the activity spectrum. from Megazyme International, Bray, Wicklow, In contrast, the total activity of limit dextrinase, Ireland. extracted and measured with DTT showed a spread of activities from 4 up to 16 mU/grain (Figure 1) or 108 to 432 mU/g. These higher Malt production activities would equate to levels of total activity found in malt (McCafferty et al., 2004). Normal aerobic germination was carried out Figure 2 shows the activities found after a using a Seeger micromalting plant (McCafferty et further day of germination (6 days). The pattern al., 2004). Aerobic malt was germinated for 5 d has changed. There are now 42% of grains with or 6 d and then freeze dried prior to analysis. ‘free’ activities between 2 and 4 mU/grain or 54 and 108 mU/g. However, there is a general The regulation of limit dextrinase activity in malting, mashing and fermentation 83

45 Extraction with DTT 40 Extraction without DTT 35 30 25 20 15

Number of grains 10 5 0 0 to 1 1 to 2 2 to 3 3 to 4 4 to 5 5 to 6 6 to 7 7 to 8 8 to 9 9 to 1 0 10 to 11 11 to 12 12 to 13 13 to 14 14 to 15 15 to 16 Limit dextrinase activity (mU/grain)

Figure 1. Limit dextrinase activity of 5 day aerobically germinated Optic grains (100).

30 Extraction without DTT

25 Extraction with DTT 20

15

10 Number of grains 5

0 0 to 1 1 to 2 2 to 3 3 to 4 4 to 5 5 to 6 6 to 7 7 to 8 8 to 9 9 to 10 10 to 11 11 to 12 12 to 13 13 to 14 14 to 15 15 to 16 18 to 19 19 to 20 17 to 18 Limit dextrinase activity (mU/grain)

Figure 2. Limit dextrinase activity of 6 day aerobically germinated Optic grains (100). spread in activities between 5 and 16 mU/ individual grain during germination was also grain or 135 and 432 mU/g. There was also seen by de Sá and Palmer (2004) who malted an increase in the ‘total’ activities, with these the varieties Decanter and Chariot. increasing from 5 to 19 mU/grain or 135 to 514 Limit dextrinase has been found to increase mU/g. as the pH is lowered from 5.5 to 4.4 and it has been hypothesised that this is due to unbinding of inhibitor from the enzyme (McCafferty et al., Discussion 2004). The inhibitor binds at the active site of the enzyme with a stoichiometry of 1:1 (MacGregor, The activities of ‘free’ and ‘total’ limit dextrinase 2004). Therefore, the increasing activity of increased with an additional day of germination ‘free’ enzyme in individual grains over time is a (Figures 1 and 2). This increase in free limit reflection of two processes. These processes are dextrinase activity of some grains led to a the breakdown of inhibitor and the synthesis of distinctly bimodal activity distribution pattern. new enzyme. The significantly higher activities An increase in activity of ß-glucanase from of ‘total’ limit dextrinase compared to ‘free’ 84 H.R. Jenkinson et al. enzyme of individual grains at 6 days (Figure 2) References suggests that the ‘free’ activities measured are a reflection of both inhibited and uninhibited Bryce, J.H. (2003). Limit dextrinase. In: Handbook enzyme. However, whereas almost 70% of of Food Enzymology. Edited by Whitaker, grains had activities of 0 to 2 mU/grain after 5 J.R., Voragen A.G.J. and Wong D.W.S., days, this was true for only 4% of grains at 6 Marcel Dekker, New York, pp. 751–759 days. The ability of normal malt to inhibit ‘free’ Cooper, C.S., Spouge, J.W., Stewart, G.G. limit dextrinase of an anaerobic malt (McCafferty and Bryce, J.H. (2004). The effect of et al., 2000), however, suggests that inhibitor distillery backset on hydrolytic enzymes may often be in excess of that required to inhibit in mashing and fermentation. In: Distilled limit dextrinase. This means that at 5 days, Spirits, Tradition and Innovation. Edited by many of those grains with low activity, probably Bryce, J.H. and Stewart, G.G., Nottingham contained inhibitor in excess of that required to University Press, Nottingham, UK, pp. inhibit their own limit dextrinase. 79–88 Cooper et al. (2003) showed that limit de Sá, R.M. and Palmer, G.H. (2004). Assessment dextrinase activity increased during the of enzymatic endosperm modification fermentation of an unboiled all-malt mash, this of malting barley using individual grain increase was not seen where the mash pH was analysis. Journal of the Institute of Brewing lowered by backset (Cooper et al., 2003). It 110: 43-50 has since been shown that if the pH is lowered Institute of Brewing (1991). Recommended during the extraction of limit dextrinase, the Methods of Analysis. Institute of Brewing, limit dextrinase is more sensitive to inactivation London, UK by mashing temperatures (McCafferty et al., Longstaff, M.A. and Bryce, J.H. (1993). 2004). Therefore, if a mash was at a pH close Development of limit dextrinase in to 5.5, then the excess inhibitor from the malt germinating barley. Evidence of proteolytic of 5 days could have bound to ‘free’ enzyme activation. Plant physiology 101: 881-889 during mashing and protected this enzyme from MacGregor, A.W., Macri, L.J., Schroeder, S.W. inactivation. and Bazin, S.L.(1994). Purification and The activity of limit dextrinase during characterisation of limit dextrinase inhibitors mashing and fermentation is thus a compromise from barley. Journal of Cereal Science 20: between ‘free’ and inhibited enzyme. Inhibitor 33-41 present during mashing will reduce the activity MacGregor, A.W., Macri, L.J., Bazin, S.L. and of limit dextrinase, but the protective effect of Sadler, G.W. (1995). Limit dextrinase this inhibitor will allow enzyme to become inhibitor in barley and malt and its possible active during fermentation as the pH declines. role in malting and brewing. In: Proceedings A knowledge of the activities of limit dextrinase of the European Brewing Convention present in individual grains will thus allow an Congress, Brussels, Oxford University Press, understanding to be developed of limit dextrinase Oxford, pp. 185-192 activities during mashing and fermentation in the MacGregor, E.A. (2003). Limit dextrinase/ distilling industries. pullulanases In: Handbook of Food Enzymology. Edited by Whitaker, J.R., Voragen A.G.J. and Wong D.W.S., Marcel Acknowledgement Dekker, New York, pp. 739–749 MacGregor, E.A. (2004). The proteinaceous H.R. Jenkinson thanks the Lindisfarne Trust for inhibitor of limit dextrinase in barley and funding of a studentship. malt. Biochimica et Biophysica Acta 1696: 165–170 The regulation of limit dextrinase activity in malting, mashing and fermentation 85

Macri, L.J., MacGregor, A.W., Schroeder, S.W. extraction, heat stability and activation and Bazin, S.L.(1993). Detection of a limit during malting and mashing Journal of the dextrinase inhibitor in barley. Journal of American Society Brewing Chemists 53: Cereal Science 18: 103–106 104-110 McCafferty, C.A., Perch-Neilson, N. and Bryce, Stahl, Y., Alexander, R.D., Coates S., Bryce, J.H., J.H. (2000). Effects of aerobic and anaerobic Jenkinson, H.R. and Morris, P.C. (2007). germination on debranching enzyme, limit The barley limit dextrinase inhibitor: Gene dextrinase, in barley malt. Journal of the expression, protein location and interaction American Society Brewing Chemists 44: with 14-3-3 protein. Plant Science 172: 47–50 452-461 McCafferty, C.A., Jenkinson, H.R., Brosnan, J.M. Stahl, Y., Coates, S., Bryce, J.H. and Morris, P.C. and Bryce, J.H. (2004). Limit dextrinase – (2004). Antisense downregulation of the Does its malt activity relate to its activity barley limit dextrinase inhibitor modulates during brewing? Journal of the Institute of starch granule size distribution, starch Brewing 110: 284-296 composition and amylopectin structure. The McCleary, B.V. (1992). Measurement of the Plant Journal 39: 599-611 content of limit dextrinase in cereal flours. Stenholm, K. and Home, S. (1999). A new Carbohydrate Research 227: 257-268 approach to limit dextrinase and its role in Ross, H.A., Sungurtas, J., Ducreux, L., Swanston, mashing. Journal of the Institute of Brewing J.S., Davies, H.V. and McDougall, G.J. 105: 205–210 (2003). Limit dextrinase in barley cultivars of Walker, J. W., Bringhirst, T. A., Broadhead, A. different malting quality: activity, inhibitors L., Brosnan, J. M. and Pearson, S. Y. (2001). and limit dextrin profiles. Journal of Cereal The survival of limit dextrinase during Science 38: 325–334 fermentation in the production of Scotch Sissons, M.L., Taylor, M. and Proudlove, whisky. Journal of the Institute of Brewing M. (1995). Barley malt limit dextrinase, 107: 99–106. 86 H.R. Jenkinson et al. The global crisis of energy and grain 87 Chapter 13 The global crisis of energy and grain

B. J. Hoskins and M. P. Lyons Alltech, Inc., Nicholasville, Kentucky, USA and International Centre for Brewing and Distilling, Heriot-Watt University, Edinburgh, UK

The sun has set on the day we could use natural consumed daily. At the current rate, the world’s resources without giving serious consideration proven oil reserves of approximately 1.3 trillion to how. We understand that resources that had barrels will be completely depleted in less than once seemed limitless, are indeed finite, and that 42 years. Similarly, proven natural gas and coal we are consuming them at an alarming rate. It reserves will be exhausted in approximately 60 is also apparent that what we are leaving in our and 155 years, respectively. All three of these wake is out of balance with the natural cycles sources of energy are fossil fuels and are non- on Earth. As the human population grows, and renewable because they take millions of years nations develop, science is continually enabling to form. a better understanding of the world in which we Not only are non-renewable energy reserves live and allowing us to make informed decisions being rapidly depleted, our ability to access as we improve our standards of living. With them is unpredictable. Over 65% of remaining the grave threat of climate change looming, oil reserves are controlled by seven countries, the global community faces the unprecedented and the same seven are responsible for 45% of challenges of feeding a hungry population and global production. Dependence of countries supplying the world with adequate energy. As on oil imports creates heavy economic and we strive to live sustainably, there are countless security burdens, as political instability and opportunities on the horizon, but we owe careful unpredictable international relations abound in consideration to how we will meet the demands most of the regions where oil is abundant. of our global economy to our children and future Furthermore, whenever fossil fuels are burned generations. they release carbon dioxide (CO2), which Oil, the most coveted natural resource in had been previously contained for millions of history, and petrochemicals shaped the politics years, into the atmosphere. CO2 is a critical and economics of the 20th century. Diesel greenhouse gas due to its ability to absorb and gasoline fuel our vehicles; polymers made heat. Climate scientists have been continually from petroleum are used to produce textiles, gathering information, and as their models packaging, and building materials; and many and equipment have become more and more of our chemicals including cosmetics and sophisticated, they have been able to improve pharmaceuticals are derived from oil. As a the information they report to policy makers and result, globally, over 85 million barrels of oil are citizens regarding the costs and consequences 88 B.J. Hoskins and M.P. Lyons of continued human contributions of carbon they all have solar energy and wind that can be dioxide to the atmosphere. The changes to the harnessed. Earth’s surface and the atmosphere brought on Another alternative that offers significant by the Industrial Revolution have undoubtedly potential is the use of biotechnology for the altered the landscape of human existence on conversion of . Plants and plant- Earth. based materials produced by photosynthesis In the 21st century, we have already begun within biological rather than geological time to transform the ways in which resources are cannot completely replace the vast volumes used to build, travel, and communicate, and this of petroleum and other fossil fuels currently innovation will become even more widespread used, but they can provide alternative fuels and in a movement that is continually gaining chemicals comparable to those derived from momentum. Restructuring the global economy petroleum. As a result, we are witnessing the according to ecological principles represents emergence of biorefineries throughout the world the greatest investment opportunity in history. at an extraordinary rate. Such a revolution will be as monumental in how Yeasts convert simple sugars into ethanol it is regarded as the Agricultural and Industrial during fermentation. The feedstocks used for Revolutions. Oil and gasoline prices continue ethanol production currently are primarily to climb to record levels as energy prices refuse sugar cane, sugar beets, and starch-containing to show any signs of relief. Conservation, grains such as corn, wheat, barley, rye, and efficiency, and alternatives are becoming sorghum. As an oxygenate containing 35% increasingly important as more and more people oxygen, the addition of ethanol to fuel results are recognizing the value of a clean environment; in more complete fuel combustion, which clean air and water, as well as the importance reduces harmful tailpipe emissions. Ethanol of nutrition and healthy lifestyles. Improved also displaces the use of carcinogenic gasoline nutrition for humans entails healthy animals and components such as benzene and methyl natural produce free of toxins and chemicals. tertiary-butyl ether (MTBE). Ethanol is non-toxic, The plants and animals we consume, the air we water soluble, and quickly biodegradable. breathe, and the water we drink are all products Despite these positive attributes, it is important of our environment, and it is clear our health and to account for the energy and chemicals required prosperity are directly related. by the production of grain and ethanol as well We already see innovations in our utilization as co-products in order for it to be considered a of fossil fuels. Hybrid engines that run cleaner sustainable source of energy. Grain is renewable than typical internal combustion engines and and consumes carbon dioxide as it grows; conserve gasoline and clean coal technology however, its production currently relies heavily including carbon capture and storage are on fossil fuels for fertiliser, operation of field examples of how we can use our resources machinery, irrigation, drying, transportation, in more efficient and more responsible ways. and pesticides. Furthermore, conversion to Harvesting energy from the earth (geothermal), ethanol and distillation requires energy, which wind, sun (solar), and oceans, rivers, and streams often comes from coal or natural gas. The (hydro-electric) will also play an increasingly production of ethanol yields two co-products: greater role in the global economy, as new distiller’s grains and CO2. Distiller’s grains ways of utilizing these alternatives will be are an important product for the animal feed developed and perfected. In countries that industry, because the remaining grain and rely on foreign imports for oil or other sources yeast after fermentation contain essentially all of energy, renewables represent a tremendous of the nutrients of corn with the starch fraction opportunity for investment domestically. While removed. Distribution and utilization of these few countries have oil fields at their disposal, spent grains is extremely important to ethanol The global crisis of energy and grain 89 producers to achieve the maximum value out the United Sates by 2017. However, unlike of biorefineries. Brazil, ethanol in the U.S. is currently produced

CO2 is generally captured for use in primarily from corn. If the stated goal of 35 applications such as carbonated beverages and billion gallons were to come from ethanol, refrigeration, though not in all cases. Releasing it would require more than 300 million tons the CO2 produced by yeast during fermentation of corn, which is greater than the current into the atmosphere offsets CO2 that was absorbed annual production level in the United States. by the grain by photosynthesis. Another use Elsewhere, Europe is targeting 200 million tons for CO2 with staggering potential is for the of corn for renewable fuel production, China is cultivation of algae. There are many different targeting 50 million tons of corn, and Argentina types of algae and possible uses, and we are only is targeting 26% of its soybean production for beginning to understand their potential. They biodiesel production. The demand for biofuels are extremely efficient at converting CO2 into compounds an already increasing demand for a high density liquid form of energy as natural corn and soybeans as feed for livestock and as oil. Petroleum itself is formed from the remains a source of food, oil, sweeteners such as high of algae over millions of years. Certain types of fructose corn syrup, etc. The feed and biofuel algae contain valuable nutritional characteristics industries are intimately linked. Therefore, such as vitamins and minerals, antioxidants, corn prices like gasoline are also experiencing and fatty acids for numerous human and animal record highs. This has rippling effects in the feed health applications. Some types of algae contain industry worldwide. greater than 50% oil, which translates into yields Of the six billion people on Earth, 20% are of oil per unit area far greater than plants such without electricity. Imagine the demands of a as corn, soybeans, rapeseed, palm, sunflowers, world population of nearly nine billion predicted and even jatropha. Orders of magnitude greater. in 2050. Global feed requirements are growing Technical hurdles exist, but the potential of algae faster than ever with China and India leading for biodiesel production from a source of oil that the way. There is a direct correlation between would not compete with grains supplied to the wealth and meat consumption. The wealthier food and feed industries is tremendous. a population, the more meat they consume. As ethanol producers continue to discover Development and improved standards of unique ways of becoming more efficient, from living in places such as China and India have the production of grain to the utilization of their monumental implications on global grain co-products, total global production is at an all production. Feed requirements for livestock time high. In Brazil, ethanol is blended into are expected to essentially double in less than every gallon of fuel sold. At each of the 30,000 50 years, from 1.05 billion tons in 2000, to 2.08 gasoline stations in the country, there are two fuel billion by the year 2050. This rapid increase is options: hydrous ethanol or a gasoline-hydrous occurring while arable land is disappearing at ethanol blend, usually of 25% ethanol. Every an astonishing rate, estimated at five to seven petroleum marketer supplies ethanol. Flexible million hectares per year. The extraordinary fuel vehicle (FFV) technology enables motorists demand for grain and alternative energy presents to put gasoline, ethanol, or a blend of the two a challenge unlike any other that people have into their vehicles. Currently, FFV sales account encountered, and it will require us to think and for over 75% of total auto sales in Brazil. live differently. In the United States, the fuel ethanol industry When we think about grain as an energy is booming. In 2007, in the State of the Union source for animals or for conversion to biofuels, address, President Bush called for setting a the hydrocarbons that provide the energy are mandatory fuels standard of 35 billion gallons found in polysaccharides or carbohydrates. of renewable and alternative fuels annually in Starch, for instance, is composed primarily 90 B.J. Hoskins and M.P. Lyons of two glucose polymers: amylose, a linear decompose dead organic matter by a process molecule with α-1,4 linkages, and amylopectin, called solid state fermentation (SSF). SSF is which is a branched molecule that contains the nature’s bioconversion process, and is generally same α-1,4 linkages, but has α-1,6 linkages characterised as the growth of microorganisms as well. Amylolytic enzymes, found in the on insoluble substrates, in the absence of free digestive tracts of animals or that are added to an water. Bacteria and fungi are capable of growing ethanol fermentation, are able to hydrolyze these on solid substrates, but filamentous fungi are the chemical bonds to produce simple sugars that best adapted for SSF due to their physiological can be utilised in cellular respiration as energy capabilities. Biotechnology and SSF have played for animals or that can be converted to ethanol an important role for centuries in the industrial by yeast glycolysis. Another glucose chain with preparation of various foods and beverages a very similar structure is . It is found throughout the world, particularly in Asia. In in plant cell walls and many types of algae addition to food and beverage applications, and is the most abundant organic compound the production of hydrolytic enzymes using on Earth. It is the major constituent of paper SSF is becoming increasingly important and and and textiles made from cotton, is responsible for the growing interest in the linen, and other plant fibers. The difference technology. Enzymes are proteins that accelerate between amylose in starch and cellulose is the a particular chemical reaction by lowering the bond that gives cellulose its structural strength. activation energy required. They are exploited Instead of a linear chain with glucose molecules commercially for their catalytic power for many bonded with α-1,4 linkages, cellulose is a linear applications in the food, feed, beverage, fuel, polysaccharide consisting of numerous β-1,4 and textile industries. Most manufacturers linked glucose units. It is the way the same produce enzymes using submerged fermentation glucose molecules are twisted and joined that (SmF) techniques; however, SSF offers some prevents animals from utilizing the simple sugars distinct advantages such as i) the medium is bound in cellulose. Specific enzymes found simple, often an unrefined agricultural product in nature known as cellulases have the ability which requires less pretreatment; ii) restricted to break this bond. Understanding how nature availability of water selects against undesirable accomplishes this is the key to a vast supply of contaminants; iii) production of specific products energy. that would otherwise not be produced by SmF; Scientists are rapidly developing technology iv) smaller reactor volumes result in lower capital for the production of ethanol from lignocellulosic and operating costs; v) aeration is easier; vi) biomass by thermochemically and enzymatically and downstream processing and waste disposal hydrolyzing cellulose and . problems are generally simplified or minimised. Hemicellulose is related to cellulose, only it Microorganisms are able to break the β-1,4 contains shorter branched chains comprised of bonds of cellulose in nature. Biotechnology glucose in addition to simple sugars other than and SSF allow us to harness nature unlocking glucose such as xylose, mannose, galactose, the potential of cellulose for fuel and animal and arabinose. Utilizing these materials has feed applications. the potential to relieve the demand on grain The challenges before us are great, yet the and also to dramatically reduce the cost of opportunities, endless. Dedication, creativity, producing ethanol, which is vital for the future and innovation are required of policy makers and of the ethanol industry. every sector in the new global economy. Our Microorganisms are already utilizing health, our security, and our survival depend these materials, for example when they assist on it. The movement to a clean environment termites or cows with digestion, or when they and sustainable future is well underway, and complete the carbon cycle in nature as they the natural world in which we live holds the The global crisis of energy and grain 91 blueprint. As an enzyme accelerates a chemical Loftas, T. (1995). Dimensions of Need : An Atlas reaction that would otherwise occur at a slower of Food and Agriculture. Rome, Food and rate, we ourselves can be catalysts. If we come Agriculture Organization. UN Library, New together with a common goal: to leave our York children and future generations a healthy planet, Mitchell, D.A., Krieger, N. and Berovic, M. we can accelerate achieving this worthy goal. (2006). Solid-State Fermentation Bioreactors: Fundamentals of Design and Operation. Springer-Verlag, Berlin Further reading Pandey, A., Soccol, C.R., Rodriguez-Leon, J.A. and Nigam, P. (2001). Solid-state Fermentation Bourne, J.K. (2007). Green dreams. Growing in Biotechnology. Fundamentals and fuel: The wrong way, the right way. Biofuels: Applications. Asiatech Publishers, New boon or booddoggle? National Geographic Delhi 212: 38-59 Roberts, P. (2005). The End of Oil: On the Edge Brown, L.R. (2006). Building a new economy. of a Perilous New World. Houghton Mifflin In: Plan B 2.0 Rescuing a Planet Under Company, New York Stress and a Civilization in Trouble. W.W. Sheehan, J., Dunahay, T.G., Benemann, J.R., Norton and Co., New York, pp. 227-248 Roessler, P.G. and Weissman , J. C. (1998). Doelle, H. W., Mitchell, D. A. and Rolz, C. E. A Look Back at the U.S. Department of (1992). Solid Substrate Cultivation. Elsevier Energy’s Aquatic Species Program: Biodiesel Applied Science, London from Algae. National Renewable Energy Energy Information Administration (2008). Laboratory, Golden, Colorado. NREL/ Official energy statistics from the U.S. TP-580-24190. http://govdocs.aquake. Government. http://www.eia.doe.gov/ org/cgi/reprint/2004/915/9150010.pdf (accessed 12 March 2008) (accessed 12 March 2008) Krishna, C. (2005). Solid-state fermentation Wald, M.L. ( 2007). Is ethanol for the long haul? systems - An overview. Critical Reviews in Scientific American January, 42-49. Biotechnology 25: 1-30 92 B.J. Hoskins and M.P. Lyons Proteomic analysis of distillers’ yeast 93 Chapter 14 Proteomic analysis of distillers’ yeast

R. Hansen1, A. M. Ferguson1, S.Y. Pearson2, J.M. Brosnan2, P. G. Meaden1, D. J. Jamieson1 1International Centre for Brewing and Distilling, Heriot-Watt University, Edinburgh, UK; 2The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK

Introduction

In recent years much information has been cerevisiae genome sequence and using this obtained about the molecular response of information methods have been developed to laboratory yeast and to some extent ale, measure levels of expression of all genes in lager and wine yeast during fermentation the cell at any given point in time, for example (Brejning, Arneborg and Jesperson, 2005; microarrays, which can quantitatively measure James, Campbell, Donnelly and Bond, 2003; relative mRNA levels in cells. Although cDNA Kobi, Zugmeyer, Potier and Jaquet-Gutfreund, microarray methodology is very informative, 2004; Rossignol, Dulau, Julien and Blondin, the results do not always reflect actual protein 2003). However, most of these studies used levels and enzyme activity because of post- small-scale fermentations and in some cases translational effects. For example, the rate of laboratory media making it difficult to ascertain synthesis and half-life of a given protein can, how the findings apply to the more typical in some cases, be altered. Therefore, it is often large-scale industrial fermentations. In marked difficult to predict protein levels from cDNA contrast, there have been few molecular studies microarray measurements, as there can be a on yeast used for fermentations in either the limited correlation between gene expression Scotch whisky or grain distillation industries. and protein levels (Gygi, Corthals,Zhang, In such fermentations the distillers yeast strain Rochon and Aebersold, 2000). Additionally, named ‘M’ is one of the most widely used, as microarray experiments provide no information it has a reputation for robust performance with on other post-transcriptional modifications, such minimal off-flavor development (Russell, 2003). as phosphorylation, oxidation and proteolysis, The ‘M’ yeast was thought to be a hybrid of which are often involved in regulating protein Saccharomyces cerevisiae and Saccharomyces activity (Oda, Nagasu and Chait, 2001). diastaticus, a strain that differs from S. cerevisiae, It is important therefore, to characterize at in that it contains a gene for ß-amylase (Kunze, a protein level the proteins present and under Kunze, Barner and Schulz, 1993). specific growth conditions. A very powerful One of the key tools available to yeast tool to probe the problem of large-scale researchers is the availability of the Saccharomyces protein identification is the combination of two 94 R. Hansen et al. dimensional gel electrophoresis (2D-PAGE) and Sampling was done from the bottom of the matrix assisted laser desorption ionization-time washback and 50 ml samples were collected of flight mass spectrometry (MALDI-TOF MS) and filtered though a 0.5 mm mesh to remove (Griffin, MacCoss, Eng, Blevins, Aaronson and solids and centrifuged at 3000 g for 5 min. The Yates 1995). Gel electrophoretic techniques cell pellet was washed once with cold phosphate such as 2D-PAGE have an increased resolving buffered saline (PBS), while the supernatant was power compared to that of conventional SDS- stored frozen at -20oC until required for HPLC PAGE and are capable of resolving thousands analysis of carbohydrate, acetic acid and lactic of proteins in a single experiment. Moreover, acid content. as the intensity of staining of the protein spot on the gel is a direct measure of protein level in the cell, these experiments can also determine HPLC analysis of carbohydrates, lactic acid, the levels of particular proteins at various points acetic acid and ethanol throughout the fermentation. In addition, post- transcriptional modifications to proteins can Supernatants from the fermentation samples were also be identified (Fey and Larsen, 2001). To diluted and used for detection of carbohydrates, date most of the proteomic work has focused ethanol and lactic- and acetic- acids. Carbohydrate on either laboratory or brewing yeast and most analysis was performed by high performance of these use small-scale laboratory fermentations anion exchange (HPAE) separation using a with laboratory media, although some have Dionex Carbopac PA-100 column coupled to performed pilot scale industrial fermentations a Dionex PAD detector. Lactic and Acetic acid on brewing yeast (Kobi et al., 2004). was extracted from samples using a Phenomenex In this study we present an analysis of the Strata SI-2 column and a Varian SAX column proteome of the distillers yeast strain ‘M’ during and separated using a Phenomenex Synergi 4u industrial grain fermentation. This study is the hydro-RP 80A column connected to a Waters first step towards understanding industrial scale 484 tunable wavelength detector. Ethanol fermentations at a molecular level. was measured by direct sample injection into a Chrompack CP9000 Gas Chromatography instrument with a FID detector using a CP SIL Materials and methods 5CB column.

Strains, media and growth conditions Free amino nitrogen (FAN) analysis The distilling yeast strain ‘M’ (Kerry Bioscience) was used throughout this study as well as the Samples withdrawn from the fermentations were laboratory yeast, S150-2B (MATa leu2-3, 112 diluted 100-fold, and free amino nitrogen was ura3-52 trp1-289 his3-∆1) (Jamieson, 1992). measured with ninhydrin (Sigma) using glycine Distillers yeast was supplied in compressed as a reference (Moore and Stein, 1954). format. Fermentations were carried out in 400,000 L mechanically agitated steel washbacks. The wort contained 15% malted barley and 85% pre- Yeast and bacterial enumeration boiled maize which had been heated to 62°C. After 2 h, the wort was cooled. At the time of Samples withdrawn from the fermentations transfer to the washbacks, the wort had a specific were diluted in PBS buffer and yeast viability gravity of 1.060 kg l-1. Fermentations were measured using methylene blue. Yeast cell pitched with a yeast cell density of 1 g l-1. counts were also performed by plating serial Proteomic analysis of distillers’ yeast 95 dilutions onto YPD plates containing 20 g l-1 were analyzed using ImageMaster 2D Platinum glucose, 20 g l-1 peptone, 10 g l-1 yeast extract software (GE healthcare). The intensity of each and 10 mg l-1 tetracycline. Lactic acid bacteria protein spot was obtained by background were enumerated by plating on MRS plates (52 subtraction followed by normalization to the g l-1 MRS 10 g l-1 brain heart infusion, 10 g l-1 total sum of pixel units in the gel and fold maltose) containing 100 mg l-1 cycloheximide. changes were calculated as the ratio of spot intensities by measuring pixel densities.

Protein extraction Peptide mass fingerprinting Cells were washed once in cold PBS buffer and the cell pellet re-suspended in 1 ml breaking Spots were excised from Coomassie-stained buffer (50mM Tris-HCl, pH 8, 50mM NaCl, 2D PAGE gels using an Ettan spot picker (GE 1% NP-40, 1mM Pefabloc). Glass beads were healthcare) adjusted to pick gel plugs with a added, and cell suspension was vigorously diameter of 1.5 mm. The plugs were washed vortexed for 4 min. Cell debris were removed twice with Milli-Q water, followed by incubation by centrifugation at 10000 g for 5 min at 4°C. in 50% acetonitrile in 50 mM ammonium Protein concentration was measured using a bicarbonate to destain the plugs. After de-staining, Bradford assay (Bradford, 1976). Nucleic acid the plugs were dehydrated in 70% acetonitrile was removed by incubation for 30 min at for 15 min in two subsequent steps where after room temperature with 100 units Benzonase acetonitrile was removed by evaporation. The gel

(Novagen) to which MgCl2 was added to a plugs were then rehydrated in 10 µl of Trypsin final concentration of 1 mM. Protein was then solution (Trypsin Gold, Promega), prepared by precipitated with acetone. adding 25 mM ammonium bicarbonate and 10% acetonitrile to a concentration of 40 ng/µl. Digestion was performed at 37°C for 4 h. The 2D gel electrophoresis supernatant of the trypsin digest was mixed 1:1 with re-crystallized α-cyano-4-hydroxycinnamic 600 µg protein was precipitated over-night in acid 10 mg/ml (LaserBio Labs) prepared in 50% 80% (v/v) acetone and precipitated proteins acetonitrile and 0.5% Trifluoroacetic acid. The solubilised in 8 M Urea, 2% CHAPS, 0.5% IPG mixture was then spotted directly onto a MALDI- buffer pH 3-10 (GE healthcare), 25 mM DTT and TOF target plate, and run on an Ettan™ MALDI- bromophenole blue. Isoelectric focusing was TOF (GE healthcare). The identified peptide performed using 24cm Immobiline IPGstrips masses were then compared to those in the NCBI 3-10NL (Amersham Biosciences) on an Ettan™ sequence data-base. IPGphor cell (GE healthcare). Gel strips were rehydrated with the protein extracts for 12-14 h, and then focused at 40-45 kVh. After focusing, Results gel strips were equilibrated for 15 min each with 100 mM DTT and 250 mM IAA, respectively. In order to characterise the proteins produced by The second dimension was run on 26 x 26 cm distillers yeast during industrial fermentations it 12 % SDS-PAGE gels using an Ettan™ DALTsix was first necessary to examine the fermentations (GE healthcare) over-night at 3 W. Gels were with respect to yeast cell numbers, sugar and stained using Coomassie brilliant blue G250 ethanol concentrations from all three washbacks (Candiano et al., 2004). Digital images of each used. These experiments made it possible gel were obtained by scanning the gel with an to relate our findings to the appearance and ImageScanner™ II (GE healthcare) and images disappearance of ethanol and key sugars. The 96 R. Hansen et al.

Figure 1. Fermentation profile. Samples were collected, after 0, 24, 48 and 72 h fermentation, from the washbacks and analysed for sugars, lactic acid, ethanol and FAN content. Yeast cell number, viability, fermentation pH and temperature were also measured. The results shown are from one of three washbacks. fermentation profile in respect to sugar usage of lactic acid and acetic acid increased during and ethanol production was similar for the the fermentations, especially in the later part of three washbacks tested (Figure 1). Maltose the fermentations (Figure 1b). Yeast cell number was the most abundant sugar with an initial increased exponentially to around 8.5 x106 concentration of 101 ± 3 g/L followed by cells per ml in the first 24 h of fermentation, maltotriose with an initial concentration of remaining unchanged until 72 h, where it then 16 ± 1 g/L. After 48 h of fermentation, only decreased to 4.0 x106 cells per ml. Yeast viability small amounts of maltose and maltotriose as measured by methylene blue remained high were detected, whereas the other sugars were throughout the fermentations, even after 72 h, substantially depleted from the media. The where viability was 92 ± 4 %. The temperature concentration of maltodextrins decreased profile was also similar for all three washbacks, throughout the fermentations, as dextrins were with temperature increasing from 17°C to 34°C converted into maltose and maltotriose. All the over the course of fermentation. Some variation fermentations resulted in an average ethanol in pH between the washbacks was observed yield of 9%. Free Amino Nitrogen (FAN) was 120 in the late stages of fermentation, which was ± 13 mg/L at the beginning of fermentation and presumably due to different loads of lactic acid after 24 h the FAN concentration had decreased bacteria contamination in the three washbacks to 6 ± 13 mg/L, where-after it stayed at a low (Figure 1d and data not shown). level until a slight increase after 72 h. The levels Proteomic analysis of distillers’ yeast 97

Preparation of a proteome reference map for Protein expression during grain fermentation ‘M’ yeast In order to quantify the levels of specific proteins As no proteomic work has been described during fermentation, proteins were isolated from for the distillers yeast ‘M’ it was essential to yeast taken from three different washbacks after construct a reference map for this strain, using 0, 24, 48 and 72 h and separated by 2D-PAGE. 2D-PAGE. This map was then used for matching The levels of 85 distinct yeast proteins were protein spots in other gels using image analysis quantified by image analysis software. Many software. The reference gel was made using proteins were only present at the beginning or protein extracts from samples withdrawn from end of fermentation, and significant changes in mechanically agitated washbacks after 24 h protein levels were observed for many proteins, fermentation. This time point was chosen as the confirming that ‘M’ yeast responded to the reference point as the yeast was actively growing multitude of environmental changes taking place and the ethanol concentration was still relatively over the course of fermentation. Similar trends low. Proteins were characterised by peptide in yeast protein expression were found for all mass fingerprinting, resulting in more than three washbacks. The major changes in protein 100 positive identifications with the majority expression observed during the progression of of abundant proteins present on the gels being the fermentations are listed in Table 1. identified (Figure 2 and Table 1).

Figure 2. Proteome reference map of M-yeast. A representative 2D-PAGE gel prepared using protein extracted after 24 h, from an industrial grain fermentation is shown. Gels were stained by colloidal Coomassie Blue and proteins identified by MALDI-TOF mass spectrometry. 98 R. Hansen et al.

Table 1. Proteins identified by MALDI-MS from 2D-PAGE gels of M-yeast.

Gene ORF Gene description Induction fold Abundance* 0 h 24 h 48 h 72 h (%)

Heat shock Proteins Hsp104 YLL026W Chaperone - - - Present 0.19 Ssa1 YAL005C Chaperone with ATPase activity 1 - - 9.4 0.36 Ssa1* - - 1 2.5 5.6 6 1.93 Ssb1 YDL229W Cytoplasmic chaperone ATPase 11.4 1 - 9.3 0.30 Ssc1 YJR045C Mitochondrial with ATPase activity 1 - - 1.5 0.30 Ssc1* - - 1 1.7 2 2 0.16 Hsp60 YLR259C Mitochondrial Chaperone 2.6 1 1 2.2 0.65 Hsp31 YDR533C Chaperone and cysteine protease 1 3.2 5.3 5.7 0.21 Sba1 YKL117W Co-chaperone - 1 1.4 - 0.25 *proteolytic C-terminal fragments

Maltose metabolism Mal12 YGR292W Maltase 1 10.6 5 1.8 0.77 Ygr287c YGR287C Similar to maltase 1 17.5 12 4.9 1.23

Glycolysis Hxk1 YFR053C Hexokinase isoenzyme - Present - - 0.04 Hxk2 YGL253W Hexokinase isoenzyme 1 5.2 1.1 - 0.25 Pyk1 YAL038W Pyruvate kinase 1.5 1.3 2 1 0.87 Eno1 YGR254W Enolase 1 3.3 4.5 3.7 7.38 Eno2 YHR174W Enolase 1.2 1.2 1.4 1 7.43 Fba1 YKL060C Fructose 1,6-bisphosphate aldolase 1 2.1 2.1 1.8 2.54 Gpm1 YKL152C phosphoglycerate mutase Present Present Present Present Pdc1 YLR044C pyruvate decarboxylase 23.6 1 2.3 6.3 2.04 Pgi1 YBR196C phosphoglucose isomerase 3.8 2.3 1.2 1 0.32 Pgk1 YCR012W 3-phosphoglycerate kinase 1 1.3 2 1.2 4.15 Tdh1 YJL052W Glyceraldehyde dehydrogenase 1 15.1 13.2 22.8 0.85 Tdh2 YJR009C Glyceraldehyde dehydrogenase 1.1 1 1.3 1 3.48 Tdh3 YGR192C Glyceraldehyde dehydrogenase 1.6 1 1.2 1 5.54 Tpi1 YDR050C Triose phosphate isomerase 1 1.4 1.6 2.3 4.81 Adh1 YOL086C Alcohol dehydrogenase 1 11.1 15.2 8.7 2.49 Adh2 YMR303C Alcohol dehydrogenase Present - - -

TCA cycle Aco1 YLR304C Aconitase 29.8 1 2.3 6.1 0.45 Idh2 YOR136W Isocitrate dehydrogenase 1.4 2.1 2.8 1 0.13 Mdh1 YKL085W Malate dehydrogenase 1.1 1 1.1 1.2 0.18 Mdh3 YDL078C Malate dehydrogenase 4.4 1 1 - 0.11 Sdh1 YKL148C Succinate dehydrogenase 5.1 1 - - 0.12

Other proteins in carbohydrate metabolism Tkl1 YPR074C Transketolase 2.4 1.5 1 1 0.39 Zwf1 YNL241C Glucose-6-phosphate dehydrogenase 1.8 1.2 1 - 0.09

Amino acid metabolism Cys3 YAL012W Cystathionine gamma-lyase 1.4 1.6 1 - 0.43 Proteomic analysis of distillers’ yeast 99

Table 1. Contd.

Gene ORF Gene description Induction fold Abundance* 0 h 24 h 48 h 72 h (%)

Amino acid metabolism (contd) Ilv3 YJR016C Dihydroxyacid dehydratase Present - - - 0.05 Ilv5 YLR355C Acetohydroxyacid reductoisomerase Present - - - 0.11 Hom6* YJR139C Homoserine dehydrogenase Present Present - - - Leu4 YNL104C Alpha-isopropylmalate synthase Present - - - 0.07 Lys9 YNR050C Saccharopine dehydrogenase 2.8 2.5 1 - 0.15 Met6 YER091C Methionine synthase 3.7 3.2 2 1 0.52 Arg1 See Gcn4 activity Gdh1 See TOR pathway regulated proteins Hom2 See Gcn4 activity Lpd1 See Gcn4 activity Met17 See Oxidative stress proteins

TOR pathway regulated proteins Pep4 YPL154C Vacuolar aspartyl protease 1 8.2 17.8 20.7 1.66 Prb1 YEL060C Vacuolar serine protease 1 2.9 2.9 4.6 1.36 Prc1 YMR297W Vacuolar carboxypeptidase Y 1 2.5 3.3 5.1 0.22 Gdh1 YOR375C Glutamate dehydrogenase 2.4 3.4 2.2 1 1.99

Gcn4p activity (Amino acid synthesis control) Asc1 YMR116C WD repeat (repressor of Gcn4) 2.5 1.9 1.5 1 1.37 Arg1 YOL058W Arginosuccinate synthetase 1 1.6 1.8 2.2 0.32 Hom2 YDR158W Aspartic aldehyde dehydrogenase 1.6 4.4 1 - 0.4 Lpd1 YFL018C Dihydrolipoamide dehydrogenase 2.8 1 - - 0.16

ATPases Atp1 YBL099W Mitochondrial ATP synthase alpha 15 1.2 1 - 0.52 Atp2 YJR121W Mitochondrial ATP synthase beta 2.8 2 1.4 - 0.73 Vma1 YDL185W Vacuolar ATPase subunit A 7.9 1 - - 0.37 Vma2 YBR127C Vacuolar ATPase subunit B 13.4 2.4 1 - 0.70 Vma7 YGR020C Vacuolar ATPase subunit F Present - - - 0.06 Cdc48 YDL126C ATPase in ER Present - - - 0.34 *see heat shock proteins for other chaperones with ATPase activity

Oxidative Stress Ahp1 YLR109W Thiol-specific peroxiredoxin 1 1.8 1.9 2.6 0.35 Gpp1 YIL053W DL-glycerol-3-phosphatase 1 1.1 1.2 1 0.90 Met17 YLR303W Homoserine/Serine sulfhydrylase 3.2 1.8 1.2 1 1.0 Sfa1 YDL168W Formaldehyde dehydrogenase 1.9 1 2.5 - 0.21 Sod1 YJR104C Cu, Zn superoxide dismutase 1.9 1 1 1.1 0.34 Sod2 YHR008C Mn superoxide dismutase 2 1 1.2 1.6 0.30

Cytoskeleton and ribosomal Act1 YFL039C Actin Cof1 YLL050C Cofilin 1 1.1 2.8 2.5 0.58 Cpr1 YDR155C Cyclophilin 1 1.9 2.2 1.1 1.07 Pfy1 YOR122C Profilin - 1 2.5 4.8 0.43 100 R. Hansen et al.

Table 1. Contd.

Gene ORF Gene description Induction fold Abundance* 0 h 24 h 48 h 72 h (%) Cytoskeleton and ribosomal (contd) Vik1 YPL253C microtubule-mediated processes 3.2 1 1 1.3 0.18

Gene transcription and translation Eft1 YOR133W Elongation factor 2 2.5 1.1 1 1 0.58 Yef3 YLR249W Elongation factor (Eft3) - Present - - 0.08 Rpo0 15.3 1 1.4 - 0.37 Rpo21 YDL140C RNA polymerase II subunit 1.2 1 1.6 - 0.11 Rpl22 YLR061W Part of the 60S ribosomal subunit Present - - - 0.40 Rpo26 YPR187W RNA polymerase subunit Present - - - 0.17 Rrn5 YLR141W transcription factor Present - - - 0.16 Tif1 YKR059W Translation initiation factor, eIF4A 9.7 1 - - 0.37

Non-classified proteins Ade17 YMR120C 'de novo' purine biosynthesis 8.8 1.3 1 - 0.18 Adk1 YDR226W Adenylate kinase 1.6 4.1 1.2 1 0.77 Ado1 YJR105W Adenosine kinase 2.1 1.2 1 1.1 0.15 Ald4 YOR374W Aldehyde dehydrogenase 12.8 1 - - 0.91 Bmh1 YER177W 14-3-3 protein, major isoform 2.5 3.4 2 1 0.51 Bmh2 YDR099W 14-3-3 protein, minor isoform 2.8 2.8 3.5 1 0.21 Cor1 YBL045C ubiquinol-cytochrome c reductase 7.2 1 1 - 0.18 Erg10 YPL028W Acetyltransferase 7.8 1 1.6 2.3 0.34 Gsp1 YLR293C GTP binding protein 3.3 2.7 1 - 0.20 Hem13* YDR044W Coproporphyrinogen 3 oxidase Present - - - 0.50 Ipp1 YBR011C Inorganic pyrophosphatase 1 1.7 1.6 2.2 1.48 Oye2 YHR179W NADPH oxidoreductase Pdb1 YBR221C Pyruvate dehydrogenase 2.4 1.9 1 - 0.15 Pdi1 YCL043C Protein disulfide isomerase 2.5 1.3 1 1 0.06 Ubi4 YLL039C Ubiquitin 1 ND ND 1.5 1.52 Wtm1* YOR230W Transcriptional repressor - 1 1.7 2.2 1.17 Ybr139w YBR139W Carboxypeptidase C activity 1 1.5 2.7 2 0.10 Yel047c Yel047c Fumurate reductase 1 2.1 1.6 3.4 0.26 Yhr033w Yhr033w Induced by starvation - - - Present 0.34 Ylr301w Ylr301w uncharacterized 1 1.4 1.2 1.3 0.28 Ynl134c YNL134C Alcohol dehydrogenase activity 1 1.5 2.7 2 0.49 Ypr1 YDR368W 2-methylbutyraldehyde reductase 1 3.2 1.4 - 0.19 * Abundance is measured as the volume of the protein spot divided with the total volume of all protein spots in the gel (time), where the normalised protein volume was highest.

Present indicates that the protein was identified but that the levels were too low for accurate quantification.

Perhaps not surprisingly the major changes the presence of glucose (~4% w/v). Levels of involve metabolism in general and in particular the Mal12 peak at 24 h and then decline in parallel enzymes required for maltose utilisation. Mal12, with the declining maltose concentration an inducible maltase (alpha-D-glucosidase) (Figure 3, see 3A). In addition to Mal12, another encoded by the MAL12 gene at the MAL1 locus protein, Ygr287c, displayed a similar pattern of is present early on in the fermentation, despite expression, though it was more abundant than Proteomic analysis of distillers’ yeast 101

Figure 3. Fermentation regulated protein. Portions of 2D-PAGE gels and quantification of the levels of Mal12 (A-B), Hsp31 (C-D), and Pep4 (E-F) proteins after 0, 24, 48 and 72 h fermentation.

Mal12. Other proteins whose levels declined enzymes involved in amino acid metabolism throughout fermentation include the vacuolar and sterol biosynthesis also decreased in level ATPase and mitochondrial ATP synthase; both of as the fermentation progressed, indicative of the these proteins were present initially in the yeast fermentation becoming anaerobic with time. but declined rapidly during the fermentation As the fermentations advanced, and particularly and were undetectable after 48 h. Many in the later stages, there was a marked increase 102 R. Hansen et al. in the levels of some proteins involved in the the fermentations. The induction of Ahp1 suggests cells response towards stress, such as heat-shock that organic peroxides are a significant cause of proteins and some proteins involved in protecting oxidative stress in industrial fermentations. The cells’ against oxidative stress. The levels of two effect of oxidative stress was also observed in protein chaperones, heat shock proteins Ssa1 and the form of oxidative modifications, in particular Hsp31, increased steadily as the fermentations peptides containing cysteines, to peptides derived progressed (Figure 3B). Interestingly, Ssa1 was from many proteins. also frequently found as a specifically cleaved In addition to the stress response proteins, proteolytic fragment and the levels of this the levels of proteins involved in protein fragment increased significantly at the end of turnover increased steadily as the fermentations fermentation (Figure 4). The levels of the other progressed. The levels of Proteinase A, heat shock and stress proteins, including Ssc1 and Proteinase B and Proteinase C all increased Hsp104 were only detectable at the very end of during fermentation (Figure 3C). The induction the fermentations, after 72 h. of proteases presumably indicates nitrogen While the majority of proteins involved in limitation and cells are forced to recycle amino stress protection were only detected very late acids by protein degradation. Consistent with in the fermentations, a thiol-specific peroxidase this notion the number of proteolytic protein (Ahp1), responsible for protecting yeast cells against fragments observed on the gels was also found organic hydroperoxides was induced earlier during to increase as the fermentation advanced.

Figure 4. Fragments of Ssa1 accumulate during grain fermentations. The relevant sections of 2D-PAGE gels are shown illustrating the increase in Ssa1 proteolytic fragments throughout fermentation A- after 24 h, B- after 72 h. Proteomic analysis of distillers’ yeast 103

Discussion Revuelta, 2000). After 48 h fermentation increasing numbers of yeast cells were found to Ethanol yield is the most important parameter be smaller in size and possessed granular-looking in the grain distilling industry. In the three intracellular structures. This distinct phenotype is analysed industrial fermentations, the yield possibly a response to stress and yeast survival in varied from 8 to 10%, which is acceptable the increasingly harsh environment in the later according to industry standards (Russell, 2003). part of the fermentation (data not shown). With the exception of dextrins, no significant The proteome is defined as the expressed concentration of residual sugars was found in any proteins under a given condition and will of the fermentations. The fermentation profile therefore be unique for each strain. As a result, showed that the yeast was growing exponentially despite the availability of a protein reference during the first 24 h and thereafter reduced map for laboratory strains of S. cerevisiae, it levels of free amino nitrogen and possibly ATP was necessary to create a reference map for resulted in a decrease in the number of yeast ‘M’ yeast, because yeast strains often show cell divisions. High levels of yeast cell division differences in their proteomes as a result of slight during fermentation is undesirable as it means changes in the genetic background (Boucherie, that carbon sources, such as sugars are converted Sagliocco, Joubert, Maillet, Labarre and Perrot, into biomass, thereby lowering the concentration 1996; Rogowska-Wrzesinska, Larsen, Blomberg, of available sugars for ethanol production. Some Gorg, Roepstorff, Norbeck and Fey, 2001). yeast growth, however, is essential for optimal The reference map for ‘M’ yeast was prepared yields of ethanol (Van Hoek, Van Dijken, and using protein extracted from yeast growing Pronk, 1998). Therefore, pitching rate must exponentially in 400,000 L washbacks containing be balanced to achieve yeast growth without a grain wort. A large number of proteins were too much biomass formation. Interestingly, positively identified and alterations to the levels ‘M’ yeast growth was observed to be limited of specific proteins throughout the fermentation after 24 h, probably due to nitrogen starvation. process were noted. As sugar concentrations were still reasonably As the genomic structure of industrial high at this stage, ‘M’ yeast appears to induce yeast is often complex with different genomes proteins required for protein turn-over and represented in hybrid strains we expected that the recycling of amino acids. In other words, this would complicate our analysis of distillers biomass formation is reduced while ethanol is yeast. For example, the presence of hybrid still being produced, a scenario which is ideal genomes in strains often results in “duplet” spots for a high specific ethanol yield (Van Hoek et al., on the 2D gel due to differences in the peptide 1998). The increase in FAN levels together with sequences (Joubert, Strub, Zugmeyer, Kobi, the decrease in yeast cell numbers in the later Carte, Van Dorsselaer, Boucherie and Jaquet- part of fermentation is suggestive of autolysis Gutfreund 2001). However, such “duplet” spots of yeast. were not observed on 2D-PAGE gels from ‘M’ Changes in the morphology of yeast were yeast, which indicates that only one genome observed during the industrial fermentations, (S. cerevisiae) is present, although the work which were replicated in laboratory scale presented here does not give any indication of experiments (data not shown). However, ploidy. This conclusion is further supported by attempts to examine the structures and presence the fact that throughout this study no non- S. of various organelles in yeast during the cerevisiae proteins were identified by peptide industrial fermentations proved unsuccessful. mass fingerprinting of ‘M’ yeast proteins. The This was likely due to a reduction in membrane high identification rate of proteins from the potential and ATP levels to drive the uptake distilling yeast ‘M’ suggests that the proteome of of the lipophilic dyes (Santos, Jimenez and this yeast is very similar to that of the sequenced 104 R. Hansen et al. strain of S. cerevisiae, S288C . However, as with Maltose metabolism is repressed by high other proteomic studies the proteins identified in concentrations of glucose as a result of repression this study are relatively abundant and in the main of gene expression and targeted degradation of soluble. Therefore it is not possible to rule out maltose permeases (Han, Cotty, Sottas, Jiang and that there are poorly abundant proteins present Michels, 1995). Based on our results it would in ‘M’ that are not represented in laboratory appear that there was little glucose repression yeast, such as S288c. Indeed it is already known of the genes involved in maltose metabolism in that ‘M’ possesses a gene encoding a glucan ‘M’ during the early stages of the fermentations 1,4-alpha-glucosidase (the STA2 gene) which is where glucose concentrations were high. It is not present in S288c (Lyness, Jones and Meaden, possible that this is a key attribute of ‘M’ yeast as 1993). it could allow for rapid wort fermentation. Quantification of protein expression in ‘M’ The work presented here is in agreement yeast during industrial fermentation revealed with earlier studies using micro-arrays on major changes, which were related to the cells’ brewers yeast in industrial settings, showing nutritional state and the increased levels of stress that the expression of many heat shock proteins experienced. For example, sterol synthesis, was repressed during fermentation (Brosnan, Erg10 was only expressed in the beginning of Donnelly, James and Bond, 2000; James et al., fermentation. This is in accordance with a study 2003). However, upon entering stationary phase, which showed a 26-fold induction of the ERG10 at the end of fermentation, the complete set of gene in the first hour of fermentation compared heat shock proteins was detected, in agreement to 24 h (Higgins, Beckhouse, Oliver, Rogers, and with previous findings in laboratory yeast Dawes, 2003). Enzymes and proteins involved (Sanchez, Taulien, Borkovich and Lindquist, in amino acid biosynthesis, gene transcription 1992). The lack of heat shock proteins might and translation, such as elongation factors and make yeast prone to dying, because of the polymerases were also mainly present at the ability of heat shock proteins to prevent beginning of fermentation. This confirms that protein aggregation, and thereby buffer against cell division and yeast growth were largely environmental stresses, which yeast faces confined to the first 24 h of the fermentation. throughout fermentation. The rate of maltose and maltotriose uptake Unlike other HSPs, the levels of the heat and conversion to glucose has a major impact shock protein Ssa1 and Hsp31 showed a gradual on fermentation due to their high abundance increase during fermentation. Interestingly, Ssa1 in maize wort (Jespersen, Cesar, Meaden and was found as discrete proteolytic fragments, Jakobsen, 1999). A similar expression pattern which accumulated as the fermentations was observed for Mal12, a known maltase, and progressed. The activity of this Ssa1 fragment will Ygr287c, which is a protein with high sequence be significantly affected because the N-terminal similarity to Mal12. Therefore, it seems likely ATPase region is completely cleaved off and that Ygr287c has maltase activity although its it is possible that cleavage of Ssa1 might be a substrate specificity and/or kinetic properties mechanism to negatively regulate Ssa1 function. may be different from those of Mal12. It would The appearance of full-length Ssa1 after 72 h of be very interesting to determine the affinity of fermentation could possibly be due to a lower Ygr287c for maltose and maltotriose. It was not specific protease activity or higher levels of possible to determine the expression of maltose Ssa1 production, which saturate the protease(s) permeases, as many membrane proteins are not responsible. easily detected by 2D gel electrophoresis due Transcription of proteinase genes is regulated to their limited solubility (Santoni, Molloy and by the TOR pathway. Specifically, deactivation of Rabilloud, 2000). TOR kinases releases the repressor Ure2 from the Proteomic analysis of distillers’ yeast 105 transcription factor Gln3, which then transcribes Conclusion its target genes, which include proteinase A (PEP4) and proteinase B (PRB1) (Kuruvilla, Samji The use of 2D gels and Peptide Mass Fingerprinting and Schreiber, 2001). Moreover, growth arrest has allowed quantification and identification, caused by nitrogen depletion has been shown to respectively, of proteins up and down regulated trigger de-activation of the TOR pathway, leading during industrial grain fermentations. It was to transcription of PEP4 and PRB1. The significant found that stress proteins such as Ssa1 were increase in the levels of Pep4 and Prb1 during highly induced, confirming that distiller’s yeast fermentation in this study is another indicator of is increasingly challenged as the fermentation nitrogen depletion. A similar up-regulation of PEP4 progresses. However, a complete stress response and PRB1 upon growth arrest has been observed was only observed as cells entered stationary during wine yeast fermentations (Rossignol et phase, late in fermentation. The levels of al., 2003). The increase in the levels of Pep4 and proteases increased, probably as a result of Prb1 correlated well with an observed increase nitrogen limitation, after 24 h as revealed by in proteolytic fragments on the 2D-gels from late FAN analysis. A protein designated Ygr287c with in the fermentation. The recycling of amino acids high sequence similarity to MAL12, a maltase, by Pep4 and Prb1 is known to be essential for was found to be induced by high maltose yeast survival upon nitrogen starvation (Teichert, concentrations, which suggests that Ygr287c Mechler, Muller and Wolf, 1989). In brewing plays a significant role in maltose metabolism in however, high proteinase A activity has been distillers yeast. Induction of Ahp1, a thiol-specific correlated with yeast cell autolysis and low foam peroxidase, indicates that oxidative stress during stability in the product (Hutter, Meidl, Kuhmann, grain fermentations may be a result of a build up Nitzsche, Bryce and Stewart, 2005). of organic peroxides. Further studies are required An optimal pH in the vacuole is vital for to examine the precise roles these proteins play degradation of proteins and acidification of the in industrial grain fermentations, potentially vacuole is carried out by an H+- ATPase, Vma1 allowing conventional strain development and Vma2 (Horst, Knecht and Schu, 1999). In programmes to produce a yeast with enhanced this study Vma1 and Vma2 were only detected at performance in terms of increased ethanol yield the beginning of fermentation, where the pH was and fermentation robustness. higher. Also the mitochondrial ATPase subunits, Atp1 and Atp2, were present at higher levels at the beginning of fermentation. This may reflect Acknowledgments changes in the mitochondrial function with a shift from aerobic to anaerobic fermentation. This paper is dedicated to the memory of Exposure of yeast to ethanol, organic acids our colleague Anita Ferguson who passed and other metabolites can result in oxidative away in June 2007. The funding given by the stress, which might result in growth inhibition Scotch Whisky Research Institute is gratefully or cell death. Ahp1 is an important enzyme acknowledged. Many thanks are also due to required for the protection against organic Carole Judd from North British Distillery Ltd. for hydroperoxides (Lee, Spector, Godon, Labarre assistance with sampling and data collection. and Toledano, 1999). Ahp1 levels were shown to be elevated during industrial grain fermentations, indicating that organic peroxides may be a References major cause oxidative stress. Interestingly, it has been previously shown that AHP1 was induced Alexandre, H., Ansanay-Galeote, V., Dequin, 5.3-fold after addition of 7 % ethanol (Alexandre, S. and Blondin, B. (2001). Global gene Ansanay-Galeote, Dequin and Blondin, 2001). expression during short-term ethanol stress 106 R. Hansen et al.

in Saccharomyces cerevisiae. FEBS Letters Microbiology 17: 1093-1107 498: 98-103 Higgins, V.J., Beckhouse, A.G., Oliver, A.D., Boucherie, H., Sagliocco, F., Joubert, R., Rogers, P.J. and Dawes, I.W. (2003). Maillet, I., Labarre, J. and Perrot, M. (1996). Yeast genome-wide expression analysis Two-dimensional gel protein database of identifies a strong ergosterol and oxidative Saccharomyces cerevisiae. Electrophoresis stress response during the initial stages of 17: 1683-1699 an industrial lager fermentation. Applied Bradford, M.M. (1976). A rapid and sensitive and Environmental Microbiology 69: method for the quantitation of microgram 4777-4787 quantities of protein utilizing the principle of Horst, M., Knecht, E.C. and Schu, P.V. (1999). protein-dye binding. Analytical Biochemistry Import into and degradation of cytosolic 72: 248-254 proteins by isolated yeast vacuoles. Molecular Brejning, J., Arneborg, N. and Jespersen, L. Biology of the Cell 10: 2879-2889 (2005). Identification of genes and proteins Hutter, K.J., Miedl, M., Kuhmann, B., Nitzsche, induced during the lag and early exponential F., Bryce, J.H. and Stewart, G.G. (2005). phase of lager brewing yeasts. Journal of Detection of proteinases in Saccharomyces Applied Microbiology 98: 261-271 cerevisiae by flow cytometry. Journal of the Brosnan, M.P., Donnelly, D., James, T.C. and Institute of Brewing 111: 26-32 Bond, U. (2000). The stress response is James, T.C., Campbell, S., Donnelly, D. and repressed during fermentation in brewery Bond, U. (2003). Transcription profile strains of yeast. Journal of Applied of brewery yeast under fermentation Microbiology 88: 746-755 conditions. Journal of Applied Microbiology Candiano, G., Bruschi, M., Musante, L., 94: 432-448 Santucci, L., Ghiggeri, G.M., Carnemolla, Jamieson, D.J. (1992). Saccharomyces cerevisiae B., Orecchia, P., Zardi, L. and Righetti, P.G. has distinct adaptive responses to both (2004). Blue silver: a very sensitive colloidal hydrogen peroxide and menadione. Journal Coomassie G-250 staining for proteome of Bacteriology 174: 6678-6681 analysis. Electrophoresis 25: 1327-1333 Jespersen, L., Cesar, L.B., Meaden, P.G. and Fey, S.J. and Larsen, P.M. (2001). 2D or not Jakobsen, M. (1999). Multiple alpha- 2D. Two-dimensional gel electrophoresis. glucoside transporter genes in brewer’s yeast. Current Opinion in Chemicl Biology 5: Applied and Environmental Microbiology 26-33 65: 450-456 Griffin, P.R., MacCoss, M.J., Eng, J.K., Blevins, Joubert, R., Strub, J.M., Zugmeyer, S., Kobi, D., R.A., Aaronson, J.S. and Yates, J.R.r. (1995) Carte, N., Van Dorsselaer, A., Boucherie, Direct database searching with MALDI-PSD H. and Jaquet-Gutfreund, L. (2001). spectra of peptides. Rapid Communication Identification by mass spectrometry of two- in Mass Spectrometry 9: 1546-1551 dimensional gel electrophoresis-separated Gygi, S.P., Corthals, G.L., Zhang, Y., Rochon, proteins extracted from lager brewing yeast. Y. & Aebersold, R. (2000) Evaluation of Electrophoresis 22: 2969-2982 two-dimensional gel electrophoresis-based Kobi, D., Zugmeyer, S., Potier, S. & Jaquet- proteome analysis technology. Procedings Gutfreund, L. (2004). Two-dimensional of the Natlional Academy of Sciences USA protein map of an “ale”-brewing yeast strain: 97: 9390-9395 proteome dynamics during fermentation. Han, E.K., Cotty, F., Sottas, C., Jiang, H. & FEMS Yeast Research 5: 213-230 Michels, C.A. (1995). Characterization of Kunze, G., Kunze, I., Barner, A. & Schulz, R. AGT1 encoding a general alpha-glucoside (1993) Classification of Saccharomyces transporter from Saccharomyces. Molecular cerevisiae strains by genetic and biochemical Proteomic analysis of distillers’ yeast 107

methods. Monatsschrift fur Brauwissenschaft 1054-1070 46: 132-136 Santos, M.A., Jimenez, A. and Revuelta, J.L. Kuruvilla, F.G., Shamji, A.F. and Schreiber, (2000). Molecular characterization of FMN1, S.L. (2001). Carbon- and nitrogen-quality the structural gene for the monofunctional signaling to translation are mediated by flavokinase of Saccharomyces cerevisiae. distinct GATA-type transcription factors. Journal of Biological Chemistry 275: Proceedings of the National Academy of 28618-28624 Science USA 98: 7283-7288 Teichert, U., Mechler, B., Muller, H. and Lee, J., Spector, D., Godon, C., Labarre, J. and Wolf, D.H. (1989). Lysosomal (vacuolar) Toledano, M.B. (1999). A new antioxidant proteinases of yeast are essential catalysts with alkyl hydroperoxide defense properties for protein degradation, differentiation, and in yeast. Journal of Biological Chemistry cell survival. Journal of Biological Chemistry 274: 4537-4544 264: 16037-16045 Lyness, C.A., Jones, C.R. and Meaden, P.G. Van Hoek, P., Van Dijken, J.P. and Pronk, (1993). The STA2 and MEL1 genes of J.T. (1998). Effect of specific growth rate Saccharomyces cerevisiae are idiomorphic. on fermentative capacity of baker’s yeast. Current Genetics 23: 92-94 Applied and Environmental Microbiology Moore, S. and Stein, W.H. (1954). A modified 64: 4226-4233. ninhydrin reagent for the photometric determination of amino acids and related compounds. Journal of Biological Chemistry 211: 907-913 Oda, Y., Nagasu, T. and Chait, B.T. (2001). Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nature Biotechnology 19: 379-382 Rogowska-Wrzesinska, A., Larsen, P.M., Blomberg, A., Gorg, A., Roepstorff, P., Norbeck, J. and Fey, S.J. (2001). Comparison of the proteomes of three yeast wild type strains: CEN.PK2, FY1679 and W303. Comparative and Functional Genomics 2: 207-225 Rossignol, T., Dulau, L., Julien, A. and Blondin, B. (2003). Genome-wide monitoring of wine yeast gene expression during alcoholic fermentation. Yeast 20: 1369-1385 Russell, I.(2003). Whisky: Technology, production and marketing. Academic Press, London Sanchez, Y., Taulien, J., Borkovich, K.A. and Lindquist, S. (1992). Hsp104 is required for tolerance to many forms of stress. The EMBO Journal 11: 2357-2364 Santoni, V., Molloy, M. and Rabilloud, T. (2000). Membrane proteins and proteomics: un amour impossible? Electrophoresis 21: 108 R. Hansen et al. Effect on new-make character due to the performance of brewer's yeast (I) 109 Chapter 15 Effect on new-make spirit character due to the performance of brewer’s yeast – (I) physiological changes of yeast during propagation and brewing

H. Yomo, Y. Noguchi and T. Yonezawa Suntory Ltd., Technical Development Center, Osaka, Japan

Introduction out in our pilot plant. Mashing was carried out with 70 kg of malted barley and 400 L of Traditionally, two types of yeast are used in wort was obtained. Original gravity of wort whisky brewing, distiller’s yeast and brewer’s was 1.056. Fermentation was carried out on a yeast. Recently, consumption of ale has decreased 120 L scale. The fermentation temperature was in the U.K. It is difficult to obtain ale yeast from programmed to range from 23 to 34 degrees breweries. In addition, its activity and vitality is Celsius. The fermentation period was 72 h. not stable compared to distiller’s yeast. Thus, Yeast pitching rate was an experimental variable. many distilleries have stopped using ale yeast for Distiller’s yeast was 5.0 g/L; brewer’s yeast was whisky brewing. On the other hand, it is said that 10 g/L, mixed fermentation was with 5.0 g/L of some whisky products have changed to a lighter brewer’s yeast and 2.5 g/L of distiller’s yeast. character than those in the 1970s and 1980s. First distillation was carried out with 120 L. The We believe this was caused by changes in yeast second distillation was carried out with 5 L and management at the distillery; namely, brewer’s repeated six times. yeast is no longer used for whisky brewing. In this paper, we would like to explain the function of brewer’s yeast; to this end, we Yeast cultures employed observed yeast performance at all points of the fermentation process. Our results show the Two types of yeast were used: a distiller’s strain, special function of the brewer’s yeast, and its a brewer’s strain. The distiller’s strain was necessity for new spirits of full body character. DCL ’M’ strain. This was a pressed yeast. The And we also shows that the physiological state brewer’s strain was ale type AH310 stocked by of brewer’s yeast could affect its fermentation Suntory. This was cultivated in a pilot plant and performance and the character of new spirits. collected by centrifuge before pitching.

Materials and methods Yeast propagation Propagation was carried out on a 30 L scale. Fermentation and distillation Wort was autoclaved, pitching rate was 5 g/L, Fermentation and distillation were carried the temperature was 30 degrees Celsius. 110 H. Yomo et al.

Chemical analysis Results and discussion

Chemical analyses of new-make spirits were Effects of brewer’s yeast on the character of carried out with GC (Shimadzu, Kyoto, Japan). new-make spirits

In order to clearly demonstrate in a laboratory Dielectric monitoring of yeast behaviour setting the effects of brewer’s yeast on new spirit character, three fermentation series were carried Dielectric monitoring of permittivity (conductivity out. One was pitched with distiller’s yeast only, of wash) was carried out using an E5051A another one with only brewer’s yeast, and the dialectic sensor (Hewlett-Packard Japan, Osaka, last one was made with both strains. Estimation Japan). of the new spirit character was carried out with sensory analysis and chemical analysis. Results are shown in Figures 1-3. Six highly In vivo 31P-NMR spectrum trained individuals carried out sensory analysis (Figure 1). Each dot represents a score from an Sample preparation and data accumulation were individual, and lines show the average. The carried out as previously reported (Furukubo et mixed fermentation sample clearly displayed al.).

Estery 5 4 3 Yeasty Fruity 2

1 Distiller's 0 Mix Brewer's

Full body Clean

Complicated Figure 1. Comparison of the three new-make spirit characters by sensory analysis.

n-ProOH I-BuOH I-AmOH a-AmOH -PheOH Et C8 Et C10 Et C12 Et C16 Et C16:1

4500 400 4000 350 3500 300 3000 250 2500 200 2000 150 PPM (at 100%) 1500 PPM (at 100%) 1000 100 500 50 0 0 Brewer's Mix Distiller's Brewer's Mix Distiller's Figure 2. Amount of fusel alcohols and esters in three new-make spirits. Effect on new-make character due to the performance of brewer's yeast (I) 111

DMTS DTPA DTPOH

1400

1200

1000

800

600

400

200 S compounds (nmols/L at 100%)

0 Brewer's MixDistiller's

Figure 3. Amount of sulphur compounds in three new-make spirits. Dithiapentyl alcohol (DTPOH), dithiapentyl acetate (DTPA), dimethyl trisulfide (DMTS). a full body and more complex aroma than the we reported before, total cell volume of viable single strain samples. yeast can be precisely measured. During the first Fusel alcohols and long chain fatty acid 24 h, the three series of fermentations showed esters were generated more by the distiller’s almost the same growth, cell volumes increased yeast and less by the brewer’s yeast. The and sugar consumption was completed. In the mixed fermentation samples contained levels next phase, each fermentation series showed between the two pure fermentations. On the different patterns. Distiller’s yeast was strong, other hand, sulphur compounds, dithiapentyl keeping total cell volume and cell components alcohol (DTPOH), dithiapentyl acetate (DTPA), almost at a maximum for approximately 36 h. dimethyl trisulfide (DMTS) were markers for On the other hand, ale yeast was weak; cell the full body character; the new-make spirits of volume quickly declined, and cell components mixed fermentation had the largest amount of such as amino acids leaked rapidly. The mixed these compounds. one showed unique patterns, declining step To investigate the reason for this, we monitored by step. The first decline might be due to ale fermentation behaviour with the dielectric yeast, although overall the mixed fermentation sensor. The result is as shown in Figure 4. As was stronger than the pure ale fermentation.

Mix Brewer's Distiller's Total cell volume Relative permittivity @ 300kHz

02436 48 60 72

Figure 4. Time course of relative permittivity in wort. 112 H. Yomo et al.

On the other hand, perhaps distiller’s yeast laboratory, we carried out propagation (Figure 6). became slightly weaker compared to its pure Fresh yeast (A) was collected from propagation fermentation. It could be considered that some by centrifuge when sugar consumption was interaction occurred between the two yeasts finished; taking about 24 h, and starved yeast that modified their physiological states under (B) was collected two days after sugar depletion. starvation. For example, early death of ale yeast After extended propagation periods, the yeast provides nutrient for lactic acid bacteria, and it did not die, and the ratio of methylene blue causes enhancement of the generation of lactic stained cells remained almost zero. Laboratory acid. Lactic acid would decrease pH of wash and fermentation and distillation were then carried enhance the death of distiller’s yeast. out with using these propagated yeasts mixed The time courses of total amino acids in wort with distiller’s yeast. are shown in Figure 5. The final amounts of amino Estimation of the new-make spirit character acids in mixed fermentations were higher than was carried out with sensory analysis and for distiller’s yeast. We consider that amino acids chemical analysis. The results are shown in are the barometer of intracellular components, Figures 7-9. Result of sensory analysis clearly and act as substrates of reactions during the showed that new spirits made using starved yeast distillation process. The wash components of a (B) with distiller’s yeast showed a more complex mixed fermentation became richer than that of aroma and full body character than that of fresh a pure fermentation by distiller’s yeast. These yeast (A). The amounts of fusel alcohols and esters results suggest that by using brewer’s yeast of long chain fatty acids were almost the same together with distiller’s yeast, amounts of sulphur for both of the new-make spirits. Ethyl esters of components can be raised, new spirits have a fatty acids were slightly larger for fresh yeast (A) fuller body character, and wash composition than for starved yeast (B). The amounts of sulphur becomes richer than distiller’s yeast, changing compounds were the marker for the heavy and the spirits’ composition to a complex aroma. full body character. New-make spirit made with starved yeast (B) had larger amounts of these Effects of the “state” of brewer’s yeast compounds than when made with fresh yeast (A) (Figure 9). These results show that physiological In order to clearly demonstrate the effects of the differences of ale yeast during mixed fermentation “state” of brewer’s yeast on new spirits in the affect sulphur compounds in new spirits.

Brewer's Mix Distiller's 14

12

10

8

6

4 Amino acids (mM) 2

0 0102030405060 Fermentation periods (hrs) Figure 5. Time course of total amino acids in wort. Effect on new-make character due to the performance of brewer's yeast (I) 113

Cell number

Dry cell weight

Amino acids Dead cell ratio (Methylene blue) Apparent ex. 0 24 72 Fermentation periods (hrs)

A; Fresh B; Starved Figure 6. Time course of yeast growth and wort composition.

Estery 5 4 3 Yeasty Fruity 2 1 Fresh (A) 0 Starved (B)

Full body Clean

Complicated

Figure 7. Comparison between the two new-make spirit characters by sensory analysis.

n-ProOH I-BuOH I-AmOH a-AmOH -PheOH Et C8 Et C10 Et C12 Et C16 Et C16:1

4500 400 4000 350 3500 300 3000 250 2500 200 2000 150 1500

1000 Esters (PPM at 100%) 100

Fusel alcohol (PPM at 100%) 500 50 0 0 Fresh Starved Fresh Starved

Figure 8. Amount of fusel alcohols and esters in two new-make spirits. 114 H. Yomo et al.

800 700 600 500 DTPOH 400 DTPA 300 DMTS

nmolS/L at 100% 200 100 0 FreshFresh StarvedStarved

Figure 9. Amount of sulphur compounds in two new-make spirits. Dithiapentyl alcohol (DTPOH), dithiapentyl acetate (DTPA), dimethyl trisulfide (DMTS).

Estimation of the physiological “state” of phase. We investigated intracellular phosphate yeast compounds with an in vivo 31P-NMR spectrum. The typical pattern for the NMR spectrum of We tried to investigate the states of the yeast in brewing yeast is shown in Figure 11. Major detail. Figure 10 shows electron micrographs compounds were detected all at once. Figure 12 of yeasts. Micrographs were taken with an shows the spectrum for propagated yeast in this electron microscope after thin sectioning. Fresh report. The fresh yeast sample (A) displayed a yeast (A) had enormous amounts of glycogen normal pattern. Starved cell (B) showed a special granules, as well as small vacuoles. Starved pattern. An especially big tri-metaphosphate peak yeast (B) had unique physiological features. Its was apparent. We believe that this phenomenon cells had a large central vacuole which contained is the marker of a special physiological change characteristic features. in the starved condition in propagated yeast. As demonstrated in Figure 6, if the starved yeast was still alive, this phase should not result in autolysis. We believe that this phenomenon occurred as a result of the biological function of “autophagy”. Our results suggest that physiological states of brewer’s yeast reflect the character of new spirits and can lead to a complex aroma and full body.

Nuclear Nuclear Conclusions Vacuole X In this paper, we focused on the function of Vacuole brewer’s yeast with regard to the character of new-make spirits. By using brewer’s yeast Glycogen granules with distiller’s yeast, new-make spirit character Figure 10. Electron micrographs showing different “states” of yeast. becomes full body type, amounts of sulphur components increased, and wash composition It is well known that central vacuoles are storage became richer, changing the spirit’s character to organelle for phosphates during the stationary a complex aroma. Effect on new-make character due to the performance of brewer's yeast (I) 115

a. Pi (Cytosolic) Standard b. Pi (Vacuolar)

c. Sugar phosphates d. Phospholipid l e. Phosphomannan a f. ATPγ + ADPß g. ATPα + ADPα b h. ATPß i. NAD(H) d c j i j. PPi (terminal) g k k. PPi (penultimate) e f h l. PPi (internal)

Pi : Ortho phosphate Chemical shift (ppm) PPi : Polyphosphate

Figure 11. Typical in vivo 31P-NMR spectrum of brewing yeast.

A;Fresh yeast B;Starved yeast

Chemical Shift (ppm) Chemical Shift (ppm)

Figure 12. Intracellular phosphate profiles of the two “states” of yeastsPeak of monitored trimetaphosphate by in vivo 31P-NMR spectrum. Physiological states of ale yeast also reflect important role in making new spirits with full the character of new-make spirits for complex body and complex aroma. Such new-make aroma and full body. Starved ale yeast showed a spirits could develop into excellent malt whisky specific character. The fact that starved yeast may after maturation. go into the phase of autophagy, as opposed to autolysis, demonstrates its unique fermentation performance. Acknowledgements Traditionally, even although yeast was in an uncontrolled physiological condition, it The authors wish to thank Dr. Inatomi for was surely the “starved state” of brewer’s yeast significant discussion. Thanks are also due to that has been used in whisky production. Thus the members of the pilot plant of Suntory Ltd. brewer’s yeast would play quite a unique and for their kind help in this work. 116 H. Yomo et al.

Bibliography Yonezawa, T., Hatanaka, T., Yomo, H., and Kojima, K. (1998). The control of yeast Boulton, C. and Quain, D. (2001). Highly during whisky fermentation; changes in growing yeast produce much fusel alcohols. yeast during fermentation and a new on-line Esterase is activated under many lipids monitoring technique. In: Proceedings of condition. Brewing yeast and Fermentation the Fifth Aviemore Conference on Malting, Blackwell Science, U.K. Brewing and Distilling, Institute of Brewing, Furukubo, S., Matsumoto, T., Yomo, H., Fukui, London, pp. 271-274 N., Ashikari, T. and Kakimi,Y. (1997). The Yonezawa, T., and Stewart, G. G., (2004). in vivo 31P-NMR analysis of brewing yeast Changes in higher chain fatty acid ethyl ester and its practical application to brewing. content of low wine at different stages of In: Proceedings of the European Brewing yeast death during whisky fermentation. In: Convention Congress, Oxford University Distilled Spirits - Tradition and Innovation, Press, Oxford, pp. 423-430 Nottingham University Press, Nottingham, Klionsky, D. J. and Ohsumi, Y. (1999). Vacuolar UK, pp. 103-111. impact of proteins and organelles from the cytoplasm. Annual Reviews of Cell and Developmental Biology 15: 1-32 Effect on new-make spirit character due to the performance of brewer's yeast (II) 117 Chapter 16 Effect on new-make spirit character due to the performance of brewer’s yeast – (II) various yeast strains containing commercial strains

Y. Noguchi, K. Urasaki, H. Yomo and T. Yonezawa Suntory Ltd., Technical Development Center, Osaka, Japan

Introduction The characters of new spirits produced by 13 brewer’s yeast strains that can be obtained Brewer’s yeast can influence the character of practically were investigated. Four strains were new-make spirit for whisky, therefore this yeast ale type yeasts collected from breweries in is one of the most important ingredients. Figure the U.K.. It is getting to be difficult to obtain 1 shows that 3 sulphur compounds that are supplies of such yeast because ale consumption related to the character of new-make spirit can is decreasing. Three strains were lager yeasts be changed by changing the combination of collected from breweries of Suntory. It is yeasts. Using a mix of distiller’s and brewer’s necessary to investigate the potential of these yeast yields results that are never achieved with because lager consumption is increasing. Six either of these yeasts acting alone, a synergistic strains were commercially available active dry “multiplier” effect that has never been seen ale yeasts for ale brewing. For this type, it is before. We investigated the potential of using necessary to investigate the potential because of various brewer’s yeasts including dry ale yeasts, their easy storage and transportation. Herein we instead of traditional ale yeasts from breweries. report that using 13 brewer’s yeasts as above, we DMTS DTPA DTPOH

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600 nmol 100% S/L 400

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0 Brewer's Brewer's + distiller's Distiller's Figure 1. Sulphur compound contents related to the characters of new-make spirit, dithiapentyl alcohol (DTPOH), dithiapentyl acetate (DTPA), dimethyl trisulfide (DMTS). 118 Y. Noguchi et al. made various new spirits in a pilot distillery, and wort was carried out with GC (Shimadzu, Kyoto, performed sensory and chemical analysis. Japan). The component concentrations of new spirits were based on 100% (v/v) alcohol.

Materials and methods Sensory analysis Making new spirits Sensory analysis was carried out by panels New spirits were made in a pilot distillery. composed 9 highly trained members who had Mashing was carried out with 400 kg of malted undergone extensive sensory training. Names of barley, and 2400 L of wort was obtained. the samples were disclosed before evaluation. The target original gravity of wort was 1.056. Fermentation was carried out using a stainless wash back. Distiller’s yeast and brewer’s yeasts Results were used, and pitching rate was 2.1g/L and 3.3g/L, respectively (the concentration was 2.1 Fermentations g/L only when dry ale yeast was used). The fermentation temperature was programmed First, the data for specific gravity of the 13 to range from 23 to 34 degrees Celsius. The strains during fermentation are shown in Figure fermentation period was 72 h. After wash 2. Although the speed of fermentation differs distillation (2400 L) and spirit distillation (1200 between strains, all fermentations of each strain L), 250 L of new spirits was obtained in one were completed. The new spirits samples of each batch. The final sample of each new-make spirit strain were obtained successfully. was obtained after 6 batches. Fermentation Periods Strain 0hrs 24hrs 48hrs 72hrs Yeasts Standard 1.054 1.011 <1.000 <1.000 A-11.054 1.012 <1.000 <1.000 A-21.055 1.011 1.001 <1.000 The strains of distiller’s yeast were supplied from Ale Kerry and Mauri in the U.K.. The strains of ale A-31.055 1.010 <1.000 <1.000 yeast were supplied from breweries in the U.K. A-41.054 1.010 <1.000 <1.000 The strains of dry ale yeast were supplied from L-11.055 1.006 <1.000 <1.000 Lallmand Inc., Montreal, Canada, and Fermentis, Lager L-21.055 1.009 1.000 <1.000 L-31.055 1.007 <1.000 <1.000 Lille, France. The strains of lager yeast were D-11.054 1.007 1.002 <1.000 supplied from the Suntory breweries in Japan. D-21.054 1.007 1.001 <1.000 D-31.054 1.006 <1.000 <1.000 Dry ale D-41.054 1.005 <1.000 <1.000 Analysis of fermentation and contents of new D-51.056 1.008 1.001 <1.000 spirits D-61.056 1.003 1.001 <1.000

Specific gravity was measured by sampling Figure 2. Changes in specific gravities during at certain stages. Dielectric monitoring of the fermentation. permittivity was carried out using an E5051A dialectic sensor (Hewlett-Packard Japan, Osaka, Sensory analysis of new spirits Japan) (Yonezawa,, Hatanaka, Yomo and Kojima, 1998). The yeast behaviour, growth, and death All sensory data were plotted on this diagram phase of yeast was monitored in real time using (Figure 3). As all of the ale strains looked similar, this sensor. Chemical analysis of new spirits and three types of yeast could be categorised from Effect on new-make spirit character due to the performance of brewer's yeast (II) 119

Estery Ale 1 5 Ale 2

4 Ale 3 Ale 4 3 Yeasty Fruity Ale avarage 2 Lager 1

1 Lager 2 0 Lager 3

Lager avarage Dry Ale 1 Full body Clean Dry Ale 2 Dry Ale 3

Dry Ale 4

Dry Ale 5 Complicated Dry Ale 6

Dry ale avarage

Figure 3. Diagram of sensory analysis of new-make spirits. the viewpoint of spirit character. The averages ale yeast was caused by lower amounts of fatty of each type were shown as lines. The line of ale acid esters. Thus, dry ale yeasts showed unique type showed more characters of estery, yeasty, features with regard to its components. and full body, although less complicated. The line of lager type was centred in all characters, and it means that these spirits had less character Yeast behaviour of fermentation than others. The line of dry ale type showed a highly complicated character. When we focus The yeast behaviour during fermentation is on dry ale yeast, the characters of new spirits shown in Figure 5. This figure shows the yeast were never seen in any ale nor lager yeasts. In behaviour during fermentation monitored the future, dry ale yeast will be the effective using dielectric permittivity monitoring. For solution to make various new spirits, if the less lager yeast, during the growth phase, yeast estery character can be solved. permittivity increased very slowly, and at the death phase decreased slowly. On the other hand, for dry ale yeast, during the growth phase, Components of new spirits the yeast permittivity increased very rapidly, and at the death phase decreased slowly. Thus, the The component analysis of the new spirits using big differences in fermentation were observed each type of typical strain of the 3 types are between the 3 types. shown in Figure 4. The amounts of fusel alcohols, and fatty acid ethyl esters were compared with these of ale yeast. In the case of lager yeast, Relationships between components of new fusel alcohols and fatty acid esters were slightly spirits and fermentation decreased. On the other hand, in the case of dry ale yeast, fusel alcohols were greatly We focused on dry ale yeast in order to consider increased and fatty acid esters were greatly the relation between components of new spirits decreased. Considering the relation between and yeast behaviour during fermentation, the components and the sensory analysis, it is because of its complicated character in terms thought that the less estery character using dry of sensory analysis and the big differences in 120 Y. Noguchi et al.

ß-PheOH Et-C16 Fusel Alcohols Fatty Acid Esters a-AmOH 250 Et-C12 5000 i-AmOH 200 Et-C10 4000 i-BuOH n-PrOH Et-C8 3000 150 mg/ L mg/ L 2000 100 1000 50

0 0 AleLager Dry Ale AleLager Dry Ale

Figure 4. Comparison of fusel alcohols and fatty acid esters of new spirits from ale, lager and dried ale yeast fermentations.

260 Ale Lager 210 Dry Ale

160

110 Relative permittivity (@250kHz) 60 020406080 Fermentation periods (hrs) Figure 5. Yeast behaviour during fermentation monitored using dielectric permittivity monitoring. components and yeast behaviour. The first is the Discussion growth phase of the fermentation. Dry ale yeast grew much more, therefore this high growth of Proposed method for increasing total fatty yeast could produce high fusel alcohols. On the acid esters in the growth phase other hand, dry ale yeast produced less esters because the yeast would be propagated under Although the amounts of total fatty acid esters aerobic conditions (Boulton and Quain, 2001). were less, the complicated new spirits were The second is the death phase. The autolysis obtained using dry ale yeasts. Thus, the three of yeast could not proceed because of the methods of increasing these esters are proposed survival of the yeast. Thus, the degradation of by changing the growth phase. The first method intracellular components would be lower, and is changing the pitching rate to be lower than the amounts of long chain fatty acid esters were usual. It will prevent a rapid depletion of the in consequence less. Thus, we have established a acetyl-CoA (Boulton and Quain, 2001). The relationship between components of new spirits second is to propagate again under anaerobic and yeast behaviour, especially the cause of less conditions. This would decrease unsaturated esters using dry ale yeast. This consideration is fatty acids contents of yeast’s membrane at important for increasing esters, i.e. by changing the beginning of the growth phase (Boulton the growth or death phases. and Quain, 2001). The third is the use of clear Effect on new-make spirit character due to the performance of brewer's yeast (II) 121 wort. The use of clear wort is the most effective arise in the case of overly long fermentations. for increasing esters. Clear wort means lower The second method is to raise the maximum contents of lipids, and can be obtained by less fermentation temperature by raising the initial raking in mashing (Boulton and Quain, 2001). fermentation temperature. However, one must Figure 6 shows the difference of fatty acid ester take care to prevent sugars remaining in the contents in new spirits between the conditions wash at the end, as the autolysis of yeast at high of cloudy and clear wort. When yeast ferments temperatures may occur before all sugars have using clear wort, the synthesis of esters would been consumed. The third method is to promote be activated and esters would be increased. the fermentation of lactic acid in the latter phase. Obviously esters were increased (Figure 6). If If conditions are set up, pH would fall due to the this method of clear wort is applied to dry ale increased lactic acid, and the autolysis of yeast yeast, it will be possible to increase total fatty would be promoted as a result. acid esters in new spirits.

Lipids in worwortt C 18 : 2 LongLong chain chain fatty Fatty acid Acid esters Este Et-C16 200 C 18 : 1 inin spirits spirits Et-C12 70 150 C 18 60 C 16 50 100

mg/ L 40

mg/ L 30 50 20 10 0 0 cloudyclear cloudy clear

Figure 6. The difference in fatty acid ester contents in new spirits comparing cloudy and clear worts using a mix of distiller’s yeast and ale yeast.

Proposal method for increasing long chain Conclusions fatty acid esters at the death phase Various new spirits were obtained by using Because of the survival of yeast at the death brewer’s yeasts in the pilot distillery. The phase, amounts of long chain fatty acid esters in character of new spirits were categorised new spirits were less ( Yonezawa and Stewart, according to three types of yeast. Especially 2004). We suggest three methods to increase the using dry ale yeasts, a complicated character of autolysis of yeast at the death phase. The first new spirit was obtained which has never been method is expansion of the fermentation period, seen in any ale nor lager yeasts, although total since the stained cell ratio of yeast at the end of fatty acid esters were less. On the other hand, fermentation is low. Thus, the yeast will be dead, a process control point at the mashing process and the degradation of intracellular components was proposed for dry yeast. In the future, we could be higher. However, there is a possibility will build these characters into new spirits, that excessive degradation of the wash would and design the optimal processes for all steps 122 Y. Noguchi et al. from mashing to distillation using each dry Yonezawa, T., Hatanaka, T., Yomo, H., and yeast, furthermore, we will investigate the other Kojima, K. (1998). The control of yeast dry ale yeasts that produce various strong or during whisky fermentation; changes in complicated characters. yeast during fermentation and a new on- line monitoring technique. In: Proceedings of the Fifth Aviemore Conference on Acknowledgements Malting, Brewing and Distilling, Edited by Campbell, I., Institute of brewing, London, The authors would like to thank Dr. Inatomi for pp. 271-274 significant discussions, Fermentis and Lallmand Yonezawa, T. and Stewart, G. G., (2004). Inc. for supplying yeast. Thanks are also due to Changes in higher chain fatty acid ethyl the members of the pilot distillery for their kind ester content of low wine at different stages help in this work. of yeast death during whisky fermentation. In: Distilled Spirits Tradition and Innovation, Edited by Bryce J.H and Stewart G.G., References Nottingham University Press, Nottingham, U.K., pp. 103-111. Boulton, C., and Quain, D. (2001). Brewing Yeast and Fermentation, Blackwell Science, U.K. The use of CO2 evolution monitoring as an indicator of yeast fermentation performance 123 Chapter 17

The use of CO2 evolution monitoring as an indicator of yeast fermentation performance

S.Y. Pearson, J.W. Walker, T.A. Bringhurst and J.M. Brosnan The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK

Introduction Materials and methods

The assessment of fermentation performance is Due to the fact that this report collates data from an essential step in determining the suitability of a range of experiments, the precise experimental yeast strains for use in distillery fermentations. conditions employed vary. However, all wort This assessment typically focuses on alcohol used in this experiment was collected from a production and fermentation rate as the measures malt distillery from a sample point on the cold of performance, since these are the key factors side of the wort cooler, prior to the addition in determining fermentation efficiency in the of yeast. This was stored frozen until required, distillery. However, these analyses are relatively when it was diluted with distilled water to the time-consuming to perform. Also, the number of appropriate gravity. samples that can be collected over the course of All fermentations were carried out using 1 a fermentation is limited. litre of 1060° wort in a temperature-controlled

Since carbon dioxide (CO2) is formed as water bath. The temperature profile employed a by-product of ethanol formation during depended on the experiment being carried out. fermentation, it should be possible to monitor Yeast strains and pitching rates also varied, with both alcohol production and sugar utilisation both compressed and laboratory-propagated by measuring the production of CO2. With the yeast used. The fermentation time in all cases, use of a digital gas flow meter, gas production however, was 45 hours. This was chosen as can be monitored continuously throughout the CO2 production after this point appears to reach course of fermentation. Also, the data obtained a baseline level and thereafter is minimal in can be automatically downloaded to a PC for comparison to the total volume produced. Also, ease of processing. alcohol production will be largely, although not In order to investigate whether it is possible completely, finished by this stage. to use CO2 production as an indicator of yeast Carbon dioxide production during fermentation performance, we have collated fermentation was monitored continuously using the data obtained from a number of different an Agilent ADM2000 digital flow meter (Figure experiments and studied the correlation between 1). Volumetric flow rate data were downloaded these two variables. automatically to a PC using the ADM Trender 124 S.Y. Pearson et al. software (Agilent Technologies) supplied, and the Wash alcohol strength was determined using total CO2 volume produced was calculated. distillation and densitometry. Separate fermentations were carried out for the assessment of wash specific gravity and alcohol strength. Samples were removed at 20 and 45 Results and discussion hours. In order to determine specific gravity, wash samples were filtered using Whatman 2V A typical CO2 production profile obtained from filter paper. The density of the filtered wash was a laboratory fermentation using conditions then determined using a digital density meter designed to simulate those in the distillery is (DMA-5000, Anton Paar, Graz, Austria) and this shown in Figure 2. value was then converted to specific gravity.

Figure 1. Agilent ADM2000 flow meter.

40 35 CO2 Flowrate (ml/min) Total CO Volume (L) 30 2 30 25 CO 2 20 volume (L) 20 15 flow rate (ml/min) 2 10

CO 10 5

0 0 0 5 10 15 20 25 30 35 40 45

Fermentation Time (h)

Figure 2. Typical CO2 production profile. The use of CO2 evolution monitoring as an indicator of yeast fermentation performance 125

In the initial stages of fermentation there Table 1. Correlation coefficients of the relationships between CO volume and wash is a delay before CO2 production appears to 2 commence. This is due to the fact that the initial analysis data (n = 32). CO generated will dissolve into the wort until 2 Wash Specific Gravity CO it is saturated before the rest is released into 2 alcohol gravity lost the headspace. After this initial lag, the flow volume rate increases sharply before reaching a peak (% v/v) (°) (°) (L) at approximately 15 hours. The flow rate then declines until a constant value, just above zero, Wash 1 is reached at approximately 40 hours. The exact Alcohol (% v/v) profile of CO2 production will vary depending on the yeast strain and fermentation conditions Specific -0.848 1 employed, but this general pattern has been Gravity (°) observed in all yeasts tested to date. Gravity 0.998 -0.857 1 The results of the correlation analysis, Lost (°) CO 0.976 -0.887 0.982 1 investigating the link between CO production, in 2 2 Volume (L) terms of volume, and fermentation performance, are shown in Table 1 (this includes data from both 20 and 45 hour samples). In order to determine if CO2 production It can be seen that there are a number of in the early stages of fermentation can be statistically significant correlations between used to predict subsequent performance, the the data obtained from CO2 monitoring and relationship between CO2 volume at 20 hours gravity and alcohol analyses. In particular, the and wash alcohol strength at 45 hours was volume of CO produced by a yeast was found to 2 studied. However, it was found that there was correlate closely with the alcohol strength of the no significant correlation between them (r = corresponding wash (Figure 3). The volume of 0.062, Figure 4). CO was also found to correlate strongly with the 2 When the relationship between the peak degrees of wort gravity fermented. These findings CO2 flow rate produced by a yeast and its agree with previous work in this area studying fermentation performance is assessed, it can brewery fermentations (Daoud and Searle, 1990; be seen that the correlations are not as strong Corrieu, Trelea and Perret, 2000). as was found with CO2 volume (Table 2). At 12 R2 = 0.9535 10

8

6

4 Wash alcohol strength (% v/v ) 2

0 0510 15 20 25 30 35 40 CO2 volume (L)

Figure 3. Relationship between wash alcohol strength (45 hours) and total CO2 volume. 126 S.Y. Pearson et al.

12

R2 = 0.0039 11

10

9

8

Wash alcohol strength (% v/v ) 7

6 0510 15 20 25 30

CO2 volume (L)

Figure 4. Relationship between CO2 volume (20 hours) and wash alcohol (45 hours).

20 hours, fermentation performance was found produced by a yeast during fermentation and to correlate to some degree with peak flow its fermentation performance, as measured by rate. However, at 45 hours no correlation was alcohol production and gravity loss. However, in apparent. This is probably due to the fact that the contrast, peak CO2 flow rate could not be used peak flow rate will have a greater influence over to accurately predict fermentation performance the volume of CO2 produced in the earlier stages over the course of a complete fermentation. of fermentation, i.e. the 20 hour sample point is It appears, therefore, that monitoring of CO2 closest to when the peak flow rate occurs. production is a useful, and relatively simple, tool for assessing yeast fermentation performance Table 2. Correlation coefficients of relationships in the laboratory under conditions simulating between peak CO2 flow rate and fermentation distillery fermentations. performance at 20 and 45 hours (n = 16).

Wash Specific Gravity CO2 alcohol gravity lost volume References (% v/v) (°) (°) (L) Corrieu, G., Trelea, I.C. and Perret, B. (2000). 20 Hours 0.717 -0.353 0.731 0.608 On-line Estimation and Prediction of Density and Ethanol Evolution in the Brewery. 45 Hours 0.268 -0.028 0.295 0.488 MBAA Technical Quarterly 37: 173-181 Daoud, I.S. and Searle, B.A. (1990). On-line Monitoring of Brewery Fermentation by Measurement of CO2 Evolution Rate. Conclusions Journal of the Institute of Brewing 96: 297-302. It was found that there was a highly significant correlation between the volume of CO2 Towards improving distilling yeast 127 Chapter 18 Towards improved distilling yeast: effect of wort gravity and pitching rate on fermentation performance

J. W. Walker, S. Y. Pearson, T. A. Bringhurst and J. M. Brosnan The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK

Introduction Data collected during this study provides information regarding the fermentation In recent years, yeast manufacturers, academics performance of standard commercial distilling and distilling industry representatives have yeast strains under different conditions to discussed the possibility of selecting new yeast provide a benchmark against which potential strains for use in the Scotch whisky production new distilling yeast strains can be measured. process. Scotch Whisky Industry members have developed a ‘wish list’ for selection of future distilling yeasts where the ability to efficiently Materials and methods ferment high gravity wort is rated as one of the key characters. Malt distillery wort with an original gravity (OG) Due to changes taking place in the distilling of 1085º IOB was diluted to provide standard industry where a reduction in yeast pitching rate gravity wort of 1060º (15º Plato) and high and increase in wort original gravity is the current gravity wort of 1080º (20º Plato). Sub-samples trend, research was carried out to determine the of wort (350 mL) were inoculated with yeast and effect of different pitching rates and wort gravities fermentations were temperature-controlled over on distilling yeast fermentation performance. a range of 19-33ºC based on a typical commercial distillery temperature profile (Figure 1).

35

30

25

Temperature (°C) 20

15 024487296 Fermentation time (h) Figure 1. Fermentation temperature profile. 128 J.W. Walker et al.

Performance of distilling yeast at different pitching rate (0.4% (w/v)) fermented more rapidly pitching rates and original gravities than lower pitching rate fermentations. This observation is demonstrated by a rapid decrease in Fermentation performance of Kerry M-type wash specific gravity (Figure 2) and corresponding yeast (compressed) was assessed using three increase in alcohol strength (Figure 3). yeast pitching rates: 0.1% and 0.2% (w/v) (g High gravity fermentations produced per 100 mL) selected to represent the lower significantly more alcohol than corresponding inoculation rates used commercially and 0.4% standard gravity fermentations and showed more (w/v) to represent the pitching rate used in small- pronounced differences in specific gravity and scale laboratory fermentations. All fermentation alcohol strength profiles than the standard gravity experiments were performed in duplicate and fermentations. These observed differences may be separate fermentations were carried out for each influenced by a number of factors. In the high sample point. gravity fermentations, more substrate is available Traditional fermentation performance for conversion to alcohol and more substrate parameters such as wash specific gravity and has to be converted to achieve comparable alcohol strength were determined daily over a final gravities. The slightly higher final gravities 4-day period and compared with on-line carbon observed for the 1080º fermentations may also dioxide production profiles. Wash specific gravity be attributed to higher levels of residual non- was measured using a Paar DMA 5000 density fermentable material present in the wash at the meter. Alcohol strength was determined by end of fermentation. Also, in fermentations with distillation followed by density measurement on lower yeast pitching rates more sugar is converted a Paar DMA 5000 density meter. to yeast cell biomass rather than production of ethanol, therefore spirit yield may be sacrificed at the expense of increased yeast growth (Walker, Performance of distilling yeast at different 1998; Campbell, 2003). original gravities Yeast cells are subject to different stress factors (Walker, 1998) such as physical stress (e.g. Three commercially available distilling yeast osmotic shock), chemical stress (e.g. ethanol and strains (compressed), Mauri Pinnacle, Kerry other metabolite toxicity) and biological stress M-type and Kerry MX, were pitched at 0.4%w/v (e.g. competition from other organisms). In the and fermented at both standard (1060º) and high current study, where a combination of high gravity gravity (1080º) wort. Yeast viability was greater wort and decreased pitching rate are used, the than 95% in each case. Fermentation performance high gravity wort provides a greater substrate was monitored using continuous carbon dioxide concentration and increased osmotic pressure production with an ADM 2000 (Agilent) flow which may stress the yeast. Also, as fermentation meter and on-line data acquisition (Pearson et progresses more alcohol is produced with the al., 2008). high gravity wort than the standard gravity wort which can increase the chemical stress on the yeast cells. Additionally, lowering the yeast Results and discussion pitching rate provides a further stress factor which allows micro-organisms present in the non-sterile Performance of distilling yeast at different wort the opportunity to compete with yeast for pitching rates and original gravities: specific available nutrients. In this study, lower pitching gravity and alcohol strength rate fermentations demonstrated a marked decrease in wash pH and a perceived increase Traditional fermentation performance data in bacterial contamination with a corresponding confirmed that wort inoculated at a higher decrease in yeast viability (data not shown). Towards improving distilling yeast 129

1060 (0.1%) 1060 (0.2%) 1060 (0.4%) 1080 1080 (0.1%) 1080 (0.2%) 1080 (0.4%) 1070 1060 1050 1040 1030 1020 Specific gravity (º ) 1010 1000 990 01234 Fermentation time (days) Figure 2. Specific gravity profiles from Kerry M-type yeast at different wort original gravities (1060º and 1080º) and pitching rates (0.1, 0.2 and 0.4% (w/v)).

1060 (0.1%) 1060 (0.2%) 1060 (0.4%) 1080 (0.4%) 12 1080 (0.1%) 1080 (0.2%)

10

8

6

4 Alcohol strength (%v/v ) 2

0 0 1 234 Fermentation time (days)

Figure 3. Alcohol strength profiles from Kerry M-type yeast at different wort original gravities (1060º and 1080º) and pitching rates (0.1, 0.2 and 0.4% (w/v)).

Performance of distilling yeast at different pronounced effect on the CO2 production profile pitching rates and original gravities: carbon during fermentation with the high gravity wort dioxide production profiles than during standard gravity fermentation.

Traditional fermentation performance data Performance of distilling yeast at different was supported by carbon dioxide production original gravities profiles (Figures 4 and 5) which confirmed that the higher pitching rate fermentations not only Carbon dioxide production profiles highlighting fermented more rapidly than the lower pitching differences in fermentation performance of three rate fermentations but also peaked earlier and commercial distilling yeast strains at different produced larger volumes of carbon dioxide (and original gravities are illustrated in Figures 6 alcohol). and 7. As can be seen from Figure 5, decreasing Figure 6 shows that differences were observed the pitching rate (on a laboratory scale) from between the three commercial yeast strains 0.4% (w/v) to 0.1% or 0.2% (w/v) has a more fermented at standard gravity. Mauri Pinnacle 130 J.W. Walker et al.

Flow rate - 0.4% Flow rate - 0.2% Flow rate - 0.1% Total Volume - 0.4% Total Volume - 0.2% Total Volume - 0.1% 40 40

30 30

20 20 volume (L) 2 flow rate (mL/min) 2 10 10 Total CO CO

0 0 0510 15 20 25 30 35 40 45 Fermentation time (h)

Figure 4. CO2 flow rate and total CO2 production from Kerry M-type yeast: OG 1060° at three different pitching rates (0.1, 0.2 and 0.4% (w/v)).

Flow rate - 0.4% Flow rate - 0.2% Flow rate - 0.1% Total Volume - 0.4% Total Volume - 0.2% Total Volume - 0.1% 40 40

30 30

20 20 volume (L) 2 flow rate (mL/min) 2 10 10 Total CO CO

0 0 0510 15 20 25 30 35 40 45 Fermentation time (h)

Figure 5. CO2 flow rate and total CO2 production from Kerry M-type yeast: OG 1080° at three different pitching rates (0.1, 0.2 and 0.4% (w/v)). appears to have a slightly shorter lag phase and Mauri Pinnacle performs better than the other ferments more rapidly than either Kerry M-type two yeast strains at the higher gravity. or MX yeast which peak slightly later. Kerry MX From these fermentation experiments it is yeast demonstrates the highest CO2 flow rate evident that there are qualitative differences (mL/min) at standard gravity, followed by Mauri in the fermentation profiles of the commercial Pinnacle then Kerry M-type yeast. distilling yeasts tested. All three yeast strains A different pattern emerges in Figure 7 where fermented the standard and high gravity wort at fermentation performance at high gravity is different rates with Mauri Pinnacle fermenting compared. Again, Mauri Pinnacle appears to faster at high gravity than the other two yeast have a shorter lag phase and ferments more strains. rapidly than Kerry M-Type and MX yeast. In While it was quite difficult to distinguish this case, Mauri Pinnacle also demonstrates the between the ultimate performance of the highest CO2 flow rate (mL/min), followed by yeasts studied using wort at normal Scotch Kerry MX then M-Type yeast, suggesting that Towards improving distilling yeast 131

MX 1060º Mauri 1060º M-Type 1060º 50

40

30

20 flow rate (mL/min ) 2

CO 10

0 0510 15 20 25 30 35 40 Fermentation time (h)

Figure 6. CO2 production by 3 distilling yeast strains (Mauri Pinnacle, Kerry M-type and Kerry MX) at standard wort gravity (OG 1060°).

Mauri 1080º MX 1080º M-Type 1080º 50

40

30

20 flow rate (ml/min) 2

CO 10

0 0510 15 20 25 30 35 40 Fermentation time (h)

Figure 7. CO2 production by 3 distilling yeast strains (Mauri Pinnacle, Kerry M-type and Kerry MX) at high wort gravity (OG 1080°). whisky distillery original gravity (1060°), (e.g. at high wort gravity) they complement each the increased stresses induced at the higher other and are all considered to be acceptable original gravity (1080°) provided a more high quality Scotch whisky distilling yeasts. challenging environment for comparing the Within the Scotch whisky distilling industry, relative performance of different yeasts. This there are different applications for different yeast provides us with a useful tool in our search for strains depending on whether they are required alternative yeasts in the context of Scotch whisky for malt whisky fermentation or for grain distillery production. production. Also, Scotch whisky fermentations vary markedly in length of fermentation where ‘short fermentations’ (e.g. 48 hours) may Conclusions require yeast with the ability to ferment rapidly to ‘long fermentations’ (approximately 100 Although the commercial distilling yeasts studied hours) where perhaps slower fermenting yeast have different properties and perform differently may be more desirable. Other factors such as 132 J.W. Walker et al. substrate utilisation, metabolite production and References alteration in congener and flavour profiles must be taken into consideration when assessing Campbell, I. (2003). Yeast and fermentation. yeast in our quest to find suitable new distilling In: Whisky: Technology, Production and yeast strains in the future. The work which has Marketing. Handbook of alcoholic Beverages been described has shown that by providing a Series, Edited by Russell, I., Academic Press, stressed environment, it is possible to gain a London, pp.117-152 clearer differentiation of the relative performance Pearson, S.Y., Walker, J.W., Bringhurst, T.A. of potential new distilling yeasts for use in the and Brosnan, J.M. (2008). The use of production of Scotch whisky. CO2 evolution monitoring as an indicator This study demonstrates that a combination of yeast fermentation performance. In: of traditional and recent technology can provide Distilled Spirits: Production, Technology valuable information regarding the behaviour and Innovation. Edited by J.H. Bryce, J.R. of yeast strains under different fermentation Piggott, and G.G. Stewart, Nottingham conditions and that this information can be University Press, Nottingham, pp. 123-126 used to assess fermentation performance Walker, G.M. (1998). Yeast Physiology and of established distilling yeast and provide a Biotechnology. John Wiley and Sons, ‘baseline’ against which future distilling yeast U.K. strains may be assessed. Opportunities for improved yeast supplies in whisky and related spirits production 133 Chapter 19 Opportunities for improved yeast supplies in whisky and related spirits production

R. Munro Fermentis, Division of S.I. Lesaffre, 59703 Marcq-en-Baroeul Cedex, France

Current yeast management practices Bought in from a yeast manufacturer used for whisky production Yeast is made in dedicated manufacturing Propagated or grown on site facilities under optimal aerobic conditions for yeast growth. Yeast is generated under less than optimal conditions for biomass production. The risk of • Non specialist yeast strain used for baking infection from other yeast strains and bacteria and distilling (e.g. India). threaten both spirit quality and yield. • Specialty yeast manufactured for the purpose of spirit production (e.g. Scotland, India, • A yeast strain propagated on site is considered Japan, South Africa). to be key to the character of the brand. • Commercial yeast bought in and multiplied Direct pitching to the fermenting vessel of at the Distillery (bubbing). non specialist yeast reduces risks associated with propagation procedures at the distillery Bubbing is not true propagation as it tends to be but may not be optimal in terms of alcohol anaerobic in part, leading to alcohol production production, and stress tolerance of the distilling as well as yeast . Maximum increase likely to be process leading to a potential loss of alcohol x8 in nine hours. As bacteria can grow much yield. Specialty yeast strains are selected to give faster, risk of contamination is very high even in specific advantages to the distiller, i.e. enhanced a dedicated yeast plant. Low cell counts risk loss congener production, resistance to stress caused of alcohol due to contamination of the wash. by the process. Commercial yeast plus bubbing is less of a risk as all propagation stages are removed. This reduces yeast volume purchased but the risk of Brewer’s Yeast contamination and loss of yield is still a concern. Dedicated vessels and plant are required for the Brewer’s ale yeast historically used for whisky operation. manufacture in Scotland and Japan (up to 134 R. Munro

30%), the supply has now almost ceased in the Compressed yeast United Kingdom due to changes in the brewing industry. Compressed (25-28% solids): common in Scotland and India. • Recycled brewer’s yeast used as part replacement (30 to 50%) of Distiller’s normal Positives yeast. • New, dry brewer’s yeast. – Baking yeast strains readily available. – Speciality strains available in some areas. It is said to enhance the performance of whisky fermentations. The exact method of action Negatives is unclear but may be based on the addition of yeast both from fermenting and also as a – Manual handling of 25 kg sacks. yeast nutrient to the distillery yeast. Variable – Short shelf life ( 2-3 weeks). performance and lack of supply has drastically – Cooled distribution needed. limited its application. New viable dry yeast – Yeast quality decreases on storage. enables the distiller to have the advantages of – Requires refrigeration. consistency and is leading to a revival of this – Re-slurry stage common prior to . practice in Japan and Scotland. Dry yeast

Yeast formats available Dry (95% solids): Common in Scotland, Japan, Thailand, USA, Canada, Finland, Australia Cream yeast Positives Cream (17-23% solids): this is in common use in Scotland and in some parts of the USA. – High solids content. – Long shelf life (2 years). Positives – Ambient storage and distribution possible. – No bacterial deterioration on storage. – Automation of pitching. No manual – No designated plant required to pitch yeast. handling. – Specialty Distillers yeast strains. – High viability. – Full certificate of analysis available on – Specialty strains available. delivery.

Negatives Negatives

– Requires capital for receipt tanks ( – Higher cost of manufacture. £20,000). – Yeast viability lower – Requires refrigerated storage. – Yeast quality decreases on storage. – Short shelf life (2-3 weeks). Improvements to yeast management – Locations of distillery important as distribution costs are high. There are good reasons to consider improving – Additional receipt plant needs CIP and yeast management Table 1. maintenance Opportunities for improved yeast supplies in whisky and related spirits production 135

Table 1. Current yeast management practices and potential improvements.

Current yeast management Potential improvements

Own strain of yeast propagated on site. 1) Review method & frequency of propagation. 2) Toll Manufacture of strain. 3) Change to a similar commercial strain. Yeast bought in ready to use. a) Non specialty strain 1) Change to a specialty yeast strain. b) Specialty strain 1) Review format of yeast ( cream, compressed or dry).

Multiple types of spirit produced at same site. Review production procedures to separate the streams. Use specialty yeast strains for each product (more than 1 format).

Reasons to review current practice: Scotland - today

• Changes to plant /automation. Yeast format under review: • Handling issues and storage (yeast format). • Improvement of yield (yeast strain / • Manual handling regulations. application). • De-manning of distillery operations. • Improvement to quality/flavour (yeast strain • Automation of yeast addition. and process). • Quality and yield requirements. • Need to show energy savings/reduced The need to reduce production costs is obvious. operating costs. Yeast automation/reduced handling is an area that can save time and manpower. Repairs and A review of yeast addition and costs has been upgrades to refrigeration plant can also prompt a driven by the need to comply with manual review of the yeast handling procedures. To use handling regulations together with reduction cream yeast may need capital expenditure and in manufacturing costs by automation and not be justified on smaller sites. Any improvement de-manning. Consistent yeast performance to of yield is welcome and a change to a specialty optimise spirit yield and quality is required as yeast may provide this. Flavour profiles can also is the need to reduce energy consumption in be altered /enhanced by changing yeast strain to the process. a specialty one. New specialty yeast strains Example: yeast developments in Scotland Dry distiller’s yeast • 1940s yeast was usually generated at distillery. Opportunity for specialty dry yeast: • 1950s yeast was outsourced to yeast manufacturers and Brewer’s yeast. • Manual handling regulations. • 1960s all distilleries use very similar strains • Remote distilleries - transport costs. of yeast. • Smaller, centralised distilleries due to the • 1990s cream yeast introduced. cost of cream reception equipment. • 2000s dry yeast introduced to remote and • Spirit quality must remain true to type for smaller distilleries. whisky produced. 136 R. Munro

As cream yeast is potentially requiring capital for Laboratory conditions investment and then requires dedicated transport for delivery, there is an opportunity for a dry • Fermentations carried out at high gravity and yeast for whisky. It is considered key that the standard gravity (1060o and 1080o). spirit quality is not drastically changed to allow • Wort from a malt distillery was collected and for product matching during both distilling and frozen. maturation. The Distiller and Master Blender • Fresh compressed yeast used as a control. must be confident about the spirit being matured • Yeast pitched at same rate. for future blending operations. • Dry yeast re-slurried before pitching. • Fermentation temperature controlled to a standard profile. Manufacturing stages • Fermentations was assessed after 96 hours.

All Fermentis yeasts are single strain, and not Assessment genetically modified or amplified. Fermentation is totally aerobic and optimal for the generation • Fermentation profiles: The rates of of biomass in the shortest time. Yeast is first fermentation and maximum alcohol yield washed and centrifuged to remove excess water. were measured. Rotary driers are used to produce a paste. To • Assessment of spirit by sensory panel (20% preserve maximum viability, the yeast is carefully v/v dilution): the nose of the spirit was dried using the latest fluid bed technology to assessed by a trained assessment panel. minimise damage to the yeast. This process has • GC analysis of major congener groups. been developed with other specialty yeasts and GC values for the major congeners were baking yeasts to maintain high levels of cell checked. recovery. Not all dry yeasts are dried in the same way; Fermentis use methods appropriate to the final application of the yeast. Fermentation

As the trials were carried out in small flasks, Laboratory assessment it was important that a standard temperature profile was adopted by means of a water bath. • Safwhisky M-1 dry yeast assessed at ICBD and The profiles were typical of that expected in a SWRI against commercial strains available in distillery wash. The gravity profile was very Scotland. similar for both the M-1 and the control samples. • Trials with distillery groups. The alcohol strength for the different yeasts was • Objective to show the effects on congener similar. There were slight differences in the profiles and spirit yields. congener profile (Table 2), but no significant ones. All fell within the expected range. To show that M-1 is indeed suitable for whisky The sensory analysis indicated that the production a series of laboratory assessments congener perception was very similar to that have been made against yeasts commonly used expected of new made spirit and is typical of that for whisky production. The primary objective seen in a new spirit (Figure 1). Slight process was to introduce acceptable dry yeast to the adjustments to the distillation parameters will be whisky distilling market. able to control such variations depending on the desired. character sought. Opportunities for improved yeast supplies in whisky and related spirits production 137

Table 2. New spirit congener analysis (g/100 litre@ absolute alcohol).

1080o 1060o M-1 Control M-1 Control Acetaldehyde 10.9 10.9 5.5 6.5 Acetal 3.4 4.1 1.3 2.1 Methanol 3.7 3.8 3.7 3.6 N-Porpanol 40.4 34.6 48.4 40.1 Iso-Butanol 52.5 43.1 77.9 57.5 Iso-Amyl Acetate 5.4 3.0 4.5 2.8 Ethyl Acetate 54.4 40.8 37.5 30.5 n-Butanol 0.9 0.5 1.4 0.9 2-Methyl-1-Butanol 54.3 41.0 68.0 46.7 3-Methyl-1-Butanol 127.3 106.1 158.2 121.2 Furfural 0.2 0.2 0.4 0.3 Total Higher alcohols 274.5 224.8 352.3 265.5

Pungent Pungent Clean Clean Phenolic 1.2 Phenolic 1.2 Stale Stale Feinty Feinty 0.8 0.8

0.4 Cereal 0.4 Cereal

0 0 Sulfury Floral Sulfury Floral

Aldehydic (grassy/leafy) Soapy Aldehydic (grassy/leafy) Soapy

Estery (fruity) Sour Sour Estery (fruity) Oily Solventy Oily Solventy

Figure 1. Sensory analysis (M-1 yeast 1060 left, and control yeast 1060 right).

Safwhisky M-1 yeast was comparable in a stock of dry yeast at even large distilleries as performance to compressed yeast for both a safeguard to yeast supply or contamination, fermentation and distillation parameters. thus enabling production to carry on without any New made spirit flavour fell within the range disruption in the event of delivery problems. expected for all major congener groups. The sensory flavour profile is consistent with flavours expected of “M” strain yeasts. Dry yeast

Distillery trials are required to determine Cream yeast optimal methods of pitching. Whisky distilleries are not designed around dry yeast, the yeast Safwhisky M-1 is the only yeast available as both pitching methods need to be optimised given dry and cream yeast. As M-1 is available as both the limitations of both plant and process cream yeast and dry there is the potential to hold conditions required for successful production. 138 R. Munro

The temperature of re-hydration was wide range Yeast is only available to the distillery that depending on the method of pitching and is supplied the yeast culture. Manufacture can only usually dictated by limitations of plant cooling be achieved with pure cultures. A small pilot capacity. Laboratory work shows that generally is made and supplied for laboratory assessment increasing the temperature at re-hydration before the yeast batch is made. Yeast is stable for improves cell recovery from the dry state. 2 years from manufacture in ambient conditions. The re-slurry stage before pitching has the The bacterial quality is assured to manufacturer’s advantage of being able to optimise re-hydration specifications agreed before production of the temperature to maximise cell revival, but it yeast with the distillery. requires a vessel (may be existing if compressed yeast is slurred). Re-slurry pitching can be done at a slightly higher temperature ( 27 to 30 Future opportunities for speciality degrees centigrade) to optimise cell recovery in yeast strains advance of wort running down. Sufficient yeast for several wash backs can be prepared and • Adding value to processes by optimisation of stored during the production day for pitching yeast use. on run down to wash back. Very little change • New product development with existing in the process is involved to use dry yeast in brewing and distilling strains for the global this way. Direct dry pitching is potentially the market. simplest system. Run down parameters need to • High gravity fermentations (1080o). be optimised by trials, but there is no plant to • High Temperature fermentations (34-38 oC). maintain or clean. For direct dry pitching; trials are needed to optimise both cell recovery (and The overall objective is to optimise the distiller’s therefore the amount of yeast required) and the fermentation for spirit yield within the bounds best temperature. This may well be dictated by of the Scotch Whisky Act and customer limitations on cooling at the distillery and may expectations. Existing strains can be further not be optimal for yeast re-hydration. Trials show improved by alteration to the manufacturing that the temperature range is between 20 and 30 process parameters. There are many yeast degrees centigrade. strains available some as dry yeast that could be of interest (e.g. bourbon whisky yeasts for use in Scotland?). Also, finding a yeast that is Toll manufacturing osmo tolerant and capable of high temperature fermentations. • D i s t i l l e r s o w n s t r a i n d r i e d b y a In some distilleries, the optimum alcohol manufacturer. production is almost achieved under the given • A confidentiality agreement. conditions. Further savings can be made by • Strain assessment clone isolation. increasing the stress conditions in the fermenting • Pilot yeast production/acceptance by stage e.g. increasing the temperature or possibly distillery. the gravity. Under these conditions yeast with a • Full scale batch production 1500 kg minimum higher potential will be required. to an agreed specification. Application of novel yeast strains to the scotch whisky fermentation process 139 Chapter 20 Application of novel yeast strains to the Scotch whisky fermentation process

H. B. de Amorim Neto1, S. Y. Pearson2, J. W. Walker2, G. M. Walker1 and J. M. Brosnan2 1School of Contemporary Sciences, University of Abertay, Dundee, UK; 2The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK

Introduction determine whether they provide any benefits over existing yeast strains used by the industry. The small number of yeast strains that are These yeasts are adapted to the industrial process used by the Scotch whisky industry to produce for fuel ethanol production, where the choice alcohol for distillation has remained relatively of yeast strain is fundamental to obtaining high unchanged for many years. Recent changes, such alcohol yields. These strains are very resistant as a decline in availability of brewers’ yeast as a to stress conditions, such as high temperature secondary yeast strain and the development of and high alcohol concentration, similar to those yeast in different formats (e.g. dried and cream found during whisky fermentations. yeasts as alternatives to compressed yeast), have prompted a resurgence of interest in yeast within the distilling industry. As a result of this, Materials and methods there have been a number of initiatives aimed at finding alternative or improved distilling yeast Malt distillery wort with an original gravity strains for the future. of 1085º was diluted with distilled water to Research has been carried out to assess the provide a relatively high gravity (1080º) wort. fermentation performance of established distilling Six yeast strains were assessed in terms of yeast strains, under a range of fermentation their fermentation performance; two distilling conditions, in order to provide the data against yeast controls (M Type and MX; Kerry Bio- which potential new strains may be compared. Science, Menstrie, UK) and four Brazilian fuel Although the main driver for developing new ethanol yeasts (PE-2, CAT-1, VR-1 and BG-1; yeast strains is to maximise conversion efficiency Fermentec, Piracicaba, Brazil). The Brazilian by increasing alcohol yield, maintaining yeasts were isolated from industrial fuel ethanol consistent spirit flavour and quality is also of fermentations using karyotyping techniques and major importance to the whisky industry. evaluated in the laboratory prior to shipping to In this present study, a selection of new Scotland. They were supplied as lyophilised yeasts, specifically Brazilian fuel ethanol yeast cultures in glass ampoules. To reactivate the strains, were assessed for their suitability for yeast strains, the ampoules were opened under use in Scotch whisky fermentation, in order to aseptic conditions and the lyophilised cells were 140 H.B. de Amorim Neto et al. then inoculated into 5 ml MYGP medium (3 g assessed each day, at 24h intervals, over a 4-day malt extract, 3 g yeast extract, 10 g glucose and period. Specific gravity, alcohol strength, pH and 5 g peptone per litre distilled water). yeast viability (methylene blue staining method) All yeasts, including the controls which measurements were carried out in duplicate on had been supplied as compressed yeast, were all samples. propagated prior to testing. A loop of broth was spread onto a plate containing malt extract agar. The plates were then incubated for 48 h at 32°C Spirit quality assessment in order to obtain isolated colonies. Selected colonies were then inoculated into 10 ml MYGP Larger volume fermentations were carried out broth and incubated for 24 h at 32°C. The to allow for the assessment of new make spirit resultant yeast suspension was then inoculated quality using 3 of the Brazilian yeast strains into 490 ml MYGP broth and incubated at 32°C (CAT-1, PE-2 and VR-1) and M Type as a control. for 72 h, with shaking (80 rpm). Two litres of 1080° wort were fermented for 72 After incubation the resultant broth was h, in duplicate, using the temperature profile centrifuged and the yeast recovered was shown in Figure 1. After fermentation was rinsed with sterile, distilled water before being complete, the wash was distilled into low wines centrifuged again. The yeast pellet obtained was (700 ml). These low wines were then further stored at 4°C until use. distilled to give foreshots (5 ml), new make spirit (155 ml) and feints (235 ml) fractions. All distillations were carried out using glass stills Fermentation performance assessment containing copper wool (10 g) placed in the lyne arm. Wort (350 ml) was inoculated with the propagated Sensory analysis of the new make spirit yeast at a pitching rate of 0.5% w/v (approx. cell samples produced was carried out using count of 3 x 107/ml) and carried out in duplicate Quantitative Descriptive Analysis (QDA) after in a temperature-controlled water bath (separate dilution of the samples to nosing strength (20% fermentations were carried out for each sample (v/v)) with water. The duplicate spirits produced point). The fermentation temperature varied by each yeast strain were combined prior to over a range of 19-33ºC, based on a typical analysis. The samples were assessed by trained commercial Scotch whisky distillery profile panellists in relation to the following attributes; (Figure 1). Fermentation performance was pungent, peaty (phenolic), feinty, cereal, floral,

35 33 31 29 27 25

Temperature (°C ) 23 21 19 17 01020304050607080 Time (hours)

Figure 1. Fermentation temperature profile. Application of novel yeast strains to the scotch whisky fermentation process 141 green/grassy, fruity/estery, solventy, oily, sour, efficiently ferment maltose. The Brazilian yeasts soapy, sulphury, meaty, stale and clean. were originally isolated from fermentation substrates containing high concentrations of sucrose. However, the predominant sugar Results and discussion present in malt whisky wort is maltose and it is likely that some of the strains tested are not able Fermentation characteristics to metabolise high concentrations of maltose efficiently. It was observed that there were large differences It was found that the control yeasts between the yeast strains in terms of their outperformed the test strains in terms of alcohol fermentation performance (Figures 2-5). In terms production over the first 48 h of fermentation. of specific gravity, CAT-1 gave a comparable At 72 and 96 h, however, CAT-1 has produced fermentation performance to the two control an equivalent amount of alcohol to the controls yeasts, M Type and MX (Figure 2), i.e. the specific (Figure 3). The other Brazilian yeast strains gave gravity decreased rapidly and the wort was fully much lower yields of alcohol compared to the fermented out. In contrast, BG-1 performed controls, BG-1 in particular. Again, this may poorly. This may be as a result of microbial be due to a reduced ability of these strains to contamination slowing the fermentation rate, efficiently ferment the high levels of maltose and or may be due to an inability of this strain to maltotriose present in malt whisky wort.

1090

1070

1050 M Type PE-2 VR-1 1030 CAT-1 MX BG-1 Specific Gravity (° ) 1010

990 01234 Fermentation Time (days) Figure 2. Specific gravity profiles of high gravity fermentations.

12

10

M Type 8 PE-2 VR-1 6 CAT-1 MX BG-1 4

Alcohol Strength (% v/v) 2

0 01234 Fermentation Time (days) Figure 3. Alcohol production during high gravity fermentation. 142 H.B. de Amorim Neto et al.

Figure 4 shows the pH profiles during level of ethanol rise over the course of the fermentation. The profiles for CAT-1, PE-2, fermentation. Interestingly CAT-1, and to a lesser VR-1 and the controls were all similar to those extent PE-2, maintained a high level of viability normally expected for whisky distilling yeasts. into the later stages of fermentation, indicating The wort had an initial pH of 5.6, decreasing a greater degree of stress tolerance. Sustained to around 4.0 and then rising again to attain a viability is an important attribute of yeast in value of 4.5. The poorest performing yeast, BG-1, terms of fuel ethanol production and may also showed a different trend, falling to pH 3.3 by the have potential significance in terms of Scotch third day. The low final pH of this fermentation whisky production. is indicative of the presence of high levels of bacterial contamination. After propagation, all yeasts exhibited a Spirit quality high level of viability and this was maintained over the first 24 h of fermentation (Figure 5). It was found that the Brazilian yeast strains all By day 3, the viability of all the yeasts, with produced spirit of a character that was consistent the exception of CAT-1 and PE-2 had declined with Scotch whisky new make. Despite its sharply. This drop would be expected in malt relatively poor fermentation performance, PE-2 whisky fermentations, as the temperature and produced a spirit with a flavour profile that

6.0

5.5

5.0 M Type PE-2 VR-1 pH 4.5 CAT-1 MX 4.0 BG-1

3.5

3.0 01234 Fermentation Time (days)

Figure 4. Profile of pH during high gravity fermentation.

100

80

60 M Type PE-2 VR-1 CAT-1 40 MX Yeast Viability (% ) BG-1

20

0 01234 Fermentation Time (days) Figure 5. Yeast viability during high gravity fermentation. Application of novel yeast strains to the scotch whisky fermentation process 143 closely matched the control (‘M’ Type) (Figure 6). whisky fermentations. The spirits produced CAT-1 also gave a similar flavour profile, but was using the PE-1, VR-1 and CAT-1 strains all “cleaner” in character. The spirit produced using had acceptable flavour profiles, exhibiting no VR-1 exhibited relatively high levels of “feinty”, sensory characteristics that were atypical of “cereal”, “sour” and “sulphury” notes, though the Scotch whisky new make. CAT-1 gave a good intensities of these aromas were not out with the fermentation performance, comparable with expected levels for Scotch new make. current Scotch whisky distilling yeasts. This, coupled with acceptable spirit quality and high Conclusions viability, indicates that this strain has potential for use as a new, and even possibly improved, It is likely that the poor performance of some yeast for Scotch whisky production. However, of the novel yeast strains was related to their validation of its performance in industrial scale preference for sucrose as a substrate, rather fermentations is necessary before it can be than maltose which characterises Scotch recommended for use by distillers.

Pungent 2.0 Clean Peaty 1.5 Stale Feinty 1.0

Meaty Cereal 0.5

0.0 Sulfury Floral

Soapy Green/grassy

Sour Fruity/estery Oily Solventy

M Type PE-2 VR-1 CAT-1

Figure 6. QDA analysis of new make spirits. 144 H.B. de Amorim Neto et al. Speciality yeast and optimal yeast management practices 145 Chapter 21 Speciality yeast and optimal yeast management practices for maximising fermentation efficiency in the fuel ethanol industry

C. L’Helgoualc’h Fermentis, Division of S.I. Lesaffre, 59703 Marcq-en-Baroeul Cedex, France

Introduction

Compared to the 150 years of experience in yeast learn that fermentation was still considered, by production of the Lesaffre group, the involvement recognized specialists, as the “black box” i.e. the of Fermentis in the fuel ethanol industry is quite least understood segment of the alcohol process. recent. Three years ago, Fermentis launched In only three years, fermentation and yeast have the first generation of yeast tolerant to high been positioned at the forefront of technological ethanol concentration, high sugar concentration advancement with the development of new and high temperature, in the United States of technologies. America (USA). Most common practices developed by the Ever since, Fermentis rapidly gained fuel ethanol industry are: knowledge thanks to visiting and developing collaboration with ethanol plants and institutes • Very High Gravity Process to become the market leader in the USA, Europe • Simultaneous saccharification and and Asia. The purpose of this presentation is to fermentation share the expertise we developed on fuel ethanol • Fermentation from non-cooked granular with the potable ethanol industry. Based on the starch. practical experience we have met in fuel ethanol facilities on various substrates and on innovative Very high gravity process processes, this paper explains how specialty yeast and optimal yeast management practices Processes such as Very High Gravity have can influence the fermentation efficiency. become the most common practice for producers using corn in batch facilities. This process Choice of yeast utilizes highly concentrated mash of greater than 30% (w/v) solids. This technology is now a Technological advancements and common practice in the USA on corn substrates opportunities for new yeast where plants with 92% efficiency compared to theoretical yield are achieved. When Fermentis first attended a biofuels With initial sugar concentrations of 380g/ conference in 2002, we were very surprised to hl, alcohol concentrations in fermenters rise to 146 C. L'Helgoualc'h over 18% (v/v) on corn substrate. This technology higher temperatures than standard existing yeast has now extended to Europe on wheat and rye to limit bacterial infection, reduce cooling costs based substrates despite viscosity concerns and and increase enzymes efficiency in cases of molasses in Europe, Asia and Australia. Ethanol simultaneous saccharification and fermentation. concentration is expected to reach 14% (v/v). The yeast we developed is able to multiply two times faster than our reference and has proved to Simultaneous saccharification and be still active at 40°C. fermentation The industry is also seeking strains able to resist high ethanol concentrations. Ethanol Simultaneous saccharification and fermentation concentrations of over 21% (v/v) with no have become the technology of choice to reach residual sugar were obtained in the laboratory high ethanol concentration in both batch and of the University of Minnesota with our yeast cascade processes. Dextrose is generated directly at 32°C. When we tried to combine high sugar in the fermenter by enzymes and is removed concentration and high temperature, the capacity as fast as it is formed by the yeast to produce of the yeast to produce high ethanol concentration ethanol. was reduced: 19% (v/v) at 36°C, 16% (v/v) at 40°C. The major concern is the increase of Fermentation from non cooked granular residual sugar that remained unfermented in starch the mash, 0.36% (w/v) at 36°C, 2.7% (w/v) at 40°C. This proves that there is still potential One of the most promising concepts is fermentation for improvement in the selection and the from non cooked granular starch. Cold hydrolysis development of new strains, more resistant to will eliminate the liquefaction and cooking high temperature. processes and will reduce energy consumption For batch producers who work at very high in the plant, along with capital cleaning and gravity, the production of glycerol represents a maintenance costs associated with the operations. significant source of loss. It is clearly understood Both Novozymes and Genencor companies have that yeast produces glycerol in response of osmotic now produced enzymes capable of converting stress, mostly due to the high concentrations of starch directly to ethanol in combination with sugar in the media. In our laboratories, we have yeast. The engineering company Broin & identified strains able to produce less glycerol Associates Inc has developed a new process and more ethanol than the current commercial currently piloted in three ethanol plants in the yeast. Our results, based on trials made on cane USA. molasses, clearly proved that the new identified strain can produce 10% less glycerol than the best available strain. Efficiency comparison of different yeast Other requirements came from distillers strains producing ethanol for potable use. Distillers are now more and more concerned by the use of Yeast is a micro organism with endless varieties. yeast with lower abilities to produce undesirable Seven thousand strains are available in the congeners because specifications are getting Lesaffre bank, and seven hundred species are stricter for alcohol quality. This can be achieved identified. As a yeast producer, our responsibility with specialty yeast without substantial investment is to select, isolate and produce pure yeast with in expensive distillation equipment. Five different specific properties that bring technical and/or strains were tested on C-Starch substrate in order economic advantages to the industry. The first to select the yeast able to produce a lower quantity requirement we received from the industry was of acetaldehyde. Variations from 1 to 6 have been to identify strains that could grow faster and at reported in the same fermentation conditions. Speciality yeast and optimal yeast management practices 147

Quality comparison of commercial dry yeast These results indicated that not only the strain is important but also the way it is Considering yeast as a living organism, it is produced. Producers can prepare the yeast difficult to precisely compare one strain from during multiplication in order to make it more another, and one commercial yeast strain from resistant to identified stress. This test has now another. Fermentis therefore rapidly identified been integrated in our production facilities the necessity to develop a laboratory procedure in addition to existing quality control tests in order to compare yeast efficiency. The first to evaluate the yeast efficiency and organize goal of this test was to have fast and direct positive release of our products. It is also of observation of the fermentation efficiency. Then great value for our Research and Development we precisely defined parameters to guarantee department to evaluate the performances of our minimal variation and a good reproducibility on yeast compared to competitor’s products. the culture media with the working method. The Bacteria and wild yeast contamination is challenge was to have a rapid test with adequate a major cause of reduction in ethanol yield sensitivity that was representative of industrial during fermentation. Among the bacterial performances. contaminants, the lactic acid bacteria are the This test takes into consideration: most troublesome because of their tolerance to high temperature and low pH and their ability • The fermentation rate to grow fast. Active dry yeasts generally have • Ethanol concentration after 48 hours low levels of contaminant. However, an analysis • Final ethanol concentration. of similar strains produced in dry form and collected in Europe, Asia and in North America The experimentation material and sample has shown a big variability of their bacterial preparation was defined as follows: contamination. Bacterial loads vary from 2E+2 to 6E+8 depending on the origin. The efficiency Synthetic media containing all suitable of the yeast has been reported to be decreased nutrients to ensure optimum growth accordingly. and fermentation performance is used, including sucrose as sugar source, nitrogen and phosphorus, minerals and vitamins. Nutritional availability and environmental factors Fermentation was carried out in flask with strict temperature control, a constant The second part of this paper focuses on stirring and controlled pH. recommendations for good nutrition and optimum environmental factors. The fermentation follow up was made by measuring the flasks weight loss (carbon dioxide production) and ethanol production. The value Managing available nutrition can be substantiated by ethanol measured by HPLC. Using this test, Fermentis compared six With all substrates considered for the production commercial yeasts collected in the USA, Europe of fuel ethanol, assimilable nitrogen has been and China and with similar genetic profiles identified to be the main limiting nutrient for their performance in alcohol production. in fermentation, especially at high sugar Significant differences were noticed which have concentration. Most of the fuel ethanol been confirmed to be representative of industrial producers know that adequate concentrations of performance. assimilable nitrogen must be present for a normal 148 C. L'Helgoualc'h fermentation but have ignored the possibility of Importance of adopting good yeast increasing productivity through supply of excess management practices assimilable nitrogen. A minimum of 200 ppm of nitrogen has been estimated to stimulate the Through this last part of the paper, I will focus on maximum rate of ethanol production. recommendations for the best yeast management Different sources of assimilable nitrogen are practices in fuel ethanol plants. available on the market and are economically feasible. For the industrial production of bio- fuel, urea is known as the most economically Rehydration attractive. But its usage may lead to the production of carcinogenic ethyl carbamate, which is In plants that use active dry yeast, the difficulties unacceptable in alcoholic beverages. Urea is not of propagating yeast in the plant is eliminated toxic for the yeast even at high concentration. as dry yeast can be rehydrated and used directly It could therefore be supplemented at the early in the fermenters. A rehydration step however stage of the process (during rehydration or is crucial to the functioning of the yeast. When propagation of the yeast). The maximum rate of managed in suitable conditions it enables the dissolved solids utilization was observed with 16 yeast to quickly regain membrane integrity and mM of urea. However, the industry is presently reduce the lag phase during fermentation. For using a quantity two times lower. optimum performances, the yeast should be Ammonium sulphate is also frequently used rehydrated in ten times its weight of water or by the industry at quantity varying from 8 to 40 wort at 95°F – 105°F during 25–30 minutes. mM. Yet, the use of ammonium sulphate can Higher temperatures will have damaging effect cause scaling problems when molasses is used as on the yeast. Lower temperatures will decrease substrate. Mono or di-ammonium phosphate is the amount of non-viable cells. frequently used by facilities running on molasses substrate as it is a common source of nitrogen Propagation and phosphorus. The optimum concentration to use varies from 8 to 40 mM depending on the Once rehydrated, the yeast can be pitched substrate and the process. straight into the fermenters. Many ethanol producers still consider this practice to reduce to a minimum the risk of bacteria and wild yeast Managing pH contamination and to ensure consistent and optimum yield. Other producers prefer to run a Initial pH also has a great impact on fermentation short propagation to reduce the amount of dry efficiency. Many ethanol producers work at yeast to be pitched in the fermenter. This requires low pH to inhibit the growth of lactobacilli, state of the art facilities and strict control over without clearly understanding the impact on contamination. However, laboratory trials and fermentation efficiency. With 20% dry solid, an experiences carried out in production facilities initial pH of 4.0 on wheat substrate leads to a confirm that propagation time, also called maximum ethanol concentration below 9% (v/v). conditioning, should be limited to a short period When the initial pH is raised to 5.5 on the same (maximum 10 hours). media, 13% v/v can be achieved. The gap is more impressive when high gravity processes are considered. Furthermore, yeast was identified Re-inoculation as tolerant to higher lactic acid and acetic acid concentrations when it is fermented at high pH Many ethanol plants are willing to propagate (above 4.5). the yeast continuously to reduce yeast purchase Speciality yeast and optimal yeast management practices 149 costs. It is being more and more admitted by One argument we generally faced with the industry that continuous yeast propagation plants running continuous propagation is that leads to an increase in bacterial numbers and contaminant yeasts are actually more efficient organic acid levels that become stressful for the than initial yeast because they are acclimatised yeast. However, very few studies have analysed to the substrate. We have therefore compared the the change occurring in the yeast population growing abilities and fermentation performance during a given period, and the best frequency of the initial yeast and the majority yeast to restart with a new batch of yeast has still not population appeared after fifteen days. been optimised by the industry. It confirms that contaminant yeasts have a We are currently conducting trials with better ability to grow on the selected substrate, several plants using different substrates to but have lower ability to produce ethanol, with evaluate what could be the optimum time for lower fermentation rates. the plant to change the yeast. The first results we obtained come from an ethanol plant in France using sugar beet juice as substrate. Summary The first day, fermenters were inoculated with a high amount of yeast. Eight days later the Yeast has a complex role in fermentation yeast population was analysed and genetic finger that is understood more and more today by printing concluded that the population was still the industry. It is a living organism and it is a very similar. One week after, the same analysis question of balance and compromise. revealed that one third of the yeast population Today it is commonly accepted that the best was replaced by two contaminant yeasts and fermentation efficiency can be obtained by: variants. After four weeks, the yeast population had nothing to compare • Using a selected strain adapted to individual with the initial inoculums. Further trials have fuel ethanol producer requirements revealed that genetic mutation and cell ageing appear in the yeast after 13 to 30 cell divisions. • By controlling the environment, to reduce This is equivalent to 3-4 days. yeast stresses These results give comfort and lead to the following conclusion: • By adopting experienced yeast management practices. In state of the art facilities exerting strict control over contamination, continuous propagation could be run for 3-4 days before a new batch of freshly conditioned yeast is re-inoculated.

This period should never exceed one week in order to avoid development of less efficient contaminant yeast and variant. 150 C. L'Helgoualc'h Spirit flavour release under mouth conditions 151 Chapter 22 Spirit flavour release under mouth conditions

M. Maçatelli, A. Paterson and J.R. Piggott Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK

Introduction 1995); for Cognac, this minimum is of two years (Cantagrel, Lurton, Vidal and Galy, 1995); for Flavour perception of beverages is strictly red wine, a period of 12-18 months is typical dependent on the release of volatiles to the (Boulton, 1995). vapour phase. The headspace concentration Many changes occur during maturation. of these compounds mainly depends on the Spirit congeners are concentrated as water temperature, pressure, chemical properties of and ethanol are lost by evaporation, several the liquid medium and volatile concentration materials are extracted from the oak cask, and and solubility. However, interactions between many reactions happen between and within aroma compounds, ethanol concentration and components of the distillate and wood-derived behaviour of non-volatiles may influence the compounds. Organic acids, for example, are solubility of the flavour compounds (Da Porto accumulated through the maturation time as a and Nicoli, 2002; Escalona, Homman-Ludiye, result of ethanol oxidation and breakdown of Piggott and Paterson, 2001; Escalona, Piggott, cask hemicelluloses (Nishimura and Matsuyama, Conner and Paterson 1999; Conner, Birkmyre, 1989). Considering the sensory aspects, the non- Paterson and Piggott, 1998a; Conner, Paterson, volatile wood compounds influence the release Piggott and Whateley, 1998b; Conner, Paterson of aroma compounds (Conner et al., 1994a,b), and Piggott, 1999a; Conner, Paterson, Birkmyre and this is one probable mechanism to attenuate and Piggott 1999b; Kappatos, Gordon and Birch, the perception of unpleasant flavours in matured 1996; Conner, Paterson and Piggott, 1994a,b; spirits. Piggott, Conner, Clyne and Paterson, 1992). Ethyl esters are important molecules in An important stage for determining the flavour alcoholic beverages (Salo, Nykänen and profile of alcoholic beverages is the maturation Soumalainen, 1972). Their aroma descriptors process. New make spirits are assessed as vary from pleasant notes (fruity), to soap- pungent, sour, soapy and harsh, but the sensory like characteristics, as the aliphatic chain descriptors related to aged spirits are smooth, length increases (Ribereau-Gayon, 1978). vanilla, spicy, woody and sweet (Piggott, Ethyl decanoate, for example, has been used Conner, Paterson and Clyne, 1993). Hence, for as a standard molecule for compounds with Scotch whisky, a minimum maturation period similar polarity in studies with immature odours of three years is required (Piggott and Conner, (Piggott et al., 1992; Conner et al., 1999a). 152 M. Maçatelli et al.

The presence of a polar head and an aliphatic spirits were lower than those of aqueous ethanol hydrocarbon chain characterize ethyl esters as solutions. Furthermore, by differential scanning amphiphilic compounds. Depending on the calorimetry (DSC) (Nishimura et al., 1983; Koga solution conditions, this chemical property and Yoshizumi, 1977), analysis of the aged enables micelle formation (Tanford, 1980; Conner whisky thermograms could detect some evidence et al., 1994a,b), which seems to be crucial to of strong interaction between water and ethanol, liquid-gas partition. and non-volatile extracts of oak wood, containing As a co-solvent with water in the matrix of inorganic salts and compounds such as alcoholic beverages, ethanol alters the solubility and , largely contributed to these solvent of non-polar compounds, which are related to interactions. In addition, multidimensional the flavour. The release of aroma components nuclear magnetic resonance (NMR) spectroscopy decreases when ethanol is added to an aqueous showed that the strength of H bonds was directly solution (Lubbers, Charpentier, Feuillat and proportional to the content of acids and phenols Voilley, 1994). However, the molecular structure coming from wood cask. of ethanol-water solutions may be modified by The flavour of alcoholic beverages then reflects changes in the proportions of the mixture. A a strict relationship with the matrix solution consequence of this is a change in the pattern of behaviour. Using static headspace studies, ethyl volatile release in beverages with different ethanol esters showed a log linear decrease in their strengths, and also changes in the aroma profile activity coefficient above 17% ethanol (v/v), with when diluting spirits for sensory assessment. volatility suppression inversely proportional to Changes in the physico-chemical properties ester acid chain length. This can be explained of solutions depending on the molar fraction of by the incorporation of these volatiles into the ethanol and water have been reported, but there ethanol clusters, which reduces their availability is still a great degree of uncertainty about the to be released from the solution (Conner et al., mechanism involved. One hypothesis (D’Angelo, 1998a). The same behaviour was observed in Onori and Santucci, 1994) considers that, at studies with aldehydes. However, higher alcohol ethanol mole fractions below 0.05-0.06 (about activity coefficients did not fit this pattern, which 15-17% v/v), the alcohol forms a monodispersed seems to be related not only to their incorporation aqueous solution. However, above this range into agglomerates, but also to the influence of ethanol molecules cluster to reduce hydrophobic the alcohol hydroxyl group in favour of higher hydration and a continuing increase in ethanol solubility (Escalona et al., 1999). proportion leads water to lose its hydrogen However, most studies reflect nosing bonded network completely, establishing an assessment of spirits, as static systems are generally ethanolic solution. In the water-rich region, used. In order to analyse flavour release during strengthened hydrogen bonds were found to be consumption, the effects of human physiology and formed between water molecules surrounding the mouth environment need to be considered. the ethanol alkyl group, whilst in the ethanol-rich Respiration rate, pH, mouth temperature, saliva region stronger hydrogen bonds were established volume and composition are parameters which between the water hydrogen and the hydroxyl have been found to influence volatile behaviour, oxygen of ethanol aggregates (Mizuno, Miyashita with changes in binding equilibria, volatile release and Shindo, 1995). kinetics and agglomerate size and stability (van In fact, some volatile and non-volatile Ruth and Buhr, 2003; van Ruth and Roozen, 2000; compounds may assist in the formation of Harrison, 1998; Harrison and Hills, 1997). ethanol-water clusters in matured spirits. Results The need to establish the relationship between from small angle X-ray scattering suggested the oral release of volatile compounds and changes in existence of a greater degree of non-uniform flavour perception is evident. The effects of non- structure (Aishima, Matsushita and Nishimura, volatile components on ethanol aggregation, with 1992) and the dielectric constants of the matured implications for aroma behaviour, should also be Spirit flavour release under mouth conditions 153 investigated. This work was, therefore, an initial ethyl esters were measured at different ethanol attempt to determine whether volatile behaviour concentrations. Then, the effect of organic acid during tasting was similar to that observed addition on ethyl decanoate headspace was under equilibrium conditions. A simulated determined. mouth model developed by Margomenou, Model solutions were prepared with 10 ml Birkmyre, Piggott and Paterson (2000) was used of ethanol-water at different ethanol strengths to determine the effects of different alcoholic (5, 10, 17, 25, 30, 35 and 40% (v/v)), plus 2 strengths and organic acids on the volatility of ml of artificial saliva and 0.1 µl of ethyl ester. ethyl esters in model solutions. In the second set of experiments, the same amount of ethyl decanoate, artificial saliva and water-ethanol solution at 23% (v/v) were Material and methods analysed with the addition of organic acid at a series of concentrations (at least 4) as required. Materials The solutions were placed in a Buchner flask. A Chrompack Tenax trap (Chrompack UK Ltd, Ethanol (HPLC grade, Rathburn Ltd, Walkerburn, London, E14 9TN) was inserted into the top of UK) and distilled water, filtered using a the flask using a special adapter. The flask was Millipore-Q system (Millipore UK Ltd, Watford, sealed with parafilm and placed in a water bath UK), were used to prepare model solutions. at 37°C for 30 min. Volatiles were then trapped Ethyl butyrate, ethyl hexanoate, ethyl octanoate, onto the Tenax by a flow of hydrated air at 40 and ethyl decanoate were all at least 98% pure cm3 min-1 for 7 min. Traps were then washed (Fluka Chemical Co and Sigma-Aldrich Co Ltd., into a capped bottle with 2 ml of diethyl ether Gillingham, Dorset, UK). 3,4 Dimethyl phenol containing 9.3 µg ml-1 3,4-dimethylphenol. The (99% pure, Sigma-Aldrich) was used as internal solvent was evaporated with nitrogen to 10 µl standard. Diethyl ether was especially dry and and 1 µl of this solution was injected into a Carlo filtered (BDH Laboratory Supplies, Poole, Dorset, Erba gas chromatograph with a split-splitless UK). Organic acids were all purer than 98% injector and flame ionization detector (FID). A (BDH, Sigma-Aldrich). Artificial saliva consisted 0.25 mm x 30 m CP WAX 52 CB (df = 0.25 of sodium hydrogen carbonate, dipotassium µm) column (Chrompack) was used with helium hydrogen orthophosphate 3-hydrate, sodium carrier gas at 1.8 ml min-1. Injection temperature was 40°C, held for 5 min, thereafter increasing chloride, sodium azide,,calcium chloride 2-hydrate, mucin (Sigma Chemicals, Poole, at 10°C min-1 to 240°C, which temperature Dorset BH12 4XA) and a-amylase (Sigma) was finally held for 3 min. The injector and (van Ruth, Roozen, Nahon, Cozijnsen and detector temperatures were 230°C and 250°C, Posthumus, 1996). respectively. All analyses were carried out in duplicate. Response factors for ethyl esters were determined by injection of a solution of equal Methods concentrations of the ester, under the same chromatographic conditions, and calculation Headspace concentrations of ethyl esters were of the relative response factors from peak areas. determined in the Strathclyde Simulated Mouth (Margomenou et al., 2000) by gas chromatography (FID-GC), operating under previously determined Results optimal conditions. Conditions were chosen so that relative peak areas closely matched peak areas Headspace concentrations of ethyl esters (in of headspace samples taken from the consumer’s terms of the corrected peak areas relative to the nose. First, the headspace concentrations of internal standard) were plotted against ethanol 154 M. Maçatelli et al. concentration typical of a number of alcoholic ethyl octanoate) showed maxima of headspace beverages (Figure 1). The analysed concentrations concentrations at intermediate ethanol content. were 5% (beer), 10% (table wine), 17% (fortified The effect of increasing chain length of organic wine), 25% (approximately sensory assessments acids was investigated using a homologous of distilled beverages) and 40% (v/v) ethanol series of monocarboxylic acids. The relative (typical bottle strength of distilled drinks), and concentration of ethyl decanoate over 23% additional determinations were made at 30% and ethanol (v/v) was plotted against the concentration 35% (v/v) ethanol. of organic acid. The concentration of acid Under simulated mouth condition, certain required to decrease ethyl decanoate headspace ester (ethyl acetate, ethyl butyrate and ethyl concentration to 50% of the original was dodecanoate) concentrations in the headspace calculated from the regression (SD 1.12 to 0.39). showed an inverse relationship with ethanol This showed a log-linear correlation (adjusted content. One of the esters, ethyl decanoate, R2=0.95) with the number of carbon atoms in showed no relationship between headspace the acid (Figure 2). The relationship held up to concentration and ethanol content, and finally, decanoic acid; longer chain acids were not used the other two esters (ethyl hexanoate and as Conner et al. (1999a) reported that dodecanoic,

1.0

0.8

0.6

0.4 Ester headspace 0.2 (relative corrected peak area)

0.0 0510 17 25 30 35 40 Ethanol concentration (% v/v) Figure 1. The effect of increasing ethanol concentration on model solutions of the headspace concentration of ethyl esters. ( ) ethyl acetate, ( ) ethyl butyrate, ( ) ethyl hexanoate, ( ) ethyl octanoate, ( ) ethyl decanoate, ( ) ethyl dodecanoate.

0.5

0.0

-0.5

-1.0

-1.5

-2.0 reduction in ester headspace

-2.5 0510 15 Log molar acid concentration required for 50% Number of carbons in acid Figure 2. The logarithm of the organic acid concentration required to halve the activity of ethyl decanoate standard solution against the number of carbons in the acid. Adjusted R2 = 0.95. Spirit flavour release under mouth conditions 155 tetradecanoic and hexadecanoic acids, even at headspace concentration increased until 25% saturation concentrations, had no effect on ethyl ethanol (v/v) and then had an abrupt decrease decanoate activity. with the following ethanol strengths. Ethyl decanoate, a standard molecule to represent some immature characteristics in Discussion spirits (Piggott et al., 1992), had its headspace concentration unchanged by different ethanol Over all the analyses, it was possible to verify solutions. It indicates that the attenuation of that headspace concentrations of ethyl esters unpleasant flavours during consumption is were broadly changed under simulated mouth not obtained by the isolated effect of ethanol environment, depending on the ethanol strength strength. and organic acids addition. In general terms, this Considering the solutions with organic acids, outcome was in accordance with reported results static headspace measurements have shown in the literature, when static conditions were that these compounds appear to influence the applied in the ester headspace analysis (Conner aggregation of ethanol in aqueous solution et al., 1998a; Conner et al., 1999a). (Conner et al., 1999a). This was demonstrated Compared with the static study (Conner et al., by marked reductions in the headspace 1998a), the first set of experiments showed that concentrations of ethyl decanoate. The same the influence of increasing ethanol molar fraction effect was observed here using simulated mouth on ester headspace concentration changed more conditions (Figure 2). During maturation in oak or less intensively under mouth conditions casks, there are increases in both volatile and depending on the ethyl esters considered. non-volatile (fixed) acidity (Liebman and Scherl, In the case of the most volatile esters and the 1949). Increase in acetic acid concentration least volatile (ethyl acetate, ethyl butyrate and from oxidation of ethanol and also extraction ethyl dodecanoate, respectively), a decrease in from the wood composes the largest addition their release over the whole range of ethanol (Reazin, 1983). Further increase in concentration concentration was shown. However, with the of organic acids in distillates must occur as a static system analysis, Conner et al. (1998a) found result of ethanol and water evaporation (Reid significant reductions of activity coefficients and Ward, 1994). Extractions from stave wood only from 17% (v/v) ethanol. They related also yield octanoic and decanoic acids, from the fact with the formation of ethanol clusters breakdown of wood lipids, and the dicarboxylic above that specific ethanol concentration, acids succinic, adipic and methylsuccinic acids which would increase ethyl ester solubility and (Nykänen and Suomalainen, 1983). Gallic, decrease headspace concentration. Therefore, vanillic, syringic and ellagic acids are also this indicates that release of ethyl esters was extracted from the lignin and components influenced by the medium existing in the of wood (Piggott et al., 1993). The dicarboxylic mouth, possibly by changes in the composition and other acids have not been studied yet, but of the surface layer of ethanol-water solution. Conner et al. (1999b) recorded that oxalic acid Additional changes in the structure of the solution had no effect. might also alter the incorporation of hydrophobic compounds into agglomerates. Viscosity is another parameter that, altered by the addition of Conclusions artificial saliva, could lead to a possible decrease in esters volatility. So, even more evident was It is clear that the use of a simulated mouth the effect of the new analysis environment on dynamic headspace apparatus is necessary the release of esters with intermediate volatility for reliable studies of release of spirit flavour (ethyl hexanoate and ethyl octanoate) as their compounds during consumption. As changes 156 M. Maçatelli et al. in headspace of ethyl esters also occurred under Conner, J.M., Paterson, A., Piggott, J.R. and simulated mouth conditions, they are more likely Whateley, T.L. (1998b). Contributions of to actually happen when alcoholic drinks are distillate components to disperse phase consumed. Then, the volatiles’ concentration structures in model spirit solutions. Journal reaching the retro-nasal cavity and consequently of Agricultural and Food Chemistry 46: the consumer’s perception of flavour will 1292-1296 be affected. The increase of organic acid Conner, J.M., Paterson, A. and Piggott, J.R. concentration observed during maturation could (1999a). Release of distillate flavour be one of the mechanisms for immature flavour compounds in Scotch malt whisky. Journal attenuation, and this effect was independent of of the Science of Food and Agriculture 79: equilibration time, and mouth physiology and 1015-1020 environment. Conner, J.M., Paterson, A., Birkmyre, L. and Piggott, J.R. (1999b). Role of organic acids in maturation of distilled spirits in oak casks. References Journal of the Institute of Brewing 105: 287-291 Aishima, T., Matsushita, K. and Nishimura, K. D’Angelo, M., Onori, G. and Santucci, A. (1992). Measurements of brandy ageing (1994). Self-Association of Monohydric using O17 NMR and small angle X-ray Alcohols in Water - Compressibility and scaterring. In: Elaboration et Connaissance Infrared-Absorption Measurements. Journal des Spiritueux, Edited by Cantagrel, R., of Chemical Physics 100: 3107-3113 Lavoisier-Tec & Doc, Paris, France, pp. Da Porto, C. and Nicoli, M.C. (2002). A 473-478 study of the physico-chemical behavior of Boulton, R. (1995). Red Wines. In: Fermented diacetyl in hydroalcoholic solution with Beverage Production, Edited by Lea A.G.H. and without added catechin and wood and Piggott J.R., Blackie Academic and extract. Lebensmittel-Wissenschaft Und- Professional, London, pp. 208-228 Technologie-Food Science and Technology Cantagrel, R., Lurton, L., Vidal, J.P. and Galy, B. 35: 466-471 (1995). From wine to Cognac. In: Fermented Escalona, H., Homman-Ludiye, H., Piggott, J.R. Beverage Production, Edited by Lea A.G.H. and Paterson, A. (2001). Effect of potassium and Piggott J.R., Blackie Academic and bitartrate, (+)-catechin and wood extracts on Professional, London, pp. 208-228 the volatility of ethyl hexanaote and octanal Conner, J.M., Paterson, A. and Piggott, J.R. in ethanol/water solutions. Lebensmittel- (1994a). Interactions between Ethyl-Esters Wissenschaft Und-Technologie-Food and Aroma Compounds in Model Spirit Science and Technology 34: 76-80 Solutions. Journal of Agricultural and Food Escalona, H., Piggott, J.R., Conner, J.M. and Chemistry 42: 2231-2234 Paterson, A. (1999). Effect of ethanol Conner, J.M., Paterson, A. and Piggott, J.R. strength on the volatility of higher alcohols (1994b) Agglomeration of Ethyl-Esters in and aldehydes. Italian Journal of Food Model Spirit Solutions and Malt Whiskeys. Science 11: 241-248 Journal of the Science of Food and Agriculture Harrison, M. (1998). Effect of breathing and 66: 45-53 saliva flow on flavor release from liquid Conner, J.M., Birkmyre, L., Paterson, A. foods. Journal of Agricultural and Food and Piggott, J.R. (1998a). Headspace Chemistry 46: 2727-2735 concentrations of ethyl esters at different Harrison, M. and Hills, B.P. (1997). Effects of alcoholic strengths. Journal of the Science air flow-rate on flavour release from liquid of Food and Agriculture 77: 121-126 emulsions in the mouth. International Spirit flavour release under mouth conditions 157

Journal of Food Science and Technology A. (1992). The Influence of Nonvolatile 32: 1-9 Constituents on the Extraction of Ethyl-Esters Kappatos, T., Gordon, M.H. and Birch, G.G. from Brandies. Journal of the Science of (1996). Solution Properties of Vanillin and Food and Agriculture 59: 477-482 Diacetyl in Aqueous-Ethanol Solutions. Piggott, J.R., Conner, J.M., Paterson, A. and Food Chemistry 57: 275-282 Clyne, J. (1993). Effects on Scotch whisky Koga, K. and Yoshizumi, H. (1977). Differential composition and flavour in oak casks with scanning calorimetry (DSC) studies on the varying histories. International Journal structures of water-ethanol mixtures and of Food Science and Technology 28: aged whiskey. Journal of Food Science 42: 303-318 1213-1217 Piggott, J.R. and Conner, J.M. (1995). Whiskies. Liebman, A.J. and Scherl, B. (1949). Changes In: Fermented Beverage Production, Edited in whiskey while maturing. Industrial and by Lea, A.G.H. and Piggott, J.R., Blackie Engineering Chemistry 41: 534-543 Academic and Professional, London, pp. Lubbers, S., Charpentier, C., Feuillat, M. and 247-274 Voilley, A. (1994). Influence of yeast walls Reazin, G.H. (1983). Chemical analysis of on the behaviour of aroma compounds in a whisky maturation. In: Flavour of Distilled model wine. American Journal of Enology Beverages: Origin and Development, Edited and Viticulture 45:29-33 by Piggott, J.R., Ellis Horwood, Chichester, Margomenou, L., Birkmyre, L., Piggott, J.R. UK, pp. 225-240 and Paterson, A. (2000). Optimisation and Reid, K.G. and Ward, A. (1994). Evaporation validation of the “Strathclyde simulated losses from traditional and racked mouth” for beverage flavour research. warehouses. In: Proceedings of the Fourth Journal of the Institute of Brewing 106: Aviemore Conference on Malting, Brewing 101-105 and Distilling, Edited by Campbell, I., Mizuno, K., Miyashita, Y. and Shindo, Y. (1995). Institute of Brewing, London, pp. 319-322 NMR and FT-IR Studies of Hydrogen Bonds Ribereau-Gayon, P. (1978). Wine flavour. In: in Ethanol-Water Mixtures. Journal of Flavour of Foods and Beverages, Edited Physical Chemistry 99:3225-3228 by Charalambous, G. and Inglett, G.E., Nishimura, K., Ohnishi, M., Masuda, M., Koga, Academic Press, New York, p. 335 K. and Matsuyama, R. (1983). Reactions of Salo, P., Nykänen, L. and Soumalainen, H. wood components during maturation. In: (1972). Odour thresholds and relative Flavour of Distilled Beverages: Origin and intensities of volatiles aroma components Development, Edited by Piggott, J.R., Ellis in an artificial beverage imitating whisky. Horwood, Chichester, UK, pp. 225-240 Journal of Food Sciences 37: 394-398 Nishimura, K. and Matsuyama, R. (1989). Tanford, C. (1980). The Hydrophobic Effect: Maturation and maturation chemistry. In: Formation of Micelles and Biological The Science and Technology of Whiskies, Membranes, J. Wiley and Sons, New York Edited by Piggott, J.R., Sharp, R., and van Ruth, S.M. and Roozen, J.P. (2000). Influence Duncan, R.E.B., Longman, Harlow, UK, of mastication and saliva on aroma release pp. 235-263 in a model mouth system. Food Chemistry Nykänen, L. and Suomalainen, H. (1983). 71: 339-345 Dicarboxylic acids. In: Aroma of Beer, Wine van Ruth, S.M. and Buhr, K. (2003). Influence of and Distilled Alcoholic Beverages, Edited by saliva on temporal flavour release from red Nykänen, L., and Suomalainen, H., Reidel bell peppers determined by proton transfer Publishing, Dordrecht, pp. 118-130 reaction-mass spectrometry. European Food Piggott, J.R., Conner, J.M., Clyne, J. and Paterson, Research and Technology 216: 220-223 158 M. Maçatelli et al. van Ruth, S.M., Roozen, J.P., Nahon, D.F., Cozijnsen, J.L. and Posthumus, M.A. (1996). Flavour release from rehydrated French beans (Phaseolus vulgaris) influenced by composition and volume of artificial saliva. Zeitschrift fur Lebensmittel Untersuching und Forschung 203: 1-6. From sugar to rum - the technology of rum making 159 Chapter 23 From sugar to rum - the technology of rum making

V. Persad-Doodnath Angostura Limited, Trinidad, West Indies

Introduction refining, which is the black treacle-like substance which remains after sugar crystallization, is The definition of Rum as outlined in the used as the raw material for the fermentation Caribbean Community Standard for Rum, process. The yeast, which is an integral part of (Revised March 2003) is as follows: the fermentation process, also imparts various flavours to the rum. There are four major Rum is a spirit drink – processes involved in rum making: fermentation, distillation ageing and blending. • obtained exclusively by alcoholic fermentation and distillation of sugar cane molasses, sugar cane syrups, sugar cane juices or cane sugar Angostura Limited produced during the processing of sugar cane, Angostura Limited has been in the business of • distilled at an alcohol content of less than rum making through its production company, 96.0 percent alcohol by volume at 20 degrees Trinidad Distillers Limited, since 1947. The Celsius, company ferments, distills, ages, blends and • produced in such a way that the product bottles alcoholic beverages, mainly rum, in has the organoleptic characteristics derived Laventille, Trinidad, West Indies. It started with a from the natural volatile elements contained French designed still made by Savalle capable of in the above raw materials or formed during producing 5400 litres of alcohol/day and today the fermentation or distillation process of the has a production capacity of over 65,000 litres named raw materials. of alcohol/day. It has 6 ageing warehouses with a total capacity of 80,000 casks. The bulk storage Steps in rum making facilities feature a Tank Farm on the Distillery compound with a capacity of 5.0 million litres The basic principle of rum making is quite and a dock side facility in Chaguaramas with simple. The raw materials required are molasses, 3.8 million litres of tankage. Angostura bottles water and yeast. The juice of the mature sugar over 600, 000 cases of rum/year. Exports are cane plant is extracted and refined as sugar, mainly to the US, UK, Europe and the rest of while molasses, the final by-product of sugar the Caribbean. 160 V. Persad-Doodnath

Molasses A typical Molasses composition is given in Table 1 and the comparison of composition of beet and Molasses is the most widely used raw material sugar cane molasses can be found in Table 2. for rum production. Its composition varies and depends on the quality of the cane, Table1. Typical molasses composition. composition of soil, climatic conditions, methods of harvesting cane, manufacturing Brix (total solids) 75-85 deg Total reducing sugars 45-55 % process for sugar and handling and storage Unfermentable sugars 4-5% of molasses. The composition of molasses is Fermentable sugars 40-50% referred to as the “quality of the molasses” and F/N ratio 0.9-1.6 is what contributes to quality and intensity of the Dry Sludge 5% or below rum flavour (Shete, 2000). pH 4.5-5.2 Titratable Volatile acidity 5000ppm or below The contents on “as is basis”are as follows: A. Water 15-25% (w/w) B. Total Solids 75-85% (w/w) Table 2. A comparison of the composition of sugar cane and beet molasses. Solids can be further divided into: Source of A. Organic solids: 65-70% (w/w) Molasses Glucose Fructose Sucrose Other B. Inorganic solids: 10-15% (w/w) Cane 7.0 8.5 35.5 4-5% A. Organic solids are: Beet 0 1.0 49.9 <1% 1. Sugars: Fermentable: 35-55% (w/w) Sucrose: 27-43% (w/w) The composition of molasses can affect Fructose: 5-7% (w/w) fermentation in the following way: Glucose: 3-5% (w/w) Unfermentable: • F/N ratio – this is the ratio of fermentable Pentose: 2-7% matter to the unfermentable matter. If <1, it Galactose: 1-2% affects the yeast activity. A ratio of > 1.2 is 2. Non sugar substances: desirable. Nitrogenous: Proteins • Ash content/settleable sludge/calcium Carbohydrate ploymers: Starch, gums content - if it is too high, it slows down Organic Acids: 500-25,000 ppm fermentation and indicates potential problems :acetic, butyric, of precipitation and scaling when the wash proprionic, goes to the still. valeric • Caramel content of molasses – high content Waxes reduces activity of the yeast. Colouring substances • Organic volatile acids – if more than 5000 Vitamins ppm, they retard yeast activity resulting in an Polyelectrolytes, biocides incomplete conversion of sugars. • PH/bacterial contamination – bacteria B. Inorganic solids: compete with the yeast for consumption Cations: Ca, K, Na, Mg, Si, Fe, Mn. of sugar. A pH of 4.5 is desirable for yeast ------Anions: SO4 , Cl , PO4 , CO3 , NO3 , O . fermentation. From sugar to rum - the technology of rum making 161

The composition of molasses varies based on its • Resistant to bacterial contamination – acetic geographical location e.g. acid or lactic acid bacteria inhibit fermentation as they compete with the yeast for growth and • South American molasses are generally high metabolism. in calcium content and volatile acidity while Caribbean molasses is in the normal range. Both have normal to high fermentable sugars. Fermentation • African molasses are high in fermentable sugars and have low calcium and sludge Fermentation is a living process. The molasses is content and normal volatile acidity. diluted with water to reduce the sugar content • South East Asian molasses are high in to approximately 15% (w/v) and a pure yeast fermentable sugars but have a high volatile culture is added to the mixture. The yeast cells acidity. Sludge content is normal. convert the available sucrose to ethyl alcohol

(C2H5OH) and carbon dioxide (CO2) with the release of heat energy. This mixture is called the Yeast (Saccharomyces cerevisiae) used “live wash”. Fermentation takes approximately in fermentation 30 hours to be completed during which time the yeast in the mixture uses up the available sugar Louis Pasteur in the mid 1800s discovered that in the molasses. The liquid left at the end of there was actually a single cell microscopic the fermentation process which is called “dead organism responsible for the conversion wash” is used for distillation. of fermentable barley malt into alcohol, Extracts of recent work done by Virender carbon dioxide and flavour compounds. Sheorain of National Rums of Jamaica Ltd This microorganism was named yeast – (Sheorain, 2004) suggest that fermentation can Saccharomyces cerevisiae (a single cell fungus). be classified based on the type of organism, In the biochemistry of fermentation, Gay Lussac chemical nature of the substrate and product, suggested the following biochemical pathway: requirement of oxygen and the medium used. Liquid state fermentation can be of three Sugar + Yeast = Alcohol + Carbon Dioxide types:

Saccharomyces yeast normally converts 88-90% • Batch – straight feeding or incremental of fermentable sugars into ethanol and carbon feeding, dioxide. The balance of the sugar is mainly • Semi continuous (batch fed), utilized in the fermentation process for cell • Continuous. growth (about 3-5%), glycerol formation (3-5%), and by products that are responsible for flavour The comparison of the three types of liquid state and aroma. fermentation can be seen in Table 3. Angostura Characteristics of an ideal yeast culture in the Limited uses batch fermentation. production of rum: During fermentation, a number of constituents called congeners are also manufactured. These • Fast fermenting – too much or too little sugar congeners, which are regarded as the rum flavours, will affect the rate of fermentation. are the major constituents of the heavy type rums. • Thermotolerant - the optimum temperature They are necessary when blending because they for most yeast cultures is 30-32oC. give flavour and character to the rum. • Alcohol tolerant – yeast will die at 13% (v/v) Congeners formed during fermentation: alcohol. Normal concentrations are 6-9% (v/v). • Aldehydes – by oxidation of alcohol, 162 V. Persad-Doodnath

Table 3. A comparison of batch, semi continuous and continuous fermentations.

Type Advantages Disadvantages

Batch 1. Easy to manipulate 1. Too many fermenters Yeast Conc: 1x108 2. Better control 2. High cost Retention time = 30-45 hours 3. Easy to calculate yields 3. Too many pumps and pipes Semi continuous 1. Less cumbersome 1. Difficult to deal with contamination Yeast Conc: 3x108 2. Lower costs 2. Less accuracy in calculation of yields Retention time = 16-24 hours 3. Possible to recycle yeast Continuous 1. Gives high yields, productivity 1. Very difficult to deal with Yeast Conc: 8x108 and efficiencies 2. Not suitable for smaller plants Retention time= 28-32 hours 2. Low costs and flexibility

• Acids – by oxidation of aldehydes, and the desirable ones that add significantly to • Fusel Oil – by conversion of free amino acids the taste and aroma of the raw rum are retained in water to higher alcohols, in the heavy type rum that is distilled from the • Esters – by esterification of alcohols and first distillation column. acids, Production of rum spirit utilises a number • Volatile sulphur compounds – by combination of distillation technologies ranging from simple of sulphate and sulphur with amino acids. single column atmospheric distillation to multi column multipressure distillation. Products can Contamination during fermentation should be classified as Raw or Rectified Spirit, Neutral be avoided at all costs. If pasteurisation is not Spirit or Rum Spirit (light or heavy rum). Typical possible, the use of sanitisers, antibacterial congeneric content of these types can be found agents or growth conditions must be monitored. in Table 4 (Godbole and Deshpande, 2000). PH and temperature control are critical. Losses Based on energy conservation, distillation are due to unutilised sugars and contamination schemes are broadly divided into two Other factors which affect fermentation: categories:

• Yeast culture (type, cell count, viability), • Atmospheric distillation, • Temperature of fermenter, • Energy efficient schemes. • Aeration, • Sugar concentration, In atmospheric distillation, separation of • Alcohol concentration. components is carried out at close to atmospheric pressure and is very energy intensive (Deshpande, 2000). This type of distillation is used at Angostura Distillation and the following problems are experienced:

After fermentation, the fermented wash is fed • Thermal degradation and by-product to the still. Distillation is the process of boiling formation at higher temperature in the mash the “dead wash” and condensing its vapour column, to recover/collect the alcohol that has been • Hard scale formation due to calcium sulphate produced. The distillation process is done precipitation in the presence of alcohol at mainly to separate and concentrate the alcohol temperatures above 90 degrees Celsius in the component of the liquid mixture. During this mash column, process, the undesirable congeners are removed • Higher steam consumption, From sugar to rum - the technology of rum making 163

Table 4. Typical Congener content of distillates.

Congeners Extra Neutral Mg/litre Heavy Rum Light Rum Neutral Spirit Spirit

Acetaldehyde 20-150 100-200 1-2 0-1 Ethyl acetate 50-500 10-50 1-2 0-1 N-propanol 10000-20000 10-50 1-5 0 Diacetyl 50-100 0 0 1-2 Methanol 20-40 20-40 1-2 0 Acetone 0 0 0 0 Higher alcohols 40-4000 50-1000 5 0 Furfural 0 0 0 0 Acetal 100-500 20-50 0 0 Acids 5-10 0 1-2 0

• Higher production downtime due to scaling The distilled product of the mash column or or fouling, “wash stripper” is referred to as “heavy rum”. • Less ease of operation in the absence of For production of light and neutral spirits, the proper instrumentation and controls. remaining columns are used. In the hydroselector, the raw alcohol is extracted from low and high There are many energy saving options available boiling impurities. The raw alcohol is preheated, which include: first by the preheating condenser of the rectifying column and then by a plate type heat exchanger • Indirect heat pumping, which uses the heat of effluent water, which • Split distillation or multiple effect has to be drained. From the bottom, the hot distillation, and watery alcohol is pumped through the • Multi pressure distillation. copper reactor, in order to remove mercaptans. These are impurities derived from fermentation,

carrying H2S-groups and giving an off flavour to Still design used at Angostura the alcohol. Inside the copper reactor, there is a large volume of copper particles which react

The columns use perforated sieve trays. Columns with the H2S-groups and remove the off flavour. are interconnected in sequence to be economical Over time, the shiny copper becomes dirty and in heating energy. The throughput capacity is black with copper sulfide. As the copper sulfide 60,000 lpd product alcohol calculated at 100 hinders the copper from reacting, it is necessary %. Consumption at Design Capacity is: to clean the copper periodically with diluted hydrochloric or sulfuric acid (1 to 3 % (v/v)). Steam ~ 7 200 kg The rectifying column has got 70 trays in total. Cooling water circulation ~ 250 m3/h The alcohol and water mixture flows into the rectifying column. Water goes downward in the The plant uses 5 columns: column and the alcohol rises inside to the top of the column. The alcohol can be concentrated • Mash column (wash stripper), to approximately 96,6 % alcohol by volume. • Hydroselection column, At side outlets, fusel oil is extracted and goes to • Rectifying column (70 trays), the fusel oil separator from where it leaves the • Recovery column (45 trays), process. Feints, the other higher alcohols, are • Final polishing column. extracted some trays higher then fusel oil. The 164 V. Persad-Doodnath low boiling aldehydes, acetals and esters go to the selective diffusion though the pores of the oak top where they are concentrated and sent as heads barrel and the chemical interaction between the to the recovery column. Good quality alcohol will congeners. Rum ageing has been practiced since be extracted some trays below the top. the sixteen hundreds when seafarers found that The recovery column concentrates all as rum was carried on long journeys in wooden impurities to a high concentration, so that they barrels it improved even more and also became can be separated as concentrated heads and darker in colour. Today, all the ageing of rum is feints and fusel oil. The recovery column has done in oak wood barrels that were previously used got 45 trays in total. The recovered alcohol for the ageing of cognac, wine or predominantly, contains acetals, aldehydes, higher alcohols bourbon. After the barrels are used once for the and all impurities which have originally been ageing of other liquours, they are employed in the in the raw alcohol, plus those which have been rum industry as “Once used” barrels. Regulations formed within the process. If the concentration that require producers of bourbon to use barrels of aldehyde is very high, then a reaction of only once assure a steady supply of barrels for the aldehyde and alcohol takes place. The reaction is rum industry. Oak wood barrels are used because a chemical condensation. Acetal (diethylacetal) they do not contribute offensive odours or tastes to is formed. This reaction usually takes place at the rum during the ageing process. the top part of the recovery column. The alcohol Oak wood is used for storage because it is a then flows to the top of final polishing column. tight grained wood capable of making leak proof The final polishing column is designed to remove barrels that are ideal for strong liquids. The size practically all light boiling impurities before the of the radial rays of oak wood is what gives the product leaves the plant. The fusel oil separator strength to its barrels and also allows it to meet receives the fusel oil (mainly isoamyl alcohol) the characteristics required for storage containers which forms liquid layers and separates like such as porosity, strength, resilience, workability oil drops from the remaining liquid, after the and lightweight. Oak is a chemically pure wood, mixture becomes diluted with water. All other its major constituents being cellulose, lignin, higher alcohols will not form oil drops. They will tannins and a small amount of lipids (oils, fats stay with the water and alcohol mixture which and waxes). These small amounts of lipids form returns from the bottom of the fusel oil separator oak lactones that have an intense effect on the to the recovery column. rum flavour during the ageing process. The hemi-cellulose releases wood sugars and imparts colour. The lignin produces vanilla Ageing and together with the oak tannins, promotes oxidation products. The char layer releases burnt After distillation, the rum is drawn off into large wood flavours. Oak wood has large porous stainless steel vessels for storage before being grains, and holes in the grain are small enough so barrelled off into forty gallon oak barrels and that water cannot pass through but large enough moved to the warehouse for ageing. Although so that oxygen can. This allows a small amount the ageing process is not fully understood, it is of oxygen to pass through the spirit during the considered to be the most significant aspect of ageing process. This slow aeration has the effect the rum manufacturing process because the rum of mellowing the rum. improves with age. Immediately after distillation, There are three types of reactions occurring the rum, which is a raw clear liquid with a hot simultaneously in the barrel during the ageing harsh taste and an acrid odour still contains small process. They are: amounts of hydrogen sulphide gas formed during the fermentation process. During ageing, many • An extraction of complex wood constituents changes occur as a result of the oxidation and from the wood by the liquid, From sugar to rum - the technology of rum making 165

• Oxidation of components originally present in the corresponding fill spirits to examine the the liquid as well as of the material extracted effect of ageing on the composition of current from the wood, distillates. Samples are analysed for major • Reactions between the various organic volatile congeners, ethyl esters, cask extractives substances present in the liquid that lead to and by gas chromatography-mass spectrometry the formation of new congeners. for a range of compounds that may contribute to rum aroma. Included in the latter analysis are headspace SPME (solid phase micro extraction) Chemical and sensory analysis of analysis for volatile ethyl esters and sulphur ageing rum compounds and direct injection analyses for phenols and a range of other current distillate Due to the lack of knowledge and printed and wood derived congeners (Jack, Connor and literature on the ageing of rum, Angostura Reid, 2005). Limited has commissioned a three year study in Initial Gas Chromatography-Olfactometry conjunction with the Scotch Whisky Research was done for: Institute in Edinburgh, Scotland. Almost one year of ageing has been completed and two • Analysis of current rums, samplings have taken place, one of current • Comparison of current and mature rums, rum and the second after six months of ageing. • Comparison of rums meeting/not meeting The findings so far have concentrated on the mature specifications casked at the same correlation of chemical and sensory analysis time. with a comparison to Scotch whisky, where possible. Findings for analysis of current rums (light and The aim of this study is to assess how heavy): warehouse conditions, the method of storage and handling of casks, and the re-use of casks • Light rums have the lowest level of congeners contribute to the ageing characteristics of rum and the fewest aromas. in the Caribbean, as well as the ageing patterns • Heavy rums have higher concentration of of different types of rums. Fill spirits (ranging in propanol, isoamyl alcohol and some esters strength from 55% to 85% (v/v) alcohol), cask e.g. ethyl hexanoate, and ethyl octanoate. types and warehousing are being controlled to • Both rums contains similar concentrations of examine their influence on the ageing of rums. shorter chain (up to 5 carbon) ethyl esters and The progress of the rums is being followed by acetals – fruity/solventy characteristics. sensory and chemical analyses. Two warehouses • Current rums contain similar concentrations are being assessed, one very old and one very of the sulphur compounds DMDS and DMTS. newly constructed. Both warehouses have DMTS is present at 15 to 100 times the odour similar capacity, insulated roofs and good threshold concentration. ventilation. Temperature and humidity are being monitored over the three year period to compare Findings regarding the comparison of current and the conditions in both warehouses. Each cask mature rums (already aged at Angostura): sample analysed is generally a composite of 12 individual cask samples drawn at random from • Maturation increases the complexity of rums the groups of 24 used in this study. Sensory through the introduction of a large number comparisons use Triangle Tests to determine of aromas, whether differences existed in the mature • Heavy rums appear to behave differently samples for the different fill spirits, cask types from light rums – producing more aromas and warehousing. Chemical analyses include and higher levels of congeners. 166 V. Persad-Doodnath

Maturation increases: 3. The chemical basis of citrus aroma could not be identified with no clear consensus • Cask extractives such as vanillin, cis oak on which aroma is responsible for this lactone and eugenol (woody, vanilla and character. spicy characters), • Phenolic compounds such as guaicol, Findings regarding oxidation during 4-ethylguiacol and 4-proplyguiacol (phenolic maturation: and spicy characters), • Esters such as ethyl acetate, ethyl hexanoate 1. Oxidation of alcohols to aldehydes and and ethyl octanoate (fresh fruit and floral acids. characters). 2. Oxidation of lignin breakdown products extracted from cask wood. Maturation decreases: 3. Removal of sulphur compounds. 4. Oxidation of fatty acids. • Sulphur compounds such as DMDS and DMTS (sulphury character). This has prompted a range of analyses to compare the current rum used for filling to that Findings regarding comparison of rums meeting/ aged for 6 months. Subsequent analyses will not meeting mature specifications when casked be conducted on a yearly basis for three years. at the same time: These analyses include:

• There is no evidence of off flavours in rums • Volatile esters using headspace SPME, not meeting the maturation specification, • Sulphur compounds using headspace • Rum not meeting the specification are SPME, characterized by lower levels of ethyl esters, • Cask extractives using Liquid Chromatogra- vanillin and oak lactones, phy, • Rums not meeting the specifications show • Medium and long chain ethyl esters using lower levels of in cask oxidation, direct injection GC, • No differences are observed in the levels of • Phenolic compounds using direct injection sulphur compounds and guiacols. GC/MS, • Major volatile congeners using direct injection GC, Correlation between chemical and • Possible aroma compounds using GC/MS. sensory analysis

1. A number of chemical groups correlate well Summary of sensory results after six with the sensory panel scores e.g. months of ageing

• DMDS and DMTS with sulphury, After only six months maturation clear differences • Guiacol and its homologues with could be observed between certain rums: phenolic. 1. Fill strength appeared to have a significant 2. The chemical basis of solventy aroma is impact on the flavour characteristics of complicated by the fact that a number both the light and heavy rums, with high of different types of compounds can be fill strengths resulting in more woody notes described as solvent (e.g. esters, acetals, and low fill strengths in sweet, dried fruit aldehydes). aromas. From sugar to rum - the technology of rum making 167

2. The number of times a cask is refilled has acetals, heterocyclics and other miscellaneous an impact on the flavour characteristics it compounds that contribute significantly to imparts. After six months maturation the the flavour and aroma of these spirits. We are rum matured in 3rd refill casks had distinctive hopeful that with the studies being conducted sweet, buttery and vanilla notes. we will discover new compounds present in rum that contributes to its unique characteristics. At this stage in the study some factors did not appear to be influencing sensory character. There were no aroma differences between the Summary rums stored at the top versus the bottom of the warehouse. Similarly racked as opposed to Blending is the secret of fine rum. It allows the palletised storage had no effect on flavour. master bender to use many different types and styles of rums to create a particular blend or brand. The barrels of rum used for a particular Summary of chemical analyses after 6 blend are selected with age as the major months of ageing selection criteria. The skill of blending involves the mixing together of light and heavy type 1. The chemical analyses separated the rums of different ages that have been carefully measured compounds into current distillate analysed and selected by the blender for the or cask derived congeners, with little overlap characteristics specified. Through a “marrying between the two groups. process”, the different rums are allowed to fuse 2. Current distillate congeners show highly together to give the blend a smoothing effect. significant differences between light and After the rum is blended, it is stored in bottling heavy rums and are generally unaffected vats and reduced to bottling strength by the by 6 months ageing. Exceptions to this are addition of deionised water. It is then passed the sulphur compounds DMDS and DMTS through filters and polishers before being bottled which decreased during the ageing period. and packaged for sale. Further exceptions are some higher alcohols and short and medium chain esters which increased with age as much as 100%, but Bibliography future sampling will assist us to determine the exact derivation of these components. Deshpande, G.B. (2000). Molasses to ethanol 3. For cask derived congeners the major distillation technologies. Praj Conference difference is between current and aged Godbole, J. and Deshpande, G.B. (2000). samples, with lesser or no differences Product quality, efficiency and cost of between light and heavy rums. operation. Praj Conference Jack, F. R., Conner, J.M. and Reid, K. J. G. (2005). Of all the variables examined in this project, Sensory and chemical comparison of aged the use of refill casks has the least effect on the rums. Scotch Whisky Research Institute extraction of cask derived congeners, with the Report level of most wood constituents decreased by Sheorain, V. (2004). Fermentation of Sugar the use of refill casks. Cane Molasses. West Indies Rum and Spirits Most spirits follow a similar process of Producers Association Conference fermentation, distillation and ageing using Shete, N. (2000). Cane molasses and its impact various sugar sources. The volatile compounds on ethanol fermentation process. Praj identified in these distilled and aged beverages Conference. include alcohols, aldehydes, ketones, esters, 168 V. Persad-Doodnath Discrimination between rum and cachaça 169 Chapter 24 Discrimination between rum and cachaça

L. G. Andrade-Sobrinho1, J. R. Piggott2 and D. W. Franco1 1Departamento de Química e Física Molecular, Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil; 2Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, UK

Introduction raw materials and the production processes involved (Lehtonen and Soumalainen, 1977; Cachaça and rum are alcoholic beverages Nykänen and Nykänen, 1999). The formation derived from sugar cane (Nicol, 2003; Lehtonen of volatile compounds in spirits is determined and Soumalainen, 1977), although cachaça is by the fermentation conditions: raw material, exclusively produced in Brazil (Nicol, 2003; yeast, and temperature (Nykänen and Nykänen, Piggot, 2003), and rum is produced mainly in 1999). The occurrence of volatile compounds in Central America and other countries wherever the spirit is largely determined by the distillation the climate is suitable for the cultivation of process. In the case of rum, the distillation sugar cane (Nicol, 2003; Cardoso, Andrade- process is used to distinguish two types: heavy Sobrinho, Leite-Neto, Reche, Isique, Lima-Neto rums are usually produced by batch distillation and Franco, 2004). Cachaça is produced only or in a two-column continuous still, while light from the distillation of fermented sugar cane rums are normally produced by continuous juice (Cardoso et al., 2004), while most rum distillation (Cardoso et al., 2004; Piggot, 2003). is produced from blackstrap molasses. In the For cachaça, the distillation can be by a pot French Caribbean islands, agricultural rum (rhum still (typical of small producers) or continuous agricole) can be produced by fermentation of column still (large producers), and until now it sugar cane juice (Piggot, 2003). Because of the has not been possible to determine what type of similarity in the starting materials it is necessary distillation has been used in the process. for regulatory purposes that the beverages During maturation of alcoholic beverages can be distinguished. Good discrimination many complex reactions occur (Lehtonen and between cachaça and rum has been described Soumalainen, 1977). Cachaça can be aged in using analytical data for protocatechuic acid, oak (Boscolo, Andrade-Sobrinho, Lima-Neto and propanol, isobutanol, isopentanol, manganese, Franco, 2002), typically redundant barrels from magnesium and copper when treated by whisky production (Faria, Franco and Piggott, Principal Component Analysis, Hierarchical 2004). White rum is generally not aged, and Cluster Analysis and K-nearest neighbour amber and dark rums are aged in wood barrels. analysis (Cardoso et al., 2004). Most commonly once-used Bourbon barrels are The presence of organic compounds and used, which may be charred before use (Piggot, metals in alcoholic beverages derives from the 2003). Flavour components such as syringic acid, 170 L.G. Andrade-Sobrinho et al. vanillin, syringaldehyde and gallic acid all have following cachaças were analysed: Cachaça their origin in the lignin of oak barrels (Lehtonen 21 (São Paulo), Caninha da Roça (São Paulo), and Soumalainen, 1977; Piggot, 2003). The Catedral (São Paulo), Jequity Cristal (São Paulo), content of these compounds can be affected by Pitú (Pernambuco), Santo Antônio (Rio Grande many things including the number of times the do Sul), Tietê (São Paulo), Velho Barreiro (São barrel has been used, degree of toasting, depth Paulo), Verita Silver (Rio de Janeiro), Ypioca Prata of char and maturation temperature (Piggot, (Ceará), Catedral (São Paulo), Colonial (Ceará), 2003). In some cases, white and golden rums Guaramiranga (Ceará), Jequity (São Paulo), Pitú can be produced by aging for a specific time, Gold (Pernambuco), Tiquara (São Paulo), Velho then decolorizing with activated carbon which Barreiro Gold (São Paulo), Veritas Gold (Rio will also remove some volatile compounds, de Janeiro), Vila Velha (São Paulo) and Ypioca suppressing the original aroma (Nicol, 2003). Ouro (Ceará). Fourteen samples were supplied The metals content of spirits depends on local by ABRABE (Associação Brasileira de Bebidas) geology (inevitably as affected by the distillation), and six others were purchased by producers. and reduction water where it is used to reduce These cachaça samples were from various states ethanol concentration before bottling. Copper of Brazil, named in parentheses. content particularly depends on the distillation The following rums were analysed: Anivesario process, and single-distilled pot-still products Pampero (Venezuela), Appleton Dark “De Luxe” may have a relatively high content compared (Jamaica), Appleton Estate (Jamaica), Bacardi particularly with stainless steel column-still Carta Blanca (Brazil), Bacardi Carta de Oro products (Cardoso et al., 2004). (Brazil), Bacardi Premium Black (Brazil), Bacardi Considering the fundamental similarities 1873 Solera (Mexico), Captain Morgan “White between cachaça and rum, it is likely that the seven rum” (Canada), Cartain Morgan “Dark rum” chemical descriptors mentioned previously may (Canada), El Dorado “Golden rum” (Guyana), not be sufficient to distinguish these spirits when Havana Club Añejo 7 años (Cuba), Havana other new samples are encountered. This paper Club Añejo Reserve (Cuba), Havana Club Silver reports an exploration of the use of multivariate Dry (Cuba), Jamaica (Brazil), Montila Carta statistical methods to distinguish cachaça and Branca (Brazil), Montila Carta Ouro (Brazil), rum, using previously-obtained analytical data Myers’s “Original Dark” (Jamaica-USA), Negrita (Cardoso et al., 2004) for the alcohols (methanol, Bardinet “Dry and Light” (France), Negrita propanol, isobutanol and isopentanol), ethyl Bardinet “Bardinet S. A. Bordeaux” (France), acetate, acetaldehyde, organic acids (octanoic Negrita Bardinet Old Reserve (France), Selecto acid, decanoic acid and dodecanoic acid), wood- (Venezuela), Soccaron (France) and XK Solera derived materials (gallic acid, protocatechuic (Mexico). Seventeen rum samples were supplied acid, epicatechin, vanillic acid, syringic acid, by ABRABE and a further six were purchased in syringaldehyde, vanillin, p-coumaric acid, the U.S.A. and Canada. coniferyl aldehyde, sinapaldehyde, ellagic acid, coniferyl alcohol, kaempferol and quercetin) and the mineral fraction (aluminum, calcium, cobalt, Analytical methods copper, chromium, iron, manganese, magnesium, nickel, sodium and zinc). The analytical methods used have been described previously (Cardoso et al., 2004). Alcohols, Materials and methods acetaldehyde and ethyl acetate were analysed by gas chromatography (GC) with flame ionisation Samples detection (Boscolo, Bezerra, Cardoso, Lima- Neto and Franco, 2000), in duplicate with an Samples were selected to provide both light and internal standard. Organic acids (Nascimento, dark rums, and aged and unaged cachaças. The Cardoso, Lima-Neto and Franco, 1998) were Discrimination between rum and cachaça 171 preconcentrated by solid phase extraction (SPE) loadings on the first component, associated and analysed by GC with mass selective detection, with the dark rums, and the acids, alcohols again in duplicate with an internal standard. and some metals with negative loadings on Polyphenols (Bettin, Isique, Franco, Andersen, the first component and positive loadings on Knudsen and Skibsted, 2002) were determined the second. One sample of aged cachaça was by high pressure liquid chromatography with an outlier in this plot, caused largely by very diode array detector, after extraction of spirit high levels of organic acids. However, when samples by SPE, in duplicate with an external the analysis was repeated without this sample standard; unaged samples were previously there was no substantial change in the result. evaporated and re-dissolved in a small volume of PLS, in the form of discriminant partial least ethanol. Minerals (Nascimento, Bezerra, Furuya, squares (DPLS), would be expected to perform Schultz, Polastro, Lima-Neto and Franco, 1999) better in predicting each sample’s group were determined by inductively coupled plasma membership, and thus in separating rum from atomic emission spectroscopy after digestion cachaça (Figure 3). The samples are clearly with HNO3, in triplicate with external standards, separated into three groups, though it is not clear and checked by standard addition. why the rum samples are so clearly split into two groups. Figure 4 shows the wood-derived materials associated with the group of dark Statistical methods rums, and the acids, alcohols and some metals associated with the cachaça samples. ANOVA Analysis of variance (ANOVA) was performed showed that some of the original variables did with Minitab 14 (Minitab Ltd., Coventry, UK). not discriminate between rum and cachaça, The results from the chemical analysis of 43 and so the DPLS was repeated, omitting the samples were organised in a matrix form, and non-discriminating variables. This provided a examined by Principal components analysis similar separation of the groups (Figure 5) with (PCA) and Partial least squares regression (PLS) the 20 remaining variables. It is clear that there using the Unscrambler version 7 (CAMO ASA, are two groups of variables (Figure 6), the wood- Oslo, Norway). PCA transforms a data matrix by derived materials being associated with the calculating components (linear combinations of rums, and a group consisting of alcohols, acids variables) which explain as much of the variance and metals being associated with cachaça. The in the data as possible (Piggott and Sharman, contents of wood-derived materials are normally 1986). Similarly, PLS calculates components highly correlated, sometimes with changes in from the predictor (X) data matrix, but they are the balance of compounds caused by variation calculated to explain maximum variance in in maturation conditions such as cask size and the dependent (Y) data (Martens and Martens, ethanol concentration (Withers, Piggott, Conner 1986). and Paterson, 1995). Similarly, the contents of alcohols and acids, arising from the fermentation and distillation conditions, are normally highly Results correlated, and the inclusion of all of them does not necessarily improve discrimination. A small As noted previously, the rum and cachaça set of representative variables was therefore samples could be readily separated by the present selected, and the DPLS run again. There is a set of analytical variables. An exploratory PCA good separation of the groups (Figure 7), but the (Figure 1) shows the rum samples generally to inclusion of octanoic acid (Figure 8) has again the lower right of the scatter plot. Examination of caused one of the cachaça samples to appear as the variable loadings (Figure 2) shows the wood- an outlier. Excluding the octanoic acid provides derived materials with mostly high positive a clear separation and a closer group of cachaças 172 L.G. Andrade-Sobrinho et al.

PC2 Scores 10 CA

8

6

4 C CA 2 CA RDRD C CA C RD C C CA RD 0 C CACA RDRD CA RD C CA CA RD RD RD C RD RD RD RD -2 RD RDRD RD RWRWRW RW RW -4 PC1 -4 -2 0246 pca codes all d…, X-expl: 28%,16% Figure 1. Sample scores on the first two components from principal components analysis of analytical data on 43 rum and cachaça samples. RW = white rum; RD = dark rum; C = cachaça; CA = aged cachaça.

PC2 X-loadings 0.4

decanoicoctanoic acid acid dodecanoic acid quercetin 0.3 isopentanol butanol

kaempferol propanol 0.2 methanol acetaldehydethyl acetate e MgCu Mn Ca epicatechin 0.1 vanillin Na coniferylsyringicsyringaldehydep-cumaric alcho acid acid vanillic acid coniferylaldehygallic acid lsobutanol ellagic acid 0 Fe protocatechuic synapaldehyde Zn

-0.1 Cd PC1 -0.2 -0.1 00.1 0.20.3 pca codes all d…, X-expl: 28%,16% Figure 2. Variable loadings on the first two components from principal components analysis of analytical data on 43 rum and cachaça samples.

PC2 Scores 5

4 RD

3 RD CA 2 RD CA RD CA C CA 1 CA C RD RD CA C C RD CA CACAC RD RD C 0 RD RD C CA CC -1 C

-2 RD RW RD RD -3 RWRD RWRDRW RWRD -4 PC1 -5 05 discrim codes a…, X-expl: 26%,15% Y-expl: 77%,12% Figure 3. Sample scores on the first two components from discriminant partial least squares analysis of analytical data on 43 rum and cachaça samples. RW = white rum; RD = dark rum; C = cachaça; CA = aged cachaça. Discrimination between rum and cachaça 173

PC2 X-loading Weights and Y-loadings 0.4 isopentanol epicatechin butanol 0.3 propanol syringaldehyde p-cumaric acid Na methanol 0.2 vanillic acid gallicconiferylaldeh syringicacid aciy d ethylacetaldehyd acetate e Cachaca protocatechuicellagic aci d Cu coniferyl alcho synapaldehyde 0.1 Cd octanoic acid lsobutanolFe Zn dodecanoic acid vanillin decanoic acidMg 0 Ca quercetin Mn -0.1 kaempferol

-0.2 PC1 -0.3 -0.2 -0.1 00.1 0.20.3 0.4 discrim codes a…, X-expl: 26%,15% Y-expl: 77%,12% Figure 4. Variable loadings on the first two components from discriminant partial least squares analysis of analytical data on 43 rum and cachaça samples.

PC2 Scores 4 RD CA

3 RD CA RD 2 CACA RD RD 1 CA CA RD CA CA C C C C RD CA C C CA C 0 RD RD RD RD C

-1 C

RD -2 RD RDRW RWRD RW -3 RWRDRD RW PC1 -5 05 discrim codes a…, X-expl: 41%,18% Y-expl: 74%,15% Figure 5. Sample scores on the first two components from discriminant partial least squares analysis of variables shown by ANOVA to discriminate between 43 rum and cachaça samples. RW = white rum; RD = dark rum; C = cachaça; CA = aged cachaça.

PC2 X-loading Weights and Y-loadings 0.5

0.4 isopentanol butanol epicatechin propanol 0.3 syringaldehyde p-cumaric acid

0.2 vanillic acid Cachaca gallicconiferylaldeh syringicacid aciy d octanoic acidCu protocatechuicellagic aci d coniferyl alcho 0.1 Zndecanoic acid

Mg 0 Ca

Mn -0.1 PC1 -0.3 -0.2 -0.1 00.1 0.20.3 0.4 discrim codes a…, X-expl: 41%,18% Y-expl: 74%,15% Figure 6. Variable loadings on the first two components from discriminant partial least squares analysis of variables shown by ANOVA to discriminate between 43 rum and cachaça samples. 174 L.G. Andrade-Sobrinho et al.

PC2 Scores 2 CA

RD CA 1 RD CA C RD RD RD CA RD CACA C RDRD RD RD CC CCA C CCC 0 CA C CA RD RWRDRWRD RWRDRWRW -1 RDRD

-2

-3 RD CA

-4 PC1 -3 -2 -1 01234 discrim codes s…, X-expl: 37%,16% Y-expl: 79%,6% Figure 7. Sample scores on the first two components from discriminant partial least squares analysis of a set of representative variables measured on 43 rum and cachaça samples. RW = white rum; RD = dark rum; C = cachaça; CA = aged cachaça.

PC2 X-loading Weights and Y-loadings 0.6 isopentanol 0.4 epicatechin

Cachaca 0.2 protocatechuic

0

Cu -0.2 octanoic acid

-0.4

-0.6 kaempferol -0.8 PC1 -0.6 -0.4 -0.2 00.2 0.40.6 0.8 discrim codes s…, X-expl: 37%,16% Y-expl: 79%,6%

Figure 8. Variable loadings on the first two components from discriminant partial least squares analysis of a set of representative variables measured on 43 rum and cachaça samples.

(Figure 9). Copper is a useful discriminator, Discussion being present at higher concentrations in some of the cachaças, but it is conceivable that a rum Previous work and the studies reported here could contain an equally high level if distilled confirm that a small selection of the chemical in the same way. When coppper was omitted, variables measured could discriminate between the separation of the groups was maintained the cachaça and rum samples used in the trial. It (Figure 10) with the remaining four variables is possible that other products exist which would (epicatechin, protocatechuic acid, isopentanol be wrongly classified by any of these models, but and kaempferol). The residual validation while the production processes and practices for variance (Figure 11) still shows a clear minimum rum and cachaça remain different it is likely that at two components, confirming the validity of the vast majority of examples would be correctly discrimination based on two components. classified. However, none of the variables used Discrimination between rum and cachaça 175

PC2 Scores 2 CA RD RD RD CA RD 1 RD RD RD CA CA RD CA C RD RD RD C 0 CCCACA C CA CA C CCC CA RD C

-1 RDRD RWRD RWRD RWRDRWRW

-2 RD

-3 PC1 -3 -2 -1 0123 discrim codes s…, X-expl: 42%,21% Y-expl: 78%,6% Figure 9. Sample scores on the first two components from discriminant partial least squares analysis of a set of representative variables (omitting octanoic acid) measured on 43 rum and cachaça samples. RW = white rum; RD = dark rum; C = cachaça; CA = aged cachaça.

PC2 Scores 2 CA

RD RD RD CA CA 1 RD CA RD RD CA RD C RD RD RD CCAC CAC 0 CCCCCA CCA CA RD

-1 RD RWRD RD RWRD RDRWRW

-2

RD

-3 PC1 -3 -2 -1 0123 discrim codes s…, X-expl: 44%,25% Y-expl: 74%,9% Figure 10. Sample scores on the first two components from discriminant partial least squares analysis of a set of representative variables (omitting octanoic acid and copper) measured on 43 rum and cachaça samples. RW = white rum; RD = dark rum; C = cachaça; CA = aged cachaça.

Y-variance Residual Validation Variance 1.1

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2 PCs PC_00 PC_01 PC 02 PC 03 PC_04 discrim codes s…, Variable: v.Total

Figure 11. Residual validation variance for discriminant partial least squares analysis of a set of representative variables (omitting octanoic acid and copper) measured on 43 rum and cachaça samples. 176 L.G. Andrade-Sobrinho et al. seems to provide a fundamental discrimination; Spectrophotometric determination of they discriminate because of differences in caramel content in spirits aged in oak casks. production practice. JAOAC International 85: 744 - 750 There may be other compounds which Cardoso, D. R., Andrade-Sobrinho, L. G., Leite- could provide more reliable discrimination Neto A. F., Reche, R. V., Isique, W. D., between rum and cahaça, possibly based on the Lima-Neto, B. S. and Franco, D. W. (2004). distinction between the raw materials. Rum has Comparison between cachaça and rum been reported to contain 2-ethyl 3-methyl butyric using pattern recogntion methods. Journal acid, thought to emanate from bacterial action on of Agricultural and Food Chemistry 52: molasses (Nicol, 2003), and molasses contains 3429 - 3433 heterocyclic nitrogen compounds, products of Faria, J. B., Franco, D. W.and Piggott, J. R. the Maillard Reaction, which are therefore likely (2004). The quality challenge: “Cachaça” to be more prevalent in rum. Rum also contains for export in the 21st century. In: Distilled phenols, some from ethanolysis of lignin and Spirits: Tradition and Innovation, Edited by therefore dependent on wood maturation, Bryce, J. H. and Stewart, G. G., Nottingham and others derived from fermentation and also University Press, Nottingham, UK, pp. potentially useful as discriminators (Lehtonen, 215-222 1983a; 1983b). Lehtonen, M. (1983a). Gas-liquid- chromatographic determination of volatile phenols in matured distilled alcoholic Acknowledgements beverages. Journal of the Association of Official Analytical Chemists 66: 62-70 Fundação de Amparo à Pesquisa do Estado Lehtonen, M. (1983b). High-performance liquid- de São Paulo (FAPESP), Coordenação de chromatographic determination of non- Aperfeiçoamento de Pessoal de Nível volatile phenolic-compounds in matured Superior (CAPES) and Conselho Nacional de distilled alcoholic beverages. Journal of the Desenvolvimento Científico e Tecnológico Association of Official Analytical Chemists (CNPq) are thanked for their support. 66: 71-78 Lehtonen, M. and Soumalainen, H. (1977). Rum. In: Economic Microbiology, Vol. 1. References Alcoholic beverages, Edited by Rose, A. H., Academic Press, New York, pp. 595-633 Bettin, S. M., Isique, W. D., Franco, D. W., Martens, M. and Martens, H. (1986). Partial least Andersen, M. L., Knudsen, S. and Skibsted, squares regression. In: Statistical Procedures L. H. (2002). Phenols and metals in sugar- in Food Research, Edited by Piggott, J. R., cane spirits. Quantitative analysis and effect Elsevier Applied Science, London, pp. on radical formation and radical scavenging. 293-359 European Food Research and Technology Nascimento, R. F., Cardoso, D. R., Lima-Neto, B. 215: 169 - 175 S. and Franco, D. W. (1998). Determination Boscolo, M., Bezerra, C. W. B., Cardoso, D. R., of acids in Brazilian sugar cane spirits and Lima-Neto, B. S. and Franco, D. W. (2000). other alcoholic beverages by HRGC-SPE. Identification and dosage by HRGC of minor Chromatographia 48: 751-758 alcohols and esters in Brazilian sugar-cane Nascimento, R. F., Bezerra, C. W., Furuya, spirit. Journal of the Brazilian Chemical S. M. B., Schultz, M. S., Polastro, L. R., Society 11: 86 – 90 Lima-Neto, B. S. and Franco, D. W. (1999). Boscolo, M., Andrade-Sobrinho, L. G., Lima- Mineral profile of Brazilian cachaças and Neto, B. S. and Franco, D. W. (2002). other international spirits. Journal of Food Discrimination between rum and cachaça 177

Composition and Analysis 12: 17-25 Industries. 4th Edition, Edited by Jacques, Nicol, D. A. (2003) Rum. In: Fermented K. A, Lyons, T. P. and Kelsall, D. R., Beverage Production, 2nd Edition, Edited Nottingham University Press, Nottingham, by Lea, A. G. H. and Piggott, J. R., Kluwer UK, pp. 247-253 Academic/Plenum Plubishers, New York, Piggott J. R. and Sharman K. (1986). Methods pp. 263-287 for multivariate dimensionality reduction. Nykänen, L. and Nykänen, I. (1999). Distilled In: Statistical Procedures in Food Research, beverages. In: Volatile Compounds in Foods Edited by Piggott, J. R., Elsevier Applied and Beverages, Edited by Maarse, H., Marcel Science, London, pp. 181-232 Dekker, New York, pp. 547-575 Withers, S. J., Piggott, J. R., Conner, J. M. and Piggot, J. R. (2003). Production of heavy and Paterson, A. (1995). Comparison of Scotch light rums: fermentation and maturation. malt whisky maturation in oak miniature In: The Alcohol Textbook. A Reference for casks and American standard barrels. Journal the Beverage, Fuel and Industrial Alcohol of the Institute of Brewing 101: 359-364. 178 L.G. Andrade-Sobrinho et al. Evaluation of Brazilian woods as an alternative to oak for “cachaça” aging in cask 179 Chapter 25 Evaluation of Brazilian woods as an alternative to oak for cachaça aging in cask - their antioxidant ability

D. R. Cardoso1, A. M. Frederiksen2, A. A. da Silva1, D. W. Franco1 and L. H. Skibsted2 1Departamento de Química e Física Molecular, Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil; 2Department of Food Science, KVL, Rolighdsvej 30, Frederiksberg C, Denmark

Introduction would be interesting to replace oak barrels with casks produced from Brazilian wood species. A great deal of effort has been dedicated to Sensory attributes of cachaça aged on barrels increase the export volume and qualify cachaça constructed with Brazilian wood species recently as an international beverage typical of Brazil reported by us (Faria, Cardello, Boscolo, Isique, (Vasconcelos, 2003). Great improvements were Odello, and Franco, 2003), suggest amendoim, made regarding the knowledge of cachaça’s pereiro, and jatobá as suitable replacements for chemical composition in the past decade oak in the construction of cachaça aging cask. (Cardoso, Andrade-Sobrinho, Leite-Neto, Reche, Herein, we describe the antioxidant ability of Isique, Ferreira, Lima-Neto, and Franco, 2004; the Brazilian wood species and oak toward the Nascimento, Cardoso, De Keukeleire, Lima- lipid peroxidation initiated by metMb, radical Neto, and Franco, 2000; Boscolo, Bezerra, reduction capacity, radical scavenging kinetics, Cardoso, Lima-Neto, and Franco, 2000; Cardoso, and total phenolics content. Andrade-Sobrinho, Lima-Neto, and Franco, 2004; Bettin, Isique, Franco, Andersen, and Skibsted, 2002). Consequently, quality control has been Materials and methods improved, and its chemical composition and sensory profile are now being moitored. The Brazilian wood species amendoim (Pterogyne Aging in wood casks is a well-established sp.), bálsamo (Myroxilon sp.), canela-sassafrás procedure for upgrading sensory attributes of (Ocotea pretiosa), castanheira (Castanea sp.), alcoholic beverages (Lee, Paterson, Piggott, ipê (Tabebuia chrysotricaha), jatobá (Hymenaea and Richardson, 2001). Despite the well- courbaril), and louro-canela (Aniba parviflora) known improvement of the sensory profile, were selected for the study on the basis of this procedure is not a common practice in their potential commercial usefulness (Faria the cachaça industry (Faria, Cardello, Boscolo, et al., 2003). No wood-extractable phenolic Isique, Odello, and Franco, 2003). Since Brazil compounds were found in the non-aged cachaça, has a wide flora and the imported oak had served which served as solvent and as a control. exhaustively for cognac and whisky maturing in Antioxidant ability of the wood extracts were North America and Europe (Faria et al., 2003), it evaluated as follows: total phenolics content 180 D.R. Cardoso et al. determined by the Folin-Ciocalteus method ambient temperature (25 ± 3°C). Samples were (Singleton and Rossi, 1965); the stable radical protected against light throughout. DPPH• was used to determine the reduction capacity; and the inhibition of lipid peroxidation by the wood extractives was evaluated in a Total phenolics content model system triggered by metMb. The total phenol content was measured according to the Folin-Coicalteu procedure using gallic acid Materials as standard, and the results were expressed in mol gallic acid equivalents (GAE) per liter of Folin-Ciocalteus phenol reagent, acetic acid, extract (Singleton and Rossi, 1965).

Na2CO3, Na2HPO4, and HNO3 were from Merck (Darmstadt, Germany), gallic acid, Tween-20, linoleic acid methyl ester, and horse heart Radical scavenging and reduction capacity myoglobin were from Sigma-Aldrich (Milwaulkee, USA), tris(2,2’-bipyridine)ruthenium(II)di- The radical scavenging capacity (mol DPPH• per chloride from Alfa Aesar (Newburyport, USA) litter extract) was assayed following decolouring were of analytical grade and used as purchased. of a solution of DPPH• in ethanol (Litwinienko The HPLC grade solvents: methanol and ethanol and Ingold, 2003). The spectrophotometer was from Merck (Darmstadt, Germany), acetonitrile a Shimadzu UV-2101 PC UV-Vis scanning from Tedia (Fairfield, USA), were used without spectrophotometer with a control unit (CPS-260 further purification. Water used was distilled and cell positional and EPS-controller) using 1.00 then de-ionized by a Milli-Q system (Millipore, cm-path length disposable cuvettes (Radiolab, Bedford, MA, US). Copenhagen, Denmark). The initial absorbance of 100 µL (2.24 mM DPPH•) and 3.00 ml 40% (v/v) ethanol in water was determined at 525 Wood samples nm. The quenching effect of an added volume of each extract was tested depending on the Certified Brazilian wood samples were provided total phenolics content (called extract volumes) by the Laboratório de Madeiras e Estruturas de ranging from 15.0 µl to 100 µl. The absorbance Madeira at the Universidade de São Paulo (São at 525 nm was measured 2 h after addition of Carlos, Brazil) and by the Instituto de Pesquisas extract and completion of the reaction. In order Tecnológicas (IPT, São Paulo, Brazil). The oak to compensate for the absorbance contribution samples from USA and Europe were kindly of extract, the absorbance of the extract in a provided by the Department of Bioscience at solution of 40% (v/v) aqueous ethanol served as Strathclyde University (Glasgow, Scotland). a blank. The amount of DPPH• consumed was calculated by subtracting the final absorbance and the background absorbance from the initial Wood extracts absorbance of the 100 µL-DPPH• solution in 3.0 ml 40% (v/v) aqueous ethanol solution. The A non-aged sugar cane spirit without sugar concentration of reduced DPPH• was calculated addition certified and provided by Indústrias using ε = 1.25 x 104 L mol-1cm-1 (Schwarz, Müller de Bebidas Ltda. (Pirassununga-SP, Brazil) Bertelsen, Nissen, Gardner, Heinonen, Hopia, was used as solvent for the extraction procedure. Huynh-Ba, Lambelet, McPhail, Skibsted, and Dry wood (air dried) was extracted in Tijburg, 2001). non-aged sugar cane spirit, in a ratio of 0.01g of The radical scavenging kinetics of DPPH• wood sawdust per ml, by shaking for 26 days at quenching in an aqueous emulsion by wood Evaluation of Brazilian woods as an alternative to oak for “cachaça” aging in cask 181 extractable antioxidants was assessed using the added and the initial intensity of the fluorescence 2+ stopped-flow technique. The present work was of [Ru(bipy)3] ion was measured immediately. carried out in an emulsion with water as the 34.5 µL aqueous solution of MbFe(III) (10-5 M) homogeneous phase which should overcome was added in order to initiate lipid oxidation and 2+ the often encountered problem that the uses measurements of [Ru(bipy)3] emission were of alcoholic solvents overestimate the rate done immediately after addition. The intensity constant of a given radical scavenging reaction was recorded at 10 s intervals for up to 1 h with (Litwinienko and Ingold, 2003). A Sequential a slit width of 2.5 nm. Excitation and emission Stopped-Flow Bio-Sequential DX-17MV (Applied wavelengths of [Ru(bipy)3]2+ were 450 and 605 Photophysics, UK) was used for this purpose in a nm, respectively (Sasso et al., 1986). single mixing mode. A 61.2 µM DPPH• solution The stability of the system was evaluated by was prepared in an emulsion consisting of 0.04 measuring different combinations of solvent, g ml-1 Tween-20 and 5.0 mM phosphate buffer fluorescence probe and metMyoglobin for up pH 7.0. Wood extracts dissolved in an emulsion to 24 h. No change in the probe luminescence of 0.04 g ml-1 (Tween-20) were placed in the spectra was observed. Each experiment was opposite syringe of the DPPH• solution. Four performed in duplicate at the same dilution different dilutions of each extract were analysed factor as used for the extracts. at least in triplicate. The pseudo-first-order relation between the complete scavenging of The quenching effect of molecular oxygen DPPH• by wood-extractable antioxidants and the obeys the Stern-Volmer equation, assuming a time was analyzed by exponential fitting using static quenching effect: Pro/Kineticist software (Applied Photophysics,

UK). I0 o = 1+ kq [O2 ] (1) I Oxygen consumption assay I0 and I are the emission intensities of excited [Ru(bipy) ]2+ in the absence and presence of A modification of the methods of (Hu and 3 oxygen. k is the bimolecular rate constant for the Skibsted, 2002) and of (Sasso, Quina, and q 0 Bechara, 1986) to assess the oxygen consumption overall quenching process, τ is the lifetime of the [Ru(bipy) ]2+ ion excited state in the absence in a peroxidating lipid-in-water emulsion was 3 2+ of oxygen. used. The luminescence of the [Ru(bipy)3] ion was monitored during the oxidation of From equation 1 the oxygen concentration at the initial and intermediate time ([O ] and [O ] , methyl linoleate ester using a Hitachi F4500 2 i 2 t Luminescence Spectrometer. A Tecnal TE184 respectively) can be calculated using: (Piracicaba, Brazil) was attached to the cell holder [O ] I (I − I ) in order to maintain a constant temperature of 2 t = i 0 t (2) 20.0 ± 0.1°C. An Aliquot of 345 L of methyl − µ [O2 ]i It (I 0 I i ) linoleate (28.2 mM) methanolic solution was mixed with 86.0 L of Tween-20 (0.04 g ml-1) µ where I represents the emission intensity of in methanol. The methanol was removed under t excited [Ru(bipy) ]2+ at time t. A relative measure a gentle nitrogen steam. Phosphate buffer pH 3 of the oxygen concentration at time t in relation 7.0 (2.40 ml of previous air-equilibrated) was to the initial oxygen concentration is obtained added at 20.0 ± 0.1oC. 100 µL [Ru(bipy) ]Cl in 3 2 giving a pseudo-first order decay from which phosphate buffer (1.75 mM) was added resulting the observed rate constant was calculated by in a final concentration of 50.0 M. 1.0 ml wood µ non-linear regression. extract diluted 300-fold in phosphate buffer was 182 D.R. Cardoso et al.

The influence of each wood extract on the Table 1. Quantitative data for the total phenols oxygen consumption rate was expressed as an and flavan-3-ols in the wood extracts. oxygen consumption index: Sample # Wood species Total phenols -1 (mmolGAE L ) Index = kobs/kox (3) 1 Amendoim 0.95 ± 0.02 where the k is the rate of oxygen consumption 2 Amendoim 1.26 ± 0.05 ox 3 Amendoim 1.78 ± 0.05 without extracts and kobs is the observed rate constant of oxygen consumption in the presence 4 Bálsamo 2.58 ± 0.04 5 Canela-sassafrás 0.65 ± 0.01 of the extract. 6 Canela-sassafrás 1.20 ± 0.03 Oxygen consumption index values below 7 Castanheira 2.45 ± 0.04 unity express antioxidant activity, while values 8 Ipê 1.16 ± 0.01 above unity implicate pro-oxidative behaviour. 9 Jatobá 4.71 ± 0.01 10 Jatobá 6.38 ± 0.06 11 Louro-canela 1.74 ± 0.01 Results and discussion 12 Louro-canela 0.75 ± 0.01 13 Oak (USA air-dried) 1.39 ± 0.03 14 Oak (USA kiln-dried) 2.87 ± 0.03 Content of phenolic compounds 15 Oak (Scottland) 2.25 ± 0.13 16 Oak (Poland) 2.30 ± 0.04 As shown in Table 1, the total amount of 17 Oak (Czech-Replubic) 2.02 ± 0.05 phenolics in the wood extracts of jatobá is remarkable high ranging from 4.71 to 6.38 -1 mmolGAE L . All values of the phenolic content The data in Table 2 indicate that oak extracts in jatobá exceed the content of phenolic show the best reducing capacity towards DPPH•, compounds of the oak samples (from 1.39 to quenching 3.25 up to 4.11 moles of DPPH• per -1 2.87 mmolGAE L ). The large variations in the total mole of total phenols expressed as gallic acid phenolic contents observed for species belong equivalents. In contrast, canela-sassafrás (from to the same genes may be due to the different -1 1.29 to 1.53 molDPPH• mol GAE) exhibits the least origin and to varying seasoning of the wood efficient DPPH•-reducing capacity. (Fengel and Wegener, 1983). The Brazilian Antioxidant activities of the wood extractable wood species canela-sassafrás was characterized phenolic compounds towards DPPH• were by the lowest average level of total phenolics clearly established in this study. Furthermore, the -1 ranging from 0.65 to 1.20 mmolGAE L . observed reducing capabilities were a function of the wood species and, also, a different species of the same genus, showing that the influence of Reduction capacity geographical origin of the wood and sylvocultural treatment of the tree needs also to be taken into Figure 1 shows the correlation plot between account (Fengel and Wegener, 1983). the contents of total phenols and the radical -1 quenching capacities (molDPPH• L ), that may be considered a titration of phenols present in Radical scavenging kinetics the extracts. Therefore, the deviations observed in the correlation plot suggest the presence In the presence of excess of phenols relative to of other reducing species and/or that some DPPH• in the reaction mixture (to ensure pseudo- phenolic compounds quench more than one first order conditions), quenching of DPPH• for molecule of DPPH• per molecule of phenol, each extract could be adequately described by while others quench less than one maybe due first-order kinetics. Figure 2 shows the plot of to steric hindrance. the pseudo-first order rate constant for DPPH• Evaluation of Brazilian woods as an alternative to oak for “cachaça” aging in cask 183

-1 -1 Figure 1. Correlation plot of radical quenching capacity (molDPPH• L ) versus total phenolics content (mmolGAE L ).

Table 2. Data for reducing capacity, reactivity towards DPPH• and oxygen consumption assay.

Sample # Wood species Reducing Second order aPseudo-first- Oxygen capacity/ rate constant order rate constant consumption -1 -1 -1 (molDPPH• / molGAE) (k2)/(M s ) for O2 depletion/(min ) index

1 Amendoim 3.20 ± 0.03 9.2 x103 4.04 ± 0.70 x10-2 0.22 2 Amendoim 1.26 ± 0.01 1.5 x104 2.12 ± 0.70 x10-2 0.12 3 Amendoim 0.91 ± 0.01 1.4 x104 6.85 ± 0.30 x10-2 0.38 4 Bálsamo 1.88 ± 0.07 3.5 x104 6.35 ± 0.40 x10-2 0.35 5 Canela-sassafrás 1.53 ± 0.06 4.9 x103 2.19 ± 0.06 x10-1 1.21 6 Canela-sassafrás 1.29 ± 0.01 9.4 x103 1.79 ± 0.13 x10-1 0.99 7 Castanheira 3.11 ± 0.06 2.6 x104 1.88 ± 0.07 x10-1 1.04 8 Ipê 2.21 ± 0.05 4.0 x104 1.03 ± 0.001x10-1 0.57 9 Jatobá 2.05 ± 0.18 1.6 x104 4.01 ± 0.18 x10-2 0.22 10 Jatobá 2.09 ± 0.03 1.9 x104 1.16 ± 0.09 x10-1 0.64 11 Louro-canela 2.15 ± 0.10 2.0 x104 1.14 ± 0.03 x10-1 0.63 12 Louro-canela 2.14 ± 0.46 8.4 x103 2.06 ± 0.15 x10-1 1.14 13 Oak (USA air-dried) 3.43 ± 0.08 8.0 x104 1.41 ± 0.07 x10-1 0.63 14 Oak (USA kiln-dried) 3.25 ± 0.09 6.7 x104 8.73 ± 1.38 x10-2 0.48 15 Oak (Scottland) 3.50 ± 0.13 7.0 x104 1.47 ± 0.01 x10-1 0.81 16 Oak (Poland) 4.11 ± 0.16 9.7 x104 1.04 ± 0.05 x10-1 0.57 17 Oak (Czech-Replubic) 3.86 ± 0.09 7.8 x104 8.70 ± 0.30 x10-2 0.48 a The rate of oxygen depletion on the control system (without extract) was 1.81 ± 0.03 x10-1 min-1. 184 D.R. Cardoso et al.

Figure 2. Plot of pseudo-first-order rate constant for DPPH• quenching (19.0 ± 0.10°C) by selected wood extracts versus wood extract concentration. The wood extract concentration (dilution factor) was corrected for the phenolics content (phenolics content = dilution factor x total phenols content in the wood extract) to calculate the specific rate constant from the slope of the linear plot. quenching monitored at 525 nm versus varying varying from 4.9 x 103 up to 4.0 x 104 M-1s-1. It dilution factors of the selected wood extracts. may accordingly be conclude that oak had the With the aim of calculating the specific rate highest quenching capacity (stoichiometry) and, constant for DPPH• scavenging by the wood also, the highest antioxidant activity (kinetic), extracts, the dilution factor was corrected while canela-sassafrás (4.9 x 103 – 9.4 x 103 to the phenols content (phenols content = M-1s-1) is the least effective wood extract both in dilution factor x total phenols content in the relation to capacity and the quenching rate. wood extract). The observed linearity indicate The magnitude of the second-order quenching a bimolecular reaction for which the second- rate constants of the wood extracts dealt with in order rate constant for DPPH• quenching by this study is of the same order of magnitude of the wood extracts (Table 2) were obtained from the second-order rate constants reported for the slope. quercetin, (+)-catechin, and (-)-epicatechin in The observation of a simple second-order polar protic solvents (Andersen, Lauridsen, and reaction for quenching in the heterogeneous Skibsted, 2003). However, jatobá extracts show system suggest that phase-transfer of the a less efficient radical scavenging ability than radicals to the aqueous phase is fast compared oak extracts, despite their higher content of total to a bimolecular reaction in the homogeneous phenolics. According to the DPPH• reducing aqueous phase. Oak extracts exhibited an capacities and the scavenging reaction rates, oak average rate constant of 9.2 x 104 M-1s-1, while appears to be the most efficient of antioxidants the corresponding second-order rate for the in comparison with Brazilian wood species. quenching reaction of the Brazilian wood extracts Evaluation of Brazilian woods as an alternative to oak for “cachaça” aging in cask 185

Antioxidant effect on lipid oxidation extracts were observed. Few extracts showed pro-oxidative effects as evidenced by an Some of the wood extracts exhibit anti- and increased oxygen consumption rate (oxygen others pro-oxidative behaviours, as quantified depletion index above unity). From the oxygen by the rate of oxygen consumption using a lipid consumption rate constant presented in Table 2, model system in an aqueous emulsion (20 ± lipid oxidation inhibition abilities can be ranked 0.1°C). Peroxidation was initiated by MbFe(III) as follows: amendoim > jatobá > oak. 2+ using [Ru(bipy)3] ion as the luminescent probe In contrast, as can be seen in Figure 4, the for oxygen depletion. extracts of canela-sassafrás (mean index of 1.10 As illustrated in Figure 3, no lag phase was ± 0.1), castanheira (index of 1.04), and louro- observed for lipid oxidation in the lipid-in-water canela (mean index of 0.89 ± 0.26) showed a emulsion, when wood extracts were added. pro-oxidative effect and its use as a material for However, the rate of oxygen depletion was wood barrel manufacturing should be carefully affected by the presence of wood extracts and evaluated, as an index above unity indicates that the oxygen concentration followed in each case the extracts may promote oxidative reactions. a first-order (exponential) decay. Fitting the Comparing the results of the oxygen parameters of a pseudo-first order rate expression consumption assay and the quenching capacities to the collected data provided the observed and kinetics towards DPPH•, an apparent rate constant for oxygen depletion presented contradiction crops up. In the assay using DPPH• (Table 2). Figure 4 illustrate the mean values for as a stable artificial radical, oak extracts occur the oxygen consumption index for each wood as the most efficient antioxidant. However, in species. the oxygen consumption assay, which mimic As expected, different degrees of inhibition a biological oxidation of an unsaturated fatty efficiencies of lipid oxidation by the wood acid triggered by the heme protein MbFe(III), the

Figure 3. Oxygen consumption decay curves ([O2]t/[O2]I) normalised to 100 % of initial oxygen concentration versus time for a control sample without extract () and in the presence of an extract (sample number 11) (l). 186 D.R. Cardoso et al.

Figure 4. Histogram indicating the average values for the oxygen consumption index for each wood species. Index below unit indicates an antioxidant effect.

Brazilian wood extracts (amendoim and jatobá) oak in a lipid oxidation model system trigged by showed a more pronounced antioxidant activity metMb. Conversely, Oak extracts exhibit higher with respect to those of oak extracts. radical reducing capacities and radical scavenging The results of the oxygen consumption assay rate constants than the Brazilian wood extracts. gave no simple correlation with other results However, the oxygen consumption assay used to reported here, suggesting a more complicated probe lipid peroxidation has the advantaging of reaction mechanism involved in the inhibition mimicing the lipid autooxidation in a biological of oxygen consumption during the linoleic acid system mediated by metMb, which is considered methyl ester oxidation mediated by MbFe(III). the major pathology of the myocardium diseases A pro-oxidative role of copper present in the after ischemica (Reeder and Wilson, 1998). extract could be considered, however, this effect Extracts of amendoim proved to be the is not clear so far. most potent antioxidant extract, as they show In addition, the most efficient lipid oxidation low oxygen consumption indices concomitant inhibitor was extracts of amendoim despite their with low total phenols contents. The use of the low total phenols contents. This remarkable effect wood species canela-sassafrás, castanheira, and would suggest a possible synergic antioxidant louro-canela should be used with caution, as interaction between the phenolics present in this they showed a pro-oxidant tendency in the lipid specific wood species or the presence of a potent peroxidation assay. antioxidant that has not been revealed hitherto. Alternatives to oak appear to be amendoim and jatobá with respect to both antioxidant power and the sensorial improvement. Further Conclusions studies should be carried out to identify and quantify the phenolic compounds that account The Brazilian wood extracts proved to be more for the remarkable antioxidant activity of the efficient inhibitors of lipid peroxidation than Brazilian woods (amendoim and jatobá). Also, Evaluation of Brazilian woods as an alternative to oak for “cachaça” aging in cask 187 the compounds that account for the observed Cardoso, D. R., Andrade-Sobrinho, L. G., Lima- pro-oxidative tendency of canela-sassafrás, Neto, B. S. and Franco, D. W. (2004). A rapid castanheira, and louro-canela extracts should be and sensitive method for dimethylsulphide identified. Data treatment through multivariate analysis in Brazilian sugar cane spirits and statistical analyses is a promising tool to provide other distilled beverages. Journal of the an authentication of the aged spirits with regard Brazilian Chemical Society 15: 277-281 to wood species used to make the wooden Faria, J. B., Cardello, H. A. B., Boscolo, M., barrels. Isique, W. D., Odello, L. and Franco, D. W. (2003). Evaluation of Brazilian woods as an alternative to oak for cachaças aging. Acknowledgments European Food Research and Technology 218: 1438-2375 The authors are indebted to CAPES (BEX Fengel, D. and Wegener, G. (1984). Extractives. 2476/02-9) and FAPESP for the financial support. In: Wood Chemistry, Ultra-structure and We thank Professor Dr Oswaldo Poffo (IPT-USP), Reactions. Walter de Gruyter, Berlin, pp. Professor Dr Francisco A. Rocco (EESC-USP), 182-222 and Professor Dr John R. Piggott (Strathclyde Hu, M. and Skibsted, L. H. (2002). Antioxidant University) for kindly providing the certified capacity of rhizome extract and rhizome wood samples. knot extract of edible lotus (Nelumbo Nuficera). Food Chemistry 76: 327-333 Litwinienko, G. and Ingold, K. U. (2003). References Abnormal solvent effects on hydrogen atom abstraction. The reaction of phenols Andersen, M. L., Lauridsen, R. K. and Skibsted, with 2,2-diphenyl-picrylhydrazyl (DPPH•) L. H. (2003). Optimising the use of phenolic in alcohols. Journal of Organic Chemistry compounds in foods. In: Phytochemical 68: 3433-3438 Functional Foods, CRC Press, London, pp. Lee, K.Y.M., Paterson, A., Piggott, J.R. and 315-346 Richardson, G.D. (2001). Origins of flavour Bettin, S. M., Isique, W. D., Franco, D. W., in whiskies and a revised flavour wheel: a Andersen, S. K. and Skibsted, L. H. (2002). review. Journal of the Institute of Brewing Phenols and metals in sugar-cane spirits. 107: 287-313 Quantitative analysis and effect on radical Nascimento, R. F., Cardoso, D. R., De Keukeleire, formation and radical scavenging. European D., Lima-Neto, B. S. and Franco, D. W. Food Research and Technology 215: (2000). Quantitative HPLC analysis of 169-175 acids in Brazilian cachaças and various Boscolo, M., Bezerra, C. W. B., Cardoso, D. R., spirits using fluorescence detection of Lima-Neto, B. S. and Franco, D. W. (2000). their 9-anthrylmethyl esters. Journal of Identification and dosage by HRGC of minor Agricultural and Food Chemistry 48: alcohols and esters in Brazilian sugar-cane 6070-6073 spirit. Journal of the Brazilian Chemical Reeder, B.J. and Wilson, M.T. (1998). Mechanism Society 11: 96-90 of reaction of myoglobin with the lipid Cardoso, D. R., Andrade-Sobrinho, L. G., Leite- hydroperoxide hydroperoxyoctadecadienoic Neto, A. F., Reche, R. V., Isique, W. D., acid. Biochemistry Journal 330: 1317-1323 Ferreira, M. M. C., Lima-Neto, B. S. and Sasso, M. G., Quina, F. H. and Bechara, E. H. Franco, D.W. (2004). Comparison between (1986). Ruthenium (II) tris(bipyridyl) ion as cachaça and rum using pattern recognition a luminescent probe for oxygen uptake. methods. Journal of Agricultural and Food Analytical Biochemistry 156: 239-243 Chemistry 52: 3429-3433 188 D.R. Cardoso et al.

Schwarz, K., Bertelsen, G., Nissen, L. R., Singleton, V. L. and Rossi, J. A. (1965). Colorimeter Gardner, P. T., Heinonen, M. I., Hopia, of total phenolics with phosphomolybdic- A., Huynh-Ba, T., Lambelet, P., McPhail, phosphotungstic acid reagents. American D., Skibsted, L. H. and Tijburg, L. (2001). Journal of Enology and Viticulture 16: Investigation of plant extracts for the 144-158 protection of processed foods against lipid Vancocelos, Y. (2003). Cachaça sem mistério. oxidation. Comparison of antioxidant assays Revista Pesquisa Fapesp 87: 74-77. based on radical scavenging, lipid oxidation and analysis of the principal antioxidant compounds. European Food Research and Technology 212: 329-328 Physical-chemical and sensory evaluation of brazilian sugar cane distilled alcoholic beverage 189 Chapter 26 Physical-chemical and sensory evaluation of Brazilian sugar cane distilled alcoholic beverage

P.H.A. Silva Departamento de Tecnologia de Alimentos, Universidade Federal de Viçosa, Viçosa-MG, Brazil

Introduction The concentration of a compound in a food and its odour threshold must be considered Cachaça is an alcoholic beverage made from in order to ascertain the significance of aroma yeast fermentation of sugar cane juice. Cachaça compounds in foods. In general, the core of the is widely produced and consumed in Brazil. The total odour intensity of alcoholic beverages are physical-chemical and sensory characterisation compounds present in low concentrations but and standardisation of the beverage is lacking. with very low thresholds, among them several Traditional Brazilian sugar cane brandy, esters and carbonyl compounds. commonly referred to as cachaça, is a distilled The methods of production of cachaça differ alcoholic beverage made by yeast fermentation greatly from region to region in Brazil, especially of sugar cane juice. Cachaça is a popular and with respect to material preparation, yeast generally cheap alcoholic beverage and it has types and distillation processes. The distilled been sold in great volume and widely consumed beverage is eventually matured in native wood in Brazil. According to the Brazilian Association casks for variable periods of time, and sold of Beverage Producers, the domestic production in bottles. Although produced and consumed output reached 1.2 billion liters in 1990. The widely in all of Brazil, cachaça production is international market of cachaça is very small, not standardized, mainly due to the strains of comprising only 0.2 % of the total production, Saccharomyces cerevisiae used in each batch, although in recent years, international sales have contamination levels, quality of raw materials, improved. process conditions and other technological The quality of cachaça is regulated by parameters. Brazilian federal legislation which establishes Even without standardisation, products the maximum total concentration for some originating from inductive processes or natural groups of compounds, such as ethanol, esters, processes are supposed to have distinct volatile acidity, higher alcohol, methanol, and compositions, resulting in typical beverages with copper. The purpose of these limits are mainly distinctive and pleasant flavors. In this study, to control the toxicity of the beverage but their the physical-chemical composition of several variation even in small concentrations should commercial brands of cachaça was analyzed impart a particular flavour to different brands as a way of evaluating the significance of the or samples. composition with regard to quality control and 190 P.H.A. Silva standardisation of the product. In a second part, by titration after evaporation of the total volatile a study was conducted to establish a lexicon of acids, water and other volatiles. descriptors for the aroma and flavor of Cachaça Higher alcohol was assayed spectrophoto- and then conduct a test to determine if any metrically and expressed as isoamyl alcohol. flavour and aroma differences exist between The colouring reaction was made by dimethyl- nine different brands of cachaça. amino benzaldehyde, and the results expressed as the fusel oil concentration. Ester contents was analyzed by stoichiometric titration of excess of Methods sodium hydroxide not used for saponification of esters (Lanarv). Copper content was analyzed by Physical-chemical analysis of cachaça atomic absorption spectrophotometry.

Sampling Statistical analysis

Duplicate samples of one-liter bottles were Assuming that there was no bivariate normal collected of 31 commercial brands coming from distribution, the correlation of the parameters different distilleries. The samples were collected was evaluated by Spearman’s rank correlation at the market or at the manufacturing plant. In (Conover, 1980). terms of technical specifications, labels of the brands mentioned only alcohol content. Most of the samples were from small producers in Minas Results and discussion Gerais, a state in Southeastern Brazil. The results of cachaça analysis are summarized Analytical methods in Table 1. Among the samples analysed, only two presented an ethanol content below the The methods used were the same as those minimum established by legal regulation. Most adopted by the official Brazilian survey section of the samples had ethanol concentrations in for food and beverage quality control (LANARVA the range of 42 to 50 °GL. The wide range of - Laboratório Nacional de Referência Vegetal, ethanol concentration, from 38 to 54 % (v/v) is Ministry of Agriculture, Brazil) following the established worldwide and accepted as standard Official Methods of Analysis of AOAC (1995). for most distilled alcoholic beverages. Alcohol contents were assayed by a density Parameters with contents over the maximum meter. The samples were analyzed at 15°C, and level allowed were volatile acidity (9/31), higher the results expressed the ethanol content in % alcohol (8/31) and copper (7/31). There was large (v/v). For analytical purposes, the ethanol content variation in the concentration of compounds is considered in the following parameters analysed among the samples (Table 1). The calculations, since the final concentration of volatile acidity concentration of the samples was compounds is presented by 100 mL of ethanol distributed equally in all ranges when within the at 100 % (100 mL AA). legal limits. The largest number of samples that Volatile acidity was assayed by titration contained esters fell within the range of 0.04 with standardized sodium hydroxide using and 0.16. phenolphthalein as the indicato. Acetic acid The only correlated variables were esters and was used as a reference and the results were volatile acidity (r = 0.705, two sided p-value = expressed in mg of acetic acid per 100 ml of 0.0001 from the Spearman’s rank correlation ethanol. The volatile acidity is the difference test). The acidity evaluated represents the total between the total acidity, measured for the amount of acids present in samples. The titration intact samples, and the fixed acidity, measured technique is that normally used for alcoholic Physical-chemical and sensory evaluation of brazilian sugar cane distilled alcoholic beverage 191

Table 1. Number of samples of compounds falling within different concentration levels.

Compounds (units) N Number of samples that fell within ranges specified

Ethanol 31 < 38 38.1 – 42 42.1 – 46 46.1 – 50 50.1 – 54 > 54 (% v/v) (2) (2) (14) (12) (1) (0) Volatile acidity 31 0 – 0.03 0.031 – 0.06 0.061 – 0.09 0.091 – 0.12 0.121 – 0.15 > 0.15 (mg/100 mL AA) (4) (5) (5) (4) (4) (9) Esters 31 0 – 0.04 0.041 – 0.08 0.081 – 0.11 0.121 – 0.16 0.161 – 0.2 > 0.2 (mg/100 mL AA) (1) (11) (4) (9) (4) (2) Higher Alcohols 31 0 – 0.06 0.061 – 0.12 0.121 – 0.18 0.181 – 0.24 0.241 – 0.3 > 0.3 (mg/100 mL AA) (0) (2) (11) (6) (4) (8) Copper 31 1.0 1.1 – 2.0 2.1 – 3.0 3.1 – 4.0 4.1 – 5.0 > 5.0 (ppm) (4) (3) (9) (5) (3) (7)

N = Total number of samples analysed.

beverages, and in the case of distilled beverages, should be related to the sensory attributes and the fixed acidity should be negligible. The ester technological production parameters. Other contents also represented also the total amount Brazilian quality surveys of cachaça have been of esters present in samples. carried out (Chaves and Póvoa, 1992; Vargas Cachaça composition with the minimum and and Glória, 1995; Yokota, 2005). maximum values of the compounds analysed are presented in Table 2. The variation of these parameters is vast and the organoleptic differences Sensory evaluation of cachaça between these samples should be investigated. It has been shown (Minim and Silva, 1996) that Procedures for the evaluation and the relationship between total composition and establishment of a sensory profile of cachaça sensory preference seems to be affected more by minor compounds than by total composition. Nine samples of cachaça were purchased Narrow limits of concentration of compounds in different locations. The samples were should be established to well characterize and produced in various regions throughout Brazil. standardize the product, and these standards Six individual panelists voluntarily participated

Table 2. Maximum and minimum concentrations found among the samples analyzeda.

Compound (unit) Mean concentration Minimum concentration Maximum concentration

Ethanol (% v/v) 45.5 38.0 52.0 Volatile acidity (mg/100 mL AA) 0.11 0.011 0.3 Esters (mg/100 mL AA) 0.115 0.006 0.318 Higher alcohols (mg/100 mL AA) 0.22 0.081 0.426 Copper (ppm) 3.45 0.057 8.89 a The range of ethanol content established by legislation is 38–54°GL. Maximum limits for volatile acidity, esters and higher alcohol are 0.15, 0.20 and 0.30 g/100 mL AA respectively, and for copper is 5.0 ppm. 192 P.H.A. Silva in the test, three men, and three women. The ethanol, sherry, solvent, smoky, woody, brown descriptive panel met for five one-hour training spice, nutty, woody, pine, cedar, vanilla, sessions. The training sessions were conducted musty, earthy, lactic, plastic, cardboard, estery/ within the descriptive analysis room of a Sensory tropical fruit, pear, peach, caramelised sugar, Laboratory. The panelists received a total of two dimethyl sulfide (canned corn or green beans), samples per session, served one at a time and minty, menthol, cooling, burning, sweet, and labeled with a random three-digit code. The astringent. panelists evaluated the aroma and flavour of each The most prominent aroma attributes based on sample and assigned descriptive terminology mean intensity ratings in descending order were: for the attributes of each of the samples. Aroma ethanol, smoky, solvent, woody, plastic, estery/ standards were developed that best represented tropical fruit and pear. The prominent flavour the specific attributes. Flavour standards were attributes were: ethanol, plastic, smoky, cooling, not provided. burning, solvent and woody (see Figures 1-6). During each training session, panelists discussed between themselves the appropriateness of the descriptors being used and evaluated the Conclusions quality of the descriptive standards prepared for them. Starting with the second session, panelists No standards have been established for physical- were asked to familiarize themselves with chemical and sensory qualities of cachaça. the prepared standards before evaluating the The quality of the beverage must be evaluated samples. mainly by its toxicology, but the sensory The standards were utilized as a tool for attributes can characterise and distinguish the calibrating the panelists with the selected beverage. There is concern about the ability attributes. By the third training session panelists to differentiate the sensory quality of samples were given intensity standards, allowing them to between total concentration of one or more calibrate themselves with a universally accepted groups of compounds, one class of compound or 15 point intensity scale (0 = no detection and a specific fraction of compounds. In any case, the 15 = extremely high). Panelists recorded their presence of fractions of esters, acids, aldehydes, findings on a paper ballot. alcohol, ketones, furfural and phenols are very The testing took place within the Testing important. Most of these compounds have been Booths of the Sensory Science Laboratory within identified in distilled alcoholic beverages. It had the Food Science Department at OSU. The been suggested that quality of cachaça should panelists were in private individual booths and be related to a ratio between higher alcohol and had no interaction with each other. The testing ester contents equal or near to one. The origin of was conducted over a one-week period with the cellular formation of higher alcohol is related panelists testing two samples per session and up to the keto acids formed from transamination to two sessions per day with a 30-minute break reactions and amino acid metabolism. The between sessions. The aroma standards were principal higher alcohols are formed in valine, placed in 120-ml glass jars with plastic lids and leucine and isoleucine metabolism, but the served at room temperature and stored in a 0°C amino acid group must also include the aromatic cold room between each use. family, tyrosine, phenylalanine and tryptophan. The highest levels of these alcohols have been found to occur at growth-limiting assimilable Results nitrogen levels when biosynthetic formation of amino acids is more prominent and higher The following lexicon of 26 descriptors were alcohol overflow is more intensive (Ryder and established for the aroma and flavour attributes: Masschelein, 1983). Physical-chemical and sensory evaluation of brazilian sugar cane distilled alcoholic beverage 193

a 4.5 4.0 3.5 3.0 b 2.5 2.0 bc 1.5

Mean rating bc bc bc bc 1.0 c 0.5 c 0.0 VA 5HGVVPTVBYp Samples

Figure 1. Mean attributes ratings estery/tropical fruit aroma. Samples with same letter above bars are not significantly different from each other.

4.0 a 3.5 ab 3.0 2.5 abc abc abc 2.0 1.5 bc

Mean rating 1.0 c c c 0.5 0.0 VA 5HGVVPTVBYp Samples

Figure 2. Mean attribute ratings pear aroma. Samples with same letter above bars are not significantly different from each other.

a 2.5 ab 2.0

1.5 abc 1.0

Mean rating bc c 0.5 c c c c 0.0 VA 5HGVVPT VB Yp Samples

Figure 3. Mean attribute ratings nutty aroma. Samples with same letter above bars are not significantly different from each other. 194 P.H.A. Silva

7.0 a 6.0 ab 5.0 ab c 4.0 abc ab c 3.0 bc bc b c

Mean rating 2.0 c 1.0

0.0 VA 5HGVVPTVBYp Samples

Figure 4. Mean attribute ratings smoky aroma. Samples with same letter above bars are not significantly different from each other.

3.5 a 3.0 2.5 ab 2.0 ab 1.5 b b Mean rating 1.0 b b b b 0.5 0.0 VA 5HGVVPTVBYp Samples

Figure 5. Mean attribute ratings lactic/sour flavor. Samples with same letter above bars are not significantly different from each other.

2.5 a 2.0

1.5

ab 1.0 Mean rating

b 0.5 b b b b

b b 0.0 VA 5HGVVP TVBYp Samples

Figure 6. Mean attributes ratings cedar flavor. Samples with same letter above bars are not significantly different from each other. Physical-chemical and sensory evaluation of brazilian sugar cane distilled alcoholic beverage 195

The sensory evaluation was a preliminary study References intended to establish a lexicon of descriptors for cachaça, Brazilian rum, and to test if any aroma Chaves, J. B. P. and Póvoa, M. E. B. (1992). A and flavour differences existed between the qualidade da aguardente de cana-de-açucar. In: samples. A group of six panelists were trained Aguardente de Cana:Produção e Qualidade, and tested over a five week period and were edited by M. J. R. Mutton and M. A. Mutton, able to establish a lexicon of 27 descriptors FUNEP, Jabuticabal, SP, Brazil, pp. 93-132 across nine different brands of cachaça. All of Conover, W. J. (1980). Practical Nonparametric the samples did not share all of the attributes. Statistics. John Wiley and Sons, New York, The attributes that were present at some level of USA all samples were: Lanarv, S.D. Métodos de Análise do Laboratório Nacional de Referencia de Produtos de Aroma: ethanol, sherry, solvent, smoky, woody, Origem Vegetal, Ministério de Agricultura, musty, lactic/sour, plastic, tropical fruit/estery, Brazil, www.agricultura.gov.br pear, DMS, mint and menthol. Minim, V. P. R. and Silva, P. H. A. (1996). Avaliação sensorial e das características Flavour: ethanol, sherry, solvent, smoky, lactic/ físico-químicas de aguardentes de cana sour, plastic, cardboard, tropical fruit/estery, comercializadas no Esatdo de Minas Gerais. mint, menthol, cooling, burning, sweet, and Abstracts of the I Simpósio Iberoamericano de astringent. Análise Sensorial, Campinas, SP, Brazil Official Methods of Analysis (1995). AOAC According to Analysis of Variance (ANOVA) International, 16th ed. Gaithersburg, MD and Least Significant Difference (LSD) tests, Ryder, D. S. and Masschelein, C. A. (1983). differences exist between the samples in smoky, Aspects of metabolic regulatory systems and nutty, tropical fruit/estery, and pear aroma physiological limitations with a view to the attributes, and in lactic/sour and cedar flavor improvement of brewing yeast performance. attributes. Proceedings of the EBC Symposium on Biotechnology, pp. 2-29 Vargas, E. A. and Gloria, M. B. A. (1995). Summary Qualidade da aguardente de cana (Saccharum officinarum) produzida, comercializada Enough evidence of a difference between e/ou engarrafada no Estado de Minas samples exists to warrant further research on Gerais. Ciência e Tecnologia de Alimentos the chemical and sensory profiles of cachaça 15:43-46 produced in different regions in order to attain Yokota, S. M. C. (2005). Avaliação sensorial a good standardisation and classification of descritiva de cachaça envelhecida por 18 different types of the beverage. a 24 meses: contribuição para um produto de qualidade da bebida. Thesis of Doctorat, Universidade Federal de Viçosa, Viçosa, Acknowledgement MG, Brazil.

This research was sponsored by FAPEMIG, MG, Brazil. 196 P.H.A. Silva Characterising the volatile compounds in three fractions of distillates 197 Chapter 27 Characterising the volatile compounds in three fractions of distillates commonly recovered during sugar cane spirit processing

P.H.A. Silva Departamento de Tecnologia de Alimentos, Universidade Federal de Viçosa, Viçosa-MG, Brazil

Introduction acceptance in several countries. Producers are expecting an accelerated growth until the end Cachaça is the typical and exclusive type of of this decade, with exports possibly reaching sugar cane distilled alcoholic beverage produced 42 million liters of the product. in Brazil, with alcoholic content of 38 to 48 % Cachaca production comprises three basic (v/v) at 20°C and unique sensory characteristics. operations: unrefined sugar cane grinding to Brazil has an official production of approximately obtain the juice, alcoholic fermentation and 1.5 billion liters per year. In Minas Gerais, then distillation, for separation and recovery of southeastern Brazil, there are around 8500 the volatile substances. During the distillation cachaça factories, distributed throughout the process in non-continuous stills, the distillate state. The main characteristics of these factories can be sorted out into head, heart and tail are the use of a natural fermentation, without fractions. The head fraction accounts for around chemical ingredients and distillation in simple, 10% of the total volume of the distillate, with non-continuous stills in a single stage. Such a high content of toxic volatile compounds cachaça has been considered to be of the best (acetaldehyde, methanol, higher alcohols, etc). sensory quality against the industrialised product The heart or body fraction accounts for around made in large volumes). 80% of the distilled volume, the part used for Brazilian cachaca consumption ranks consumption, while the tail fraction accounts for third among distilled worldwide, 15 to 20% of alcohol, and is characterized by after only vodka and soju or sochu. Average low ethanol and high acidity. The head and tail world consumption is 2.2 liters per inhabitant fractions may be mixed and added to wine for per year while in Brazil, the average is 11 further distillation, being thus recycled during liters, approximately, based only on cachaça the production process. consumption. Similar to wine in Italy, whisky in The secondary compounds generally present Scotland, and beer in Germany, Brazil has been in cachaça may be grouped into the following making headway in the production of cachaça. organic classes. It is the second most consumed in the country. Acids – organic acids originating from cachaça Cachaça export has attracted the attention production, namely: acetic, lactic, butyric, of entrepreneurs and the government due to its succinic, and other acids. The high acidity in 198 P.H.A. Silva cachaça may be attributed to contamination of and the alcohols turn oily. Some of them sugar cane or of the fermentative wort by acetic strongly resemble the aroma of flowers. They are acid and other bacteria, either during storage of called fusel oil, which, in excess, decrease the sugar cane or in the juice itself, metabolising part commercial value and quality of cachaça. Similar of the substrate and product and thus increasing to methanol and ethanol, these alcohols also acidity and decreasing ethanol production. have biological properties, acting as depressors of the central nervous system. However, they do Aldehydes – aldehyde production may be the not cause acidosis or lesions in the retina. result of yeast activity during the preliminary According to some workers, the degree of stages of the fermentation process. The main esterification during distillation and the content one is acetaldehyde, which tends to disappear of the higher alcohols in the distillate are due to at the end of the process due to oxidation into distillation, regardless of type of equipment used. acetic acid. These are very volatile compounds, However, other authors report that the type of of acute odour, affecting the aroma of alcoholic distillation or equipment used affects the final beverages; are intermediaries of the alcohols composition of volatiles. formed by decarboxylation of oxyacids, or by oxidation of the respective alcohols, as occurs with furfural and hydroxymethyl furfural. Objectives

Esters – esters are formed in esterification The aim of this work was to characterize reactions, between alcohols and carboxylic fermentations conducted in the laboratory acids during the oxidative process. The main using 7 different strains of Saccharomyces ester found in cachaça is ethyl acetate which, cerevisiae, 3 commercial ones and 4 isolated in small amounts, adds a pleasant fruit aroma from industrial fermentors located in Minas Gerais to the drink. However, when in large amounts, state, to evaluate the fermentative process and it confers an undesirable and distasteful flavour. composition of different fractions of distillates The typically pleasant, pungent and suave aroma collected through the conventional system. acquired by cachaça with ageing is a result mainly of the relative formation of aromatic esters, contributing to “bouquet” formation. Materials and methods

Higher Alcohols – these are alcohols containing Around 350 Kg of sugarcane early variety more than two carbon atoms, being formed during RB855156 was harvested with soluble solid the fermentative process and originating to a large content of approximately 20°Brix. The raw extent from amino acid metabolism. Their formation material was obtained at the Experimental Field is favoured by the presence of poor fermenting of the Universidade Federal de Viçosa (UVF) and yeasts, low pH and high wort temperature. immediately ground after harvest. The sugarcane juice was sent to the Laboratory of Fermentation Alcohols with up to five carbon atoms give of the Department of Food Technology of UFV, characteristic odours traditionally associated with where it was diluted to 15°Brix and subdivided distilled liquors. They are directly responsible for into stainless steel fermentation vessels of 15 the odour in the spirit and have characteristic liters for the start of the fermentative process. aromas, the most outstanding being the amyl The juice was inoculated with 7 different and propyl alcohols and their respective Saccharomyces strains, each vessel receiving a isomers. Their formation is based on amino acid different inoculum, 4 isolated from the cachaça metabolism by the yeasts. As the number of distillery of Minas Gerais and the 3 recently carbons increases, aroma changes substantially available commercial strains. Characterising the volatile compounds in three fractions of distillates 199

The yeasts were previously propagated in fractions were analyzed for their contents of 150 mL of sugarcane juice and kept at 23°C ethanol (alcohol degree), volatile acidity, higher under constant stirring for approximately 24 h. total alcohols and quantitative chromatographic They were then transferred to 500 mL and later analysis for isobutanol, isoamyl alcohol and 1500 mL, under the same conditions. The juice 1-propanol. used to prepare the ferments was autoclaved to ensure purity of the culture. Inoculation time in the 15 L vats was fermentation zero time when Results and discussion the analyses were initiated. The fermentative processes were followed As an example, data on sugar consumption, cell by measuring the consumption of the carbon growth, and ethanol and acidity production of substrates (fermentable sugars) and subsequent fermentation number 5 are presented below formation of the product(s). Fermentation (Figures 1 to 4). changes were monitored at intervals of 8 hours. After 70 h of fermentation using 7 strains, The following parameters were evaluated: the worts were selectively distilled, and the composition of their three fractions is shown in • Sugar content by refractometric index Figures 5 to 10. With the adopted methodology, (ºBrix); the average ethanol concentrations were • Microbial counting with a microscope; observed to be the following: 52.5 oGL in the • Alcoholic content by density at 20ºC head, 41.1 oGL in the heart, and 27.7 °GL in applying the picnometer method; the tail. For volatile acidity (in mg of acetic acid • Volatile acidity by titration. for 100 mL anhydrous alcohol (AA)) the values averaged 77.29, 101.15 and 175.25 in the head, For the alcoholic content and volatile acidity heart and tail fractions, respectively. The values analyses carried out during the fermentation found for the higher alcohols averaged 723.21, process, the samples were distilled in a Gibertini 306.85, and 97.30 mg by 100 mL anhydrous Vade 3 Analytical distiller. alcohol for the fractions described. After fermentation was concluded, the These values are summarised in Table 1 fermented worts were distilled with selective and lead to the following interpretations. It was collection of the head, heart and tail fractions, observed that in the heart fractions, few samples in volumes of 10%, 80% and 10% of the exceeded the maximum limits established by the estimated distillate volume. For distillation, a legislation, with such values being highlighted copper container with a cooling coil system in the Figures by a horizontal line, while in the similar to the industrial pot still was used. These respective discardable fractions, in which the

20

15

10 ºBri x

5

0 020406080 Time (h) Figure 1. Brix consumption according to fermentation time. 200 P.H.A. Silva

160

120 cells/ml) 6 80

40

Cellular growth (10 0

01020304050607080 -40 Time (h)

Figure 2. Change of cell growth according to fermentation time.

10

8 6 Amostra 5 4

2 Ethanol degree (°GL)

0 020406080 Time (h)

Figure 3. Change of ethanol according to fermentation time.

100

50

0 Acidity (mg acetic acid/100 ml AA) 020406080 Time (h)

Figure 4. Acidity increase according to fermentation time. Characterising the volatile compounds in three fractions of distillates 201

Head Heart Tail 60

50

40

30 °G L

20

10

0 1234567 Samples

Figure 5. Ethanol level in the three fractions of distillate.

Head Heart Tail 250

200

150

100

50 mg acetic acid/100 ml A

0 1234567 Samples

Figure 6. Acidity in the three fractions of distillate.

Head Heart Tail 1200

1000

800

600

400

200 Higher alcohols (mg/100 ml AA) 0 1234567 Samples Figure 7. Total higher alcohols in the three fractions of distillate. 202 P.H.A. Silva

Head Heart Tail 8000 7000 6000 5000 4000 3000 2000

Chromatogramme surface 1000 0 1234567 Samples

Figure 8. Isobutanol concentration in three fractions of distillate.

Head Heart Tail 25000

20000

15000

10000

5000 Chromatogramme surface

0 1234567 Samples

Figure 9. 1-Propanol identified by gaseous chromatography in the fractions head, heart and tail.

Head Heart Tail 80000 70000 60000 50000 40000 30000 20000

Chromatogramme surface 10000 0 1234567 Samples

Figure 10. Isoamyl alcohol identified by gaseous chromatography in the fractions head, heart and tail. Characterising the volatile compounds in three fractions of distillates 203 compound groups are concentrated, these values The concentrations found in the various fractions were clearly exceeded. must be evaluated regarding their impact on drink flavour (concentrations x threshold), Table 1. Positive or negative average variation and toxicological aspects, to verify if the in ethanol, volatile acidity and higher alcohol commonly applied practices are adequate or concentrations of the three distillate fractions. the technological process could be adjusted to improve the quality of cachaça. Some predictive Variation Head/Heart Heart/Tail mathematical models must also be established Ethanol -21.7% -32.7% regarding volatile compound recovery. Volatile acidity +23.5% +43.0% Higher alcohols -57.5% -68.0% 204 P.H.A. Silva Sensory implications of modifying distillation practice in Scotch malt whisky production 205 Chapter 28 Sensory implications of modifying distillation practice in Scotch malt whisky production

F.R. Jack, J.M. Brosnan, K.A. Campbell, O. Fagnen, R.N. Fotheringham and I.C. Goodall The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK

Introduction Finally distillers may want to change distillation parameters in order to manipulate Flavour is key to the consumer acceptability of the flavour character of their spirits, such as Scotch Whisky. It is used widely in the promotion increasing the intensity of green/grassy notes or and differentiation of products, with sales and decreasing oily characteristics. marketing departments throughout the industry There are a large number of possible changes eager to highlight the particular taste, aroma that can be made during distillation. Rather than and mouthfeel characteristics of their whiskies. trying to cover all of these, this paper gives two Every distiller wants to produce predictable, examples of parameters that might be altered controlled and consistent spirit, which when for economic or environmental reasons; 1) matured will give a whisky with the desired distillation temperature and 2) amount of copper flavour profile either for sale as a single malt or contact during distillation. for use in blending. Spirit quality is likely to be more consistent if no major changes are made to the distillation process. However, distilling, like Case Study 1: Sensory implications of all other UK manufacturing industries, is under modifying distillation temperature increased pressure to minimise the environmental impact of its production, by cutting energy use Introduction and reducing co-products. As the price of fuel continues to rise in the UK, energy is also an ever Distillation is one of the key stages in the increasing economic driver. Hence, changes in Scotch Whisky production process where distillation practice are inevitable. energy consumption is high. Stills are heated Shifts in spirit character can occur due to to a temperature to allow boiling and this factors other than distillation, e.g. due to use temperature must be maintained for several of a new type of yeast or changes in other raw hours, over the length of distillation. Since the materials. A good understanding of distillation stills are not insulated, a large amount of energy and its role in flavour may help to address such is lost in the form of radiant heat. issues, allowing distillation parameters to be One potential means of conserving energy is altered in a controlled manner to bring spirit through the modification of still heating regimes. character back in line. 206 F.R. Jack et al.

There are two main heating methods used in the effects that would occur using standard laboratory industry, direct fired stills and in-direct heating. distillation techniques. The resulting spirits were The base of the direct fired still sits on a hearth or assessed by the SWRI Sensory Panel. furnace, with either coal or gas used as the heat source. The in-direct heated still has an internal coil, pan or kettle, through which super heated Sensory evaluation of distillates steam, generated using an oil or gas fired boiler, is passed (Nicol, 2003). Direct firing results in Triangle Tests revealed that with each 20°C localised high temperatures, while with in-direct increase in temperature there was a significant heating the temperature is more uniform. Over change in the aroma of the spirit, with a rise recent years the industry has tended to favour in- in temperature corresponded to an increase in direct heating systems as these are more energy heavy, cereal, sulfury notes (Table 1). efficient. Other potential means of conserving energy are to decrease the length of time that the still is running by increasing temperature Implications of modifying distillation and hence distillation rate, or to insulate the still. temperature All of these possible modifications will cause changes in the temperature profile through the Even small differences in distillation temperature course of distillation. can have a significant flavour impact. Therefore, Industry observations have revealed that changing from direct firing to in-direct heating, changes in distillation temperature can have a altering distillation rate, or insulating the considerable impact on spirit quality. Lab-scale still/stills, is likely to result in a shift in spirit work has been carried out at the Institute to character. provide insights into the flavour changes that occur. Case Study 2: Sensory implications of reducing copper contact during Lab-scale distillations at various temperatures distillation by removal of copper from A range of temperatures (60°C, 80°C, 100°C & one of the distillation stages 120°C) were chosen to mimic the wide range of possible still surface temperatures found Introduction in current distillation practice. Wash samples were heated to each of these temperatures In Scotch Malt Whisky production both stills and held under reflux for 1 hour. The samples are constructed entirely of copper. It is widely were then vacuum distilled at 57°C. Vacuum recognised that this copper plays an essential distillation allows the spirit to distil at a reduced role during distillation in the formation and temperature, overcoming the further thermal removal of flavour congeners, and hence has

Table 1. Sensory comparisons of the distillates produced at different temperatures. Temperature Triangle Probability of Description comparison Test difference (no. correct/total)

60 vs. 80°C 68/96 P<0.001 Rise in temperature gave an increase in heavy, cereal, sulfury notes 80 vs. 100°C 48/102 P=0.003 Rise in temperature gave an increase in heavy, cereal, sulfury notes100 vs. 120°C49/90 P<0.001 Rise in temperature gave an increase in heavy, cereal, sulfury notes Sensory implications of modifying distillation practice in Scotch malt whisky production 207 a major influence on the flavour profile of the resulting spirit (Reaich, 1998). However, with Pungent environmental issues coming under increased Clean Sulfury 1 scrutiny, the high levels of residual copper in Stale Meaty distillery by-products (pot ale and spent lees) that 0.5 result from using copper wash and spirit stills Sour Fruity/estery may become a concern in the future. Although it 0 is acknowledged that copper cannot be removed Oily Solventy completely from the distillation system, this study posed the question of whether or not copper Sweet Green/grassy contact is required during both distillation stages in order to produce the desired flavour Soapy Floral characteristics of Scotch new make spirit. Cereal Feinty

Both stills Spirit still only Wash still only Lab-scale distillations to evaluate the importance of copper in the wash and spirit Figure 1. Flavour profiles of distillates produced stills with copper in both stills, copper in the spirit still only and copper in the wash still only. Three set of lab-scale distillations were set up using industrial wash. still (“spirit still only” sample) an increase in cereal, feinty, sulfury and meaty characteristics • Copper in both the wash (1st) and spirit was observed. This was not unexpected as (2nd) stills – representing current industry copper is known to play an important role in practice the formation and removal of flavour congeners • Copper in the wash (1st) still only during distillation. Although such aromas are • Copper in the spirit (2nd) still only important contributors to flavour complexity, a spirit with dominant levels of these attributes The resulting spirits were assessed by the SWRI would not be acceptable. These observations Sensory Panel. suggest that the complete removal of copper from the wash (1st) distillation would be inadvisable. However, on a more promising note, removal Sensory evaluation of distillates of copper from the spirit (2nd) still (“wash still only” sample) appeared to have minimal impact Quantitative Descriptive Analysis was used to on flavour in this lab-scale study. examine the flavour characteristics of the spirits. Panelists scored the intensities of a number of key attributes and average scores were calculated Implications of reducing copper contact across the panel. The resulting flavour profiles during distillation are displayed in Figure 1. The presence of copper in both stills, The amount of copper in distillery by-products representing industry practice, produced a clean could potentially be reduced by removing distillate with the types of flavour characteristics copper from certain parts of the still system. that might be expected in a new make Scotch However, lab-based research indicates that malt, with fruity, estery, solventy notes. However, removal from some parts of the system will have when copper was removed from the wash (1st) a much greater impact on flavour than others. 208 F.R. Jack et al.

Understanding flavour changes that Identifying the compounds responsible for occur when distillation parameters are key flavour characteristics altered Figure 2 shows the 1st tier of the Scotch Whisky Research Institute’s Flavour Wheel. A copy of the These case studies illustrate that although the full Flavour Wheel, which comprises of 3 tiers of distiller is often under pressure to make changes to attributes, can be found in Jack (2003). distillation parameters, such alterations can have a With the exception of “cask derived”, significant impact on flavour. Although lab-based which is obviously maturation related, these studies can provide a guide to the types of changes are the key flavour characteristics of new make likely to occur, the actual flavour implications in Scotch. The first stage in understanding flavour full scale distillation must be evaluated. However, is to identify the compounds responsible for industrial scale trials can be costly, particularly these attributes. Certain aroma notes are well if they result in undesirable spirit character. understood, e.g. the phenolic compounds that Alternatively, a fundamental understanding of contribute to “peaty” flavours. However, there flavour changes would allow ultimate control are other aspects of flavour, such as “feinty” of spirit quality. Flavour understanding can be and “cereal” characteristics, are not well divided into 3 areas; understood in chemical terms. Developments in instrumentation are allowing greater insights into • Identifying the compounds responsible for the chemical composition of whiskies, with GC- key flavour characteristics MS Olfactometry being a particularly useful tool • Determining the role of distillation in creating/ for the identification of flavour active congeners. modifying flavours Continuing work is allowing gaps in our flavour • U n d e r s t a n d i n g f l a v o u r c o m p o u n d knowledge to be filled in. interactions

s Mou Primary tast

thfeel y

Peat

Nasal effect es Stale Feinty

Sulfury Cereal

Green/Grass Sour y Flora d Oily ve l Este

ry eet

Cask deri Sw

Figure 2. 1st tier of the Scotch Whisky Research Institute’s Flavour Wheel. Sensory implications of modifying distillation practice in Scotch malt whisky production 209

Determining the role of distillation in Table 2. Flavours produced as a result of Maillard creating/modifying flavours reactions between wash amino acids and sugars during distillation. Since the use of artificial flavourings is prohibited in Scotch Whisky production, the flavours Aldehydic Cooked Grassy/ Sweetcorn present in the new make spirit must either come potatoes green from the raw materials or be formed/modified Biscuity Cooked Malty Sour during fermentation and/or distillation. This vegetables paper considers the role of distillation, with the Burnt Damp wool Meaty Stale case studies demonstrating that alterations in Buttery Dead roses Metallic Sulfury distillation parameters can have a considerable Cereal Faecal/ Musty Sweaty impact on flavour. manure In Case Study 1, an increase in heavy, Cheesy Farmyards Nutty Sweet cereal, sulfury notes was observed as distillation Chlorine Floral Oily Toasty temperature increased. This may be due to Chocolatey Fruity Rubbery Toffee an increase in Maillard reactions. As shown in Figure 3, these are temperature dependent Many of these flavours relate directly to the reactions that occur between amino acids and heavy, cereal, sulfury type characteristics that sugars, present in the wash, during distillation. were observed at higher distillation temperatures. Maillard reactions result in the formation of a Some of the congeners responsible for these range of highly flavour active congeners. Since flavours have been identified, such a 2-acetyl- this is heat mediated it can be hypothesised that pyroline (cereal), tri-methyl pyrazine (nutty) the higher the temperature the greater the level and 2,5 di-methyl pyrazine (nutty). Work is of Maillard reactions. Detailed information on continuing to gain further insights into the other Maillard reactions and their role in food quality congeners involved. can be found in Rizzi (1994). Similar flavour changes were observed in Amino Heat Flavour active Case Study 2, with an increase in cereal, feinty, +Sugars acids congeners sulfury and meaty characteristics resulting when copper was removed from the 1st distillation. Wash However, since no temperature alterations were Figure 3. Maillard Reactions. involved a different route of formation is likely, suggesting that similar flavour changes can occur Wash contains a wide range of different amino for different reasons. acids (methionine, cystine, cysteine, arginine, threonine, histidine, proline, tryptophan, tyrosine, phenylalanine, isoleucine, leucine, Understanding flavour compound lysine, asparagine, valine, glycine, alanine, serine, interactions glutamic acid, aspartic acid and hydroxyproline) and sugars (glucose, maltose, maltotriose, Knowledge of the types of flavour active sucrose, fructose, xylose and arabinose), giving congeners present in a spirit and their levels a huge number of possible interactions that relative to threshold (the minimum level required can occur. The range of flavours that can be to evoke a sensory response) is not enough to produced, based on observations from lab-scale fully understand flavour. Possible interactions distillations of individual amino acids and sugars, between compounds should also be taken into are summarised in Table 2. account. For example, although we have a good chemical understanding of sulfur compounds the 210 F.R. Jack et al. levels found in spirit frequently do not tie in with with a compound that smells intense to perceived sulfury character. In the past when one panelist being relatively weak or even interpreting analytical data it was assumed that undetectable to another. The solution containing spirits containing a diverse range and high level a mix of all 21 compounds had the highest of sulfur compounds would be most “sulfury”. average intensity score. This was not unexpected However, recent research suggests that this is as the mix will contain at least some compounds not always the case. that each panelist is sensitive to. Table 4 shows In a sensory experiment, panelists were the maximum score that each panelist gave to asked to score the intensities of 21 individual an individual sulfur compound (shaded boxes in sulfur compounds (presented at approximately Table 3) in relation to their score for the mix. threshold levels) alongside a mix of all 21 Two way analysis of variance, which takes compounds (all present at the same levels that differences in individual panelist’s scoring they occurred in the individual solutions). The strategies into account, showed that the scores data produced is shown in Table 3. allocated to the mix were significantly lower Results showed that panelists vary greatly in than the maximum scores given to individual their sensitivities to different sulfur compounds, compounds (p=0.0082). Therefore, although

Table 3. Intensity scores.

Compound Panelist Mean s.d. A B C D E F G H I J

DMS 1.6 1.6 5.4 1.9 5.4 0.6 3.7 8.3 2.0 0.1 3.08 2.58 DMDS 4.6 2.0 0 0 4.1 0.4 0 1.8 1.0 0.2 1.41 1.72 3-methyl thiopropanol 2.0 7.0 0.2 1.9 3.7 2.0 1.5 7.6 3.8 2.2 3.19 2.40 DMTS 7.3 2.0 5.1 0.6 6.2 1.1 0.8 7.7 2.4 0.2 3.34 2.93 MMFDS 8.0 4.1 4.1 5.9 8.0 8.0 3.3 6.2 6.8 0.8 5.52 2.40 Thiophene 0.9 0.6 0 0 0 0.4 0.8 0 0.8 0 0.35 0.39 2-methyl-thiophene 2.8 2.0 0.1 3.4 3.8 6.1 1.1 9.6 2.0 5.3 3.62 2.78 3-methyl-thiophene 0.1 6.4 0.5 0.1 2.5 1.2 1.1 8.1 0.9 2.2 2.31 2.75 2,5-dimethyl-thiophene0.6 0.2 7.6 0.1 3.1 0.8 0 0.2 1.5 1.3 1.54 2.33 Thianapthene 1.6 8.0 6.7 0 8.0 2.0 0.8 0.4 2.0 1.5 3.10 3.17 3-thiophene-c-aldehyde9.5 7.7 4.8 3.8 9.2 6.0 5.6 8.8 6.8 9.6 7.18 2.09 2-thiophene-c-aldehyde1.0 2.0 4.0 0.5 6.6 0.7 3.9 8.7 3.2 0.6 3.12 2.78 5-me-2-thiophene-c- 0.6 2.2 1.6 1.4 1.0 0.6 0 7.3 4.9 6.8 2.64 2.69 aldehyde Ethyl-2-thiophene- 0 3.3 0.3 3.0 6.5 0 2.8 7.4 3.7 0 2.70 2.70 carboxylate Thiazole 0.1 0 2.2 0 3.0 0.5 0.1 2.8 0.6 0.2 0.95 1.22 4-methyl-thiazole 8.1 8.0 6.0 3.3 5.1 6.1 6.8 8.4 5.4 4.3 6.15 1.70 5-methyl-thiazole 0.8 0.1 1.8 1.5 1.6 2.0 1.4 0.9 5.1 2.3 1.75 1.34 4,5 dimethyl thiazole 2.0 0.2 2.0 0.6 1.6 0.8 0.1 3.5 1.5 0.4 1.27 1.06 4-methyl-5-vinyl 9.6 5.9 5.8 1.0 6.4 6.0 5.3 8.9 8.4 8.3 6.56 2.47 thiazole Benzothiazole 5.2 3.0 0.5 3.4 6.1 2.0 0.6 1.5 2.5 8.0 3.28 2.46 4-methyl-5-thiazole- 0.6 0.4 0.6 0.2 0.8 0.6 6.1 1.8 0.3 0.2 1.16 1.80 ethanol Mix of all 21 compounds 9.2 8.1 5.9 4.8 8.9 6.0 5.0 8.8 8.1 9.6 7.44 1.83

Shaded boxes denote maximum score given to an individual compound by each panelist Sensory implications of modifying distillation practice in Scotch malt whisky production 211

Table 4. Maximum intensity scores for individual compounds vs. scores for mixes.

Scores Panelist A B C D E F G H I J

Maximum score for individual sulfur compound 9.6 8.0 7.6 5.9 9.2 8.0 6.8 9.6 8.4 9.6 Score for mix of all 21 sulfur compounds 9.2 8.1 5.9 4.8 8.9 6.0 5.0 8.8 8.1 9.6

more individuals are likely to identify a sulfury formation will ultimately offer improved control note if there is a greater range of sulfur compounds of spirit quality, allowing the distiller to predict in a spirit, the effect on intensity is not an additive the sensory implications of any modifications that one, i.e. presence of more sulfur compounds might be required. However, new make spirit does not equate to increased sensory response. flavour is complex, with congener interactions In fact sulfur compounds appear to suppress also playing an important role, so there is still the aromas of one another. Further research has some way to go before this is achieved. shown that other congeners in the spirit can also have a suppressive effect, e.g. 2-pentyl furan References suppresses the aroma intensity of DMTS. This example of sulfur compound interactions Jack. F.R. (2003). Understanding Scotch Whisky is unlikely to be unique. Changes in the distillation flavour. Food Science and Technology 14: process may alter the levels of congeners that 28-30 might not be flavour active themselves but Nicol, D.A. (2003). Batch distillation. In: Whisky: could impact on the perception of key flavour Technology, Production and Marketing. compounds. Such interactions must not be Edited by Russell, I., Academic Press, UK, overlooked when trying to understand new make pp.155-178 spirit flavour. Reaich, D. (1998). The influence of copper on malt whisky character. In: Proceedings of the Fifth Aviemore Conference on Malting, Conclusions Brewing & Distilling. Edited by Campbell, I., Institute of Brewing, UK, pp. 141-152 Although distillers are often under pressure to Rizzi, G.P. (1994). The Maillard reaction in modify distillation parameters, due consideration foods. In: Maillard Reactions in Chemistry, must be paid to the possible flavour impact Food and Health. Edited by Labuza, T., that any such changes might have. A greater Reineccius, G., Monnier, V., Baynes, J. and understanding of new make spirits flavours, O’Brien, J., The Royal Society of Chemistry, the congeners responsible and their routes of UK, pp. 11-19. 212 F.R. Jack et al. Characterisation and differentiation of peat used in the preparation of malt for scotch whisky production 213 Chapter 29 Characterisation and differentiation of peat used in the preparation of malt for Scotch whisky production

B.M. Harrison1, 2, K.J.G. Reid1, G. M. Steele1 and F. G. Priest2 1The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK; 2International Centre for Brewing and Distilling, Heriot-Watt University, Edinburgh, UK

Introduction

Burning peat during the kilning of malted barley phenols alone may not be a reliable indicator of produces a smoke or “peat reek”, the constituents peated character. of which are adsorbed or absorbed by the malt The peat used by the Scotch whisky industry and impart distinctive flavour characteristics is currently sourced from various locations across to the spirit. These flavours survive maturation Scotland, particularly from the north east of the and are key quality attributes for certain Scotch mainland and the islands of Islay and Orkney. whiskies (Swan and Howie, 1983). It is known The chemical composition of peat is influenced that peating introduces numerous different types by the type of vegetation from which the peat of chemical compound (Deki and Yoshimura, is derived as well as the level of decomposition 1974a; Deki and Yoshimura, 1974b; Deki and (Bozkurt, Lucisano, Moreno and Neretnieks, Yoshimura, 1974c). For example, phenols, 2001). However, prior to this study no research such as guaiacol and cresols, are particularly has been carried out to determine whether or important for the peaty flavour of malt whisky not peat from different areas of Scotland differs (Swan & Howie 1983). Indeed, investigations in chemical composition. Such knowledge is carried out on wood smoke condensate where of interest to the whisky industry as variation measures were taken to remove non phenolic in composition could potentially result in compounds showed that the essential smoke differences in the flavour of the resulting spirit. odour was retained (Fiddler, Doerr, Wasserman The aim of this study was to establish whether and Salay, 1966). As such, in the Scotch whisky peat sourced from four geographically distinct industry the level of total phenols has traditionally areas; Islay, Orkney, north east mainland (St been used as a predictor of peated character. It Fergus) and north central mainland (Speyside) must be noted however, that whatever the could be distinguished by Fourier-transform magnitude attributable to phenols, other types infrared (FT-IR) spectroscopy. Subsequently, of compound such as furans, carbonyls and these differences were characterised using nitrogen containing compounds are all known to Curie point pyrolysis-gas chromatography-mass contribute to smoke flavour (Maga, 1988) so total spectrometry (Py-GC-MS). 214 B.M. Harrison et al.

Materials and methods peat in a kiln. The pyrolysis sample carrier is a ferromagnetic material which heats up in a very Sampling short time inside a magnetic field to a defined Curie-Point temperature. In this work samples Samples were taken from the six major peat were pyrolysed at 610°C as this temperature sources used by the Scotch whisky industry: has been reported previously to facilitate the Gartbreck moss, Islay (labelled B), Glenmachrie complete pyrolysis of peat (Van Smeerdijk and moss, Islay (L), Castlehill, Islay (P), St Fergus, Boon, 1987). The products of pyrolysis were Aberdeenshire (N), Tomintoul, Speyside (T) then identified using GC-MS. and Hobbister Hill, Orkney (H). At each site, 20-24 peat samples, representative of the current extraction areas, were taken from the same depth Results and discussion along transect lines. At two sites, L and H, samples were also taken from depth profiles (Lp0- Lp3 FT-IR and Hp0- Hp4, where sample 0 was taken from the peat surface and subsequent samples were All peat samples were subjected to FT-IR analysis taken at regular intervals down to the deepest and the resulting spectral data analysed using level of peat). Also, samples of industrially cut Principal Component-Discriminant Function peat were taken at each site (labelled ind). All Analysis (PC-DFA). In PC-DFA, each spectrum samples were air dried and ground to a finely is formerly assigned to a definite class, in this divided dust prior to analysis. case sampling site, and spectral data are then treated to highlight a relationship between these data and the class considered. The first two FT-IR discriminant functions showed a clear separation of the two mainland peat sites, St. Fergus and FT-IR is a vibrational spectroscopic technique Tomintoul, from the four island sites (Figure which measures the absorbance of infrared 1). Also, samples from Orkney were partially light by molecules. FT-IR is a rapid, automated, separated from Islay samples. non-destructive technique, not biased to any Samples were taken from stocks of industrially particular group of chemicals and so gives cut peat at each site to see how their composition a ‘holistic’ overview of the sample under related to that of the transect line samples. These investigation. This method was therefore well samples were used in the PC-DFA shown in suited to fingerprinting a large set of peat Figure 1. They all co-localised with the transect samples. samples from their respective sites showing that industrially cut peat shared similar chemical characteristics with the transect samples. Py-GC-MS This suggested that the transect samples were representative of the peat currently being Analytical pyrolysis in combination with GC-MS extracted from each site. has previously proved very useful for the chemical Generally, the samples taken from different characterisaton of recalcitrant macromolecules depths at sites H and L were found to be correctly such as those found in peat (Van Smeerdijk and classified to their respective sampling sites Boon, 1987; Stout, Boon and Spackman, 1988; suggesting that peat depth has little effect on site Kuder, Kruge, Shearer and Mills, 1998). The peat specific chemical characteristics. It was notable, sample is thermally degraded during pyrolysis. however, that whilst the samples taken from the Therefore, this method, in some ways, reflects surface of the Orkney and Islay depth profiles the process undergone during the burning of (Hp0 and Lp0) were not misclassified they did Characterisation and differentiation of peat used in the preparation of malt for scotch whisky production 215

5

Htb0 *Htb0 4 Tt7 *Hta 0 Tt9 *Lp0 *Hta6 Hta0 *Tt7 Hta6*Htc 0 *Tt-2 *Tt9 *HHttaa22 Htc0 Tt-2 Tt8 Lp0 *Hta 5 Ht*NHta5t7a3Ht c4Htc7 *TTt1t100 3 Hta4 *Hta3*H*Htc4tc7 Htc5 *T t17 Tt16 Hp4 *Htc5 *Tt8 Htc6Hta1 *T t16 *Tt6 *B*Ht8 p4 Hi*Hndind Tt17 Tind Tt6 *Hta4 *H*Bttb16 Htc1*Htc 1 Tt1Tt154 Tt13 Bt 10 *HHttc3c3 Ht*Hb6tc6 *T t15 *Tind 2 *H*Btta 810 *Tt1*T2 t1*T t13 *Hp3Hp3*Ltb4 Hta8 *Hta1 Tt-Tt01 Tt1Tt1T *Tt12 1t14 Bt8 Bt15 *Tt-1 *Tt1t41 Ltc2 *LLtap13 *H*HtbHpta272 Tt3 *Bt*Bt119 *HBtHt1tc *Hb2Ht2Hta7tbb44 TTt2t4 *Lta3 *B t1Ltb34 *Htb5 *Bt4 Htb3 *TTt5t5 1 Lt b3 Bt7 Bt*LBt6 tb2 1*Bt1*B6t15 *Hp2 *Hp0Hp0 *Tt3 Lt c7 Ltc 1 *LLtata5Ltb*L*Btta22 3 Htb5 *Tt0 *Ltc*B1 t7Lp 1*L ta*L6BtBttb11Bt4*BtLt0 a29 6 BtHt16c2*Htb3 *L*Ltap20Lt b6LtLa6p2 Bt3 *B t2 *Ltc7 Lt b5 Bt 13Lt a1 *T t2 L*ta4LtaLtLtc6b14Lt*Ltb*Lc0Ltbtc506 *Bt14 0 Lta*Bt7 18*Lta1 LtaBt018 *LBttb146 Bt 12*LLttcc303Bt 19 *Bi*Pndt9 *Bt12 LtcBt05 Pt9 *Nt11 Ltb7 Lind*BBt*Lit1179nd Bind *Nind Bt5 *Lp3 *Nt0 -1 *LLtctb47 Lp3 *Pt10 *P in*Btd 17 NtNt6 5 *Bt0 *PPt10t14 Nt2 Nind *LtcPt*P4*Bt0t0 5 Pi nd PtPt*1Pt 7 1 Discriminant function 2 -2 Pt14Pt8 Nt0 *Nt6 *Nt4 Pt*P13t12 Nt3 Pt19*Pt18 *Pt6Pt 2*P t11*Pt1t73 *Pt16 Pt6 Nt-1 Nt4 Pt18 *Pt2 Pt4 PtPt1511 NtNt-37*N 11t-2 *P t3 Pt3 Pt1216 Pt5 Nt*N-4t10 *Nt1 -3 *Pt5 Nt-2 Nt*N1 t-1 *Nt3 *Nt15 *Nt9 Nt10 Nt15 Pt17 *Nt-3 Nt138 -4 Nt9 Nt14 *Nt12 Nt12 -5 -6-4-20246 810 Discriminant function 1 Figure 1. PC-DFA plot of all the peat samples encoded with knowledge of the six geographical locations from which the peat was collected. For sample labels the uppercase letter indicates the site, the lower case letter a transect (t) or depth profile (p), and the number indicates the sample number. Where more than one transect was taken (sites H and L), the transect number is indicated by the letters a, b and c. Commercial samples are labelled “ind”. Circles represent 95% χ2 confidence limits. locate outside the 95% confidence intervals (e.g. pyrroles and pyridines) and polyphenolic of their respective classes (Figure 1; indicated derivatives which could be further divided into by arrows). This finding was probably due to specific lignin markers (guaiacyl and syringlyl samples Hp0 and Lp0 consisting of relatively compounds) and non-specific compounds, undegraded plant matter found on the surface such as catechols, p-hydroxy phenols and other of the peat deposits. aromatic compounds. Principal Component Analysis (PCA) was used to summarise the differences in chemical Py-GC-MS composition. The four geographical locations were separated using the first two principal Three peat samples representing each of the components (Figure 2). By examining the PCA four clusters (Orkney, Islay, St Fergus and loadings for this plot, it was possible to show Tomintoul) identified using FT-IR were selected which compounds were characteristic of each for Py-GC-MS analysis. 130 peat pyrolysis site (Figure 2). products were initially identified using GC-MS. The first principal component, which Analysis of variance (ANOVA) was subsequently described 58.6% of the variance, separated used to identify pyrolysis products which areas predominantly according to the ratio of showed significant differences from location to polysaccharide derivatives to lignin derivatives location. 79 compounds matched this criterion. (guaiacyl and syringyl compounds). St Fergus These compounds could broadly be split samples were characterised by high percentages into three groups: polysaccharide derivatives of lignin derived compounds whereas Tomintoul (e.g. furan and pyran derivatives as well as samples were characterised by relatively high anhydrosugars), nitrogenated compounds percentages of polysaccharide derivatives. This 216 B.M. Harrison et al.

6 P P N P N N N N N P N 4 P

) C G Orkney P Islay G C P P 2 P P G G G P N G P P C S P C C 0 S GG C C C P P S P P GS S G GS SS P S G SSG Component 2 (15.7% 2 Component CCC G G St Fergus -2 C CC P P S S C CC G C C -4 Tomintoul

-6 0 10 20 Component 1 (58.6%) Figure 2. Principal Component Analysis of Py-GC-MS data for selected peat samples. Loadings are overlaid on the sample distribution. Coding for loadings: C = polysaccharide derivatives, P = polyphenolic derivatives, N = nitrogenated compounds, G = guaiacyl compounds, S = syringyl compounds. difference in chemistry may reflect the different such as toluene, styrene and ethylbenzene vegetational inputs to peat from different areas. were also found in increased proportions in the For example, Sphagnum moss contains a high Orkney and Islay samples. These compounds concentration of polysaccharides and also may represent the products of further lignin is thought to lack the guaiacyl and syringyl degradation (Stout et al., 1988). compounds characteristic of the lignin found in Also found in increased abundance in the higher plants (Vanbreemen, 1995). Thus peat island peat samples were several nitrogen derived largely from Sphagnum moss would be containing compounds. This may provide further expected to contain a correspondingly low lignin evidence for the island peat samples having to polysaccharide derivative ratio. It may also undergone increased levels of biodegradation be caused by peat from different areas having as it has been shown previously that nitrogen undergone varying degrees of decomposition compounds tend to accumulate in more as it is known that during peatification there degraded peat (Williams 1983). The source of tends to be a preferential accumulation of lignin these compounds is uncertain but one possibility derived material relative to polysaccharides is proteins and chitinous components found in (Stout et al., 1988). lignin-degrading fungi (Stankiewicz, van Bergen, The distribution along the second principal Duncan, Carter, Briggs and Evershed, 1996; van component showed that several oxidised lignin Heemst, van Bergen, Stankiewicz and de Leeuw, derivatives, such as acetovanillone and vanillin, 1999; Bennett, Lager, Russell, Love and Larter, were found to be more abundant, relative to 2004). the other lignin derivatives, in Orkney and Islay peats. This may reflect an increase in fungal degradation of lignin in these island samples Conclusions which is known to involve the oxidation of lignin monomer side chains (Kirk 1971). Furthermore, FT-IR spectroscopy coupled with DFA allowed several polyphenol derived aromatic compounds the classification of peat according to sample Characterisation and differentiation of peat used in the preparation of malt for scotch whisky production 217 origin. Curie-point pyrolysis coupled with on the volatile components of peated malt. GC-MS revealed the chemical characteristics II. Identification of neutral components. responsible for the classification. It would appear Chemical and Pharmaceutical Bulletin 22: that important characteristics include the ratio 1754-1759 of lignin derived compounds to carbohydrate Deki, M. and Yoshimura, M. (1974c). Studies derived compounds, the types of phenolic on the volatile components of peated compounds present and also the levels of malt. III. Identification of acidic and basic nitrogen compounds. The implication is that peat components. Chemical and Pharmaceutical from different origins may contribute different Bulletin 22: 1760-1764 chemical characteristics to peated malt and, Fiddler, W., Doerr, R. C., Wasserman, A. E. and therefore, to peated Scotch whisky. The next Salay, J. M. (1966). Composition of Hickory stage in this work will be to elucidate what the Sawdust Smoke. Furans and Phenols. effect of using peat from different sources for Journal of Agricultural and Food Chemistry Scotch whisky production has on the flavour of 14: 659-662 the resultant spirit. Kirk, T. K. (1971). Effects of microorganisms on lignin. Annual Review of Phytopathology 9: 185-210 Acknowledgements Kuder, T., Kruge, M. A., Shearer, J. C. and Miller, S. L. (1998) Environmental and botanical We are grateful to Suntory Ltd for financial controls on peatification - A comparative support to Barry Harrison, to Dr R. Goodacre study of two New Zealand restiad bogs using (University of Manchester) for assistance with Py- GC/MS, petrography and fungal analysis. FT-IR analyses, Dr J Conner (Scotch Whisky International Journal of Coal Geology 37: Research Institute) for assistance with Py-GC-MS 3-27 analyses and to the peat companies for access Maga, J. A. (1988). Smoke in food processing. to peat samples. CRC Press, pp. 53-54 Stankiewicz, B. A., van Bergen, P. F., Duncan, I. J., Carter, J. F., Briggs, D. E. and Evershed, References R. P. (1996). Recognition of chitin and proteins in invertebrate cuticles using Bennett, B., Lager, A., Russell, C. A., Love, G. D. analytical pyrolysis/gas chromatography and Larter, S. R. (2004). Hydropyrolysis of and pyrolysis/gas chromatography/mass algae, bacteria, archaea and lake sediments; spectrometry. Rapid Communications in insights into the origin of nitrogen compounds Mass Spectrometry: RCM 10: 1747-1757 in petroleum. Organic Geochemistry, 35: Stout, S. A., Boon, J. J. and Spackman, W. (1988). 1427-1439 Molecular aspects of the peatification Bozkurt, S., Lucisano, M., Moreno, L. and and early coalification of angiosperm Neretnieks, I. (2001). Peat as a potential and gymnosperm woods. Geochimica et analogue for the long-term evolution Cosmochimica Acta 52: 405-414 in landfills. Earth-Science Reviews 53: Swan, J. S. and Howie, D. (1983). Sensory 95-147 and analytical studies on the regional Deki, M. and Yoshimura, M. (1974a). Studies composition of Scotch malt whiskies.In: on the volatile components of peated malt. Current developments in malting, brewing I. Identification of phenolic compounds. and distilling, Edited by F. G. Priest and I. Chemical and Pharmaceutical Bulletin 22, Campbell, Institute of Brewing, London, 1748-1753 pp. 129-142 Deki, M. and Yoshimura, M. (1974b). Studies Van Heemst, J. D. H., van Bergen, P. F., 218 B.M. Harrison et al.

Stankiewicz, B. A., and de Leeuw, J. W. Vanbreemen, N. (1995). How Sphagnum bogs (1999). Multiple sources of alkylphenols down other plants. Trends in Ecology and produced upon pyrolysis of DOM, POM Evolution 10: 270-275 and recent sediments. Journal of Analytical Williams, B. L. (1983). The nitrogen-content and Applied Pyrolysis 52: 239-256 of particle-size fractions separated from Van Smeerdijk, D. G. and Boon, J. J. (1987). peat and its rate of mineralization during Characterisation of subfossil Sphagnum incubation. Journal of Soil Science 34: leaves, rootlets of Ericaceae and their 113-125. peat by pyrolysis-High-Resolution Gas Chromatography-Mass Spectrometry. Journal of Analytical and Applied Pyrolysis 11: 377-402 Understanding and enhancing cask performance 219 Chapter 30 Understanding and enhancing cask performance

K.J.G. Reid, J.M. Conner, F. Jack, M. Patterson and J. Freeman The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK

Introduction these casks are removed from the system. A proportion of these exhausted casks may be There is little documented evidence explaining sent to the cooperage for regeneration and then why, historically, the process of maturation re-introduced back into the cask population. became normal practice in the manufacture of However, the regeneration process currently most whiskies produced today. However, the does not reproduce cask activities that replicate most likely explanation must be related to the either a first fill ex-bourbon or ex-sherry cask. need for storage capacity, and the most suitable Consequently, the number of regenerated casks container at the time would have been the oak that can be used is limited. or wooden cask. In the United States where large This paper discusses the work The Scotch reserves of white oak were available, whiskies Whisky Research Institute has undertaken to were normally stored in new casks. However in understand why the maturation performance Scotland, where the supply of oak was limited, of a regenerated cask is different from a first fill second-hand containers were used. For example, cask. In addition, the paper describes a patented American bourbon and rye whiskies are matured heat treatment process known as ‘Cask Curing’, in new charred oak casks, while whiskies developed by the Institute, which is capable produced in Scotland are mainly matured in of enhancing the maturation performance of a oak casks previously used for the maturation of cask. bourbon, or the fermentation and shipment of sherry (Booth, Shaw and Morhalo, 1989). Ex- bourbon casks are in fact the most common type Understanding cask performance of container used for the maturation of whiskies today, as the United States is currently the largest The maturation performance of a cask is producer of casks worldwide. heavily influenced by the degree of heating In Scotland, ex-bourbon and ex-sherry casks it undergoes during its initial construction are used repeatedly (termed re-fills) until they can or during regeneration should the cask have no longer adequately mature whisky in a realistic reached the point of exhaustion. The main effect timescale, at which point they are regarded as of heat treatment is to degrade the polymeric exhausted. On reaching the point of exhaustion constituents of the wood, namely, lignin, 220 K.J.G. Reid et al. cellulose and hemi-cellulose, to form colour and whisky. However, while maturation of a Scotch flavouring compounds. Studies in the past have malt whisky in a new charred oak cask may focused mainly on lignin degradation to produce produce a well-matured whisky, it may not be aromatic aldehydes and acids, such as vanillin readily identifiable as Scotch (Clyne, Conner, and vanillic acid. More recent research however, Piggott and Paterson, 1993). has highlighted the importance of polysaccharide When a cask is regenerated the ‘target’ of the breakdown (cellulose and hemi-cellulose) in the process is to re-activate the cask to a condition formation of a number of flavour compounds. that is the same as, or similar to, a fresh ex- The main class of compounds produced from bourbon or ex-sherry cask. However, re-charring polysaccharide degradation are furaldehydes, the cask will thermally degrade the polymeric which in themselves have little sensory impact. constituents of the wood to produce colouring However, their formation is accompanied by that and flavouring components at concentrations of many other molecules with sweet, caramel and that exceed those available from ex-bourbon toasted aromas. Maltol and 2-hydroxy-3-methyl- or ex-sherry wood. In contrast, the ‘free 2-cyclopentenone were identified in toasted extractives’ that are available from oak wood, oak by Nishimura, Ohnishi and Masuda (1983). such as oak lactones or hydrolysable tannins, More recently, furylhydroxymethyl ketone and have been removed by previous cask use and 2,5-furandicarbaldehyde have been identified in are not available to be regenerated to any great extracts of toasted oak, being formed by the direct extent. Consequently, the availability of these pyrolysis of sugars (Cutzach, Chatonnet, Henry components will be less that those available from and Dubourdieu, 1999). Extracts also contained ex-bourbon or ex-sherry wood. This ‘flavour 2,3-dihydromaltol, furaneol, and 2,3-dihydro- imbalance’ compared to ex-bourbon and ex- 3,5-dihydroxy-6-methyl-4H-pyran-4-one, which sherry wood can be observed in the subsequent are the products of Maillard reactions between maturing spirits, hence the reason why only a amino acids and sugars (Cutzach, Chatonnet, limited amount of regenerated wood is currently Henry and Dubourdieu, 1997). used within the Scotch whisky industry. When a cask is charred or re-charred, an ‘active’ carbon layer forms on its inner surface, as a result of carbonisation of the Methodology polymeric constituents. This layer contributes little in the way of colour or extractives to Gas Chromatography–Olfactometry the maturing whisky. However, it does play an important role in the removal of immature There is not an ideal sample preparation characteristics. Previous work has shown that technique for gas chromatography–olfactometry cask char promotes the oxidation of dimethyl (GC-O) analysis of whiskies. While headspace sulphide (Fujii, Kurokawa and Saita, 1992) and techniques such as purge and trap and solid phase may reduce the concentration of other sulphur micro-extraction are more representative of the compounds by a combination of adsorption and volatiles sampled during sensory assessment, the oxidation (Philp, 1986). information obtained from the chromatographic Combining the ‘subtractive’ activity of the trace is often limited by the small amounts of cask char with the ‘additive’ activity of the colour high boiling point congeners, such as wood and flavouring compounds (present deeper in the extractives, present. An alternative is to use wood), a particular flavouring profile will have solvent extracts of whiskies. While this gives been produced which may suit the style of one greater sensitivity for wood extractives, it alters whisky better than another. For example, in the the balance of extractives present and can lead United States a new charred oak cask produces to enhanced responses for volatiles such as the desired flavour profile for bourbon or rye guaiacol, furaneol and sotolon due to thermal Understanding and enhancing cask performance 221 degradation reactions in the heated injector of mass spectrometric detection scanning 35 to the gas chromatograph. Consequently when 400 amu at 2 scans per second. extracts are used for GC-O, then further analysis To give a better representation of the levels is required to establish the concentration of the of the less volatile compounds present, such as compound present and how this relates to its cask derived extractives, extracts were prepared odour threshold, so a proper assessment of its as for the GC-O analysis. These extracts were odour impact can be made. analysed in duplicate using a programmable Extracts were used for the GC-O comparison temperature vaporising injector to minimise of whiskies matured in re-fill ex-bourbon and thermal degradation reactions in the injector. regenerated casks. These were analysed using The initial injector temperature was 40°C a HP5971 GC-MS fitted with a sniff-port. increasing to 240°C within a splitless time of Congener separation used a 60m DB-WAXetr 0.8 minutes. Congener separation used a 30m (polyethylene glycol) column with the effluent SolGel-WAX column which, due to its greater from the column split between the odour port thermal stability, allowed the detection of and the mass spectrometer in the ratio of 5 to 1 higher boiling point wood constituents such as in favour of the odour port. At the odour port, coniferaldehyde and sinapaldehyde. column effluent was mixed in the nosing cone with a make-up gas (nitrogen at 30 ml/minute) and humidified air. Each sample was assessed Results in duplicate and the time, description and an indication of aroma intensity recorded for To examine the chemistry and sensory impact aromas eluting between 7 and 40 minutes. The of the regeneration process, 3 year old grain accuracy of the times recorded at the nose port whiskies matured in both re-fill ex-bourbon compared with the retention times of peaks on and regenerated casks were analysed by gas the mass chromatogram was confirmed using a chromatography-mass spectrometry (GC-MS) number of identified peaks with a characteristic and gas chromatography-olfactometry (GC-O), aroma, e.g. isoamyl acetate, ethyl octanoate, and by sensory evaluation using Quantitative 2-phenylethanol, cis-oak lactone and vanillin. Descriptive Analysis (QDA). Figure 1 displays To reduce the possibility that wood aromas the flavour profiles of the two whisky types. On have been mis-identified due to the co-elution of the right hand side of the radar diagram flavour different congeners, the extracts were re-analysed descriptors relating to the new make spirit can using a 60m DB-1701 (cyanopropyl phenyl be observed, such as feinty, cereal, sulphury, silicone) column. soapy and oily. On the left hand side maturation related descriptors can be observed, such as spicy, woody, sweet and dried fruit. Gas Chromatography–Mass Spectrometry The profile of whisky matured in the re- fill casks appears to be balanced relatively The initial sample comparison used headspace evenly from left to right. In contrast, the profile solid phase micro-extraction (SPME) coupled of whisky matured in regenerated casks has with gas chromatography–mass spectrometry. shifted significantly to the left showing less Samples were diluted to 20% ethanol (v/v), as new make and higher maturation related for sensory analysis, internal standard added character. The severity of this flavour shift is and 6 ml sealed in a 10 ml headspace vial. probably exaggerated in this case as the flavour The samples were then analysed in duplicate. characteristics of new make grain whisky is An 85 µm polyacrylate fibre was used with an relatively light in comparison to malt whisky, adsorption time of 10 minutes at 30°C. Congener allowing the flavour impact of the cask to be separation used a 60m DB-WAXetr column with more apparent. 222 K.J.G. Reid et al.

Pungent analysis of the two whiskies. Comparison of the 1.5 total ion chromatograms showed a large number Spicy Feinty of differences, with all but 5 compounds showing 1 significant differences between regenerated and Woody Cereal refill ex-bourbon whiskies. The five compounds 0.5 showing no significant difference were all low boiling point compounds that have previously Sweet Sulphury been identified in new make grain spirits. The majority of the remaining congeners detected by this analysis were esters and alcohols that are Dried fruit Soapy common constituents of Scotch malt whiskies. The source of these esters was the residue from Fresh fruit Oily the malt whisky that had been previously held in the cask (‘in-drink’). Quantitative analysis Solventy Re-fill (ex-bourbon) showed that the most abundant congeners in the De-char/Re-char re-fill ex-bourbon samples were ethyl octanoate, Figure 1. Radar diagram showing the sensory ethyl decanoate and 2-phenylethanol, with profiles of 3 year old grain whisky matured in concentrations around 1 mg/L. Concentrations regenerated (de-char/re-char) and re-fill (ex- in the regenerated cask sample were between bourbon) casks. 0.1 and 0.3 mg/L. The lower level of these compounds in the whisky matured in the To examine the chemical differences between regenerated casks may have been a result of the whiskies, caused by the type of cask used, the de-charring and re-charring process, which GC-MS and GC-O was carried out. Figure 2 would effectively remove the majority of any displays an overlay of the headspace GC-MS spirit residue that was present. RT: 10.00 - 45.00

100 Re-fill (ex-bourbon) 95 90 De-char/Re-char 85 80 75 70 65 60 55 50 45

Relative Abundance 40 35 30 25 20 15 10 5 0 10 15 20 25 30 35 40 45 Time (min) Figure 2. Overlay of gas chromatography chromatograms from the headspace analysis of 3 year old grain whisky matured in regenerated (de-char/re-char) and re-fill (ex-bourbon) casks. Understanding and enhancing cask performance 223

A number of compounds were detected at chromatogram. Of these, half had previously been higher levels in the regenerated cask sample identified as components of new make whiskies including the wood components cis- and trans- (both grain and malt) and are most likely to be oak lactone, diethyl hydroxysuccinate, eugenol components of the new make spirit, or to have and iso-eugenol (2-methoxy-4-(1-propenyl) carried over from spirits matured in previous phenol). However, the recorded peak areas fills of the cask. Previous research has shown of these compounds were very low and other that although components that carry-over can potential wood aroma compounds (guaiacol, give rise to strong aromas in GC-O analysis, the 4-ethylguaiacol, 4-vinylguaiacol and vanillin) amount present in the matured spirit is generally were not detected by the headspace SPME not sufficient to have a significant impact on analysis. the aroma of the product (Conner, Reid, and The headspace analysis however, does not Richardson, 2001). However, a number of cask provide any significant information regarding derived compounds, which were important the levels of cask derived extractives present in contributors to aroma, were identified. Several the whiskies. To obtain this information a liquid of these are highlighted in Figure 3. extract analysis was carried out (Figure 3). Once To compare the relative amounts of these again comparing the overlays of the resulting compounds, a radar diagram was produced (in a chromatograms highlighted a considerable similar way to the sensory data displayed in Figure number of chemical differences. GC-O was then 1). However, in this case the data were based on used to examine the aroma characteristics of the peak area measurements as opposed to sensory individual chemical components. Comparison scores (Figure 4). In every case, higher levels were of the recorded aromas with the total ion present in the whiskies matured in the regenerated chromatogram tentatively identified 32 of the casks. These chemical data correlate well with sixty recorded aromas with peaks on the mass the sensory data discussed previously. However,

RT: 30.00 - 50.00

100 Re-fill (ex-bourbon) 95 90 De-char/Re-char 85 80 75 vanillin 70 65 60 55 50 45 cis oak lactone

Relative Abundance 40 35 30

25 diethyl hydroxysuccinate isoeugenol 4-vinylguaiacol 4-ethylguaiacol 20 eugeno l guaiacol trans oak lactone 15 10 5 0 30 32 34 36 38 40 42 44 46 48 50 Time (min) Figure 3. Overlay of gas chromatography chromatograms from the liquid extract analysis of 3 year old grain whisky matured in regenerated (de-char/re-char) and re-fill (ex-bourbon) casks. 224 K.J.G. Reid et al.

vanillin

11 10 cis oak lactone 9 guaiacol** 8 7 6 5 4 3 trans oak lactone* 2 4-ethylguaiacol* 1 0

diethyl hydroxysuccinate* 4-vinylguaiacol**

iso-eugenol** eugenol* Re-fill (ex-bourbon) De-char/Re-char Figure 4. Radar diagram showing the relative amounts of 9 cask derived ‘flavour active’ compounds in 3 year old grain whisky matured in regenerated (de-char/re-char) and re-fill (ex-bourbon) casks. the data displayed in Figure 4 represent only Throughout its development, the Cask a proportion of the cask derived components Curing process has had to meet a number of that are contributing towards the aroma of the different criteria. Firstly, it had to comply with whiskies. GC-O data revealed a number of other the definition of Scotch whisky and secondly flavour active compounds that contributed to the utilise existing cooperage equipment available aroma of the whisky matured in the regenerated in Scotland. After several years of development casks. Many of these were described as sweet, the process was tested in 20 separate full size caramel, toasted and are likely to have originated cask trials. The spirits being matured ranged from the thermal breakdown of the carbohydrate from new make malt and grain spirits through constituents of the oak wood. These compounds to poorly matured malt whiskies. A summary of have not as yet been identified. the results from the trials is set out below along with examples of the different applications that the Cask Curing process has been used for. Enhancing cask performance

At the Institute, research into whisky maturation Experimental cask trials has, for a number of years, been examining how the maturation potential of a whisky cask could Within the trials, two types of cask were be enhanced. These efforts have resulted in examined. The Curing process was applied to the development of a patented heat treatment casks being regenerated with a view to both process known as ‘Cask Curing’, which enables enhancing the performance and expanding the enhanced levels of natural flavouring and number of applications for which this type of colouring compounds to be released from the cask can be used. The technique was also used oak. The technique simply involves treating the to condition new ends for remade hogsheads inside surface of a cask with an aqueous solution (containing fresh ex-bourbon staves) with a view of a food grade salt catalyst prior to firing in to enhancing the performance and extending the the cooperage, allowing more efficient thermal lifetime of this type of cask (i.e. 3 parts fresh ex- breakdown of the wood and therefore release of bourbon to 1 part new Cask Cured wood). extractives into spirit. Understanding and enhancing cask performance 225

For each cask regeneration trial, the exhausted spirits as demonstrated in Figure 5. This example casks were initially de-charred using a rotating shows the level of colour in whiskies from Cured brush or flail system. The cask wood surface being and re-charred casks increasing almost three fold Cured (cask shell, or ends in the case of remade over whiskies that were matured in casks that had hogsheads) was then treated with a solution of only undergone re-charring. Re-toasting casks food-grade salt catalyst. The wood was allowed in conjunction with Curing increased the level to dry and then either charred or toasted. Charring of colour in the resultant whiskies by a factor of was carried out using standard cooperage gas four over the control spirits. burners, to the point where the cask shell ignited Shelf life studies have also shown that the for 30 seconds. Cask shells were toasted using a colour delivered by the Curing process has wood fire in a brazier for 45 minutes. Cask ends similar stability characteristics to colour derived for remade hogsheads were toasted electrically from standard maturation casks, in that the rate using medium wave infra-red heating. Prior to of colour fade was the same under ambient filling, all casks were rinsed with water to remove lighting conditions. When compared to colour any residual salt. added to whiskies through the use of spirit The standard format for the trials was to divide caramel, the rate of colour fade of the caramel the exhausted casks into three equal groups. Two was approximately five times faster that of the groups were treated with the Curing solution, and natural cask derived colour. then one group charred and the other toasted. In a similar manner to the release of colour, The remaining untreated control group either the Curing process also consistently delivers underwent the typical regeneration process for higher levels of cask derived extractives to cask regeneration trials or consisted of standard maturing spirit, as demonstrated in Figure 6. remade hogsheads with plain (non-heat treated) In this example, a poorly matured malt whisky ends for remade hogshead trials. The three groups was ‘finished’ in Cured/regenerated casks over were then filled with the appropriate spirit. a 6 month period, improving the quality of the whisky significantly. From casks that had been Cured and re-charred, Colour and cask extractives the increase in the level of extractives over the controls (re-charring only) was approximately Regenerated casks two fold in relation to the concentration of extractives on filling. However, in whiskies A typical characteristic of the Cask Curing process finished in Cured and re-toasted casks the level is its ability to deliver significantly enhanced of cask extractives increase approximately levels of natural cask derived colour to maturing five fold. This particularly high increase in the

30 Re-charred controls Cured & re-charred Cured & re-toasted 20 (EBC )

Colour 10

0 0612 18 24 30 36 42 Maturation Time (Months) Figure 5. Time-series graph of colour development in malt whisky maturing in Cured and regenerated casks. 226 K.J.G. Reid et al.

50 Re-charred controls Cured & re-charred 40 Cured & re-toasted

30

20

10 Cask extractives (mg/L)

0 0510 15 20 25 Maturation time (weeks)

Figure 6. Time-series graph of uptake of casks extractives into malt whisky ‘finished’ in Cured and regenerated casks. level of extractives produces significant flavour in this case using medium wave infra-red heating, differences in the maturing spirits which may or delivered much more colour and cask extractives may not suit the particular style of whisky being to the maturing spirit. The level of extractives produced (see Sensory Evaluation below). increasing 3 fold and colour approximately 3½ fold compared to that which was achieved by the control casks. Remade hogsheads

Figure 7 shows results from one trial where Sensory evaluation a 5 year old malt whisky was transferred into hogsheads with Cured and heat treated ends for Figure 8 displays the sensory profiles of 3 year 6 months. Casks with Cured and charred ends old grain whisky matured in regenerated casks had more than double the levels of both colour that had been de-charred and either re-charred and cask extractives in the whisky compared (controls), Cured/re-charred or Cured/re-toasted. to the increase achieved by the control casks The shifts in the profiles away from the controls constructed with plain non-heat treated ends. are typical of the flavour changes caused by the One again Curing in conjunction with toasting, Curing/heat treatment process. In this example

15 15 Fill Spirit Control (Plain Ends) Cured/Charred Ends 10 10 Cured/Toasted Ends EBC PPM

5 5

0 0 Colour Extractives

Figure 7. Bar chart showing the uptake of colour and casks extractives into a 5 year old malt whisky stored in hogshead casks with Cured and heat treated ends over a 6 month period. Understanding and enhancing cask performance 227 the Cured and re-charred treatment has only Applications for cask curing caused a minor shift in the profile, resulting in slightly lower scores for immature characteristics Through both laboratory and industrial scale such as feinty, sulphury and soapy. In contrast, studies and trials, a number of applications for the sensory scores for some of the maturation the Cask Curing process have been identified related descriptors (spicy, woody and sweet) and can be summarised as follows: increased slightly. • The Cask Curing process in general can Pungent 1.4 enhance a cask’s performance through its Spicy 1.2 Feinty ability to deliver much greater levels of both 1 colour and cask extractives to maturing spirit. 0.8 Woody Cereal These elevated levels of natural cask derived 0.6 colour may also reduce or possibly eliminate 0.4 0.2 the need to use spirit caramel, which is a Sweet 0 Sulphury much less stable alternative.

• The type of heat treatment (re-charring or Dried fruit Soapy re-toasting) after Curing can significantly influence the levels of colour and extractives Fresh fruit Oily released, and as a consequence can have a large impact on the resulting flavour Solventy characteristics of the mature spirit. The Re-charred (Control) treatment applied may therefore have to Cured/Re-charred be matched to the particular spirit being Cured/Re-toasted matured. Figure 8. Radar diagram showing the sensory profiles of 3 year old grain whisky matured in • The Curing process has been successfully regenerated casks that had been de-charred and applied to both the maturation of new make either re-charred (controls), Cured/re-charred or spirits and the quality enhancement of poorly Cured/re-toasted. matured spirits. The process may also be suitable for the maturation of other whiskies The shift in flavour profile caused by the Cured and brown spirits. and re-toasted treatment is however, much more significant. Larger reductions in scores for • The inclusion of new Cured ends in a cask immature characteristics feinty, cereal, sulphury has been shown to be a relatively easy means and soapy were observed, with even larger of enhancing its performance. It is therefore increases in scores for the maturation related likely that the process could also be applied descriptors spicy, woody, sweet and dried fruit. during the preparation of new oak casks for This correlates well with the much greater release spirit maturation. of cask extractives that is caused by the Curing/ re-toasting process (Figure 6). However, it should be noted that the degree of flavour change is Conclusions also influenced by the initial character of the spirit. Therefore it would be necessary to match Improving our ability to correlate analytical and the Curing process with the particular type of sensory data will further assist us in understanding spirit being matured and the desired maturation the impact of heat treatment processes, during characteristics of the end product. cask construction or regeneration, on the 228 K.J.G. Reid et al. flavour characteristics of maturing spirits. Journal of Food Science and Technology At the Scotch Whisky Research Institute, 28: 69–81 progress has been made in understanding the Cutzach, I., Chatonnet, P., Henry, R. and additive and subtractive processes that occur Dubourdieu, D. (1997). Identification of during maturation, and this has assisted us in volatile compounds with a ‘toasty’ aroma in developing a patented process that can enhance heated oak used in barrel making. Journal the performance of a cask through the delivery of Agricultural and Food Chemistry 45: of elevated levels of colouring and flavouring 2217–2224 constituents to maturing spirits. However, the Cutzach, I., Chatonnet, P., Henry, R. and process of maturation is complicated and much Dubourdieu, D. (1999). Identifying new has still to be understood, but through continued volatile compounds in toasted oak. Journal research further improvements in optimising or of Agricultural and Food Chemistry 47: enhancing cask performance may be possible. 1663–1667 Fujii, T., Kurokawa, M. and Saita, M. (1992). Studies of volatile compounds in whisky References during ageing. In: E´ laboration et Connaissance des Spiritueux: Recherche Booth, M., Shaw, W. and Morhalo, L. (1989). de la Qualite´, Tradition et Innovation. Blending and bottling. In: The Science and Edited by Cantagrel, R., Lavoisier-Tec and Technology of Whiskies. Edited by Piggott, Doc, pp. 543–547 J.R., Sharp, R. and Duncan, R.E.B., Longman Nishimura, K., Ohnishi, M., Masuda, M. Scientific and Technical, pp. 295–326 (1983). Reactions of wood components Conner, J.M., Reid, K. and Richardson, G. during maturation. In: Flavour of Distilled (2001). SPME analysis of flavor components Beverages: Origin and Development. in the headspace of Scotch whisky and Edited by Piggott J.R., Ellis Horwood Ltd, their subsequent correlation with sensory pp. 225–240 perception. In: Gas Chromatography – Philp, J.M. (1986). Scotch whisky flavour Olfactometry: The State of the Art. Edited development during maturation. In: by Leland, J., Schieberle, P., Buettner, A. Proceedings of the Second Aviemore and Acree, T., American Chemical Society, Conference on Malting, Brewing and Washington D.C., pp. 113–122 Distilling. Edited by Campbell, I. and Priest, Clyne, J., Conner, J.M., Piggott, J.R. and Paterson, F.G., Institute of Brewing, London, pp. A. (1993). The effect of cask charring on 148–163. Scotch whisky maturation. International Impact of brandy production processes on flavour 229 Chapter 31 Impact of brandy production processes on flavour

B. Colonna-Ceccaldi Pernod Ricard Research Centre, 94015 Créteil Cedex, France

Introduction converted to the corresponding higher alcohol: alanine to ethanol, valine to isobutanol, Grape quality is an important parameter in wine phenylalanine to phenylethyl alcohol. quality and is responsible for the huge diversity • Metabolites produced by the yeast during of wine supply. However, the situation is not fermentation (biosynthesis): part of the exactly the same with brandy as the process is higher alcohols are produced in this way via more complex. the ketoacids pathway. Production by this mechanism depends on fermentation conditions such as temperature, aeration, quantity and Impact of brandy production quality of the available nitrogen etc. processes on flavour • Distillation is mainly separation and concentration of the volatile components. Modern analytical techniques allow for the However some of them can be modified detection of hundreds of volatile compounds during this step, for example esterification in brandy, and, as in any fermented distilled occurs due to heating in an acidic medium, beverage matured in wood, flavours have sulphur compounds are adsorbed by the multiple origins: copper, residual sugar and amino acids react together to form Maillard compounds etc. • Compounds coming from the grape and • Maturation is quantitatively the most important kept unchanged up to the final product. This step: all the non-volatile components and contribution is more important in the case of many of the volatiles in the final product brandy compared to grain-made beverages are extracted from the wood by the hydro- and will be further discussed in depth by the alcoholic medium, some are adsorbed or example of terpene compounds in Pisco and modified by oxidation. All the components the influence of the cru on Cognac. are concentrated because of evaporation • Compounds coming from the grape and (“Angel’s share”). Wood extract is mainly converted to another compound during composed of phenolic compounds coming fermentation (bioconversion): for instance from the degradation of the lignin and more or free amino acids in the must are generally less modified during toasting of the wood. 230 B. Colonna-Ceccaldi

Impact of the raw material on the In Muscat grape varieties, some of the production of Pisco terpenes are in free form, but 2 to 4 times more are in the form of glycosides (Agosin, Pisco is a traditional Chilean brandy obtained Belancic, Ibacache, Baumes, Bordeu, Crawford by distillation of wine produced from Muscat and Bayonove, 2000), not volatile and thus not varieties like Moscatel de Alejandria, Moscatel detectible. It can be monoglycosides (terpene Rosada, Moscatel de Austria, Torontel, Moscatel + glucose residue) or diglycoside (with another Amarilla etc (Faria, Loyola, Lopez and Dufour, sugar linked to glucose) (Figure 2). 2003). In Muscat wine, they can be realised Production occurs in a delimited area in progressively during aging of the wine thus the dry valleys of Northern Chile (Boubals, giving a reservoir of free terpenes. In Pisco, 1979), total acreage is around 10 000 ha for a few hydrolyses occur during fermentation, the production of 4.5 million cases, of which 99% use of purified ß-glucosidase or yeast with this are sold on the domestic market (Bordeu, Formas important activity can increase the free terpene and Agosin, 2004). Grape variety and production content several times (Belancic, Gunata, Vallier area are regulated by a denomination of origin and Agosin, 2003). While these glycosides do dating from 1931. not go through distillation, it seems that some Total sales of Pisco are around $US 120 M chemical hydrolysis occurs in a hot acidic with a market share of 75% of the spirit in Chile. medium thus increasing the total terpene content The local market has been stable for some years, of the distillate (Agosin et al., 2000). with great expectations for future export. The The quantity and nature of the terpenes are very main consumption is as “pisco sour”, mixed with variable depending on the grape variety. Some lime and sugar (and sometimes an egg white), a varieties are actually very poor in terpenes and do very popular beverage in Chile and Peru. not bring much flavour to the final product (Agosin The typical flavour of Pisco is characterised by et al., 2000). For instance, Moscatel de Austria a very fruity aroma due to monoterpenes, mainly (Torrontes Sanjuanino) and Moscatel Negra (black geraniol, linalool and nerol (Figure 1) (Herraiz, Muscat) contain only very few terpenes, despite the Reglero, Herraiz and Loyola, 1990). Pisco is the name (Figure 3). Altogether (the other one being only brandy produced on a large scale from the Pedro Jimenez), these poorly aromatic varieties Muscatel grape, besides very local products like represent almost 50% of the acreage (Table 1). some Moldavian brandy. In contrast, varieties like Moscatel rosada (South

HO

HO

HO

Nérol Géraniol Linalol

Figure 1. The main monoterpenes in Pisco (from Belancic, 2005). Impact of brandy production processes on flavour 231

O CH2 O CH2 R R O O O O CH3

HOH2C

6-O-α-L-arabinofuranosyl-ß-D-glucopyranoside 6-O-α-L-rhamnopyranosyl-ß-D-glucopyranoside

O CH CH2OH 2 R R O O O

CHOH2

6-O-ß-D-apiofuranosyl-ß-D-glucopyranoside ß-D-glucopyranoside Figure 2. The structure of terpenes glycosides (from Belancic, 2005).

American variety, 26%), Moscadel de Alejandria brands or category of Pisco (Agosin et al., 2000) (Muscat of Alexandry, 18%) Torontel (local variety, (for definition of typicity see ‘Characterisation 8%) bring much of the flavour to the final product of the typicity’ in the section below). (Agosin et al., 2000).

Table 1. The acreage (ha) of the main grape Influence of grape ripening varieties used for Pisco production (from Belancic, 2005). In most of the varieties, the free terpene Atacama Coquimob Total concentration reaches a peak around 12-13 Probable Alcoholic Degree (PAD) then decreases Moscatel Rosada 75 2,532 2,607 sharply (Figure 5). A good knowledge of grape Moscatel de Alejandria 215 1,549 1,763 ripening is therefore very important to avoid loss Moscatel de Austria 185 2,106 2,291 of aromas due to over-ripeness. Pedro Jimémez 114 2,201 2,315 Torontel 5 774 779 Moscatel Amarilla 5 58 62 Otras 12 12 Influence of sun exposure Moscatel Blanca 4 19 22 Temprana A trial conducted using shading screens has shown Albilla 2 2 that the full solar light is not optimal for terpenes Total 615 9,239 9,853 formation. (Belancic, Agosin, Ibacache, Bordeu, Baumes, Razungles and Bayonove, 1997): for instance a 75% or 16% increase of the main Despite the similar concentration of terpenes terpenes (linalool, géraniol and nerol) is observed in the two preferred varieties, Moscatel Rosada with Moscatel Rosada and Moscatel de Alejandria and Moscatel de Alejandria, their patterns are respectively when sun exposure is reduced by 50% very different: Moscatel Rosada is especially (Table 2). The difference between the two varieties rich in linalool and its oxides, while Moscatel can be due to the colour of the berry: Moscatel de Alejandria has more nerol and especially Rosada is a red grape variety although Moscatel de geraniol (Figure 4). These differences can be used Alejandria is white. as a tool to bring complexity or typicity in certain 232 B. Colonna-Ceccaldi

Bounds terpenes 5000 4500 Free terpenes 4000 3500 3000 2500 2000 1500

Concentration (µg/L ) 1000 500 0

a

Torontel

Pedro Jimenez Moscatel rosada Moscatel de Austri Moscatel de Alejandría

Figure 3. Terpene concentration in the main grape varieties used for Pisco (from Agosin et al., 2000).

Moscatel de Alejandria 1400 Moscatel Rosada Moscatel de Austria Torontel 1200

1000

800

600

400 Terpens concentration (µg/L) 200

0 l

Nerol Linalo Geraniol L + N + G

Figure 4. The distribution of terpenes in the main Muscatel varieties (from Agosin et al., 2000).

Conclusions Impact of the terroir in the production of Cognac Many parameters can influence the production of terpenes in the grape berry, the most important The concept of ‘terroir’ is central to an being the grape variety, the maturation and understanding of natural wine. It is a word the light exposure. All further steps in the with no exact English equivalent, often roughly process aim to limit the loss of terpenes during translated as ‘sense of place’. Terroir is the fermentation (the slower the better), distillation word given to that part of a wine’s experience and (eventual) aging. which is determined by place. Among the Impact of brandy production processes on flavour 233

Free terpenes Free terpenes 14000 3500 Total terpenes Total terpenes 12000 3000

10000 2500

8000 2000

6000 1500 Terpens (µg/L ) Terpens (µg/L ) 4000 1000

2000 500

0 0

5 9.5 9.5 10.5 11.5 12.5 13. 14.5 10.5 11.5 12.5 13.5 14.5 P.A.D. P.A.D.

Figure 5. The flavour concentration depending on ripeness (from Belancic, 2005).

Table 2. The effect of sun exposure on terpene production in µg/l (from Belancic et al., 1997).

Moscatel rosada Moscatel de Alejandria 100% exp. 50% exp. 20% exp. 100% exp. 50% exp. 20% exp.

Linalol (L) 546 923 397 351 348 136 Nerol (N) 18.5 80 41 48 59 45 Geraniol (G) 118 223 181 332 433 303 L + N + G 682 +75% 1196 619 731 +16% 850 484 Acido geránico 116 188 274 22 332 183 Oxidos de linalol 299 263 143 119 123 48 Dioles terpénicos 166 196 40 13 5.3 7.5 Total Teprenos (13) 1232 1933 1105 891 1314 748 Total Compuestos C6 (5) 1122 2111 1635 876 655 664 Total Alcoholes (2) 402 46 1899 989 1368 668 Total Libres (34) 2872 4536 4838 2986 4311 2526

brandies, Cognac has been recognised for at accordance. All the master distillers in Cognac least 4 centuries as a particularly flavourful spirit (“maître de chai”) are able to discriminate a spirit (Moll and Guyot, 1883), most of the production based on its origin. being exported to improve the flavour of other According to these differences, the Cognac spirits. production area (now 82 000 ha planted in It has always been known that the location total) has been divided into 6 zones or “cru”, where the Cognac is produced has a strong by decrees of the 1st of May, 1909 and the 13th influence on the character of the spirit (Ravaz, of January, 1938 (Figure 6): 1900), and the price was determined in 234 B. Colonna-Ceccaldi

Figure 6. The six “crus” of Cognac (courtesy BNIC, Bureau National Interprofessionel du Cognac).

• Grande Champagne: around the city of Characterisation of the typicity Cognac, the most famous area, 13 000 ha cultivated for Cognac Typicity is a term in wine tasting used to describe • Petite Champagne: close to Grande the degree to which a wine reflects its origins, and Champagne, 15 200 ha cultivated thus demonstrates the signature characteristics • Borderie : a small area in the North of Cognac, of the area where it was produced, its mode of 13 440 ha cultivated production, or its parent grape. Two main groups are defined (Leaute, 1994): • Fin Bois and Bons Bois : respectivly 31 000 on the one side, Grande Champagne, Petite and 9500 ha cultivated Champagne and Borderies are characterised by • Bois Ordinaires: 1100 ha cultivated. a very fruity/flowery flavour, more or less intense and persistent depending on the cru, Grande While it is allowed to sell Cognac with a particular Champagne being the most characteristic. appellation, most of the commercial products are Borderies has the Champagne character but actually blends. The total production is around lighter and with a very typical violet flavour note. 11 million cases, more than 94% being exported In contrast, the Bois are simpler and faster to age, to USA, UK, Asia etc. with less fruity and more “terroir” flavours. Impact of brandy production processes on flavour 235

These differences have been confirmed by less than 1% of the acreage. As almost all the vine systematic organoleptic and chemical analysis: is now Ugni blanc, grape variety cannot be held the first category has less higher alcohols (< responsible for the diversity of Cognac (Cantagrel 3000 mg/l of spirit at 40% (v/v) alcohol), less and Galy, 2003). acetaldehyde, more heavy esters (sum of ethyl caprylate, caprate and laurate > 50 mg/l) and Fermentation: wines produced using Active furfural than the Bois. Work is in progress to Dry Yeast are generally less complex than better characterise these differences, but it those obtained by spontaneous fermentation; is obvious that it is more a matter of balance work in progress aims to produce a specific between chemical categories than a specific blend of yeast for Cognac (Cantagrel and Galy, molecule characteristic of a cru. 2003). Today most of the producers use selected ADY with particular characteristics in terms of congener production… Origin of the typicity Distillation: this step is very strictly regulated: Climate: the Champagne and Borderie area is Double pot still distillation with direct heating, rather dryer and sunnier than the Bois, with the first still being of 25-100 hl capacity, the slightly larger temperature amplitude. second of 25 hl. Some variability can be brought by the cuts and their recycling (in the wine or Soil: the seminal work of Coquand 1860 (see in the “brouilly”), and the distillations of lees or Moll and Guyot,1883) established a good otherwise. correlation between new spirit character and composition of the soil. The main parameter Aging: unlike in whisky, the spirit is transferred seems to be the proportion of chalk in the soil from cask to cask during aging. Generally, the and particularly the structure of this chalk: the first year is spent in a new cask and then the more porous and loose structure, the better. For spirit is transferred to older casks. Variability is instance, Grande Champagne is characterised by introduced by the choice of wood (difference in a layer of very porous chalk at the upper part of porosity), the toasting (heavy or light), the time the earth’s crust, similar to the Champagne wine spent in the cask and the “ouillage” (refilling), area, while in Fins bois and Bon Bois the soil is done with spirit or water. more compact and clay/sandy. This effect has been attributed more to the structure itself than to the actual chemical composition of the soil, Conclusions such a porous arrangement acting like a sponge and a water reserve in summer, thus giving the There is no definitive answer to the question of vine continuous water feeding (Lafon, Couillaud the origin of the typicity, it is clear the soil has and Gay-Bellile, 1973). a strong influence, but other parameters like winemaking, distillation and aging bring also Grape variety: grapes for Cognac production some variability. should give a wine low in alcohol (7-9%), very acidic and clean in taste and flavour (Cantagrel and Galy, 2003). Three main varieties are allowed General conclusions for Cognac production: Ugni blanc (94% of acreage), Colombard (4%), Folle blanche (1%). In the case of Brandy the raw material contribution Five auxiliary varieties are allowed at less than to the organoleptic characteristics of the product 10% of the blend: Semillon, Blanc ramé, Jurançon is more obvious than in the case of grain spirits. blanc, Montil and Select. Today, they account for Sometimes a particular class of molecule is 236 B. Colonna-Ceccaldi responsible for the specifity of the product, like terms for pisco, a young muscat wine the terpenes in Pisco, but most of the time it is distillate. American Journal of Enology and more a matter of balance of different compounds. Viticulture 55: 104-107 Like wine, the contribution of the vine itself is Boubals, D. (1979). Lessons from the present essential, being either the variety or the growth development of Chilean viticulture. Part III. conditions. (Enseignements de l’actuelle évolution de la viticulture chilienne.) Progrès Agricole et Viticole 96: 396-398 Acknowledgments Cantagrel, L. and Galy, B. (2003). From vine to Cognac. In: Fermented beverage The Pisco data is based on the work of Pr Eduardo production, 2nd edition, edited by A.G.H. Agosin and team, Centro de Aromas, DICTUC, Lea, and J.R. Piggott, Kluwer Academic/ Pontificia Universidad Católica de Chile. I am Plenum Publishers, New York, USA, pp. grateful to Stéphane Verger (Martell) for his help 335-360 in the preparation of the Cognac work. Coquand, H. (1860). Des rapports qui existent entre les diverses qualités d’eaux-de- vie et celles du sol. In : Description References physique, géologique, paléontologique et minéralogique du département de la Agosin, E., Belancic, A., Ibacache, A., Baumes, Charente, volume 2, Barlatier-Feissat et R., Bordeu, E., Crawford, A. and Bayonove, Demonchy, Marseilles, pp. 261-275 C. (2000). Aromatic potential of certain Faria, J.B., Loyola, E., Lopez M.G. and Dufour, Muscat grape varieties important for Pisco J.P. (2003). Cachaca, pisco and tequila. In: nd production in Chile. American Journal of Fermented Beverage Production, 2 edition, Enology and Viticulture 51: 404-408 edited by A.G.H. Lea, and J.R. Piggott, Belancic, A. (2005). Uva Moscatel: Materia prima Kluwer Academic/Plenum Publishers, New para la fabricacìon de Pisco. Presentation at York, USA, pp. 335-360 « Vinitech America Latina 2005 », 14th to 16th Herraiz, M., Reglero, G., Herraiz, T. and Loyola, of July, Santiago, Chile E. (1990). Analysis of wine distillates Belancic, A., Agosin, E., Ibacache, A., Bordeu, E., made from Muscat grapes (Pisco) by Baumes, R., Razungles, A., and Bayonove, multidimensional gas chromatography and C. (1997). Influence of Sun Exposure on the mass spectrometry. Journal of Agricultural Aromatic Composition of Chilean Muscat and Food Chemistry 38: 1540-1543 Grape Cultivars Moscatel de Alejandría Lafon, J., Couillaud, P. and Gay-Dellile, F. and Moscatel rosada. American Journal of (1973). Le cognac. JB Baillieres Ed., Paris, Enology and Viticulture 48: 181-186 pp. 30-40 Belancic, A., Gunata, Z., Vallier, M. J. and Leaute, R. (1994). Recherche de relations entre Agosin, E. (2003). beta-Glucosidase from the terroir et caractéristiques sensorielles des èmes grape native yeast Debaryomyces vanrijiae : eaux-de-vie de Cognac. 13 Journées purification, characterization, and its effect internationales huiles essentielles, Digne- st rd on monoterpene content of a Muscat grape les-bains, 1 to 3 September, 1994 juice. Journal of Agricultural and Food Moll, L. and Guyot, E. (1883). Le cognac. In: Chemistry 51: 1453-1459 Encyclopédie pratique de l’agriculture, Bordeu, E., Formas, G. and Agosin, E. (2004). Firmin-Didot Eds, Paris Proposal for a standardized set of sensory Ravaz, L. (1900). Le pays du cognac. Louis Coquemard Ed., Paris. Instrumentation of a pilot scale distillery with on line data recording 237 Chapter 32 Instrumentation of a pilot scale distillery with on line data recording to study the Cognac production process

G. Ferrari2, B. Galy2, A. Sommier1, C. Chipeaux1 and M. Decloux1 1ENSIA-UMR GENIA, Massy, France; 2Station Viticole du Bureau National In- terprofessionnel du Cognac, Cognac, France

Introduction

In its quest for Premium Quality, BNIC research department has developed a pilot scale unit for all the steps of Cognac production : from grape to spirit. One important step is the distillation in pot still (Figure 1). Many parameters, such as physical and chemical transformations, directly affect Cognac quality. The objective of the pilot scale distillery was to be able to study these parameters in order to Figure 1. A typical charentais pot still. know and to improve the production process. After a long phase to obtain the same spirit Distillation of wine quality with the pilot scale distillery as with an industrial one, BNIC decided to improve the Distillation control of the pilot and the traceability of the process. A collaboration with ENSIA (Massy, For of the first distillation, wine, with or without France) food process engineering research its lees in suspension, is introduced into the department has been established. This team still. The heating curve of the still carries the is recognized for its expertise in sensors, liquid to its boiling point. The alcoholic steams automation and distillation. Therefore, a fully accumulate in the ‘chapiteau’, enter into the automatised distillation process was provided swan-neck then into the streamer where they are taking into account sensors, controllers and condensed to flow out as ‘brouillis’. One stops supervision functions, specifically adapted to the the distillation when the alcohol metre indicates requirements of Cognac distillation. 2% alc/vol.. The liquid that flows out until 0% alc/vol. is called the ‘tail’. 238 G. Ferrari et al.

The ‘brouillis’ alcoholic strengh is from 24% After many preliminry tests, we decided to to 32% alc./vol according to the initial alcohol build a pilot scale distillery with 2 pot stills of content of wine. It represents about 1/3 of the 70 liters for the first step (wine distillation) and initial wine volume. At the time of the second 4 stills of 14 liters for the second distillation distillation, know as ‘bonne chauffe’, the (see Figures 2 and 3). Two of these 14 litre stills ‘brouillis’ is put in the boiler and distilled one are paired, and each pair is linked to one 70 more time. The first portions of the distillate, the litre still. This pilot scale distillery was tuned to ‘heads’ are separated and then often blended with produce the same spirit as an industrial one and another ‘brouillis’ or with wine. One collects the we verified (chemical and sensory analysis) this liquid spirit (know as ‘eau de vie’ or ‘cœur’) by distilling the same wine by the two processes. that flows out until the cut toward 60% alc./ A system of control-command and on line data vol. What flow out then until 0% alc./vol is put acquisition has been put in place in order to aside. These are the ‘secondes’, following by the characterize the charentais still working and the tails. The period that separates the ‘cœur’ from respective influence of the controls, to optimize the ‘secondes’ is called ‘la coupe’. The cycle the process reproductibility. of complete distillation is about 24 hours. The techniques, or the art, of distillation (proportion of fine lees, recycling of the ‘secondes’ in wine Still instrumentation or ‘brouillis’, curves of temperatures, cuts,…) are varied and confer on Cognac its unique premium 70 liter still. The two still’s boilers are heated quality with a large variety of tastes. by a gas burner, as are the industrial ones. Two proportional electronic valves make it possible to adjust the gas pressure and therefore the Pilot scale distillery optimization heating intensity. Software control permits work in the range from 100 to 1200 mBar and To run some tests in an industrial distillery requires to + or - 1 mBar. The essential parameter to careful organization with heavy constraints and control during the distillation of wine is the a high cost. In order to maximise the necessary change of temperatures at different points. wine quantities and to avoid blending, the idea Several thermocouples have been installed in was to design a pilot scale distillation unit that strategic places of the still. We also programmed would reproduce the processes faithfully. the gas pressure and opening or closing of

Position on the still: temp. 3 4 sensors Col de cygne 1 In the boiler (different levels) 1 6 2 In « le chapiteau » Chapiteau Sortie 3 Top of the swan-neck 2 eau froide 4 Bottom of the swan-neck 5 Cooling water input T° niveau 4 Pipe réfrigérante 7 Chaudière 6 Cooling water output T° niveau 3 7 Spirit outflow niveau 2 Porte Alcomètre niveau 1 Others Brûleur 9 10 8 Gas propotional valve 5 Pressure sensor Armoire 8 Entreé eau froide 9 Flow meter à gaz 10 Scale Figure 2. Still instrumentation (70 liters). Instrumentation of a pilot scale distillery with on line data recording 239 the smokestack register. The quantity of spirit necessary to the distillation at the appropriate flowing out is measured with a scale that also moment, and pilot various components gives outflow. (flowgates, regulating…). We chose a deported SCXI-1001, including 12 Slots. We equiped 14 liter still. The four small stills are equiped it with 2 cards SCXI 1100 (36 measures of with a 14 litre boiler heated by electrical temperatures), a card 1102 (36 measures of resistance whose intensity is controlled by the pressure) and a card (12 analogical outputs) sofware. The heating power is calculated from as well as terminal blocks to facilitate the the initial alcohol content of the ‘brouillis’, the connection of the sensors or commands. The distillate weight recovered and the temperatures SCXI 1001 receives every acquisition card that measured on the still. Every still is equipped amplifies the signal before sending it toward the with a precision scale that measures the distillate computer. This case is connected to the PC that weight. The temperature sensors have been includes an acquisition card 12 bits PCI 6023. placed on high and low points of the swan neck, The set of the signals is multiplexed by the first spirit exhaust for each still, and on the way in card of the SCXI 1001. and way out of the cooling water. LabVIEW (Laboratory Virtual Instrument Choice of DATA ACQUISITION system. Engineering Workbench) is a software application The online data acquisition system is composed development created by National Instruments. of three parts (see Figure 4). The SCXI (Signal A system of acquisition and storage of the on Conditioning eXtension for Instrumentation) is line data has been coupled to the LabVIEW a system of instrumentation and conditioning programmes. All data, as well as a card of adapted to data acquisition on a PC. It was follow-up, are written in excel format, and can necessary to choose the material according to the be exploited and visualized with other software. initial specifications : measures of temperature The card for follow-up assures the traceability on 6 stills, entry pressures (pressure sensors, gas of the process and allows one to reach the final flow…), acquisition of the 6 scales to calculate goal, that is to perfect the knowledge of the the weight and flow of the distillate. From process. The scales data are collected by RS232 these measures we do the different operations lines with a six way card added in the PC.

5 3 4 Temp. sensors Col de cygne 1 In the boiler (one level) Sortie 2 In « le chapiteau » 1 2 eau froide Entrée 3 Top of the swan-neck Chapiteau 6 eau froide 4 Bottom of the swan-neck Pipe réfrigérante 5 Cooling water input 6 Cooling water output 7 7 Spirit outflow Chaudière Porte Alcomètre Others 8 Electrical resistance Armoire de Percent of power (%) Résistance électrique régulation 9 9 Scale 8 Spirit wieght (g)

Figure 3. Still instrumentation (14 liters). 240 G. Ferrari et al.

Still, sensors, valves N.U. SCXI 1001 Acquisition card in the PC BNIC file server Box with cards supervision program

Upper arrows = acquisition; lower arrows = operation)

Figure 4. Hardware and software for control and command programmes.

Programming the 14 Litre stills while promoting the good ones, and recover a 70% spirit (value imposed by the French Cognac It was necessary to define the steps and the decree) by adjusting the speed and the regularity running parameters for the management of the of the heating. We also have to do ‘la coupe’ at the second distillation. We had to develop a special right time. The distillation has three main stages: process, at our pilot scale, in order to recover a heating before spirit flows out, distillation of “le spirit from a wine by the original “charentaise” cœur” and “la coupe”. These parameters have distillation, but without blending with ‘heads’ or been fixed by B. Galy, Enologist at the Station ‘tails’ from another wine! In an industrial distillery Viticole after many years of experiments. the imperfects (‘heads’, ‘tails’, ‘secondes’) are recycled from wine to wine. Thanks to many previous distillations achieved Heating programme before spirit flows out manually, a database permitted us to establish viable calculations. These calculations determine It is really important to drive carefully the from the ‘brouillis’ TAV (total alcohol by volume) head separation because these imperfects and the boiler load, the weight and the TAV of contain unpleasant components. The steam the spirit to berecovered. In order to recover flow temperature at the high and low swan in ‘le cœur’ 70% of the alcohol content in the neck points are measured in order to adjust the ‘broullis’, a relationship between the spirit and electrical power of the heater and to manage the ‘brouillis’ TAV has been established: TAV the heating curve. The stage ‘end of heads’ was spirit = 0.19*TAV brouillis + 65.6. Knowing more complex to programme. According to the quantity of pure alcohol and the TAV of the the boiler and the outside conditions, the spirit spirit to recover, its volume and its weight are does not flow precisely at the same time on the calculated. 4 stills. To define this stage in the programme, To manage the brouillis distillation we we determined the mean length of this stage and programmed the power (in %) applied to the we fixed it to 70 min. The stages of distillation resistances. The art of distilling consists of are summarized in the Table 1. avoiding the passage of unpleasant substances Table 1. Regulation of the electrical power versus time and temperature measured in the still (T, temperature; t, time). T top swan neck <65°C and T bottom swan neck <65°C and t <70min Power = 100% T top swan neck 65°C and T bottom swan neck <65°C and t <70min Power = 50% T top swan neck 65°C and T bottom swan neck 65°C and t <70min Power = 20% T top swan neck 65°C and T bottom swan neck 65°C and t> 70min Power = f (TAVbrouilis, t)

Instrumentation of a pilot scale distillery with on line data recording 241

Heating programm for the ‘cœur’ tuned up the 4 stills (position of the resistances, opening of the registers…),a rectification It is important to start the heating mildly in order difference of less than 1% was observed between to improve spirit outflow. Then, it is necessary the 4 stills. The law of heating power used was to increase the power regularly and to limit the the following: distillation length. Indeed, with the progression of the distillation, the boiler contains less and P (of it%) = (0.0083*TAVbr - 0.1417)*t + less alcohol, so it is necessary to heat more and (0.8333*TAVbr + 0.8333) with t in seconds. more to get a constant spirit flow. Taking into account these considerations, For each 14 litre still, the measures can be we established a power curve according to recorded to adjustable time interval (between 1 ‘brouillis’ TAV and distillation time. More TAV and 600 seconds). The recorded values are the is important, the more it is necessary to heat to ambient temperature, air pressure, temperature at extract the maximum alcohol (P = a*t + b). We top and bottom of the swan-neck, heating power, determined the evolution of the coefficients a weight of spirit, mass flow of the distillate. For and b for the Power = f(TAV’brouillis’, time). each distillation, the references of the test and The power is controlled by this equation until the command orders are archived. In case of a the approach of ‘le coeur’ ending. When this failure in the distillation, the problem can be point is reached, the program signals by a beep discovered quickly by exploiting the recorded and a visual alarm that it is necessary to begin to data. A nomenclature for the abbreviations of control the spirit alcohol content. A reduction of the measures and the names of files have been 20% of this current value has been programmed. established. At the end of every month, the files For the rest of the time, the power is constant. are saved in the directory of the corresponding After ‘la coupe’, there is the ‘secondes’ flow month and volume of the boiler, 14 or 70 (BC1 heating power is 100%) or we stop the litres. distillation for BC2. Figure 5 presents final TAV of the spirits for different initial ‘Brouillis’ distilled during 2001 with manual control. We calculated that Validation of the distillation program R²=0.59. Figure 6 presents the same results for 2002 with supervision/computer control. We To validate this distillation program, we distilled calculated that R² = 0.73. The supervision/ many 'brouillis' of a TAV range between 24 to computer improved the reproductibility and for 30% alc./vol in each 14 Litre still. After having different initial products we more nearly reached

75

73

71

69 % alc./vol final spirit 67

65 20 22 24 26 28 30 32 TAV brouillis % alc./vol

Figure 5. Final TAV of cognac for different TAV Brouillis in 2001 (manual control). 242 G. Ferrari et al.

75

73

71

69 % alc./vol final spirit 67

65 20 22 24 26 28 30 32 TAV brouillis % alc./vol Figure 6. Final TAV of cognac for different TAV Brouillis in 2002 (supervision control). the goal with a reduced range of alcohol levels this was done with empirical knowledge. (69.5 < Cognac TAV < 71.5). This knowledge has been passed down from generation to generation oraly. Benefits of Conclusions the distillery pilot scale supervision/computer control could be multiple and are the fruit of an The ‘charentaise’ batch distillation is a active collaboration betwen ENSIA and BNIC fundamental part in the Cognac production engineers. In addition, the process management process. It was tuned up for ages in order to programming allows process reproducibility. improve the extraction of characteristic volatile We also provide data traceability which will compounds from original white wines of this make the distiller’s work easier. This pilot scale country to produce the Premium quality that unit can be considered as a reliable means to secures Cognac its worldwide success. But reproduce traditional distillation and to study it in detail. Esters - the most important group of flavour-active compounds 243 Chapter 33 Esters – the most important group of flavour-active compounds in alcoholic beverages[1]

G. G. Stewart International Centre for Brewing and Distilling, Heriot-Watt University, Edinburgh, UK

Introduction • Fermentation conditions – temperature,

agitation, CO2 tension, pH, fermenter design, Esters are important flavour components which pitching rate. impart characteristic flowery and fruit-like flavours and aromas to beers, wines and spirits What is the role of ester production during yeast such as whisky and brandy (Engan, 1972). growth and fermentation? There are a number Their presence is desirable at appropriate of possibilities (Hunkova and Fencl, 1977): concentrations but failure to properly control fermentation can result in unacceptable ester • There may be spill-over products from sugar levels in the final product. Over 90 distinct esters metabolism (Figure 1). have been detected in beer, wine and whisky. • The relative cellular concentration of acetyl- The important esters include:- CoA and free CoA regulate intermediary metabolism – ester synthesis may be a • Ethyl acetate (fruity/solvent) method of controlling the “acetyl-charge” • Isoamyl acetate (banana/apple) (Figure 1). • Isobutyl acetate (banana/fruity) • Ester metabolism may be a detoxification • Ethyl caproate (apple/aniseed) process, fatty acids (C8 and C14) are toxic to • ß-Phenylethyl acetate (roses/honey). yeast cells. Esterification of these fatty acids may function as a protection system. There are many factors that influence the level of ester production during fermentation: In this paper, the influence of the following factors on ester synthesis will be discussed: • Yeast characteristics – pitching rate, yeast strain, physiological state. • Wort original gravity and sugar spectrum • Wort composition – sugar and amino • Wort clarity acid spectrum, lipids, vitamins, inorganic • Yeast strains. nutrients, dissolved oxygen, clarity (trub), original gravity.

[1] Some of the data contained in this paper was presented at the 30th European Brewing Convention Conference held in Prague, Czech Republic, 14 to 18 May 2005. 244 G.G. Stewart

Wort Sugars the sugars for the transporter (Bisson and Fraenkel, 1983). The disaccharide maltose is internalised by the cell only when 40%-50% of the glucose has Ethanol Pyruvate been removed from the wort (Stewart and Russell, 1998) and occurs via a different active transporter (Serrano, 1977). Esters Acetyl-CoA TCA Cycle Initially 4% (w/v) glucose and maltose in a synthetic medium (yeast extract – peptone medium) were fermented separately with shaking Lipids at 21ºC, in order to eliminate any possible Amino Acids inhibition of sugar uptake and the production Nucleic Acids of ethyl acetate and isoamyl acetate monitored

Biomas s (Younis and Stewart, 1998). The fermentation performance of the three ale and three lager brewing yeast strains employed in this study were Figure 1. Central role of acetyl-CoA in yeast. similar. Tables 2 and 3 show the viabilities and vitalities of the cells, respectively, following four Influence of wort sugar spectrum and days of fermentation. For all six strains studied, gravity on ester formation cells cultured in maltose consistently had greater viabilities and enhanced vitalities compared to The advantages and disadvantages of high their glucose cultured counterparts. gravity brewing has been discussed in many publications (for example Murray and Stewart, Table 2. Viability of brewer’s yeast cultures following 1991; Stewart, 1999; Stewart, 2003). One of fermentation of synthetic media* containing either the disadvantages of this process is that during 4% (w/v) glucose or maltose. fermentation high gravity worts induce the Glucose Maltose production of disproportionately high levels of + esters (Table 1) (Anderson and Kirsop, 1975; Ale 96 98 D’Amore, Celotto and Stewart, 1991). Ale 92 97 Ale 96 99 Lager 96 99 Table 1. Influence of wort gravity on beer ester Lager 94 98 levels. Lager 94 99

12° Plato 20º Plato + Methylene blue staining after 4 days fermentation Ethanol (v/v) 5.1 5.0 * Peptone – yeast extract – nutrient medium

Ethyl acetate (mg/L) 14.2 21.2 Table 3. Vitality of brewer’s yeast cultures following Isoamyl acetate (mg.L) 0.5 0.7 fermentation of synthetic media* containing either 4% (w/v) glucose or maltose. Glucose Maltose Varying the wort sugar source has been reported (Cason, Reid and Gatner, 1987) to modify the Ale 0.8+ 1.3 levels of many metabolites, including esters, Ale 0.9 1.3 although reasons for these differences are unclear. Ale 1.1 1.4 Lager 0.7 0.9 Entry of the hexose sugars, glucose and fructose, Lager 0.8 1.2 into the yeast cell is facilitated by the same protein, Lager 0.9 1.0 although utilisation of glucose occurs more quickly than fructose when the two sugars are fermented *Peptone – yeast extract – nutrient medium + separately, possibly due to the differing affinities of Acidification Power Test – Glucose driven pH change after 4 days fermentation Esters - the most important group of flavour-active compounds 245

Reasons for these differences are not immediately It has been proposed that ester production is apparent. It may be a result of the slower initial linked to lipid metabolism (Shindo, Murakami uptake rate of maltose and consequent reduced and Koshino, 1992). If this is the case, or if for growth rate. some reason maltose metabolism produces Despite the apparent sturdiness of the maltose fewer toxic fatty acids, it would be reasonable grown cells, the production of ethyl acetate was to assume that fewer toxic fatty acids would also lower than glucose grown cells (Table 4). Similar be produced. results were obtained for isoamyl acetate and It is generally agreed that a reduction in ester isobutyl acetate production (results not shown). levels (particularly ethyl acetate and isoamyl acetate) from high gravity brewed beers would Table 4. Levels of ethyl acetate produced (mg/L) be welcome (Table 1). In order to study the by brewer’s yeast cultures during fermentation influence of maltose and gluctose levels in of synthetic media* containing either 4% (w/v) high gravity worts, two 20º Plato worts were glucose or maltose. prepared, one containing 30% maltose syrup Glucose Maltose and the other containing 30% (w/v) very high Ale 4.13 2.79 maltose syrup (the sugar composition of the Ale 2.97 2.59 two brewing syrups is shown in Table 5). In Ale 3.13 2.71 addition, a 12º Plato wort containing 30% (w/v) Lager 6.02 5.82 Maltose Syrup (MS) was prepared and used as Lager 3.75 3.28 a control. Lager 4.62 3.52

Table 5. Sugar composition of brewing syrups. The lower levels of the esters produced with Maltose Very high maltose as the substrate could be due to a number syrup maltose syrup of reasons. It is possible that fermentation with (MS) (VHMS) maltose inhibits the transport of esters out of the cell, perhaps by modifying the plasma membrane, Glucose 15* 5 thus giving the impression that fewer esters are Maltose 55 70 Maltotriose 10 10 produced. However, in the light of the increased Dextrins 20 15 viability and vitality of the maltose grown cells, this is unlikely. Another possibility is that maltose *Percent Composition metabolism produces lower levels of acetyl-CoA, which has been suggested as resulting in fewer The sugar spectra of the three worts is shown esters due to lack of intermediate metabolites. in Figure 2. The maltose plus maltotriose

Glucose and fructose 200 180 Maltose and maltotriose 160 140 120 100 80 60 40 Sugar concentration (g/L ) 20 0 12°Plato (30% 20°Plato (30% 20°Plato (30% VHM) maltose syrup) maltose syrup) syrup Figure 2. Wort sugar profiles. 246 G.G. Stewart concentration in the 20º Plato Very High Maltose However, the ester concentration in the 20º Syrup (VHMS) wort had increased compared to Plato (VHMS) was approximately 25% reduced the 20º Plato (MS) wort with a corresponding compared to the 20º Plato (MS) wort (Younis decrease in the concentration of glucose plus and Stewart, 1999). These results confirm the fructose. findings employing synthetic media with single The three worts were fermented in the ICBD sugars, that maltose fermentations produce less 2hL pilot brewery with a lager yeast strain at ethyl acetate and isoamyl acetate than glucose 13ºC and the concentration of ethyl acetate fermentations. In addition, similarly to the and isoamyl acetate determined throughout synthetic media fermentations, the wort with the fermentation (Figures 3 and 4). The profiles elevated concentrations of maltose produced were similar with both esters. The concentration yeast with higher viabilities than with wort of both esters in the 20º Plato (MS) wort was containing lower levels of maltose (Table 6). twice that in the 12º Plato (MS) fermented wort.

12°P (30% MS) 40 20°P (30% MS) 20°P (30% VHMS)

30

20

10 Ethyl acetate concentration (mg/L) 0 04080120 160200 240 Fermentation time (hours) Figure 3. Ethyl acetate concentration in worts of differing gravities and sugar composition. Fermentations conducted in 2hL static fermenters at 13ºC.

12°P (30% MS) 1.0 20°P (30% MS) 20°P (30% VHMS) 0.8

0.6

0.4

0.2

Isoamyl acetate concentration (mg/L) 0.0 04080120 160200 240 Fermentation time (hours) Figure 4. Isoamyl acetate concentration in wort of differing gravities and sugar composition. Fermentations conducted in 2hL static fermenters at 13ºC. Esters - the most important group of flavour-active compounds 247

Table 6. Final viabilities (%) of brewing ale and cerevisiae “M” strain (Quest International, lager yeast strains after fermentation in 12º Plato Menstrie, Scotland). Carbon dioxide evolution and 20º Plato worts. rates during fermentation and ethyl acetate and isoamyl acetate concentrations at the end of 12º PLATO 20º PLATO fermentation were determined. The following MS* VHMS MS VHMS wort conditions were studied: Ale 95+ 98 93 96 Lager 94 97 95 98 • Cloudy wort • Clear wort *MS: Maltose (55) Syrup; VHMS: Very High • Clear wort plus 0.2 g/L diatomaceous earth Maltose (70) Syrup (DE) +Methylene blue and methylene violet stains • Clear wort plus DE and 5.5 mg/L C16:1 fatty employed acid • Clear wort plus 0.2 g/L bentonite. Influence of wort clarity on ester formation The concentration of CO2 during fermentation of the above 15º Plato wort types was determined The level of wort solids carried over from cereal (Table 7). Cloudy wort, containing trub, and mashing and grape crushing and their effect on wort containing DE acted as nucleation sites and fermentation performance has been the subject increased CO2 evolution out of the wort. Clear of considerable debate in the production of beer, wort and wort plus bentonite did not function as whisky and wine (Ancin, Ayestaran, Corroza, nucleation sites and consequently CO2 remained Garrido and Gonzalez, 1996; Merritt, 1967; in the fermenting medium to a much greater O’Connor-Cox, Lodolo, Steyn and Axcell, 1996; extent. Why was there a difference between trub, Thomas, Hayes and Ingledew, 1994). DE and bentonite? In order to visualise each type of particle and obtain data on their surface Wort solids (trub) influence: characteristics, environmental scanning electron microscopy (ESEM) was conducted (Figure 5). In DE, there was a heterogeneous mix of shapes • The removal of CO2 from solution during fermentation by acting as nucleation sites. and sizes of particles. The surface of most of the particles revealed an extremely porous structure, Removal of CO2 from solution can occur with no suspended particles but requires a great as would be expected. The micrograph of the deal of energy. cloudy wort solids showed a mix of different • Wort solids confer nutritive value. The rate structures, again porous in nature. Bentonite of fermentation is faster in the presence of had a more homogenous structure. In addition, insoluble material. Wort solids are generally it possessed a much different surface topology. associated with higher levels of fatty acids. It did not appear to possess the same porous • In some instances, yeast cells are able nature as DE or wort solids. to attach themselves to wort solids and

display enhanced growth because of the Table 7. Concentration of CO2 (g/L) present during concentration of nutrients at the particle fermentation. surface. 24 hours 48 hours In order to assess the influence of wort clarity Cloudy wort 2 4 on ester formation, 15º Plato wort was produced Clear wort 5 8 in the ICBD 2hL pilot brewery and a number of Clear wort plus DE 2 4 wort types fermented in 1 l volumes in 2 l tall Clear wort plus bentonite 5 8 tubes at 27ºC employing the Saccharomyces 248 G.G. Stewart

Figure 5. Environmental scanning electron microscopy of different solid materials.

Ester levels are also influenced by the particle Influence of yeast strain on ester concentration and type in wort. Ethyl acetate formation and isoamyl acetate concentrations are high in cloudy wort and DE containing C16:1 fatty The brewer and distiller have a large number acid (Tables 8 and 9). This reflects the fact that of yeast strains from which to select – distillers unsaturated fatty acids that would be absorbed (sugar attenuating), ales and lagers. The use to wort trub induces the synthesis of esters of alternative strains offers the prospect of (Äyräpaa and Lindström, 1973; O’Connor-Cox more efficient fermentation properties, diverse et al. 1996). characteristics and beer and spirit flavour manipulation. The characteristics of strain variation have been extensively reported but Table 8. Ethyl acetate levels (mg/L) following 160 a basic understanding of the reasons for these hours fermentation of different wort types. differences is still unclear (Stewart and Russell, Cloudy wort 30 1998). Clear wort 16 To illustrate this yeast strain diversity, ester Clear wort plus DE and C16:1 fatty acid 25 production under standard conditions has been studied. Fermentations were conducted in 1 l static fermenters with 16º Plato all-malt wort at 20ºC. When the fermentations were complete Table 9. Isoamyl acetate levels (mg/L) following ethyl acetate and isoamyl acetate levels were 160 hours fermentation of different wort types. determined in the fermented wort (Table 10). These results of differences regarding strain Cloudy wort 0.95 variation in the production of esters illustrate Clear wort 0.55 Clear wort plus DE and C16:1 fatty acid 0.75 strain differentiation within a yeast species. Although the genome of Saccharomyces Esters - the most important group of flavour-active compounds 249 cerevisiae has been sequenced and this has particularly the situation with concentrated enabled an intensive study of genetic control of worts. Under similar fermentation conditions these species our understanding of the reasons many yeast strains produce beer/spirit with for strain differences is still largely unknown! differing ester spectra.

Table 10. Ethyl acetate and isoamyl acetate (mg/L) production by distillers and brewers yeast strains. Acknowledgements Ethyl Isoamyl acetate acetate The author is grateful to James Bryce, Kenny Leiper, Stephan Martin and Omar Younis for Distillers 46.2 1.3 advice, support and assistance. Thanks are also Distillers 36.2 0.9 due to Graham McKernan and Jim MacKinlay for Ale 20.6 0.6 help with technical matters. The financial support Ale 36.2 0.8 of Suntory Limited, Corn Products Limited and Ale 25.6 3.5 The International Centre for Brewing and Lager 60.2 4.6 Distilling is acknowledged and appreciated. Lager 32.6 1.6

Fermentations conducted in 16º Plato all-malt-wort at 20ºC References

Conclusions Ancin, C., Ayestaran, B., Corroza, M., Garrido, J. and Gonzalez, A. (1996). Influence of Although considerable research has been prefermentation clarification on the higher conducted on ester synthesis in alcoholic alcohol contents of wines. Food Chemistry beverages, the exact reasons for their production 55: 241-249 by yeast during fermentation are unknown. Anderson, R.G. and Kirsop, B.H. (1975). Oxygen There are many factors that influence their as a regulator of ester accumulation during synthesis. A number of factors are considered the fermentation of wort of high specific in this paper: wort gravity, clarity and sugar gravity. Journal of the Institute of Brewing spectrum together with a consideration of yeast 81: 286-301 strain diversity regarding ester formation. All of Äyräpaa, T. and Lindström, I. (1973). Influence these factors influence ester formation which is of long-chain fatty acids on the formation of directly proportional to the inverse relationship esters by brewer’s yeast. In: Proceedings of between yeast growth and ester synthesis. the Eurpoean Brewing Convention Congress, Salzburg, Oxford University Press, Oxford, pp. 271-285 Bisson, L.F. and Fraenkel, D.G. (1983). Summary Involvement of kinases in glucose and fructose uptake by Saccharomyces Esters are an integral part of the flavour matrix of cerevisiae. Proceedings of the National many alcoholic beverages. They can contribute Academy of Science 80: 1730-1734 both positive and negative effects on product Cason, D.T., Reid, G.C. and Gatner, E.M.S. quality and stability. Wort gravity, clarity and (1987). On the differing rates of fructrose sugar spectrum have an influence on ester and glucose utilisation in Saccharomyces formation. High levels of wort maltose produce cerevisiae. Journal of the Institute of beer with lower ester concentrations compared Brewing 93: 23-25 to wort with high levels of glucose. This is D’Amore, T., Celotto, G. and Stewart, G.G. 250 G.G. Stewart

(1991). Advances in the fermentation Journal of Fermentation and Biotechnology of high gravity wort. Proceedings of the 73: 370-374 European Brewing Convention Congress, Stewart, G.G. (1999). High gravity brewing. Lisbon, Oxford University Press, Oxford, Brewers’ Guardian 128: 31-37 pp. 337-344 Stewart, G.G. (2003). Technical advances in Engan, S. (1972). Organoleptic threshold values measuring yeast stress – the implications of some alcohols and esters in beer. Journal of brewing beer with good foam stability of the Institute of Brewing 78: 33-36 and shelf life. Proceedings of the European Hunkova, Z. and Fencl, Z. (1977). Toxic effects Brewing Convention Congress, Dublin, of fatty acids on yeast cells: dependence of Oxford University Press, Oxford, pp. inhibitory effects on fatty acid concentration. 839-847 Biotechnology and Bioengineering 19: Stewart, G.G. and Russell, I. (1998). An 1623-1641 Introduction to Brewing Science and Merritt, N.R. (1967). The effect of suspended Technology, Series III: Brewer’s Yeast, The solids on the fermentation of distiller’s malt Institute of Brewing, London, England. wort. Journal of the Institute of Brewing Thomas, K.C., Hynes, S.H. and Ingledew, W.M. 73, 484-488 (1994). Effects of particulate materials Murray, C.R. and Stewart, G.G. (1991). and osmoprotectants on very-high-gravity Experience with high gravity brewing. Birra ethanolic fermentation by Saccharomyces & Malto 44: 52-66 cerevisiae. Applied Environmental O’Connor-Cox, E.S.C., Lodolo, G.J., Steyn, Microbiology 60: 1519-1524 G.J. and Axcell, B.C. (1996). High gravity Younis, O.S. and Stewart, G.G. (1998). Sugar wort clarity and its effect on brewing yeast uptake and subsequent ester and higher performance. Technical Quarterly of the alcohol production by Saccharomyces Master Brewers Association of the Americas cerevisiae (1998). Journal of the Institute 33: 20-29 of Brewing 104: 255-264 Serrano, R. (1977). Energy requirements for Younis, O.S. and Stewart, G.G. (1999). Effect of maltose transport in yeast. European Journal malt wort, very-high-gravity malt wort, and of Biochemistry 80: 97-102 very-high-gravity adjunct wort on volatile Shindo, S., Murakami, J. and Koshino, S. (1992). production in Saccharomyces cerevisiae. Control of acetate ester formation during Journal of the American Society of Brewing alcohol fermentation with immobilised yeast. Chemists 57: 38-45. Environmental solutions and threats in today’s distilling industry 251 Chapter 34 Environmental solutions and threats in today’s distilling industry

B. Higgs Diageo Plc, Elgin, UK

Introduction Given the breadth of these threats the potential solutions will come from both technical and The opinions expressed in this paper have been political/commercial avenues. influenced by the author’s work with Diageo and the environment committees of both the Malt Distillers Association of Scotland and the Scotch Threats to the industry from the Whisky Association: however, they are solely the environment opinions of the author and do not represent any corporate or association position. Whilst significant debate continues as to the Whilst the paper will concentrate on the cause of climate change, there can be no Scotch whisky industry, the themes covered are argument that climate has, and continues to, applicable and relevant to many other distilled change over time. Whilst the only evidence spirits. I can present is anecdotal, there is a view that The title of this paper is somewhat ambiguous over the last 25 years or so the Scottish winters and it is perhaps useful to initially clarify what have become milder and snowfall has reduced threats and solutions will be discussed. On the significantly. The slow release of water into threats side of the equation it is important to the hills through snowmelt has reduced and look at: the natural reservoirs of groundwater have diminished accordingly. • Factors arising from the environment that In addition, there is some evidence to suggest actually pose a threat to the distilling that whilst the summer months average rainfall industry. has not changed significantly, the propensity for sudden periods of very heavy rain and flash • The threats posed to the environment by flooding has increased. If this situation is correct, the operations carried out by the distilling and the trend continues, the industry will face industry. some difficult challenges as continuity of water supply across the production year is stretched. • The threats to the industry arising from The distilling industry is also often impacted increasing environmental legislation and by its ability to dispose of its co-products and regulation. since much of this is in the form of animal 252 B. Higgs feeds, milder winters would undoubtedly reduce The long-term viability of animal feeds demand, as farmers would have the option to production is also in question. Biofuel production field graze livestock for longer periods. is growing extremely quickly and with it will come large quantities of co-products that will play into the animal feeds markets. Distiller’s Threats to the environment from the feeds will come under even greater supply and distilling industry demand pressures and alternatives to dark grains, as a disposal route for draff and pot ale, will The production of any potable spirit produces become essential. significant volumes of by-products and If climate change is occurring as a result of wastewater streams and Scotch whisky is no energy use and greenhouse gas production, then different in that respect. Looking just at malt the distilling industry is contributing its share. whisky production we can see that for every Distilling is by its very nature an energy intensive litre of alcohol produced there is a concurrent business and the Scotch whisky industry uses, production of 8 litres of pot ale, 3.5 litres of spent on average, 12 kWh for every litre of malt spirit lees and a further 3.5 litres of washing waters. produced and 6kWh for grain whisky. Add to In addition, some 300 litres of cooling water are this the energy consumed in making the raw required for each litre of spirit distilled. material, malted barley, and handling the co- Last year the industry produced just under product production of dark grains and it is easy 150 mola (millions of litres of “original” to understand why the sector has its own climate alcohol, production volume of new-make spirit change levy agreement with the government. before evaporative losses due to maturation) of malt whisky and the consequent wastewaters produced had a potential effluent loading with Threats to the industry from a population equivalent of 2 million. That environmental legislation is greater than the combined populations of Edinburgh, Glasgow and Aberdeen. Add the Scotch whisky is internationally renowned for impact of the grain whisky production and the its quality and provenance. The industry has population equivalent is significantly greater than consistently used the messages and imagery of the entire national population of Scotland. a beautiful and pristine environment in Scotland In addition the industry saw some 60 tonnes of to build this reputation. There can, therefore, be copper dissolved from its production plants into no doubt that environmental protection should the pot ale, spent lees and washing waters. All of be a major consideration in the activities of the which makes its way, either directly or indirectly, Scotch whisky distillers. back into the environment. It is, therefore, a Environmental protection is also one of the little difficult to argue that the industry does not fastest growing political considerations of the pose some threat to the environment in which developed and developing world. More and it operates. more concern is being expressed about the long- Some of the potential environmental loading term sustainability of the earth’s resources and its from the pot ale is dealt with through animal ability to absorb the pressures put on it by human feeds production in the form of barley or wheat activity. The environment needs protection dark grains and the spent lees and washing and inevitably this means that legislation and waters are processed through aerobic effluent regulation will be brought to bear on businesses treatment plants. However, the volume handled whose operations either take things from or put in this way is considerably less than the total things into the environment; and rightly so. produced and disposal to land and/or sea is Environmental legislation and regulation is essential in many cases. not, in itself, a threat to the distilling industry. Environmental solutions and threats in today’s distilling industry 253

In fact, the industry should welcome regulation distillery discharges. Whether this can be proved that protects the environment because by its very as yet is a moot point – the removal of 2,000 nature it protects the businesses that operate tonnes of nutrient per year from the shallow within it. However, the threats come from how waters around Islay must have had some impact this additional regulation is constructed and on the biodiversity. implemented and the impact these have on In addition there are now some 5,000-tanker the way the industry approaches solutions to journeys per year moving the material to its environmental protection and improvement. discharge point. Road damage, noise and A particularly good illustration of this type of combustion fumes all adversely impact on the threat occurred on Islay with the introduction environment; but none of this was factored of the Urban Waste Water Treatment Directive into the solution the legislation was framed to (UWWTD). The high suspended solids and provide. The question has to be asked about biological oxygen demand (BOD) characteristic whether the benefits achieved have justified the of pot ale and spent lees meant that discharge of actions demanded by legislation in this case, these materials from distilleries direct to coastal or whether a holistic view to environmental waters was only permissible if sufficient initial protection would have been more appropriate. dilution could be achieved to meet standards It is not only regulation of wastewater laid down by Europe. disposal to the marine environment that causes In the case of five distilleries on Islay, difficulty for the industry. Disposal of distillery this meant that their traditional practice of wastewaters to land is regulated under waste discharging the by-products into adjacent bays management licensing provisions that only or sea lochs was brought to an end. The solution allow land disposal where an agricultural benefit was to build a sea outfall into the deep water of from the material can be demonstrated. The the vigorously tidal Sound of Islay and transport agricultural benefit of distillery wastewaters is the wastewaters there by road tanker. A further now being challenged by some of the regulators three distilleries were able to extend their as it is spread to land throughout the year – existing sea outfalls into deeper water adjacent the argument being made that an agricultural to the distilleries and hence meet the dilution benefit can not be made during the non-growing demands of the UWWTD. The total capital period of the year. The alternatives, such as cost of these mitigation measures was around road haulage to sea discharge points or energy £2.5million. intensive evaporation plants, all bring adverse There is no doubt that the area of seabed environmental impacts and again the holistic around the old discharge pipes has improved view to protection seems to be missing. The as a result of this change. Previously an area of situation also begs the question of why the test around 600 m2 was most definitely anoxic due to for permission to dispose is agricultural benefit the high BOD material. Little, if anything grew rather than environmental impact. If the actual in this area. Outwith this area, however, where impact to the environment is neutral what is the dilution of the wastewaters by seawater the issue? turned the high BOD waste into a highly Cooling water discharges are obviously nutritional organic feedstock, the flora and fauna critical to the operation of distilleries and the flourished. Micro-organisms and invertebrates Freshwater Fisheries Directive has resulted in thrived and these in turn attracted both fish and significant concerns around the possible need to wading bird populations: and this over an area install cooling towers and refrigeration systems of approximately 12 km2. at sites. The directive calls for a maximum Today some of the local population on Islay temperature uplift by discharged cooling waters believe that both fish and bird populations have of 1.5oC in rivers with salmonid fish populations decreased in the areas previously fed by the on the basis that these elevated temperatures are 254 B. Higgs always harmful to fish populations. Whilst there Governments have the ability to encourage is no doubt that warm water discharges cause industry to invest in energy efficiency but using harm in some circumstances, there is strong either “carrots”, such as capital allowances on evidence from field studies that in other cases energy efficiency improvements, or “sticks” the impact is beneficial. This badly written piece such as levys on fuel consumed. With the of legislation, however, did not take this fact introduction of the Climate Change Levy and properly into account. the European carbon trading schemes, there is Currently the Water Framework Directive little room for doubt as to which method has is being implemented in Scotland under the been preferred. Controlled Activities Regulations and this One area, which may produce results in the demands that every distillery will be licensed. long term, is the development of new cereal Eventually, when an impact assessment is carried varieties. Distillers have, historically, looked out, distilleries that are having no adverse impact principally for yield improvements from cereals on the environment will be released from the development and this has been the main factor on licensing regime and put into simple registration. the shopping list presented to the barley breeding The industry has argued, unsuccessfully to date, companies. If the emphasis was changed and that this process is the wrong way round – all low temperature processing became a desired abstractions of water from the environment key attribute, the plant-breeding programme should be registered but only those having an could potentially deliver some major changes adverse impact on the environment should be to the industries energy requirements. Malted licensed after being demonstrated to do so by barley, for example, with a starch gelatinisation an impact assessment process. temperature of 45oC would reduce the average Undoubtedly there is more to come as the energy consumption used in mashing 10%. In Soil Directive is developed through Europe and overall distilling terms, this represents around commitments made, by the heads of the EU 50% of the commitment made by the industry states, at conventions such as the Oslo and Paris in its CCL agreement for the next 5 years. (OSPAR) meetings are pushed through. Despite the amount of legislation that is relevant to the industry, it is not the legislation itself that poses Copper removal the threat, but the apparent lack of any joined up or holistic thinking. There is little doubt that the emission of copper to the environment will become further constrained under future legislation. The solutions The technologies already exist for removal of copper from spent lees and washing waters Energy and the industry has started investing in these. It is perhaps disappointing that the industry From the climate change perspective the has not decided to collectively adopt the ion distilling industry will struggle to make any major exchange system because at present this is the contributions to energy efficiency improvements only technology which has the ability to be up when set against conventional return on rated to copper recovery by the use of electro- investment criteria. Whilst the industry is energy winning systems. intensive it is also highly energy efficient and Reed bed technology is also likely to play opportunities for capital investment with sub an increasing role in treatment of distillery 5-year payback periods are virtually non-existent. wastewaters. The systems have been used for Future investment in energy efficiency will need a number of years for polishing treated effluent to be encouraged by some form of environment from conventional bio-plants and the technology improvement incentive. has been developed further in Scandinavia to Environmental solutions and threats in today’s distilling industry 255 provide a process for heavy metal removal from the distilling industry, will not invest anything bio-plant sludge. more than is absolutely necessary to meet current legislation. There is no incentive to do anything more than the minimum and this position is By-products and effluent treatment entrenched further by the generally held view that environmental legislation is “death by a This area is sufficiently broad to merit an entire thousand cuts” arising from the apparent lack of paper in its own right and can only be touched joined up thinking discussed earlier. upon here. Pot Ale evaporation and dark grains Some of the solution lies in a more drying, as already mentioned, will come under sophisticated approach to future legislation. pressure in the future from both an energy consumption and air emissions perspective. • By generating a truly holistic approach to Developments in Anaerobic Digestion (AD) environmental protection, whereby the and Biomass power generation technologies impact of mitigation measures is taken show great potential for applications in the into account in assessment of a specific distilling industry of the future. Whilst AD plants situation. handling the BOD loads of pot ale are not yet in And: operation the technology is developing rapidly. AD systems in modular format are becoming • By introducing a process of “Environment more readily available and the consequent improvement option trading” whereby an capital costs are falling. operator would be allowed to continue a Conceptually, a future where distilleries difficult to mitigate activity if they invested use their waste materials to generate gas and in a different, but environment improving, electricity that is then used in the principal activity. production process, is highly persuasive. Whilst it is not possible to balance perfectly the amount The former is perhaps just common sense. of energy required to that potentially produced Why damage the environment you are trying to from by-product treatment an integrated system protect? However, this would require a change would dramatically reduce the energy input in mindset by the regulator, as the application required from conventional sources. For of “powers of discretion” would need to replace example, the methane generation from an AD the more easily administered “precautionary plant treating clarified pot ale would provide principle”. At present, no judgement is required 30% of the total energy requirements of a as the precautionary principle is applied based distillery. on the over simplistic approach of “if in doubt, Considerable development investment will say no”. This mindset change is only conceivable be required to achieve such a future and again if the regulators political masters see the benefit it is unlikely to occur if conventional returns on and support the culture for change. investment criterion need to be met. No doubt, the latter would be difficult to establish and administer. However, for industries like distilling, where the environment Political and commercial is genuinely a business and product concern, considerations this approach would ultimately generate reasons for investing ahead of the compliance curve. Technology will provide some of the answers but If by investment in one area of the process, not all, and certainly not in the current culture of a business could offset the need to invest in “minimum compliance” investment. Businesses, other areas, the whole mindset to environment even those as environmentally conscious as in related investment would change. Agreements 256 B. Higgs between operators and regulators could result in agreed environment-improving project. Three an industrial contribution toward environmental of those 5/6-year payback projects for low wines improvement rather than simple compliance. and feints pre-heating would suddenly become An example of such a trade would be a viable and at the end of the day, instead of adding distillery faced with the installation of cooling another 75 tonnes to the CO2 emissions into the towers to meet the Freshwater Fisheries Directive atmosphere, the scheme would reduce current maximum temperature levels. With capital costs levels by around 450 tonnes as the energy of around £300 k and annual running costs of consumption of the distillery fell. Admittedly £15 k+, the benefits would surely need to be one small Scottish burn would still be devoid of great to justify such demands. In many cases the salmon, as it had been for a couple of hundred situation would be that the distillery in question years (at least), but overall the environmental sits on a small burn which has never had a run impact of man’s activities would have been of salmon but which happens to be caught by reduced. the general designation of watercourses in that area. Rather than this meaningless investment, Is this sort of change likely to happen? I don’t why not have a scheme where the operator in know – one can but hope. question can “buy” exemption by investing in an Wastewater management and energy recovery 257 Chapter 35 Wastewater treatment and energy recovery go hand in hand in the distilling industry

V. Groot Kormelinck Paques bv, 8560 AB Balk, Netherlands

Introduction provides the entire range of agricultural alcohol products from its own production for use in Today wastewater treatment and energy recovery numerous applications. The principal customers are hot issues in Europe. To recover energy from are the fuel, chemical and pharmaceutical wastewater in the form of biogas, an anaerobic industries as well as manufacturers of paints, treatment system needs to be applied. For films and plastics. With an annual output of companies with high COD loads, the biogas 200,000 hl in 2005 and storage capacity for over produced with an anaerobic system could 20,000 cubic metres, KWST is one of Germany’s replace a significant amount of natural gas or leading producers of alcohol from renewable could be used to produce electricity and heat. raw materials. Not only biogas production can be a driver, but This ethanol specialist sets great store also lower discharge costs or the permission to by energy conservation and environmental discharge wastewater. Another positive effect is protection, not only in their modern distillery the positive effect on the carbon dioxide balance and the plants for the rectification and absolution of the company; the biogas replaces part of the of alcohol, but also in the field of recycling and natural gas consumption. waste water treatment, where the company’s This paper discusses wastewater treatment plants are among the world’s most technically and energy recovery. First we will describe the advanced. technology of anaerobic digestion followed by a presentation of the wastewater treatment project of KWST in Hannover Germany. This case study The theory behind anaerobic digestion will provide details of the decision process, design and operational data. Finally we will Anaerobic digestion of biodegradable waste present some indicative figures on investment involves a large spectrum of bacteria. Generally costs (CAPEX) and the operational costs (OPEX) three main groups are distinguished, with each for a large distillery. one playing its role in the anaerobic degradation. Kraul & Wilkening u. Stelling KG-GmbH & Co. As can be seen in Figure 1, the digestion process (KWST), a midsize company based in Hannover, can be divided into four separate but closely has been producing and refining alcohol of related steps. the highest quality for over 140 years. KWST 258 V. Groot Kormelinck

POLYMERIC PROTEINS LIPIDS CARBOHYDRATES

HYDROLYSIS

MONOMERIC HIGHER VOLATILE FATTY ACIDS AMINO ACIDS METHANOL CARBOHYDRATES GLYCEROL, ETC

ACIDOGENESIS

FORMATECO 2+ H 2 ACETATE ETHANOL BUTYRATE VALERIATE PROPIONATE

ACETOGENESIS

FORMATE CO 2+ H 2 ACETATE ACETATE H2 ACETATE + H2 + CO 2 METHANOL

CO 2 METHANOGENESIS

CH 4 + CO 2 AND H 2O

Figure 1. The stages of anaerobic digestion.

Fermenting bacteria perform the first two steps. It is a fact that neither during the hydrolysis They hydrolyse (= split a molecule into two or nor during the acidogenesis and acetogenesis, more fractions with uptake of water) dissolved does any important reduction of COD takes and undissolved polymers like proteins, fats place. In fact only conversion takes place from and carbohydrates through the action of exo- one form of COD to another form of COD. The enzymes, into smaller units, which can enter the actual elimination of organic matter takes place cells. In the cells an oxidation-reduction process in the methanogenic step in which the COD results in the formation of carbon dioxide (CO2), disappears from the wastewater in the form of hydrogen (H2) and mainly volatile fatty acids methane. Of the methane formed 70 to 75% is (VFA). The environmental conditions (pH, partial derived from acetate and the rest from hydrogen pressure of H2) determine to what extent other and carbon dioxide. more reduced compounds, like for instance ethanol, are formed. Because of the production of VFA the fermenting bacteria are usually Development of wastewater treatment designated as the acidifying population. at KWST The second group of bacteria, which breaks down the products of the acidification step, are In the mid 1980s KWST decided, together with called the acetogenic bacteria after their main the university of Hannover, to perform some product acetate. Besides acetate, H and in the 2 testing with the treatment of their wastewater. case of odd numbered carbon compounds, The tests came out positively and KWST decided CO is also formed during acetogenesis. The 2 to build a low loaded anaerobic trickling filter and methanogenic bacteria form the third group. post-aeration with a clarifier (dortmundtrichter) They convert acetate or H plus CO into 2 2 as secondary clarifier. At the end of the 1990s methane. Other possible substrates for the this treatment plant could hardly handle the methanogens, such as formate (HCOOH), increased COD load of the distillery. As the methanol (CH OH), carbon monoxide (CO) and 3 technical and technological situation of the methylamines, are of minor importance in most existing WWTP (Waste Water Treatment Plant) anaerobic digestion processes. Wastewater management and energy recovery 259 was at an end, KWST decided to build a new technically advanced WWTP. Together with the university of Hannover, KWST wrote out a tender procedure for the realisation of this new plant. The contract, for the design and construction of the WWTP, was awarded to Paques in June 2001. The plant was completed in 6 months and was started up in January 2002 (Figure 2).

Wastewater composition at KWST

The distillery, using mainly molasses, has different wastewater sources. • Condensate evaporator (COD and flow) • Floor cleaning (mainly flow) • Drain pits (mainly COD) • Vacuum pumps wastewater (mainly COD) • Lutterwasser alcohol absolution and rectification plant (mainly flow)

There is a broad variation in wastewater composition, because each installation has different production patterns. The foreseen expansion of the production had to be taken into account for the design of the proposed Figure 2. Buffer tank and IC (Internal Circulation) wastewater treatment plant, but the effect on the reactor. wastewater composition was not very high. In Table 1 you will find details on the wastewater composition.

Table 1. Wastewater composition at KWST. Besides the wastewater composition the effluent requirements are important for designing the Average Maximum optimal plant. Table 2 lists the most important Flow m3/h 12 effluent requirements for KWST imposed by the Flow m3/d 200 240 city of Hannover. CODtotal mg/l 15500 75000 CODfiltered mg/l 15000 70000 CODload-filtered kg/d 3000 3600 Table 2. Effluent requirements for sewer discharge TKN mg/l 140 750 in Hanover. Nitrat mg/l 10 50 N-total mg/l 150 800 Maximum P-total mg/l 10 50 S-total mg/l 80 400 COD total mg/l 2,000 TSS mg/l 300 2000 TSS mg/l No limit Chlorides mg/l 1100 3500 Temperature °C 35 Calcium 20 100 pH 6.5 - 10 PH 4 3 (minimum) AOX mg/l 1 Temperature °C 65 90 260 V. Groot Kormelinck

Brief overview of effluent treatment at COD concentration entering the IC-reactor and KWST alkalinity is retrieved. This alkalinity recycle reduces the caustic dosing requirements to a The flow diagram (Figure 3) and process description minimum. The temperature of the wastewater can hereafter give an overview of the solution that be controlled within the required range before was developed for the WWTP at KWST. The entering the IC reactor by a heat exchanger. wastewater of the different production plants The wastewater is then pumped to the flows to the influent pit by gravity and is pumped anaerobic IC-reactor. Before start-up, the IC- to the buffer tank. If the wastewater temperature reactor has been inoculated with anaerobic sludge in the buffer tank is too high, the influent can be in order to bring the IC-reactor up to the design cooled by a heat exchanger. In the buffer tank, load within weeks. The IC-reactor, with a volume 3 hydraulic and COD-load variations are leveled. of 125 m reduces 80 to 90% of the soluble COD. Because the wastewater is lacking sufficient Due to the flexibility of the system, the variation nutrients, urea, phosphoric acid and a yeast in the wastewater composition is less critical. This extract are dosed. These nutrients are already gave us the opportunity to decrease the originally dosed in the buffer tank to be available for the projected buffer volume. acidifying bacteria as well as for the methanogenic The COD is converted into valuable biogas, bacteria in a later stage. consisting mostly of methane and carbon dioxide. After a retention time of approximately 8 This biogas is burned in a boiler to generate steam. hours, the wastewater is pumped from the buffer At KWST there are plans to use the biogas for the tank to the recirculation tank. Because of the high production of electricity. The design has already COD concentration (above 10 g/l) and the low allowed for expansion with this gas of a motor pH (average 4.5) in the influent, wastewater from unit. The anaerobically treated wastewater is the anaerobic as well as the aerobic treatment post treated in an aeration tank. In this tank part is recirculated. This recirculation reduces the of the remaining COD is converted into water

KWST Gasholder Flare HANNOVER -GERMANY 5 m³ 80 m³/h

N, P CaCl2 Biogas NaOH *

* 1 m³ 1 m³ 1 m³

* Dortmund * 46 m³ Influent Effluent

Buffer- Mix/ recirculation IC reactor Anaerobic Aeration Recirculation Preacidification tank 125 m³ sludge tank tank tank tank 32 m³ H=20m D=2.85m 30 m³ 137 m³ Sludge 12 m³ 275 m³ Air

*

Figure 3. Flow diagram for effluent treatment at KWST. Wastewater management and energy recovery 261 and carbon dioxide, ammonium is nitrified and recirculation ⇒ more influent dilution. The sulfides are oxidized to sulphate. The off-gasses recirculation flow under these circumstances is from this WWTP are used for the aeration of this approximately 2.5 times the gas flow. Alkalinity tank. In this way the anaerobic treated wastewater is formed in the reactor during COD conversion as well as the off-gasses are polished and the and is recovered for influent neutralisation. smelly components as such are oxidized. A clarifier (dortmundtrichter) prevents solids from entering the aerobic recycle to the recirculation tank. The overflow of the clarifier flows into the sewer system to a municipal wastewater treatment plant.

Operating principle of an IC-reactor (Figure 4)

The influent is pumped into the reactor via a distribution system, where influent and recycled sludge/effluent are well mixed (1). The first reactor compartment contains an expanded granular sludge bed (2), where most of the COD is converted into biogas. The biogas produced in this compartment is collected by the lower level separator (3) and is used to generate a gas lift by which water and sludge are carried upward via the ‘riser’ pipe (4) to the gas/liquid separator located on top of the reactor (5). Here the biogas is separated from the water/sludge mixture and leaves the system. The water/sludge mixture is Figure 4. IC-reactor. directed downwards to the bottom of the reactor via the (concentric) ‘downer’ pipe (6), resulting in the internal circulation flow. Results The effluent from the first compartment is post- treated in the second, low-loaded compartment Within 2 weeks after start-up, the plant was able (7), where remaining biodegradable COD is to treat all the wastewater of the distillery. The removed. The biogas produced in the upper average COD load in the wastewater at that compartment is collected in the top 3-phase time was approximately 2000 kg/d. Figure 5 separator (8), while the final effluent leaves the shows an overview of the COD load, the biogas reactor via overflow weirs. production and the efficiency of the IC-reactor as well as the overall efficiency. Although outside the design range, the Recirculation System anaerobic reactor did treat the COD-peaks of 5800 and 4100 kg/d, without sludge loss. The internal circulation is based on the gas lift Temperature during this period was 28°C and principle. Driven by the reactor gas flow, the the volumetric loading rate during these peaks recirculation flow rate depends on the influent were respectively 46 and 33 kg/m3*d. However, COD and is therefore self-regulating: higher after these peaks were noticed, the influent flow influent COD ⇒ higher gas flow ⇒ more was adjusted to within the design parameters. 262 V. Groot Kormelinck

7000 100

90 6000 80

5000 70

60 ) 4000 50 3000

40 Efficiency (% COD load (kg/d)

2000 30

20 1000 10

0 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 Date COD load efficiency Figure 5. COD load and efficiency of IC-reactor.

1800 7000

1600 6000 1400 5000 1200

1000 4000

800 3000 COD load (kg/d )

Biogasproduction (m3/d) 600 2000 400 1000 200

0 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 Date Biogas production COD load Figure 6. Biogas production and COD load.

The measurement of the biogas production did Because of the relatively short aeration time not function properly during the first period only the easily convertible COD is converted and (Figure 6). Therefore this is not shown in the the efficiency of the aerobic treatment is not very graph. At a COD-load of approx. 2,000 kg/d high. The solids and the rest of the COD washes the IC reactor produces an average of 900 m3 through. Therefore, the overall efficiency is only biogas/d (Figure 6). slightly higher then the IC-efficiency (Figure 7). Wastewater management and energy recovery 263

100

90

80

70

60

50

Efficiency (%) 40

30

20

10

0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 Date efficiency IC efficiency overall Figure 7. COD efficiencies of IC-reactor and overall. Costs and benefits Table 4. Guidelines for calculation of effluent treatment costs. To give an idea of the costs and benefits from Mixed liquor suspended 5 g/l a wastewater treatment plant in the distilling solids industry, we have compared the capital as well Food / Mass ratio 0.1 kg BOD/kg MLSS as the operational costs for an aerobic treatment Oxygen uptake 1.5 Kg O2/kg BOD plant with those for a combined anaerobic/ Aerobic sludge production 0.3 kg TSS/kg BOD aerobic treatment plant. The basis for this Energy consumption aerators 1 kg O2/kWh comparison is a distillery with the wastewater Electricity price 8 eurocent/kWh characteristics shown in Table 3. Aerobic sludge treatment 100 €/per ton and discharge costs Table 3. Wastewater characteristics of a distillery. Conversion rates from 40% electricity, biogas 50%heat, 10%loss Average Income for the supply of 11 eurocent/kWh green electricity to 3 Flow m /h 300 general network 3 Flow m /d 7200 CODtotal mg/l 9700 CODload kg/d 70000 Required for aerobic treatment only: BOD load kg/d 49000 BOD/COD 0.7 PH 4 - 5 • Aeration volume of approximately 98,000 Temperature °C 35 m3. • Aeration capacity of 5444 kg O2/h or 5444 kWh. The guidelines for calculation of costs are given • Sludge treatment for aerobic sludge of 14.7 in Table 4 and are discussed below. There are ton/d insufficient nutrients in the wastewater, so dosing • Secondary clarifier is required. 264 V. Groot Kormelinck

Required for anaerobic/aerobic treatment: • In comparison to aerobic systems, the sludge production in an anaerobic/aerobic system • Buffertank; retention time 12h is very low, whereas the anaerobic sludge • IC-reactor with a volume of 2800m3 (85% production can be “sold” to other anaerobic COD efficiency and 90% BOD efficiency) plants for start-up. • Aeration of 9800 m3 • The economic benefits for a combined • Aeration capacity: 544 kg O2/d or 544 kWh anaerobic/aerobic system for distilleries are • Sludge treatment for aerobic sludge of 1.47 huge. ton/d • Secondary clarifier Table 5. Breakdown of effluent treatment costs.

Table 5 shows that costs for electricity usage Aerobic Anaerobic / and sludge production are crucial figures. These treatment aerobic only treatment are responsible for respectively. 90% and 40% of the operational costs. The investment costs Investment costs € 4.300.000 8.500.000 for the combined anaerobic/aerobic system are Operational costs almost twice the investment costs of the aerobic system. But combined with the operational costs Interest costs (6%) € 258,000 510,000 both options are almost equal. The biogas, that Personnel € 36,000 36,000 can be used as a replacement for natural gas in Chemicals € 145,000 230,000 the boilers or converted to electricity and warm Energy € 3,815,155 451.315 € water is the decisive factor in this comparison Sludge treatment 536.650 53.665 leading to a firm preference for the anaerobic/ and discharge Total op. costs € 4,790,805 1,280,970 aerobic combination. For wastewaters with 70 Benefits tons of COD the economic benefits are very clear but also for smaller distilleries like KWST Methane production m3/a 0 7,601,125 there are enough advantages to build their own Methane Mw/a 68,410 anaerobic WWTP. Gas motor Electricity MW/a 27,365 Electricity € 3,010,046 Conclusions MJ heat MJ/h 14,057 Total benefits € 0 3,010,046

• An anaerobic system has a relatively short start-up period. • The reactor performance is stable over a longer period of time. Bibliography • High COD peaks are accepted by the IC- reactor. Sahn, H. Microbiology and Biochemistry fo • Efficiencies of 85% and more are no Anaerobic Water Treatment www.kwst. exception. com • Anaerobic treatment, and especially the use of Zehnder, A. J. B. (1988). Biology of Anaerobic the IC-reactor, for this wastewater has proven Microorganisms. John Wiley and Sons, to be highly effective. U.K. Co-products from the distilling industry 265 Chapter 36 Co-products from the distilling industry

R.W. Hall James & Son (Grain Merchants) Ltd, Wellingborough, Northants, UK

Co-products for animal feed the high moisture content. This necessitates additional drying and/or transport costs. Moist The distilling industry takes raw materials distillers’ grains can be effectively marketed, rich in starch or sugars and converts much of but customers are generally local to the site of these components into alcohol, which is then production and volumes must match animal separated. This leaves behind all the other consumption. However, dry distillers’ grains fractions of the raw material in more concentrated have a much larger target market and, due to form. This includes undistilled fermentation the loss of water, they are cheaper to transport products and yeast debris. The residues of malt and have a longer storage potential. and unmodified cereal grain are richer in protein, Additional constraints have appeared more fibre, oil and ash. The challenge for the industry recently. Of greatest concern is the low is to identify profitable markets where there is profitability of dairy farming and the low price demand for such fractions. Table 1 summarises of competitive feedstuffs. As a consequence, the volumes and physical form of co-products research is now being directed towards the for animal feed. identification and extraction of high value Traditionally, the principal outlet has been products by means of enzymes, carbon dioxide the ruminant feed market (Table 2), because extraction and other high-tech processes. Such ruminant animals have evolved a microbial high value extracts may be some way from digestive system that can make good use of plant commercialisation, but they will not represent fibre and enhance the quality of cereal-derived a complete answer unless there is concomitant protein. The greatest obstacle to the profitable effort to identify effective and economic ways use of distilling co-products has always been of utilising the residues. Provided that these

Table 1. The volumes and physical forms of co-products for animal feed.

Product 2005/06 2008/09 2009/10 2010/11

Moist distillers’ grains 1,000,000 1,250,000 1,875,000 2,500,000 Wheat starch syrup 210,000 263,000 395,000 526,000 Dried distillers’ grains 284,000 355,000 533,000 710,000 266 R.W. Hall

Table 2. UK ruminant feed market. All values in ‘000 tonnes.

Feed types 1998/99 2002/03 2004/05 2009/10 Forecast

Moist feeds 1650 1830 1827 1930 Compounds/blends 4508 4080 4060 3725 Cereals 1530 1550 1650 1650 Other straights 875 868 885 960 Total straights 2835 2788 2930 3000 Molassed 430 370 395 390 Sugarbeet feed

Total 8993 8698 8817 8655 processes do not contaminate the residue, the on availability and the supply chain - are major animal feed market may still offer the sensible issues. commercial option. Elsewhere in the world, the development of Distillers’ want guaranteed clearance – ‘Just biofuels industries has resulted in an increase of in Time’ and to be able to meet their legal and between 50% and 100% in costs of feedstock environmental responsibilities while receiving supplies. Future grain prices are already acceptable financial returns. The requirements reflecting the new demand in the UK; experience of farmers are for cost-effective feed and a elsewhere suggests that prices could increase guaranteed regular supply with sound nutritional by around 25% in the short to medium term. advice. Competition for the purchase of raw materials will increase dramatically as producers of biofuels enter the market. Co-products for biofuels In the UK, it is likely that biopetrol (bioethanol or biobutanol) will be produced from fermentation At present, 21 million tonnes of wheat and of cereal starch from wheat and biodiesel from barley are grown in the UK per annum, the rape oil, if the industry is to use home grown majority of which is used in domestic feed and crops. In order to achieve the initial 5% target human food processing. The government has the required volume of biopetrol will consume placed an obligation on fuel supply companies all or most of the UK’s current surplus of cereal; to include 5% of biofuels in road transport production of biodiesel will need an increase of fuels by 2010 with an aim to increasing this to 50% - 70% in oilseed rape production. 10-20% subject to vehicles being modified to Over the coming years, the aim will be to utilise such levels (Table 3). The implications produce biofuels from the residues of food of this – commercially, environmentally, and production, in particular, cellulose and other Table 3. Renewable transport fuel obligations (RTFOs) and the implications for bioethanol production and wheat use.

2008/09 2009/10 2010/11 EU Commission’s target for inclusion 5.75% Government’s RTFO proposals 2.5% 3.75% 5% Bioethanol required – tonnes 450,000 675,000 900,000 Wheat requirement – tonnes 1.5m 2.25m 3.0m Co-products from the distilling industry 267 cell wall material that contains sugar polymers. The distilling co-products industry will be well placed to meet this market as it develops.

Further reading

Animal Feed and Animal Health - Publications and Information (2008). Feed Facts Quaterly, http://www.simonmounsey.com (accessed 30 April 2008) DEFRA (2008). Department for Environment, Food and Rural Affairs, http://www.defra. gov.uk (accessed 30 April 2008). 268 R.W. Hall Packaging materials as a source of taints 269 Chapter 37 Packaging materials as a source of taints

J.M. Conner and K.J.G. Reid The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK

Introduction The odour thresholds of these compounds in water are 0.07, 4 and 0.008 ng/L respectively Taint is the term used to describe an alien flavour (van Gemert, 1999), so only minute amounts in food, which comes from an outside source and are required to produce a noticeable taint. In is perceived by consumers as unpleasant. The alcoholic beverages the thresholds are higher. number of complaints due to tainted products Reported thresholds for 2,4,6-trichloroanisole received by the distilled beverage industry is in wine are generally around 5 ng/L (Taylor, low, with most taints due to ‘musty’ or ‘earthy’ Young, Butzke and Ebeler, 2000). The threshold aromas. Analysis of these complaint samples by of 2,4,6-trichloroanisole in 20 % (v/v) ethanol, the Scotch Whisky Research Institute has shown determined by the Institute’s sensory panel, was that they are the result of contamination by 44 ng/L but 1,400 ng/L in a blended whisky 2,4,6-trichloroanisole, 2,3,4,6-tetrachloroanisole diluted to 20 % (v/v) ethanol for sensory analysis. or 2,4,6-tribromoanisole. The general structure The increase in thresholds between water, wine of these compounds is shown in Figure 1. and spirits reflects the greater solubility of the anisoles at higher alcoholic strengths. However OCH the differences between the neutral alcohol 3 and the blended whisky suggest that there can be significant matrix effects, which reduce the Cl 6 2 Cl amount of anisole entering the headspace and

3 consequently its perception (Figure 2). This R elevated threshold will contribute to the lower 4 incidence of taints in whisky and other spirits when compared with wine. Cl Taints by chloro- and bromo-anisoles have been the subject of much research by the food Figure 1. Structure of chloroanisole. and beverage industry and the two major sources R=H for 2,4,6-trichloroanisole; R=Cl are corks and wood/paper based packaging for 2,3,4,6-tetrachloroanisole. For materials. Their mechanism of formation has 2,4,6-tribromoanisole R=H and all chlorine atoms been identified and requires the presence of (Cl) are replaced by bromine atoms. precursor phenols in the packaging material 270 J.M. Conner and K.J.G. Reid

180000 160000 aqueous ethanol 140000 120000 100000

Area 80000 60000 mature malt whisky 40000 20000 0 0.05.0 10.0 15.0 20.0 Conc(ppb) Figure 2. Comparison of headspace peak areas for 2,4,6-trichloroanisole when dissolved in aqueous ethanol and an untainted malt whisky. that are converted to the active taint by micro- looked at the formation of chloro- and bromo- organisms growing in damp conditions. Fungi anisole taints (Gee and Peel, 1974; Curtis, Land, naturally present in the environment where the Griffiths, Gee, Robinson, Peel, Dennis and packaging materials are stored and handled have Gee, 1972; Land, Gee, Gee and Spinks, 1975; the ability to convert the available precursor to Tindale, Whitfield, Levingston and Nguyen, the anisole taint. This process is the crucial stage 1989; Whitfield, Nguyen and Tindale, 1991; in the transfer of the contaminant to product. Hill, Hocking and Whitfield, 1995) and in each The anisole is more volatile than the phenol and case it was shown that fungi isolated from the this increases the mobility of the taint within the source of the taint were capable of converting storage environment. The anisole is also much chloro- and bromo-phenol precursors to the more fat soluble than the phenol and this leads corresponding anisoles. All studies have shown to accumulation in plastic or waxy material. that the conversion of the precursor phenol This aids transfer through plastic wrapping and cannot be related to the presence of a single experiments with chloroanisoles have shown that species or strain of fungus, but in each incident a only metallised polypropylene laminates present range of fungi have been isolated with the ability a significant barrier to contamination by these to form chloro- or bromo-anisoles. Although taints (Jickells, Poulin, Mountfort and Fernandez- different taint incidents have shown a number Ocana, 2005). of species/strains in common, it is likely that In each incidence of taint, there are two factors the fungi converting the phenol precursor are to be considered. The first is the formation of the naturally present in the environment where the anisole taint from the precursor phenol and the packaging materials are stored and handled. second is the origin of the phenol precursors. This An essential trigger for microbial growth paper is based on the analysis of tainted samples is the moisture content of the packaging. A of whisky and will review the major sources of number of the anisole forming fungi are known the packaging taints encountered and conclude to be xerophilic and for these to germinate in with a summary of ways in which packaging taints packaging requires a moisture content between may be avoided. 12 and 16 % (Whitfield et al, 1991). Most paper and cardboard has moisture content between 2 and 6 %, requiring additional moisture The formation of the anisole taint ingress to initiate fungal growth. Consequently, from the precursor phenol maintaining dry conditions during the transport and storage of goods is an effective method There have been a number of studies that have of preventing the development of taints. Packaging materials as a source of taints 271

However, it is not always possible to maintain The origins of the phenol precursors dry conditions during transport. Fluctuations in temperature within transport containers have Two sources of phenolic precursors have been shown to produce condensation that is been linked to incidents of taint: paper and sufficient to promote fungal growth in packaging cardboard packaging materials and wood in or on the container walls, producing chloro- or the form of pallets, flooring and lining transport bromo-anisoles and tainting goods transported containers, etc. For both sources the chloro- and within the container (Whitfield, Nguyen, Shaw, bromo-phenols present are the result of their Last, Tindale and Stanley, 1985; Whitfield, Shaw, deliberate use as fungicides or preservatives, Lambert, Ford, Svotonos and Hill, 1994). or their inadvertent formation during cleaning, In cork taint, the formation of sterilising or bleaching operations. 2,4,6-trichloroanisole has been blamed on the Incidents of taint have been linked to the use growth of moulds and fungi that survive the of tetrachlorophenol and pentachlorophenol cork cleaning process. However, results from as preservatives for wood and pulp. For the EU funded QUERCUS project suggest that pentachlorophenol, taints are generally caused the single greatest correlation with the amount by 2,4,6-trichloroanisole and/or tetrachloro- of 2,4,6-trichloroanisole in finished corks is anisoles, which are derived from impurities or the relative level of 2,4,6-trichloroanisole in degradation products of the pentachlorophenol the original bark, though the origin of this (Gunschera, Fuhrmann, Salthammer, Schulze 2,4,6-trichloroanisole has not been identified. and Uhde, 2004). The use of pentachlorophenol Cork is a very complex ecosystem and the is now restricted or banned in many countries microbiota associated with the various steps round the world (Jensen, 1996) and this should of the manufacturing process has not been decrease the incidence of taints from this established. The growth of filamentous fungi on source. However a potential replacement for cork has been considered beneficial, contributing pentachlorophenol is 2,4,6-tribromophenol to softening of the cork, cleaning the cork surface and concerns have been raised that its use by and filling the internal lenticellular channels the wood, paper and pulp industries will lead (Silva Pereira, Figueiredo Marques, and San to a much greater incidence of musty taints due Romão, 2000). Recent research has shown that to 2,4,6-tribromoanisole (Whitfield, Hill and of 14 fungal strains isolated from cork samples Shaw, 1997). obtained at different stages of production, Chlorophenols are known to be formed eleven could produce 2,4,6-trichloroanisole during chlorine bleaching of pulp (Jensen, 1996) when grown directly on cork in the presence and chlorine disinfection of drinking water of 2,4,6-trichlorophenol (Alvarez-Rodriguez, (Hodin, Boren, Grimvall and Karlsson, 1991) Lopez-Ocana, Lopez-Coronado, Rodriguez, and their formation must also be suspected Martinez, Larriba and Coque, 2002). The study any time that active chlorine (including noted different trichlorophenol conversion hypochlorite) comes into contact with woody efficiencies for the same species of fungi isolated materials. Examples where this reaction has in different studies (e.g. Chrysonilia sitophila). been linked to the production of chloroanisole This may be due to the isolation of different taints are the cleaning of wood lined transport strains or the different growing conditions used containers with chlorine based cleaning agents in the studies and indicates that further research (Tindale and Whitfield, 1989) and in paper and into both the biochemical pathways and the cardboard based packaging materials made regulatory processes is required before the role from recycled materials (Whitfield, Tindale, of fungi in converting 2,4,6-trichlorophenol to Shaw, Last and Stanley, 1984). The latter case 2,4,6-trichloroanisole in cork production is fully is of particular interest as analysis of cardboard understood. boxes that contained tainted fruit detected the 272 J.M. Conner and K.J.G. Reid presence of trichlorophenol, from bleaching of Little is known about the sources of the pulp, and pentachlorophenol, a preservative chlorophenol precursors in cork. The use of during paper manufacture (Whitfield et al., hypochlorite for cork washing has been shown 1985). Replacement of pentachlorophenol with to increase the levels of chlorophenols, and 2,4,6-tribromophenol would greatly increase this type of wash has generally been replaced the taint potential of paper and cardboard based by processes that do not utilise chlorine or packaging materials. To assess this, the Institute hypochlorite. Another suggested source is analysed a range of packaging materials currently treatment of trees in the past with chlorinated used by the Scotch whisky industry. The analysis pesticides, but challenging a known former of found very low levels of 2,4,6-trichlorophenol TCA with a range of these compounds including (Table 1) in all the packaging materials analysed, pentachlorophenol and hexachlorobenzene with the highest concentration (35 µg/kg) in did not produce TCA (Alvarez-Rodriguez et al., packaging that contained a high concentration 2002). It is also possible that they are formed of pentachlorophenol. The low levels of these naturally in the environment around cork trees. compounds are most probably due to the Fungi with the ability to degrade woody forest decline during the 1990s in the use of chlorine litter have the capacity to produce chlorinated by the paper and pulp industry in response metabolites. A wide range of chlorinated to environmental pressures (Confederation of metabolites has been identified, which includes European Paper Industries, 1998). The amount chlorophenols and chloroanisoles and these of 2,4,6-tribromophenol in packaging samples are thought to play an important role in lignin was highly variable suggesting that its presence degradation processes (de Jong and Field, 1997; is not due to its widespread use as a preservative Verhagen, Van Assema, Boekema, Swarts, or a single instance of contamination. Little Wijnberg and Field, 1998). However, there has information could be found on the current use been only limited research on the metabolites of this compound and its sporadic occurrence produced by microbial growth in cork. in packaging may be due to its presence in the recycled materials used in the manufacture of the paper and cardboard. Summary

Packaging taints may be the result of Table 1. Concentrations (µg/Kg) of 2,4,6-trichloro- contamination by 2,4,6-trichloroanisole, and 2,4,6-tribromophenols detected in packaging 2,3,4,6-tetrachloroanisole or materials used by the Scotch Whisky industry. 2,4,6-tribromoanisole. Moisture is the essential Total number of samples analysed was 54. trigger for the growth of fungi that can form Sample 2,4,6- 2,4,6- these anisoles from their precursor phenols. trichlorophenol tribromophenol The fungi responsible for conversion may come from the microbial environment where materials Mean Range Mean Range are packaged, stored and transported. Potential Layer pad 4 2 – 6 25 0 – 110 sources of the precursor phenols are paper and Divider 10 3 – 35 28 5 – 62 cardboard based packaging materials and wood Cork case 3 1 – 5 19 1 – 65 surfaces in transport containers and storage areas. Packing case 3 2 – 4 6 4 – 10 Once formed the taints are pervasive and volatile. Case divider 4 2 – 6 41 12 – 84 Contamination may occur during the transport Bottle carton 2 1 – 5 3 1 – 5 and storage of packaging components such as Bottle tube 3 2 – 4 13 11 – 20 bottles and caps, or may occur during the storage of cased goods. Standard screw cap closures Packaging materials as a source of taints 273 do not prevent chloro- and bromoanisole taints Avoid the formation of chlorophenols in entering the product. storage areas Cork taint in spirits is exclusively caused by 2,4,6-trichloroanisole. Taints due to This can be achieved by making sure there is 2,3,4,6-tetrachloroanisole or 2,4,6-tribromoanisole no contact between wood and paper products are most likely to have been acquired during the and chlorine based cleaning agents. This is transport and storage of raw materials and finished particularly true for all wood products. Chlorine corks. The origin(s) of 2,4,6-trichloroanisole in will react with both soft and hard woods to cork is not fully understood, but a number of produce di- and trichlorophenols. Timber-lined potential sources have been identified and Good transport containers should not be cleaned with Manufacturing Practice guidelines drawn up chlorine based cleaning agents and timber pallets to eliminate these sources. These guidelines and cardboard cartons should be removed from and innovations by cork manufacturers should storage areas before cleaning with hypochlorite reduce the incidence of cork taint. agents and not returned until the areas are completely dry.

Avoiding packaging taints Where possible eliminate chloro- and These measures should be applied to both the bromophenols from packaging and storage storage of packaging components (bottles, corks, areas screw caps etc) and to finished products. There have been cases where chloroanisoles have Avoid cardboard and paper products containing been adsorbed by the dry goods, such as bottles, chloro- and bromophenols. This however may cans and closures, and this resulted in a tainted not be easily achieved as raw materials may be product. Also it should be noted that wrapping treated prior to processing by the cardboard goods in polythene gives no protection against manufacturers. contamination by chloro- or bromoanisoles, indeed it may actually assist the transfer of anisole to the product within. Avoid the environmental conditions required for fungi / mould growth

Cork taint Most anisole producing fungi require damp humid conditions (water activity greater than The incidence of cork taint should decline 0.75) for growth. As long as precursors persist in in the coming years as a Code of Good packaging materials, producers should minimise Manufacturing Practice for Cork has been drawn the risk from taints by storing packaging, up and will have been in use ‘by all responsible dry goods and bottled products in dry, well- manufacturers’ from the 1997 harvest. There ventilated conditions, which will prevent the have also been technological improvements by growth of micro-organisms responsible for taint the major cork manufactures (use of hydrogen formation. However the risk of taint formation peroxide, hyrodynamic extraction) that will also remains, when products are stored in adverse reduce the incidence of taint. Moisture is an storage conditions outwith the control of the important trigger for fungal growth on the cork producer. surface and the transport and storage of corks in a clean dry atmosphere will prevent any taint formation post-production. 274 J.M. Conner and K.J.G. Reid

References Jickells, S.M., Poulin, J. Mountfort, K.A. and Fernandez-Ocana, M. (2005). Migration of Alvarez-Rodriguez, M., Lopez-Ocana, L., contaminants by gas phase transfer from Lopez-Coronado, J., Rodriguez, E., carton board and corrugated board box Martinez, M., Larriba, G. and Coque, J-J. secondary packaging into foods. Food (2002). Cork taint of wines: Role of the Additives and Contaminants 22: 768 – filamentous fungi isolated from cork in 782 the formation of 2,4,6-trichloroanisole by Land, D.G., Gee, M.G., Gee J.M. and Spinks, O-methylation of 2,4,6-trichlorophenol. C.A. (1975). 2,4,6-trichloroanisole in broiler Applied Environmental Microbiology 68: house litter: a further cause of musty taint 5860 – 5869 in chickens. Journal of the Science of Food Confederation of European Paper Industries (1998). and Agriculture 26: 1585 – 1591 Environmental Report 1998 Confederation Silva Pereira, C., Figueiredo Marques, J.J. and of European Paper Industries San Romão, M.V. (2000). Cork taint in wine: Curtis, RF., Land, D.G., Griffiths, N.M., Gee, scientific knowledge and public perception M., Robinson, D., Peel, J.L., Dennis, C. and – a critical review. Critical Reviews in Gee J.M. (1972). 2,3,4,6-tetrachloroanisole Microbiology 26: 147 – 162 association with musty taint in chickens and Taylor, M.K., Young, T.M., Butzke, C.E. and microbiological formation. Nature (London) Ebeler, S.E. (2000). Supercritical Fluid 235: 223 – 224 Extraction of 2,4,6-Trichloroanisole from de Jong, E. and Field, J.A. (1997). Sulfur tuft and Cork Stoppers. Journal of Agricultural and turkey tail: Biosynthesis and biodegradation Food Chemistry 48: 2208 -2211 of organohalogens by Basidiomycetes. Tindale, C.R. and Whitfield, F.B. (1989). Annual Reviews of Microbiology 51: 375 Production of chlorophenols by the reaction – 414 of fibreboard and timber components with Gee, J.M. and Peel, J.L. (1974). Metabolism chlorine-based cleaning agents. Chemistry of 2,3,4,6-tetrachlorophenol by micro- and Industry (London), 835 - 836 organisms from broiler house litter. Journal Tindale, C.R., Whitfield, F.B., Levingston, S.D. of General Microbiology 85: 237 – 243 and Nguyen, T.H. (1989). Fungi isolated Gunschera, J., Fuhrmann, F., Salthammer, T., from packaging materials: their role in the Schulze A. and Uhde, E. (2004). Formation production of 2,4,6-trichloroanisole. Journal and emission of chloroanisoles as indoor of the Science of Food and Agriculture 49: pollutants. Environmental Science and 437 - 447 Pollution Research 11: 147 – 151 van Gemert, L.J. (1996). Compilations of odour Hill, J.L., Hocking, A.D. and Whitfield, F.B. threshold values in air and water. TNO (1995). The role of fungi in the production Nutrition and Food Research Institute, of chloroanisoles in general purpose freight published by Boelens Aroma Chemical containers. Food Chemistry 54: 161-166 Information Service Hodin, F., Boren, H., Grimvall, A. and Karlsson, Verhagen, F., Van Assema, F., Boekema, B., S. (1991). Formation of chlorophenols and Swarts, H., Wijnberg, J. and Field, J. (1998) related compounds in natural and technical Dynamics of organohalogen production chlorination processes. Water Science and by the ecologically important fungus Technology 24: 403 – 410 Hypholoma fasciculare. FEMS Microbiology Jensen, J. (1996). Chlorophenols in the terrestrial Letters 158: 167-178 environment. Reviews of Environmental Whitfield, F.B., Tindale, C.R., Shaw, K.J., Last, Contamination and Toxicology 146: 25 – J.H. and Stanley, G. (1984). Contamination 51 of cocoa powder by chlorophenols and Packaging materials as a source of taints 275

chloroanisoles adsorbed from packaging Whitfield, F.B., Shaw, K.J., Lambert, D.E., materials. Chemistry and Industry (London), Ford, G.L., Svotonos, D. and Hill, J.L. 772 – 774 (1994). Freight containers: major sources Whitfield, F.B., Nguyen, T.H., Shaw, K.J., of chloroanisoles and chlorophenols in Last, J.H., Tindale, C.R. and Stanley, foodstuffs. In: Trends in Flavour Research. G. (1985). Contamination of dried Edited by Maarse, H. and van der Heij, D.G. fruit by 2,4,6-trichloroanisole and Elsevier Science B.V., pp. 401 – 407 2,3,4,6-tetrachloroanisole adsorbed from Whitfield, F.B., Hill, J.L. and Shaw, K.J. (1997). packaging materials. Chemistry and Industry 2,4,6-tribromoanisole: a potential cause (London), 661 - 663 of mustiness in packaged food. Journal of Whitfield, F.B., Nguyen, T.H. and Tindale, Agricultural and Food Chemistry 45: 889 C.R. (1991). Effect of relative humidity and - 893. incubation time on the O-methylation of chlorophenols in fibreboard by Paecilomyces variotii. Journal of the Science of Food and Agriculture 55: 19-26 276 J.M. Conner and K.J.G. Reid Proving that alcoholic beverage distillates are free from cereal, nut & milk derived allergens 277 Chapter 38 Proving that alcoholic beverage distillates are free from cereal, nut and milk derived allergens

I.C. Goodall, J.M. Brosnan, K.A. Campbell, C.D. Owen, G.M. Steele and J.W. Walker The Scotch Whisky Research Institute, The Robertson Trust Building, Riccarton, Edinburgh, UK

Introduction Whisky Research Institute (SWRI) to carry out such scientific studies to support the deletion of From November 25th 2005, Directive 2003/89/ the indicated alcoholic distillate raw materials EC of the European Union requires that alcoholic from Annex IIIa. beverages must be labelled to indicate the presence of certain ingredients, or any products thereof, known to cause allergic reactions or Initial work intolerances in consumers. Three of the listed ingredients, cereals, nuts Background and milk products, are used in the distilled spirits industry prior to distillate collection: cereals and Directive 2003/89/EC stated that the European whey as substrates for the production of alcohol, Commission could be notified, prior to August nuts as natural flavourings. Whilst the EU spirits 25th 2004, of studies being conducted to establish industry supports the protection of consumer that ingredients or substances, derived from the health and the provision of information to specific ingredients listed in Annex IIIa, are not sensitive consumers, these principles would not likely under specific circumstances to trigger be upheld by labelling distilled spirits to indicate adverse reactions. Following this notification, the use of these three ingredients. Distillation European Food Safety Authority (EFSA) would be should not allow the non-volatile allergens of the consulted and a list of ingredients adopted which cereal, nut and whey raw materials to transfer would temporarily be excluded from Annex IIIa into the final product. As a result, labelling until November 25th 2007, pending the results spirits to indicate the use of cereal, nuts or milk from the full studies. products could lead to consumer confusion and Three dossiers were prepared by the SWRI, diminution of choice. individually requesting exemption of the Directive 2003/89/EC provides a means by following ingredients from Annex IIIa of which an ingredient can be deleted from the Directive 2003/89/EC: list in Annex IIIa when it has been scientifically established that it is not possible for it to 1. Distillates where cereals are used as raw cause adverse reactions. The European Spirits materials in their manufacture before Organisation (CEPS) commissioned the Scotch distillation. 278 I.C. Goodall et al.

2. Distillates where nuts, either whole or transfer from the raw materials to the alcoholic processed (for example, nut oils), have been distillate, a range of distilled spirit samples were added to the process prior to distillation to analysed to test this theory. Two techniques were act as a flavouring. applied. The first technique was to analyse the 3. Distillates where whey is used as a raw material samples for specific allergens that are present in in their manufacture before distillation. the raw materials. ELISA based methods were used to measure for levels of specific proteins. In Beverages which contain such alcoholic the case of cereals, the ELISA method was shown distillates are wide ranging and include whiskies, to reliably detect gluten down to 10 mg/l, based gins, vodka, flavoured spirit drinks such as pastis on tests where spirits were spiked with known and ready to drink alcoholic beverages. Cereal or levels of this protein. For nuts, the method was whey derived neutral alcohol can also be used shown to detect almond proteins equivalent to as a solvent for flavourings as used in a range of 1 mg almond/l. For whey, the ELISA method food and beverage products. could detect the whey protein ß-lactoglobulin down to 5 mg/l. Lactose was measured using ion-exchange chromatography with pulsed Rationale amperometric detection with a limit of detection of 0.2 mg/l. Similar arguments were put forward to defend Samples were also analysed using a non- the exemption of all three distillate ingredients specific total protein method. By use of from allergen labelling requirements. appropriate recoveries it was demonstrated that, Although commercial distillation systems can depending on the alcoholic matrix, the method vary, the physical principles they are based on could determine protein concentrations down are the same. All spirit drinks, by definition and to levels of 5-10 mg/l. law, undergo various distillation and rectification The following results were obtained from steps. Due to the non-volatility of the cereal, the analyses undertaken on a range of branded nut or whey derived allergenic or intolerance bottled products and production samples causing proteins and polypeptides, they will not obtained from manufacturers. distil over into the resultant spirit. This argument 46 cereal derived alcoholic distillate samples also applies to lactose, which is contained were analysed for the presence of gluten – in whey and to which some individuals are none was detected; 39 cereal derived alcoholic intolerant. distillate samples were analysed for total protein A search of the scientific literature has not – none was detected. highlighted any recorded links between the 13 samples were analysed for the presence of ingredients requested to be exempted from Annex almond protein - these included gins, liqueurs IIIa and any allergic reactions or intolerances. In and a sample of distilled almond oil – none was addition, any medical information and advice detected; 9 gins were analysed for protein – none available also supports the argument that such was detected. distillates are safe for consumption by sensitive A whey derived neutral spirit was analysed individuals. for the presence of ß-lactoglobulin, lactose and total protein – none of these substances was detected. Analytical data The data obtained so far confirm that allergens derived from the raw materials of interest are not To support the contention that allergenic proteins transferred to alcoholic beverage distillates. and lactose do not distil and will therefore not Proving that alcoholic beverage distillates are free from cereal, nut & milk derived allergens 279

Results samples is being conducted. This process, coupled with the development of more The EFSA has, on the basis of three initial sensitive analytical techniques for proteins and documents presented to them containing the lactose, will result in detailed data designed to arguments and analytical data summarised above, demonstrate the absence of specific allergenic indicated that the distillates produced using ingredients in alcoholic distillates. In addition, cereals, nuts and whey used prior to distillation work is being undertaken to support the are unlikely to cause an adverse reaction in argument that proteins and lactose do not distil. sensitive consumers. These ingredients have This includes a commissioned analysis from an been temporarily removed from labelling independent expert of the physical process of requirements until 25th November 2007. distillation and its effect on these allergens, as This information can be found in European well as experiments designed to demonstrate Commission Directive 2005/26/EC. their non-volatile nature. The combined work However, EFSA has indicated that a more will be submitted to EFSA during 2006. extensive sampling regime and more sensitive analysis is required to further prove that alcoholic distillates will not trigger adverse reactions as a References result of specific raw ingredients known to cause allergic reactions or food intolerance. Directive 2003/89/EC of the European Parliament By the 25th November 2007, following the and of the Council amending Directive submission of further data, EFSA will give a final 2000/13/EC as regards indication of the opinion on the likelihood of adverse reactions ingredients present in foodstuffs. Official from the ingredients requested for deletion Journal of the European Communities No. from Annex IIIa and the Health and Consumer L308: 15-18 Protection Directorate General of the European Commission Directive 2005/26/EC establishing Commission (DG SANCO) will legislate on the a list of food ingredients or substances requested deletions based on the assessed risk. provisionally excluded from Annex IIIa of Directive 2000/13/EC of the European Parliament and of the Council. Official Current and future work Journal of the European Communities No. L75: 33-34. A wide ranging sampling regime of bottled alcoholic beverages and distillery production 280 I.C. Goodall et al. Authenticity indicators - enhancing consumer and brand protection Chapter 39 Authenticity indicators – enhancing consumer and brand protection

R. I. Aylott Diageo Plc, Brand Technical Centre, Menstrie, Clackmannanshire, UK

Introduction to the issue of distilled seen as an issue in Asia, Latin America and Eastern spirits counterfeiting Europe. However, counterfeiters’ methods are increasingly professional with fake materials Counterfeit goods are estimated at between 7 and sourced internationally. Whilst the incidence 9% of world trade. The impact on the UK economy of organised counterfeiting in well-regulated alone is estimated at £11 billion, 4000 jobs and countries is relatively low, illegal products can £2 billion lost Value Added Tax (Anti-counterfeit now be encountered almost anywhere. Group 2005). The distilled spirits industry is no On-trade bar substitution takes place, as the exception and suffers from counterfeiting. This words suggest, in pubs, clubs and hotel bars. illegal and potentially hazardous activity takes Genuine branded bottles, once emptied, are many forms and occurs throughout the world. simply refilled with cheaper inferior product by Counterfeiters can make easy profits. Any brand bar staff operating either singly or in small groups. or spirit category is at risk. Bar substitution is encountered globally. Counterfeit distilled spirits are encountered in two distinct forms: organised criminal counterfeiting and on-trade bar substitution. Effects of counterfeit spirits Organised criminal counterfeiting involves the production, distribution and sale of fake So what are the effects of counterfeit spirits on spirits. Organised counterfeiters minimise society? Firstly, consumers are deceived and their production costs by recovering as many may be placed at potential health risk. The original packaging components as possible. deception may leave consumers not realising Their processes normally start with the recovery they have been sold counterfeit, but simply of genuine empty bottles from bars and even disappointed with the quality of their chosen domestic waste. Counterfeiters give their goods brand. This deception is also an illegal act in false authenticity by using combinations of fake many jurisdictions contravening, for example closures, labels, cartons and shipping cases and, in the UK, the Trade Descriptions Act 1966 and of course, fake liquids. Sometimes counterfeiters the Food Safety Act 1990. even have fake bottles manufactured. The Secondly, governments lose tax revenue organised counterfeiting of spirits was historically resulting in contravention of fiscal laws. 282 R.I. Aylott

Counterfeit prices are normally a little less that Effective action against counterfeiters is of genuine product, just enough to encourage dependent upon the availability of appropriate customers to buy illegal product. However, laws, active enforcement agencies and accurate counterfeiters pay no tax and as alcohol tax methods for brand authenticity analysis. Whilst is invariably a large proportion of the final laws and enforcement agency capabilities vary price of genuine product, it can be seen how according to jurisdiction, the spirits industry counterfeiting results in good profit margins. is proactive in setting standards for both Thirdly, genuine spirit manufacturers, brand and generic authenticity analyses. The distributors and retailers lose revenue. Not only authenticity of congener rich spirits such as is this a simple loss of a genuine sale, it can result Scotch whisky is checked by well-established gas is loss of future sales as consumers perceive chromatographic methods (Aylott, Clyne, Fox a high risk that future purchases may also be and Walker, 1994, Aylott 2003b). The analyst counterfeit. This undermines brand equity and determines alcoholic strength and major volatile the spirit category in general. Lastly, profits congeners in a suspect sample and compares from counterfeiting often go to serious organised results with the normal ranges for the genuine criminals and even terrorists. brand. Checking the authenticity of white spirits such as gin and vodka is more problematic due to volatile gin botanical congeners exhibiting wide Action against counterfeit of distilled analytical ranges (Aylott, 1995) and most vodka spirits containing few analytical attributes that may be used in authenticity analysis (Aylott, 2003a). As the geographical occurrence of counterfeit Counterfeit investigation is enhanced when distilled spirits has spread, so has the range of supported by fast and reliable forensic information, counterfeit products detected. Premium spirit ideally based on quick and easy field-tests rather categories such as Scotch whisky and cognac than slower laboratory tests. A portable brand were the first identified counterfeit targets along authenticator was introduced for Scotch whisky with gin and vodka in the on-trade. Risks vary during the last two years (MacKenzie and Aylott, from country to country with other international 2004). This novel application uses ultraviolet/ brands, local products such as cachaça in visible absorbance spectra and enables brand Brazil; aguardiente elsewhere in South America, authenticity analysis to be made by enforcement locally made whisky in India and admixtures officers and investigators working in the field all suffering from counterfeit activity. The and at remote sites where gas chromatography fake liquids themselves are invariably cheaper is unavailable. Opportunities exist to extend products, often extended with locally obtained this spectroscopic technology to other liquids neutral alcohol. Deluxe Scotch whiskies are such as other matured spirits, beers, wines and often counterfeited with cheaper whiskies non-alcoholic beverages. However, alternative diluted with alcohol or with locally produced strategies are required for white spirits that admixtures. The second bottle may then be may not normally have strong spectroscopic reused providing base packaging materials for or congeneric fingerprints. This has led to the another counterfeit that uses inferior liquids and development of brand authenticity indicators. so on. Nothing goes to waste. In recent times Europe has seen the organised counterfeiting of vodka employing fake closures, labels and even The design and deployment of bottles. Some of these counterfeits contained authenticity indicators illicit alcohol contaminated with methanol, a dangerous combination that can put consumer So what is an authenticity indicator? It is a health at risk (Paine and Dayan, 2001). unique component in a product whose detection Authenticity indicators - enhancing consumer and brand protection shows that the product is what it purports to be. Key criteria were set for ingredients selected for Firstly, a unique flavouring material can find use in authenticity indicators. application as an authenticity indicator. For example, one brand of gin has cassia bark as a • Ingredients must be permitted for use in botanical ingredient. This results in a peak for foodstuffs. cinnamaldehyde in its chromatogram that offers • Ingredients must be permitted in the chosen a unique analytical characteristic applicable to distilled spirit. authentication. Many brands of vodka contain • Ingredients must not cause any change to additives for flavour purposes, such as glycerol, the organoleptic properties of the protected propylene glycol, and sugars; all of which may be product. used to facilitate brand authenticity analysis. • Ingredients should be covert, yet easily and A specifically designed brand authenticity cheaply detectable in the host product ideally indicator is an extra ingredient in a product, by field operators following basic training. normally present at trace concentrations. Such indicators are particularly useful in well- When designing brand authenticity indicators, established products whose popularity makes careful reference to spirit drink definitions and them counterfeit targets. It is also sensible to related regulations is required. Spirit drinks are design authenticity indicators into new products defined in the European Union under Council during the innovation process. Authenticity Regulation No1576/89 and other regulations indicators may be proprietary products from a define permitted food additives, processing aids, specialist supplier or may be invented in-house. colourings and flavourings. Reference should The next section describes our approach to in- also be made to relevant national legislation as house activity. this may restrict options. For example, whilst Authenticity indicators were designed in Scotch whisky authenticity is well protected by the Diageo PLC, Brand Technical Centre with other technologies, authenticity indicators cannot key performance characteristics in mind. First, be used for regulatory reasons - UK legislation indicators should be simple to detect under field on Scotch whisky only allows addition of water conditions by operators following basic training. and spirit caramel (The Scotch Whisky Order, Next, indicators should enable enforcement 1990). officers to decide whether a test sample is either genuine or suspect. A suspect result then enables an officer to take action, such as seizing The use of authenticity indicators in the tested product for confirmatory authenticity regulatory enforcement analysis at either a public or brand owner’s laboratory. Vodka provides a good working Field and confirmatory tests are completed example of brand authenticity indicators in using a combination of primary and secondary action. Smirnoff is very pure and contains very indicators. The primary indicator triggers a little if any congeneric material. Chromatographic response in coloured dipsticks used by the authenticity analysis is a non-starter. The enforcement officer in the field. The secondary authenticity indicator designed for Smirnoff has indictor enables a confirmatory test at a UK a formulation based on three trace compounds Public Analyst’s laboratory and a non-authentic that provide primary and secondary indicators. result can then form part of prosecution’s The primary indicator is used in the field test to evidence. Other field detection technologies trigger a dipstick test whose colour changes from for primary indicators may be considered as yellow to light green for the genuine product (as alternatives to dipsticks. shown in Figure 1). No colour change indicates 284 R.I. Aylott

1

2 3

4

Instructions for Smirnoff field authenticity test.

• Pour sample of test liquid into a clean container. • Take two new dipsticks. • Immerse one dipstick in test liquid for 10 seconds. • Allow colour to develop for further 30 seconds and compare to the unused dipstick. Dipstick colour for genuine product appears light green; suspect product stays yellow.

yellow (fake liquid) light green (genuine Smirnoff)

Copyright Diageo PLC 2003. Not to be reproduced or copied without consent. May 2003

Figure 1. The field test for the Smirnoff primary brand authenticity indicator. the absence of the primary indicator and is thus The strategy also provides data for brand owners’ a suspect result, leading to the enforcement surveys, information for counterfeit investigators officer taking a formal sample for secondary and forensic evidence for use in the prosecution of indicator analysis in the laboratory by liquid offenders. The range of authenticity indicators has chromatography (Figure 2). This analysis is both been extended to create unique brand fingerprints qualitative and quantitative as lower than normal applied to the protection of Gordon’s, Tanqueray indicator concentrations may indicate that the and Gilbey’s gins. Their role in regulatory genuine brand has been partially substituted by enforcement is now well established. another product. It is also relevant to determine Whilst the formulation of each authenticity alcoholic strength (in order to check for dilution indicator should be unique and confidential with water) and to look for the presence of to the brand owner, it is desirable that details components not normally present in the genuine of its composition be made available to the brand. enforcement laboratory for analytical and This strategy now successfully provides evidence purposes. In a similar way, it is consumer protection officers with a means of desirable that precise indicator formulations are checking brand authenticity in the on-trade. Local not released during court proceedings. Courts authority trading standards and environmental are usually sympathetic to this need when an heath staff in the UK conduct this work and Figure expert witness is available to speak on behalf of 3 shows a typical sampling and testing process. the brand owner. Ingredients’ listing is being Authenticity indicators - enhancing consumer and brand protection

6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 A 1.5 B C 1.0 0.5 0.0

Response - MilliVolts (span=7.0) -0.5 -1.0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Time - Minutes (span=15)

Instructions for the Smirnoff laboratory authenticity test. • Determine alcoholic strength. • Determine secondary indicators A, B & C. Check for their presence and correct concentrations. • Check for components not normally present in product.

Figure 2. Typical forensic analysis for Smirnoff secondary brand authenticity indicator, for the presence of abnormal components not present in genuine product and for alcoholic strength.

Enforcement officer visits Pub and requests sample of on-trade brand.

Is the sample authentic by field test on YES If genuine, no further primary indicator? action

NO

If suspect, officer takes formal sample for Public Analyst

Is the sample authentic in laboratory tests on secondary YES If genuine, no further indicator? Is alcoholic strength correct? action

NO

Enforcement officer uses Public Analyst's result as part of prosecution evidence

Figure 3. A typical sampling and testing process applicable to checking brand authenticity in the on-trade. 286 R.I. Aylott considered for alcoholic beverages at European A proposed regulatory framework for Union level. It is important that authenticity authenticity indicators indicator formulations are not revealed in ingredients’ lists, a position well understood at Finally, with the current review of EU Council government level. Regulation No1576/89 European Union When there is a risk of organised counterfeiting, spirit drink definitions (1576/89); there is an the authenticity of packaging also requires opportunity to propose a regulatory framework appropriate tests. Investigators should make for authenticity indicators. The Gin and Vodka themselves familiar with the visual and material Association of Great Britain (GVA) working attributes of genuine packaging components through the Confederation of European Spirits and, as a matter of routine procedure, check the Producers (CESP) have proposed the following authenticity of suspect bottles, closures, labels, paragraph for use in the revised regulations. cartons and shipping cases. These packaging authenticity tests should ideally precede liquid “…For the purposes of consumer and brand authenticity tests. In particular, break rings security, all distilled spirit drinks, except and seals on closures should be confirmed as those for which additions of substances are either intact or broken before opening bottles prohibited by the definition of the spirit in for liquid analyses. Figure 4 shows a typical question, may contain trace substances to sampling and testing process applicable to act as brand authenticity indicators. Such organised counterfeiting where fake packaging substances must be food grade materials is suspected.

Investigator receives sample

Are the break rings and seals on closure intact? Continue.

Is the liquid genuine? Is the packaging genuine? Conduct field test for liquid authenticity. Check the bottle, its labels carton and shipping case?

No Yes

If any answer is NO, take formal sample to public laboratory Only if both answers are YES, take no or brand owner for liquid and packaging analysis. further action.

If fake, develop results and report for use as part of potential prosecution evidence.

Figure 4. A typical sampling and testing process applicable to the investigation of organised counterfeit spirits where fake packaging is suspected. Authenticity indicators - enhancing consumer and brand protection

that satisfy food law and be present at Aylott, R. I. (2003a). Vodka, gin and other flavored concentrations less than 0.1% (w/v) in spirits. In Fermented Beverage Production. a product without imparting distinctive Second edition, Edited by A.G.H. Lea and character.” J.R. Piggott, Kluwer Academic/Plenum Publishers, New York, pp. 289-308 In conclusion, brand authenticity indicator Aylott, R. I. (2003b). Whisky analysis. In: Whisky. technology has extended authentication to a Technology, Production and Marketing. wider range of distilled spirits by offering rapid Handbook of Alcoholic Beverages Series, field tests backed up by confirmatory laboratory edited by I. Russell, Academic Press, tests. It now plays a key role in enhancing London, pp. 277-309 consumer and brand protection. Opportunities Aylott, R. I., Clyne, A. H., Fox, A. P. and Walker, exist to extend this technology into more brands D. A. (1994). Analytical strategies to confirm and other categories of food and drink. In this way Scotch whisky authenticity. Analyst 119: the interests of the consumer, government and 1741-1746 brand owner can be protected and enhanced. MacKenzie, W.M. and Aylott, R.I. (2004). Analytical strategies to confirm Scotch whisky authenticity, Part II, Mobile brand References authentication. Analyst 129: 607-612 Paine A. J. and Dayan A. D. (2001). Defining Aylott, R. I. (1995). Analytical strategies to confirm a tolerable concentration of methanol in gin authenticity. Journal of the Association alcoholic drinks. Human and Experimental of Public Analysts 31: 179-192 Toxicology 20: 563-568 Aylott, R. I. (1999). That’s the spirit: product The Scotch Whisky Order (1990). Statutory authenticity in the Scotch whisky industry. Instuments 1990 No. 998. HM Stationary Chemistry Review 8: 2-6 Office, London, UK. 288 R.I. Aylott Meeting the challenges of tomorrow 289 Chapter 40 Meeting the challenges of tomorrow

G. Hewitt The Scotch Whisky Association, Atholl Crescent, Edinburgh, UK

Introduction of Scotch Whisky. Apart from the broad (and important) definition of Scotch Whisky in the I cannot claim a long exposure to or experience Scotch Whisky Order of 1990, the industry has of the industry; nor do I have some special got by following established conventions. These academic or technical qualification, but a few conventions are not written down anywhere and years working close to the Scotch Whisky only serve to protect Scotch Whisky as long as industry has given me some insights. I would everybody knows what they are and follows like to address some issues that are vital for them. ensuring the future of the UK distilling industry and creating the best environment for building further success. Most of my discussion will relate Malt whisky definitions to the Scotch Whisky industry, but they all have a resonance for the wider distilling industry at Everyone within the industry knows the home and abroad, and in many ways for the difference between a blended Scotch Whisky drinks industry as a whole. and a single malt. Within the industry everyone I took up the role of Chief Executive of the knows what vatting means, and what therefore Scotch Whisky Association in late 2003 after a vatted (or, in new parlance, a blended) malt 35 years in the British Diplomatic Service. I Scotch Whisky is. looked forward to the new challenge. It proved But go out and ask the ordinary consumer to be even a more interesting time than I had what these terms mean. Consumers particularly bargained for. I had not expected to be faced outside the UK (and remember that over 90% in the very early months of the new job with of Scotch is exported) are confused about the trying to address two major problems – UK different varieties of Scotch Whisky blends and duty stamps and a discussion within the Scotch malts. Do some sampling yourselves. The fact Whisky industry over the terms to be used for that some of the best selling premium whiskies blended malt whisky. are blended Scotch Whiskies does not jump off On taking up the job, it seemed strange to me the face of the bottle. that with the long history of Scotch Whisky there Some might argue that it does not matter. I do was no definition in law of the various categories not agree. If we have different types of whisky, all 290 G. Hewitt of them of great value and quality whether they I can appreciate why. Some have suggested are malts or blends, it is in everyone’s interests that the term “blended” before “Malt” might that the consumer knows what they are buying. cause confusion with “Blended Scotch Whisky”, One of the first jobs is therefore clearly to identify which of course everyone in the industry knows by category the different whiskies and make sure is a blend of Malt and Grain whisky - but without that they are presented to the consumer in terms constraints on the proportions of malt and grain. of their proper category. We recognise that some people have grown up The SWA Council decided in December 2003 with and are comfortable with the term vatted that we needed to address the issue of Scotch malt, but there is a danger for all of us close to the Whisky definitions with the emphasis on better industry that we fail to address the perceptions protecting Single Malt Scotch Whiskies. That led and knowledge of the consumer in the 200 or to the early establishment of a Working Group so markets to which we export. comprising very senior figures from a cross Let us look at “Blended Malt Scotch Whisky” section of companies in the industry to look at rationally, and in the context of current EU (and the issues and come up with recommendations. therefore British) law. This input from companies was vital, and I am A “blend” is defined as a combination of two delighted that the Group made it their priority spirit drinks that are the same: ie two or more to ensure that we reported back to Council a whiskies, or two or more cognacs or rums. If year later on our findings, which the Council you mix cognac and rum or cognac and whisky, endorsed in March 2005. The Working Group the resulting drink by law is a mix or mixture also took the opportunity to address other issues of spirits. which needed to be looked at in the changing So we have a legal framework within which international environment in which we are we have to operate. It would be very difficult to now working, with the aim of better protecting try to amend EU law. We would have to convince the names of Scottish regions where whisky is the Commission, the European Parliament and produced. all the other member states. And from my own We went out to consultation with our experience I know how difficult that would be. It members in March 2005 and have been follows that a combination of two or more malt consulting non-members more recently. Many Scotch Whiskies is therefore - in law - a “blend” of you may be familiar with our proposals, but whatever we might want to call it for marketing I would like to highlight one issue. purposes. We listed the five category definitions that Within the Working group we looked at many all bottlers must use on their labels so that alternatives for naming a category that has not the consumer knows exactly what they are perhaps been all that common in the past but is, buying. I suspect, going to be a growing whisky category The five categories are: of the future. First we considered retaining the name “Vatted”. It is a concept and name well Single Malt Scotch Whisky known in the industry. But it was quickly pointed Blended Malt Scotch Whisky out that most companies shied away from using Blended Scotch Whisky the term in their marketing to the consumer Single Grain Scotch Whisky as the term sounded rather industrial, with Blended Grain Scotch Whisky connotations of stainless steel and large tubs, and certainly lacking glamour and appeal. There is no controversy on most of these We thought long and hard about just using categories, but we have had considerable the term “Malt”. But then we ran into exactly comment and feedback about the term Blended the problem we were trying to address, and the Malt Scotch Whisky. Meeting the challenges of tomorrow 291 origin of our work. It is very easy to put a nice to discussion between the Commission and the bit of romance text in front of the term “Malt” Indian authorities. If the Indians do not sort out with the result that you would have the addition their tariff and internal tax arrangements to the of “Pure” in front of the term. And we are being satisfaction of the European Spirits industry, the told very clearly that the term “pure malt” is Scotch Whisky industry is determined to take its something industry wants outlawed. complaint all the way to Geneva. The industry As I stated, we have consulted widely. To hopes that dialogue will deliver a resolution date, no phrase has better met the requirements without the need for this, but its case is strong of, or encapsulated the term of the category in and the industry has much to gain if successful. question, than Blended Malt Scotch Whisky. We are committed to securing a fair outcome But I am happy to set a challenge. If anyone for Scotch Whisky. can come up with a neater solution, a better India is one of the larger spirits markets in name than “blended malt” for a defined category the world with about 90 million cases sold a of Scotch Whisky, that we could adopt without year. Scotch Whisky has only about 0.6% of causing many of the problems that we were that market with sales of about 600,000 cases a trying to address in the Working Group, we year. If we can get the Indian restrictions lifted, would be pleased to hear it and consider it. we believe that we could sell some 5 million cases a year among the affluent and aspiring middle classes of India. That would make International markets some difference to the turnover of our member companies. It is certainly a prize worth working The Scotch Whisky industry has had remarkable for. success over the years in opening up markets and The interesting thing for me, given my ensuring that Scotch Whisky is not discriminated Diplomatic Service background, is the extent against in favour of local products. It has a unique to which the UK authorities leave it to the SWA record with the WTO in Geneva. I think it is true to fight our corner on trade issues. I do not say to say that the Scotch Whisky industry is the only that in any way to criticise the Government. industry ever to have won three consecutive We enjoy exceptional support, as we should. WTO dispute settlement cases, against Japan, Scotch Whisky has been among the top five Korea and Chile where we were facing serious manufactured British exports for many years and discrimination in the local market. it brings a major contribution to the UK balance It won because it prepared its cases of payments. meticulously and had a clear case to prosecute. It is an unusual compliment to the staff of the Our successes in Geneva have been a warning SWA and our member companies that the DTI to others. Earlier this year the Scotch Whisky recognises that we have a competence that they industry won another success against Uruguay. cannot begin to compete with. I intend to keep it The industry did not have to go the whole way that way. India is the immediate issue, but we are to Geneva. Discussions led by the European also preparing a case against Turkey – another Commission were enough to secure sufficient market of considerable potential - and will improvement in market access. But success is prosecute that as urgently and as energetically not assured nor does it persuade others to desist as we have done in all the other market access from discriminating against Scotch Whisky and cases we have tackled. other European spirits. By contrast, we can report positively about The Scotch Whisky industry lodged a market access to China. As part of its negotiations complaint against India with the European to join the WTO, China agreed to lower its Commission in July 2005 and our complaint tariffs to 10% with effect from the beginning was endorsed by the Commission. That will lead of 2005, and they have kept their promise. 292 G. Hewitt

As a result, we are seeing a huge upsurge in society. It is a topic that is certainly is taking a exports to China, 150% increase in 2004 and much larger slice of management attention in the in 2005 another 150%. That is good work by Executive and filling more of the Association’s the companies taking advantage of the market agenda at home, in Europe and internationally openings. Importantly, China in 2004 featured at the WHO. for the first time in our top 20 markets by value The debate in the UK is largely being driven and we intend to do everything to ensure that it at the moment by those concerned about remains that way. Early indications this year are disorder and misbehaviour on the streets rather that it is climbing even higher up the ranks. than concern about the misuse of alcohol The biggest threat to success of Scotch and the possible harm to health, family and Whisky is probably counterfeit whisky or society generally. As a result, the Home Office passing-off an inferior whisky product as Scotch is currently in the lead in the UK, with the Whisky. It is hardly surprising that we have to Department of Health a long way behind. defend our corner hard. If we set ourselves up Unfortunately the social disorder debate is giving as producing the world’s premium spirit drink, encouragement to those who would like to treat it is little wonder that fraudsters try to cash in on alcohol in much the same way as they have dealt our success. It may come as a surprise to you, with tobacco. The Scotch Whisky industry needs but my five lawyers in the Executive have on to set an example, promote best practice, and their books on any one day in the year some 50 engage in this major debate, if we are going to be cases which they are working on somewhere in able to continue to trade, promote and advertise the world. our great product without undue constraint. It is And they are also assiduous in pursuing not surprising therefore that the Association has and blocking trademark applications which been at the forefront of alcoholic beverage trade may encroach on the intellectual property that associations in the UK. Scotch Whisky represents. Their work often It was also brought home to me strongly goes unsung. But the task gets bigger, the greater as I made my initial calls on company Chief our success. What is amazing is the network Executives some two years ago that social aspects of people out in the market who bring to our of alcohol were firmly on their agendas and they attention the many examples of fraudulent wanted to bring some coherence to policy and practice. We receive messages from around practice throughout the industry. I decided that the world giving us reports and sending photos we needed to come forward with some firm of the retail shelves of their local shops and proposals. supermarkets. We follow up most in one way Last year we drew up a Code of Practice or another, but the work is rather like that of on the Responsible Promotion and Marketing Sisyphus – never-ending. And we are of course of Scotch Whisky. There are other guidelines only looking after non-branded Scotch Whisky. around but it is significant that the Scotch Whisky We leave it to the companies and the IFSP Association Code is the only one that lays down (International Federation of Spirit Producers) to sanctions for not respecting its terms, with an pursue those who are counterfeiting brands. independent Panel to adjudicate if we have been unsuccessful within the industry to persuade one or other of our members to come into line Alcohol and society with the Code. The Code has been live since February 2005. I would like to finish this paper on an issue The Code does not have mandatory application which has been rising up the agenda and is now overseas as yet, but we have made clear to our probably top of our concerns, the social aspects member companies that it should be seen and of alcohol: alcohol and health and alcohol and used by them as a minimum standard to guide Meeting the challenges of tomorrow 293 promotion and advertising practices where a law and should be required to demonstrate that stricter national code does not exist. proficiency. That means that even casual staff So far we have not had any formal complaints in a bar should have been given training in the but we have had many approaches for guidance law by the licensee before he or she is allowed and have commented on some overseas to serve a drink. I think most teenagers know promotions. We have also taken the Code to that it is illegal for them to buy a drink before companies to provide in-house briefings. But they are eighteen (not that many of them seem the Code is only one way of demonstrating that to care) but how many of them know that the we mean business. person who serves them a drink is committing It is not the producers and bottlers who usually a criminal offence. have the final say on the way our excellent More needs to be done. We have to find matured products are sold in the retail market. some way of ensuring that teenagers are given We have therefore been active in the debates a much better understanding of the risks they in England and Scotland relating to the Alcohol take with over-consumption of alcohol. Schools Harm Reduction Strategy and the new licensing usually have programmes and lessons on drugs, laws. In doing so, we have made quite clear that sex education and STDs, but very little is said we want to see the powers of the law and local about alcohol. So we must start to try to shift the regulation used more effectively and regularly agenda and the focus of “civic” lessons. Children against those who break the law, or otherwise should also be given the material to engage with fail to respect the terms of their licence. their parents - to be able to challenge them about More particularly, we are on record for calling their drinking habits. on the police and other enforcement authorities I believe that social change is possible if we to ensure that those who sell to under age work at it hard enough and consistently enough. drinkers and continue to serve an intoxicated Drink driving in the UK is a good example. person feel the full force of sanctions against The law is clear; it is enforced systematically them. A football analogy might be useful; a and consistently. No-one is in any doubt what yellow card for a first offence and a red card for happens if you are stopped by the police and the second. So I believe it should be for pubs, found to be over the limit. The drink driving supermarkets and corner shops if drink is sold law, supported by the industry, has done the to an under age person. Loss or suspension of a drinks trade no harm. The consequence of the licence with the inevitable loss of income will campaigning over the last 25 years or so is that the be a penalty enough to get the retail trade to younger generation are probably more observant tighten up its procedures. of the law than the older generation. I think we Why should we be concerned about what can do the same in educating everybody about is happening in the retail trade? It is obvious the consequences of misuse of alcohol. really. If we allow unacceptable practices on Why do this? It is in our self interest - to the High Street, it is not just the pubs and the allow the industry to grow and continue to be retailers who are targeted by those attacking the successful. But we must take our responsibilities drinks industry but the producers and bottlers seriously. If we do not and merely pass on to themselves. We will all be brought down by others the problems which misuse of alcohol our weakest link. creates (because the individual is ultimately There are plenty of training courses to help responsible for his or her own behaviour), we an understanding of alcohol, Drinkwise, WSET, will be cut out, and rightly so, from the debates Servewise etc; they are all doing good work, that are going to shape the industry’s future but again we need to do more. I believe that and the environment in which we go about our anyone handling alcohol in a retail setting should business. That is not a good recipe for success at the very least know the fundamentals of the or for business growth and continuity. 294 G. Hewitt

Acknowledgements

I am proud to be able to represent the Scotch Whisky industry. I probably have one of the best jobs in the world and I would like to thank all those in the Scotch Whisky industry who have given me such support over the last few years. Thank you also to the organising committee of the Second Worldwide Distilled Spirits Conference for the opportunity to make this presentation Whisk(e)y production 295 Chapter 41 Whisk(e)y production: raw materials, malting, cooking, mashing, fermentation and beyond

I.C. Goodall The Scotch Whisky Research Institute, The Roberton Trust Building, Riccarton, Edinburgh, UK

Introduction Malting

Whilst the market leaders in whisky (or whiskey) The breakdown of cereal starch into fermentable production herald from Scotland, the United sugars during mashing is typically achieved States, Canada and Ireland, whisky distilleries by enzymes present in the cereals. To ensure can be found distributed across the globe. The enough enzyme activity, a percentage of the process varies nationally, regionally and even cereal used must be malted. Malting is a process from distillery to distillery, but certain elements by which the cereal is germinated in a controlled are common across all whisky production. In fashion (usually barley, but rye is sometimes many instances, national legislation prescribes malted). During this process the cereal undergoes elements of the process and can act as a means modification, allowing enzymes to develop and of achieving a geographical designation, such making the starch more accessible. The cereal is as Scotch whisky. first hydrated, after which germination proceeds. When the desired level of modification has been achieved the cereal is heated or kilned, to arrest Raw materials biological activity. In the production of malted barely, peat may be burned during kilning to Cereals provide the source of fermentable sugars produce a smoke. This is adsorbed by the malt which will be converted into alcohol. This and contributes a distinctive smoky, medicinal imparts at least some of the characteristic aroma flavour to the final product. and taste of all whiskies. Scotch malt whisky is produced from 100% malted barley, but wheat and maize are used in the production of Scotch Mashing grain whisky. The same distinction is true for Irish malt and grain whiskey. Maize (corn) also Mashing is the process of releasing starch from tends to form the backbone of Canadian and the cereal and converting it into fermentable US products, but malted barley, rye and wheat sugars. After milling, malted cereals are mixed will contribute to varying degrees. ‘Pot Still’ with hot water in a vessel called a mash tun. Irish whiskey includes a percentage of unmalted This allows the enzymes present in the malt to barley with its malted equivalent 296 I.C. Goodall break down the starch into fermentable sugars Fermentation (Figure 1). For unmalted cereals, such as wheat and After mashing, the sugar-rich solution is cooled maize, an initial cooking step is required after and yeast (Saccharomyces cerevisiae) is added milling. This is necessary to separate the cereal to begin fermentation. The yeast employed is starch from its surrounding protein matrix. Two typically a specific strain of a high-performance cooking techniques are traditionally employed; distilling yeast. In a small number of Scotch atmospheric batch cooking or pressure cooking. malt whisky distilleries, brewer’s yeast may be Malted cereals are never cooked, since the elevated used as a supplement because it is believed to temperatures would destroy the developed impart additional flavour. In Scotland, yeast is enzymes. However, in distilleries which use supplied from commercial yeast manufacturers; unmalted cereals, a proportion of malted cereal in the US, distilleries tend to propagate their own is usually required to provide sufficient enzyme yeast strains. Fermentation typically takes 40 to activity to breakdown all the cereal starch. This 48 hours (Table 1), but industrial fermentation will be added as malt slurry to the cooked cereal times are often longer than this, ranging up to slurry (sufficiently cooled to preserve the enzyme 120 hours. The resulting solution will typically activity of the malt), in a mash tun or some other vary from 8-10% alcohol by volume. conversion vessel. Table 1. Differences in fermentation apparatus US distillers all use ‘backset’ (centrifuged or between small scale and large scale production. screened stillage from the base of the still) in the mashing process. It adjusts the acidity of the mash Fermentation apparatus - general differences in scale and can provide nutrition during fermentation. Small scale production Large scale production This practice has also been employed in the Wood vessels Stainless steel vessels production of other whisky types. No temperature control Temperature control

No CO2 collection CO2 collection

Corn mill Cooker Unmalted cereals slurry

Rye mill

Malt mill Malt slurry Mash tun/ conversion vessel

US bourbon mash schematic

Malt mill Malt slurry Mash tun

Scotch malt whisky mash schematic Figure 1. Flow diagram illustrating the production of mash. Whisk(e)y production 297

Distillation Maturation

The two methods of distillation used in whisky Following distillation, water is usually added production are batch pot still and continuous to reduce the spirit to a desired strength and column. Generally, pot still distillations are the product is matured in casks made from used in the production of malt whisky (Figure either American white oak or European oak. 2). Scotch malt whisky production typically During maturation, alterations take place in the uses two successive pot still distillations whilst composition and flavour of the stored spirit due Irish malt whiskey uses three. Column stills to interactions with the wood, to produce what tend to produce a lighter flavoured whisky is regarded as a more acceptable product. Oak due to increased rectification. In Scotland and type, barrel size, maturation period, climate Ireland they are used to create grain whiskies for and cask history will have an impact on how blending with the heavier malt whiskies. US and maturation affects the whisky distillate. Some Canadian distilleries’ whiskies are dominated conditions, such as a minimum three year by the use of the column still, the flavour of the maturation period for Scotch and Irish whiskies, product being dictated by the number and design are prescribed in national legislation. of the column still(s) and the strength at which By law, US bourbon, rye, wheat, malt and the spirit is collected. rye malt whiskies must all be matured in new

Figure 2. Examples of pot stills (left) and continuous column stills (right). (Photographs are used with the kind permission of the Scotch Whisky Association (left) and the North British Distillery Company (right). 298 I.C. Goodall charred casks. The rest of the whisky distilling Blending, flavouring, colouring, world principally employs previously used bottling barrels and the legal requirement that most US whiskies must be matured in new casks ensures Blending is used in the whisky industry to ensure a plentiful supply of used charred American flavour consistency in the final product. Generally oak casks. The other principal source of casks this involves mixing different proportions of is the Spanish sherry industry which uses both lighter flavoured spirits with their heavier American and Spanish oak. Sherry casks will counterparts. Although some US and Canadian have been toasted prior to initial use. Aside from whiskies may contain added flavouring, most the production of most US whiskies, casks will regulations prevent such practice. Addition of be re-used until they have lost the capability to caramel to achieve colour standardisation is effect a desired sensory change in an economic common, but some regulations prevent its use. timescale. Whisky usually has its strength adjusted by the addition of water and will be filtered prior to bottling. Nearly all countries which set regulations require whisky to be bottled at a strength of at least 40% alcohol by volume. Gin and vodka 299 Chapter 42 Gin and vodka

R.I. Aylott Diageo Plc, Brand Technical Centre, Menstrie, Clackmannanshire, UK

Introduction Neutral spirit production

Gin and vodka are made from neutral spirits, Neutral spirits may be fermented using raw pure alcohol otherwise known as “ethyl alcohol materials from any agricultural source, the most of agricultural origin”. They are often known in common being cereals (particularly wheat and the industry as white spirits and, unlike brown corn) and molasses (derived from cane and spirits, undergo no maturation in wood. sugar beet). Other cereals such as barley and rice, together with potatoes, grapes and fruits • Gin is a clear distilled spirit flavoured to give are also used for neutral spirit production. The a predominate taste of juniper. Distilled gin key component required from the raw materials is produced when the alcohol is redistilled for fermentation into ethanol is carbohydrate. in stills traditionally used for gin in the Other elements such as protein and fibre may presence of juniper berries and other natural be recovered as useful by-products. Starch may botanicals. also be gelatinised by milling and steeping or by • Vodka is clear distilled spirit without cooking under pressure. Wort may be produced distinctive character, aroma or taste. It is by the action of malt enzymes or by the action produced by either rectifying neutral spirits of added artificial enzymes and then fermented or by passing them through activated by the action of distillers’ yeast. so that the organoleptic characteristics of the The resulting fermented wash or beer is raw materials are selectively reduced. then purified and concentrated by distillation into neutral spirits. Distillation processes Both spirits are defined in law. Current definitions are normally continuous and employ at within the European Union may be found within least two and often up to five columns, with Regulation 1576/89, although these definitions each column fulfilling different functions of will be subject to new proposals during 2005. removing unwanted fermentation by-products Their minimum bottling strengths vary from and increasing the alcoholic strength of the market to market and are 37.5% (v/v) in the EU, distillate up to >96% (v/v) at the top of the 40% (v/v) (80° US proof) in the USA and Canada final still. Trace congeners are removed using and 37% (v/v) in Australia. Flavoured the technique of hydroselection or extractive have separate regulations. distillation and trace methanol is reduced on 300 R.I. Aylott a demethylising column. The resulting neutral Gin production spirit is then ready for further processing into gin or vodka. Figure 1 shows a schematic diagram Distilled gin is made in a copper pot still (similar of a neutral alcohol distillation process and to that used for malt whisky) (Figure 2). Juniper Table 1 gives the characteristics required for berries and other botanical ingredients (see ethyl alcohol of agricultural origin required in Table 2) such as coriander seeds, angelica root, the European Union.

Table 1. Characteristics of ethyl alcohol of agricultural origin in the European Union (Council Regulation (EC) No.1576/89, 1989).

Organoleptic characteristics: No discernible taste other than that of the raw material Minimum alcoholic strength by volume: 96.0% (v/v) Maximum level of residues [measured as g/l00 I alcohol at l00% (v/v)] Acidity: acetic acid 1.5 Esters: ethyl acetate 1.3 Aldehydes: acetaldehyde 0.5 Higher alcohols: 2-methyl 1-propanol 0.5 Methanol 50 Dry extract 1.5 Volatile nitrogen bases: nitrogen 0.1 Furfural Not detectable

Fermented wash

IN

Neutral alcohol

OUT

Primary Hydro-selective Final Demethylation By-product distillation distillation rectification distillation

Figure 1. Schematic diagram for a neutral spirit distillation process. Gin and vodka 301

Figure 2. Schematic diagram of a typical gin still (from Aylott, 2003b, Figure 2). orange and lemon peels, are placed within the Vodka production still along with neutral spirit and water to bring the alcoholic strength to about 60% (v/v). Heat Neutral alcohol destined for vodka production is applied to the still by means of steam coils and is normally subject to further processing in order as the temperature of the liquid increases, flavour to reduce the concentrations of trace congeners compounds in the botanical materials co-distil that may impart organoleptic character. Vodka with the alcohol. The initial and final portions definitions in some countries require additional of the distillate (heads and tails) are collected processing or neutral spirit. This may involve separately as feints and the middle portion is further distillation or treatment of the alcohol collected as high strength gin at about 80% (v/v) with activated carbon. alcoholic strength. In certain markets vodkas contain trace London Gin is a type of distilled gin. Plymouth additives such as sugars, glycerol and propylene Gin carries a geographical designation. A glycol for sensory and/or authenticity indicator “compounded gin” requires the simplest method purposes. In some other countries no additions of manufacture and involves adding essences or are allowed. Finally, some vodka is given special flavours to alcohol. Table 2. Some common botanical materials used in gin distillation.

Common name Botanical name Principal origins Juniper berries Juniperus conimunis Italy, Central Europe Coriander seed Coriandrum Sativum Morocco, Eastern Europe Angelica root Archangelica officinalis Germany Sweet orange peel Citrus sinensis Italy Bitter orange peel Citrus aurantiun Spain Lemon peel Citrus limon Mediterranean 302 R.I. Aylott organoleptic characteristics, such as a mellow from glass or PET (polyethylene terephthalate), taste, by the addition of flavourings. the most popular sizes being 70 cl, 75 cl and 1 litre. 5cl PET bottles are particularly popular in the duty-free and airline trades. Packaging and marketing Tables 3 and 4 list the major gin and vodka brands with production data for the year 2003. High strength gin and vodka are then reduced to Whist the well-known gins and vodkas sell bottling strength with high purity demineralised internationally; there are certain regions of the water. Gin and vodka bottling strengths vary from world, such as the Philippines for gin and Russia market to market but normally range from 37.5% and Poland for vodka, where local brands sell (in the European Union) up to 47.3% (in certain very large volumes. duty-free markets). Most containers are made

Table 3. The major 7 gin brands, their brand owners, major markets and sales in 2003 (from: Drinks International 2003, Millionaires Club, website: www.drinksint.com).

Brand Brand owner Major markets Sales (millions of 9 litre cases)

Ginebra San Miguel La Tondena Distillers Philippines 27.3 Gordon’s Diageo International 5.05 Seagram’s Extra Dry Pernod Ricard USA 3.3 Beefeater Allied Domecq International 2.4 Tanqueray Diageo International 2 Larios Pernod Ricard Spain 1.8 Bombay Sapphire Bacardi Martini International 1.5

Table 4. The major 15 vodka brands, their key markets and sales in 2003 (from: Drinks International 2003, Millionaires Club, website: www.drinksint.com). Brand Brand owner Major markets Sales (millions of 9 litre cases) Sojuzplodimport Russia 55 Moskovskaya Sojuzplodimport Russia 32.5 Smirnoff Diageo International 18.7 Russkaya Sojuzplodimport Russia 18.1 Absolut Vin & Sprit International 8.1 Nemiroff Ukranian Vodka Ukraine 5.1 Sibirskaya Sojuzplodimport Russia 4.0 Polmos Bialystok Poland 3.3 Russkiy Rasmer Veda JSC Russia 3.3 Flagship Russian Wine & Vodka Russia 3.0 Bols Remy Cointreau Europe 2.3 Skyy Gruppo Campari Regional 2.0 Diageo USA 1.8 McCormick McCormick USA 1.8 Brown Foreman International 1.6 Gin and vodka 303

Bibliography Sciences and Nutrition, edited by Caballero, B., Trugo, L., and Finglas, P., Academic Aylott, R.I. (2003a). Vodka, gin and other Press, London, pp. 2889-2893 flavoured spirits. In: Fermented Beverage Clutton, D.W. (1979). The Production of Gin and Production. Second edition, edited by Lea, Vodka, Brewers Guardian, 108, 25-30 A.G.H. and Piggott, J.R., Kluwer Academic/ Murtagh, J.E. (1995), Neutral Spirit Production. Plenum Publishers, New York, U.S.A., pp. In The Alcohol Textbook. edited by Lyons, 289-308 T. P., Kersall, D. R. and Murtagh, J E., Aylott, R I., (2003b). Gin. The Product and its Nottingham. University Press, U.K., pp. Manufacture. In: Encyclopaedia of Food 193-212. 304 R.I. Aylott Shochu 305 Chapter 43 Shochu

K. Hashimoto and S. Matsumoto Sanwa Shurui Co. Ltd., Usa-shi, Oita 879-0495, Japan

Shochu market Production of shochu

Shochu sales have been rising so that by 2003 the Figure 5 contrasts the production of shochu volume consumed was greater than that of Sake with that of malt whisky. One of the main (Figure 1). It can be drunk either straight or on differences in the production process involves the rocks, with fruit juice, with soda, with tea or the production of koji. with hot water. There are two types of Japanese shochu, Honkaku shochu and Korui shochu. Honkaku shochu is equivalent to malt whisky Production of barley koji and Korui shochu to grain whisky (Figure 2). The main regions of Honkaku shochu production are One type of koji is produced specifically for in the south of Japan (Figure 3) with the types and barley shochu (barley shochu is a Japanese their consumption shown in Figure 4. clear distilled beverage with an alcohol content

1500 Sake 1300 Shochu

1100

900

700

500

Consumption volume (×1000 KL) 300 1999 2000 2001 2002 2003 Year Figure 1. Consumption of sake and shochu from 1999-2003. 306 K. Hashimoto and S. Matsumoto

Category 1.Honkaku (genuine) shochu; Malt whisky type

•Japanese traditional spirits •Ingredients: Barley, sweet potato, rice, buckwheat •Pot still: Single batch distillation •Alcohol of content of product: 20~25% (v/v) •Original flavor and taste derived from ingredients

Category 2. Korui shochu; Grain whisky type

•Patent still: Continuous distillation •Alcohol content of product: 20~25% (v/v) •Ethanol taste

Figure 2. Comparison between Honkaku shochu and Korui shochu.

300 distillers in Kyushu

Usa Tokyo Osaka

Rice Kome-Shochu Barley Mugi-Shochu

Sweet potato Buckwheat imo-Shochu Soba-Shochu

Figure 3. The Honkaku shochu regions of Japan. of approximately 25% (v/v). The manufacture 3. Inoculate the steamed barley with white koji of barley koji consists of the following steps fungus (Aspergillus kawachii) and incubate (Figure 6): at a temperature between 30- 40 degrees centigrade for 40-48 h. The mold-covered 1. Pearl barley grain to approximately 60% barley produced by the process is known as (pearled barley) “barley koji”. 2. Steep and steam the pearled barley Shochu 307

Products Company (KL) iichiko Barley SANWA SHURUI CO.,Ltd 87,840 iitomo, unkai Barley Buckwheat UNKAI SHUZO CO., LTD 32,763 Kirishima, kurokirishima Sweet potato Sweet potato KIRISHIMA SHUZO CO., LTD 27,540 Shiranami, kannoko SATSUMASHUZO CO., LTD Sweet potato Barley 27,000 Nikaido, kichomu NikaidoSHUZO.inc. 26,640 Barley Barley Hakutake, shiro TAKAHASHI SHUZOCO.,Ltd Rice Rice 17,370 hakatanohana FUKUTOKUCHO SHURUI CO.,Ltd Barley Buckwheat Rice 16,437 Kurouma, tensho KAGURA SHUZO CO.,Ltd Barley Buckwheat 15,480 Kaido, kakushigura HAMADA SHUZO CO.,Ltd 13,050 Sweet potato Barley Yokaichi TAKARA SHUZO CO.,Ltd Barley Rice 8,355 Year 2003 Figure 4. Types of Honkaku shochu.

“”“Barley shochu” “Malt“” whisky”

Ingredient Two-ro- wed Barley

Ingredient treatment Polishing Malting

Saccharification Barley koji Malt source by Koji strain

Fermentation Saccharomyces

Distillation Batch distillation by Pot still

Alcohol after distillation 40~45% 60%

Aging No aging Cask

Figure 5. The differences between the production of barley shochu and whisky.

What is Koji? ancient times. In liquor production, koji is a source of saccharification enzymes, provides The production of barley koji is the most nutrition for yeast multiplication and gives important step in barley shochu manufacturing flavour to an alcoholic product. Its role in liquor and has been the subject of a significant amount production is analogous to the role of malt in of research. “Koji” is a mold adapted to grow on beer and whisky manufacturing. The use of koji cereals and cereal by-products (rice, bran). Koji in alcohol manufacturing can be seen in a wide has been used in the production of fermented geographical area, from north eastern China to foods (liquor, seasoning) in Eastern Asia from Japan, south eastern Asia and Indonesia. 308 K. Hashimoto and S. Matsumoto

Two-rowed barley

Polished barley

Polishing to About 60%

Koji

Temperature: 30~40 ºC Humidity: 95~100% Time: 40~42hr

Figure 6. The production of Koji.

Mashing to bottling

The seed mash is made from barley Koji, water, starch with Koji and fermentation with yeast take and yeast. During the seed mash, yeast proliferates place at the same time. Following fermentation rather than the production of alcohol occurring and pot distillation, young shochu is aged before (Figure 7). In the main mash, saccharification of blending and bottling.

Koji Strain

PEARLED BARLEY STEEPING STEAMING

BARLEY KOJI

Water Water Yeast

DISTILLATION Young by Pot still Shochu SEED MASH at 20~30 ºC for 5 days MAIN MASH Alcohol: 45% Alcohol 17 ~ 18% Taste: fresh & rough at 20~30 ºC for 10 days

Figure 7. The conversion of polished barley to young shochu. The production of cognac 309 Chapter 44 The production of Cognac

G. Ferrari, B. Galy, V. Dumot, and R. Cantagrel Station Viticole du Bureau National Interprofessionnel du Cognac, Cognac, France

Introduction nowadays the vines are mainly in Charente and Charente maritime. Later, six “crus” (area Cognac, the worldwide well-known premium producing spirit of characterised quality) were quality grape spirit, is the oldest French A.O.C. specified by a French decree in January 1938: (appellation d’origine contrôlée = label of “Grande Champagne”, “Petite Champagne”, controlled origin). The French law fixed the “Borderies”, “Fins Bois”, “Bons Bois”, “Bois geographical area of Cognac production in Ordinaires” (Figure 1). The 1936 decree May 1909 and May 1936. Only four counties described the authorised vine varieties and were authorised to produce Cognac, but process specifications.

Figure 1. Map of the six ‘‘Crus’’ of the Cognac delimited area. 310 G. Ferrari et al.

The professional organisation B.N.I.C. quantities of wines that they became difficult (Bureau National Interprofessionnel du Cognac) to dispose of, particularly because these low has been allowed by the French government to alcohol wines often deteriorated during the long control movements and ages of Cognac since sea shipping. At the end of 16th century, Dutch 1946. B.N.I.C. aims are also to promote Cognac, merchants then began to use them in their own to defend it from counterfeiting (imitation), to new distilleries, transforming them into “burnt ensure traditional production and to protect wine” – brandwijn or brandywine, that became quality (monitoring technical development, “brandy”. In the 17th century, the wines were scientific research, experimentation). Cognac transformed into spirits before shipping from vine growers and merchants have looked for the Charente, then matured in oak casks to become best spirit quality through the ages, continuously Cognac. That was the start of a worldwide improving their processes with respect to adventure for a town which was to become the traditions. capital of a world famous trade. The market became more established from the end of the 17th century. Owing to the History demand for Cognac, “Comptoirs” or agencies were created in the main towns of the area, some The Saintonge vineyards (all around the roman of which are still in existence. They collected city of Saintes, Charente maritime) are believe the Cognac produced and established regular to date back to the Roman emperor Probus who links with the buyers in Holland, England extended the privilege of owning vines and and Northern Europe, later extending links to making wine to all the Gauls, in the 3rd century. America and the Far East. With the encouragement of William X, Duke Many more trading companies were set up, of Guyenne and Count of Poitiers, a large wine and in the mid-19th century, they began to ship producing area was established, known as the their spirits in bottles rather than in cask. This “Vignoble de Poitou”. new way of trade furthered other industries: The wines from Poitou, Aunis (around La glassworks (the manufacturing of the glass bottle Rochelle) and Angoumois (around the cities of was invented by C. Bouchet in Cognac, 1898), Angoulême and Cognac), produced from high manufacturing of cases and corks, labels printing quality vineyards, were shipped to Northern and cooperage. Europe where they were enjoyed by the English, Dutch and Scandinavians, as early as the 13th century. The Charente river played an important Cognac production process part: it furthered the wine trade as the wine barrels were shipped from there to La Rochelle Raw material harbour, using specific ships with flat bottoms: the “gabare”. Cognac is a wine spirit, distilled from wines With such a widespread interest in wine produced only in the Cognac delimited area production from Cognac country in the Middle (about 80,000 ha). The vine varieties allowed Ages, people developed this new trade, which were defined by the 1936 decree. The main followed the previous salt trade. They were varieties are: Ugni blanc, Colombard, Folle helped by foreign wine merchants (Dutch and blanche. But nowadays, the Ugni blanc variety English). The Dutch ships came to the Cognac represents more than 95% of the vineyards. It is area to collect the well-known wines of the a late vine (harvested in October), vigorous and “Champagne” and “Borderies” growing areas. productive. It gives an acidic and low alcohol The Aunis vineyards were producing such great white wine, with a mild aroma. The production of cognac 311

Must production start to fermentation. Commercial dry yeast strains (Saccharomyces cerevisiae) specific for The grapes are picked during October, usually Cognac production are now available: “FC9”, just before maturity. The average content of “FA1” selected by B.N.I.C., and “SM102”, must required is about 130-160 g/l of sugar, for example. The fermentation is monitored

8-9 g/l (as g/l H2SO4) of total acidity, and pH (temperature, density) in order to prevent = 3. Must nitrogen content is also checked in problems. The fermentation time varies from order to prevent fermentation problems. Grapes 4 to 8 days, depending on temperature, must attacked by the “grey mould” (Botrytis cinerea) nitrogen content and yeast efficiency. When are not selected for Cognac production to avoid the fermentation is finished, the wines are either off flavours. kept on lees or racked. The tanks are filled up Grapes are now mainly picked with harvesting to avoid air contact with wine. After, malolactic machines, and brought back to the cellar where fermentation may take place. Once alcoholic they are lightly crushed with pneumatic or fermentation is completed, the white wine mechanical presses to get the juice, which is has to be distilled immediately to obtain the pumped to the fermentation tank. Screw-presses spirit. Therefore, the pot stills are full on during are forbidden by the decree, as it is forbidden November. Wine production from different areas to add sugar to the juice. Also, no preservatives is shown in Figure 2. are added. So, it is necessary to start the alcoholic fermentation very quickly to prevent Pot still distillation juice damage. In some case, the juice is rapidly decanted (during about 2–3 h) to discard the The “charentais” pot still is defined by the heavier solids. decree. It is made of copper and heated by open fire (gas burners are the most used) (Figure 3). Its capacity is limited to 10,000 litres for the Fermentation first wine distillation and to 3000 litres for the second step (2500 l useful load). The Charente The must is sometimes heated if necessary (to distillation process for Cognac production is also reach at least 18°C, ideally 22°C) and yeasts governed by a number of precise regulations are often added to provide a good and fast described by the decree.

Bois Ordinairos 97 701 Bons Bois Grande Champagne 953 394 1 334 973

Petite Champagne 1 648 598

Fins Bois 3 315 236 Borderies 414 076 Figure 2. Cognac wine production in hl, vintage 2003: 7 763 978 hl. 312 G. Ferrari et al.

It is a two stage distillation : first the wine is Maturation and ageing distilled to obtain a spirit with an alcoholic strength of about 27% (known as the “brouillis”) (Figure 4), After the distillation process, the spirit is pumped then the “brouillis” is distilled in several fractions into oak barrels for aging. Oakwood comes only (this is the “bonne chauffe”). The head (1–2% of from France (Limousin and Tronçais forests the volume) and tails are separated (in order to be mainly) and the staves are dried in the open air redistilled with another wine or brouillis). Only for three years. Barrels containing from 270 l to the “heart” is kept to become the Cognac and it 450 l are lined up in many rows in cellars. These may have an alcoholic strength of 70-72% (v/v). are specific for Cognac aging and characterized The regulation obliges the pot stills to be switched by their balance between temperature and off before 31 March. Today, about 500 000 hl of humidity. alcohol are produced for Cognac annually.

Figure 3. Charentais style distillation.

The distillation of cognac WINE

Brouillis Heads 26 - 32% Tails 0 - 0.25% 0 - 5%

BROUILLIS Heads Tails 0 - 2% HEART Seconds 0 - 5% 27 - 29% 24 - 26%

Figure 4. The “Charentais” pot still (volumes (in percent) of distilled fractions). The production of cognac 313

During maturation in cellars, there is a loss of Packaging and marketing water and alcohol (know as “la part des anges”, the “angels’ share”) of about 20 million bottles Today, the majority of Cognac is shipped in per year!. On the other hand, there is a slight bottles to about 160 countries. Cognac has a entry of oxygen into the barrels producing a worldwide and unique reputation, which needs moderate oxidation of the spirit components. to be protected. Over many years, rules have Over many years, the spirit gets its colour and been established to eliminate any copies, both specific aroma (Figure 5). in production and in presentation (counterfeit). After aging, the cellar master (known as French regulations are very strict about the sales “le maître de chai”) blends many spirits from rules. Cognac may not be sold to the public various origins and ages to get a new unique unless it has been aged for at least two years, spirit corresponding to the taste of its own counting from the end of the distillation (1st April office. Therefore, every Cognac keeps its own following the year the grapes were harvested). personality throughout the ages. Before bottling, The age of the Cognac is that of the youngest the blend is matured for 6 to 10 months in spirit used for the blend. big barrels to let its components marry and Each case of Cognac is exported with an become stabilized. During this time, the spirits age and origin certificate, delivered only by are checked by tasting and chemical analysis to B.N.I.C., and an analysis certificate to prove its verify the quality. compliance with quality requirements.

Figure 5. Age and selling name. 314 G. Ferrari et al. Grappa: the Italian distillate 315 Chapter 45 Grappa: the Italian distillate

C. Da Porto1 and M. Longo2 1Dipartimento di Scienze degli Alimenti, Università di Udine, Udine, Italy; 2Distilleria Bonaventura Maschio S.r.L, Gaiarine (TV), Italy

Introduction of ethanol from the raw material is very low: 3.8 litres of pure alcohol from 100 kg of marcs, According to the European Union (EC, 2008) compared with 37 litres of pure alcohol from 100 grappa is the spirit beverage made in Italy from kg of cereals. Grappa together with the Spanish direct steam marcs distillation or distilled after ‘aguardente’, mainly from the Galice region, the adding water or wine lees to marcs. Any use of Portuguese product ‘bagacerias’, mostly from liquids from pressing of marcs, from washing the Vinho Verde region, the Greek ‘tsipouro’ it with water, and from diffusion of marcs is from the northern part of the country, named forbidden. The greatest amount of wine lees that ‘tsikoudia’ in the isle of Crete and ‘raki’ in the can be added to marcs is 25 kg per 100 kg of the other areas of Greece, is an alcoholic beverage latter, corresponding at most to 35% (v/v) of the obtained from marcs distillation. final alcoholic amount. The highest proof of raw distillate is 86% (v/v), and that of the commercial product must be higher than 37.5% (v/v). The Raw materials lowest volatile compounds content of grappa, different from ethanol and methanol, is 140 mg Grape marcs /100 ml p.a. (pure alcohol) and the legal limit of methanol is 1 g /100 ml p.a. Moreover, Italian Grape marcs are the mass of skins, stalks, and seeds regulations (GU, 1997) permit the addition of left after the winemaking process. This material a maximum sucrose content of 2% (w/v), the can contain residual sugar (unfermented marcs) addition of botanical ingredients and, only for or ethanol from the fermentation (fermented grappa put into wood for a period of at least marcs). The quality of marcs depends not only 18 months - the addition of caramel for colour on the winemaking process used for grapes (mild enhancement. and soft technologies are the best), but also on Italian regions in which grappa is granted the factors affecting berry composition, such as the Geographic Appellation are Piemonte, environmental conditions (temperature, rain-fall, Lombardia, Trentino Alto-Adige, Friuli Venezia- humidity, wind, soil, etc.), vine variety, date of Giulia, and Veneto (EC, 1989). Grappa is subject harvest, viticultural practices, soundness, and to several government taxes and the recovery residues of agro-chemicals. 316 C. Da Porto and M. Longo

The mean chemical composition (% of total) bacteria. Also, higher alcohols do not increase, of unfermented marcs is: water 50 to 60%, except 1-propanol, while 2-butanol formation sugars 6 to 10%, organic acids 1 to 2%, phenolic is the result of contamination by anaerobic compounds 1 to 2%, mineral compounds 1 to bacteria. In these conditions, the ethanol losses 2%, cellulose 10 to 20%, lipids 2 to 4% besides for acetification were estimated to be about 10 proteins, vitamins, aromatic substances, pectins, to 20%. yeasts, and bacteria (De Rosa and Castagner, In order to get a better and limited in amount 1994). The moisture of the marcs (optimum value storage of marcs, plastic containers (capacity about 50%) is related to the systems of pressing ca.130-150 kg) may be used (Figure 1). Actually, used in winemaking. Marcs of aromatic grapes some distilleries use a cylindrical-shaped plastic (Muscat, Traminer, Prosecco, etc.) produce grappa container (capacity ca. 300 tons) in which the subtly exhibiting the sensory characteristics of the marcs are strongly pressed. This storage method starting grape. is an excellent technical solution for marcs storage because the salami-like containers, laid horizontally on the ground, permit the maximum Marcs storage and fermentation exploitation of the distillery’s available space during the period when marcs are carried to the Fermented grape marcs could be distilled distillery in large amounts. Furthermore, this immediately, but are often stored to extend the system creates stricter anaerobic conditions than use of industrial scale distilleries. Problems of the others ensilage methods and this can improve setting up and managing both the distillation the volatile composition of grappa (Da Porto, plant and the distillery wastewater plant give rise 2002). Stainless steel containers filled with ca. to this practice. Various storage systems are used 200-300 tons of marcs are the storage systems for grape marcs. Traditionally, grape marcs are used by the new large distilleries. stored in large concrete earthen tanks filled with 50-100 tons of marcs. Concrete rooms are also used, they are closed on three sides and the fourth side is opened to allow filling with 400-500 tons of grape marcs both by trucks and mechanical shovels. At the end of this storage operation, the marcs are covered with plastic sheets to limit the entrance of oxygen and the development of aerobic bacteria such as acetic acid bacteria. Figure 1. Plastic containers used to store marcs. However, during storage, bacteria or mould contamination of the marcs can take place, leading to off-flavour production in the resulting Usually the distilleries do not inoculate a spirit (Usseglio-Tomasset, 1971; Williams and specific strain of yeast but let the fermentation Strauss, 1978). In order to improve the quality process be carried out by natural fermentation. of grappa, the marcs may undergo several Others inoculate the unfermented marcs with treatments such as acidification to lower the pH, commercial dried yeast used in winemaking compression to reduce the presence of oxygen, in order to improve the chemical composition addition of potassium metabisulfite usually just at of grappa. In this case, the substrate for yeast the surface towards the outside, mostly to exploit growth is obtained using the dripping liquid of the pressed marcs with added SO and enriched the antimicrobial activity of sulphur dioxide. 2 Usseglio-Tomasset (1985) found during marcs with commercial yeast nutrients. This liquid is storage that acetic acid and methanol increase, stored in a tank, acidified, and sprayed on other while acetaldehyde and ethyl acetate remain marcs during their ensilage. By lowering the quite constant because they are metabolised by pH level of the marcs, methanol formation is Grappa: the Italian distillate 317 also reduced because the pectin methyl esterase extremely limited because of technical problems, activity decreases at acidic pH. According to the the water bath alambic, which is widespread composition of the marcs (acidity, pH, sugars, etc.) over the country because it can produce fine and the fermentation conditions (temperature, quality distillates (Figure 2a), and the direct oxygen), different aromatic constituents are steam alambic, which is the most common synthesized by yeast. (Figure 2b). This last kind of still consists of The fermentation temperature of grape marcs three major parts, two or more overturned cone can often exceed 30°C, affecting yeast growth and frustum-shaped copper boilers connected in metabolism. The poor heat transfer which occurs series, the rectifying column, and the condenser. in grape marcs during storage is due to various Each copper boiler holds about 180 to 200 kg factors such as the high content of cellulose in of marcs and a hermetic seal hatch is on the the grape marcs and the material and size of the upper part. Marcs are loaded into special copper containers used. Besides stuck fermentations that baskets that are laid one on the other, in order can lead to the reduction of ethanol recovery, to prevent marcs compression and to improve volatile compounds detracting from overall grappa marcs unloading. Heating of each copper boiler quality can be produced in appreciable amounts is done indirectly by steam injection. as a result of bacterial spoilage. Recently, by Steam passes through marcs and strips the applying cooling to the steel containers for marcs phlegm vapours (liquid at about 20% (v/v) storage, a consistent improvement of chemical ethanol), which are then fed into the base of the composition of grappa has been obtained (Da rectifying column (Figure 3a) usually equipped Porto, Cortella and Freschet, 2004). with a few bubble-cap plates (Figure 3b).

Distillation Continuous distillation

Two distinct distillation systems are used for The distillation plant is normally constructed grappa production: the batch still and the of stainless steel. Distillation is carried out continuous still. continuously in two steps. First, marcs are introduced into a de-alcoholising plant in which ethanol is steam-stripped to give phlegm Batch distillation vapour at about 10 to 20% (v/v) (Figure 4a). Second, the condensed phlegm vapours feed Traditional systems of grappa batch distillation the column for ethanol concentration and are: the direct flame alambic, which is actually rectification. The addition of further columns to

(a) (b)

Figure 2. Water bath alambic (a) and direct steam alambic (b). 318 C. Da Porto and M. Longo

(a) (b)

Figure 3. Rectifying column (a) and a bubble-cap plate (b). the system improves the separation of undesirable of spirit collection, and it is a critical operation compounds such as methanol (Figure 4b). for the quality of the product. Depending on the variation of proof level, which can be read on the hydrometer port of the still, the ‘heads’ Rectifìcation cutting (usually between 70% (v/v) ethanol and 80% (v/v)) and ‘tails’ cutting (between 35% (v/v) Among the alcoholic beverages, grappa is the ethanol and 50% (v/v)) are made. These fractions richest in volatile compounds (about 1%). contain, respectively, the undesirable highly Grappa is obtained by distilling a raw material volatile and less volatile compounds together with a high percentage of solids (from 70 to 80%), with a large amount of ethanol (heads contain consequently, a rectification procedure to lower 5% of total ethanol from the raw material and the concentration of undesirable components tails 10%). They can be recycled at the end of (especially methanol) is required. distillation. In batch stills, rectification is made by In batch stills, methanol distils throughout selection of the cut points at the start and the end the entire distillation process and shows a slight

(a) (b)

Figure 4. De-alcoholising plant (a) and columns system (b). Grappa: the Italian distillate 319 increase in the tail fraction. In discontinuous components responsible are primarily the ethyl equipment, methanol is the volatile compound esters of long chain fatty acids. Refrigeration most difficult to separate because the rectifying followed by filtration provides stabilization of column is fed by liquid at low proof and the grappa at the low temperature of consumption. relative volatility of methanol exceeds unity for Grappa is chill filtered in the temperature range an alcoholic strength higher than 40% (v/v). By 0°C to -15°C. In some processes, the refrigerated cutting a greater amount of ‘tails’, which are not grappa is held at 0°C or -5°C for 1 to 2 days to be recycled, but distilled again separately, the before filtration. methanol content is lowered by about 20%. In continuous distillation plants, methanol is separated by the ‘demethylization column’, Blending and bottling even though a loss of volatiles occurs with this procedure. The demethylization column is a Prior to being marketed, several marcs spirits are rectifying column fed by liquid at high proof, blended. When the blend is correct (evaluation is equipped with several plates and characterized made by sensory assessment), deproofìng water by a high reflux ratio. is added to the blend to reduce the strength for bottling. Blending can be followed by a further period of maturation. The naturally golden Expended marcs yellow colour of grappa aging in wood can be enhanced with caramel. Fresh grappa can The expended marcs, completely exhausted be flavoured with botanical ingredients. Sugar after distillation, are dried to separate the skins solutions are sometimes added to attenuate the and stems of the grape from the seeds. The skins ‘burn’ of the alcohol. and stems, rich in cellulose, can be employed by livestock feed producers or by the distillery as a source of energy for the production of the Chemical composition steam necessary to feed the distillation process. The seeds, on the other hand, are sold to oil Aroma compounds found in grappa originate producers who, from these, produce one of from a number of sources, including the varietal the most dietetic oils available. Lastly, many origin of the grape-marcs, the storage times and distilleries, with respect to the environment, have methods, and the distillation technology used. installed filters for smoke purification. Table 1 shows a set of average values established from 51 commercial samples of grappa, produced with autochthonous non-aromatic marcs in six Maturation Italian regions and not aged in wooden barrels. Gas chromatographic analysis was performed by Traditionally, grappa is a spirit ready to drink. direct injection of the distillate in a glass column Consequently, maturation in wood, absolutely (2 m x 2 mm i.d) packed with 0.2 CW 1500 necessary for other kinds of alcoholic beverages on Carbopack C 60-80 mesh and fused silica such as brandy, cognac, whisky, is for grappa capillary column (Supelcowax 30 m x 0.32 mm more a demand of the market than a real need. i.d. x 0.2 µm) (Versini, Monetti, Dalla Serra and Freshly distilled grappa is matured in vats Inama,1991). of inert material (stainless steel, etc.) for 5 to 6 months or it is slightly reduced in strength prior Grappa categories to filling into wood casks for at least 6 months. Grappa is subject to the formation of cloudiness The most important classification of grappa is when the alcohol concentration is ‘reduced’ to based on its sensory characteristics (taste and approximately 40% (v/v) with distilled water. The smell): 320 C. Da Porto and M. Longo

Table 1. Volatile compounds of commercial samples of grappa produced with non- aromatic marcs (results expressed as mg/100 mL pa (pure alcohol). Average Standard deviation Maximum Minimum Ethanol (%vol) 45.04 3.6985 60.00 39.44 Methanol 0.51 0.2244 0.92 0.12 1-Propanol 50.02 22.3040 117.50 18.50 2-Butanol 30.22 52.9223 212.00 1.00 2-Methyl-1-propanol 69.98 18.6580 119.00 25.50 1-Butanol 2.55 1.3002 7.50 0.90 2-Methyl-1-butanol 48.04 14.4020 95.00 19.00 3-Methyl-1-butanol 181.52 60.0078 377.00 65.00 Acetaldehyde 114.91 128.3713 540.00 2.00 Ethyl acetate 162.99 133.4181 529.00 4.50 Isoamyl acetate 2.54 1.9418 9.20 0.39 Hexyl acetate 0.20 0.1693 0.72 0.01 2-Phenylethylacetate 0.18 0.1613 0.69 0.01 Ethyl hexanoate 1.85 0.9449 4.14 0.10 Ethyl octanoate 7.08 4.7826 23.00 0.86 Ethyl decanoate 11.97 9.2318 42.90 1.35 Ethyl dodeconoate 3.42 3.2513 15.30 0.09 Ethyl myristate 0.30 0.04972 2.40 0.01 Ethyl palmitate 0.69 1.3010 7.73 0.01 Ethyl oleate 0.08 0.2130 1.33 0.01 Ethyl linoleate 0.35 0.7130 3.70 0.01 Ethyl linolenate 0.08 0.1837 0.96 0.01 Ethyl lactate 32.16 7.7991 98.80 0.01 Isoamyl lactate 0.30 0.3276 1.24 0.01 Diethyl succinate 2.98 2.8238 11.80 0.01 1-Hexanol 8.97 3.4959 17.00 3.69 Trans-3-hexen-1-ol 0.18 0.1223 0.44 0.01 Cis-3-hexen-1-ol 0.38 0.2325 1.21 0.06 Trans-2-hexen-1-ol 0.19 0.1591 0.63 0.01 1-Eptanol 0.14 0.0951 0.45 0.01 1-Octanol 0.12 0.0667 0.25 0.01 1-Nonanol 0.12 0.0817 0.37 0.01 Benzyl alcohol 0.07 0.0789 0.40 0.01 2-Phenylethanol 2.30 2.1702 7.20 0.01 3-Ethoxyl-propanol 0.12 0.2111 0.88 0.01 Linalool 0.07 0.0847 0.52 0.01 Nerol 0.06 0.2082 1.50 0.01 a-Terpineol 0.08 0.1586 0.70 0.01 Furfural 0.45 0.2944 1.20 0.01 Benzaldehyde 0.81 0.8460 3.28 0.04 1-Decanol 0.10 0.0586 0.25 0.01 Isoamyl octanoate 0.14 0.1872 0.10 0.01 Isoamyl decanoate 0.15 0.1629 0.88 0.01

- young grappa is characterized only by means - aged grappa is distinguished by colour (tendency of the aromas derived, during distillation, from towards a yellowish/gold) and for its spicy tones the vine and from fermentation. It is preserved acquired after an aging period equal to at least in containers other than wood; 12 months in oak or other type of wooden barrels; Grappa: the Italian distillate 321

- aromatic grappa derived from aromatic grape tonality of colour provides evidence of the category varieties (grappa of Moscato, of Malvasia etc.); to which the ‘grappa’ belongs. Young grappa is - flavoured grappa derived from the effect of without any colour and is crystalline, aged grappa maceration (soaking) with herbs, the grappa tends to lean towards a yellowish-gold colour, while acquires a particular aroma (grappa with rue, flavoured grappa is in part charged with the colour with gentian etc.). pigment of the herb or fruit used in its flavouring. Nosing is done in tulip-shaped glasses to Popular in recent years is grappa ‘monovitigno’ concentrate the aroma and the glass is swirled which is obtained by marcs coming from a single before nosing to release the volatiles. It is of utmost grape variety. The law allows for the indication of importance to verify the absence of off-odours. the grape variety on the grappa label when 85% Among the off-odours, those of fodder (livestock of primary materials used to make the grappa feed), rancid, wax or pungent are due to bad marcs in question hail from the variety vine indicated storage and distillation. When the absence of all on the label. In the case of grappa named for its defects has been verified, the final step is to search Geographical Appellation, the law provides for the for pleasant aromas that recall floral, fruity or spicy guardianship and the careful definition of product notes. characteristics within the single area in which it is The taste evaluation is the last one. Of course, manufactured. the first sensation is that of alcohol, followed by the sensations of sweet, sour, acid and then the tactile sensations. Those of particular importance are of Sensory evaluation clearness or of oiliness. The sensation of oiliness means poor distillation, and as a result, a grappa Very important in the quality assessment of grappa of a poor quality. is the sensory evaluation of distillate samples. In Italy, the Association, which gets together many trained grappa testers is A.N.A.G. (Associazione Marketing Nazionale Assaggiatori Grappa). An ANAG sensory test consists of an evaluation procedure based on Grappa accounts for about 57% of Italian spirit scores given to colour, taste and smell of grappa. production, with 35% being brandy, 4% grape- Examination for clarity verifies the absence of distillate and 4% fruit spirits (AssoDistil, 2004). In veiling or of powdery suspensions in the distillate, the year 2003 grappa production was of 120.000 which if discovered would provide evidence hl p.a. (pure alcohol) and grappa export, expressed of some production problems. Examination for as hl p.a., is that reported in Figure 5.

Other countries 580 United Kingdom 52 Netherlands 61 France 94 Spain 133 Belgium 146 Austria 177 Canada 263 Swtizerland 702 Germany 2771

Figure 5. Grappa export 2003, data expressed as hl p.a. 322 C. Da Porto and M. Longo

References Usseglio-Tomasset, L. (1971). Le caratteristiche delle grappe derivanti dall’evoluzione dei AssoDistil (2004). Relazione annuale 58ma costituenti volatili delle vinacce durante il Assemblea AssoDistil; Roma, 28 maggio periodo di insilamento e dalle modificazioni Da Porto, C. (2002). Volatile composition of apportate dal processo di distillazione. Vini ‘grappa low-wines’ using different storage d’Italia 13: 453-462 methods and conditions on an industrial Usseglio-Tomasset, L. (1985). La grappa nella scale. International Journal of Food Science sua composizione e nei suoi sistemi and Technology 37: 395-402 di elaborazione. Atti del IX Convegno Da Porto, C., Cortella, G. and Freschet, G. Nazionale della Grappa, Treviso, 20-21 (2004). Preliminary study on a cooling settembre practice of grape pomace during storage on Versini, G., Monetti, A., Dalla Serra, A. and an industrial scale. Italian Journal of Food Inama, S. (1991). Caratterizzazione analitico- Science 16: 87-95 statistica su base regionale della grappa. De Rosa, T. and Castagner, R. (1994). Tecnologia Bollettino del CIDEO 11: 132-150 delle grappe e dei distillati d’uva. Edagricole, Williams, P.J. and Strauss, C.R. (1978). Spirit Bologna recovered from heap-fermented grape marc: EC (2008). Council Regulation (EEC) nature, origin and removal of the off-odour. No.110/2008 Journal of Science and Food Agriculture 29: GU (1997). Gazzetta Ufficiale della Repubblica 527-533. Italiana No. 213 of 12 September 1997. DPR No.297 of 16 July 1997 Ouzo and raki 323 Chapter 46 Ouzo and raki

A. Varvagiannis, L. Margomenou and I. Zabetakis Laboratory of Food Chemistry, Department of Chemistry, University of Athens, Athens, Greece

Introduction reducing sugars must be between 65-75% (w/w) and materials must be used within a year of The production of the spirits ouzo and raki is harvest), anise seed (Pimpinella anisum) (which straightforward. The quality of the raw materials contains 90% anethole), water and sugar. is a key issue affecting the quality of the end products. A detailed methodology for the two products, which have similar raw materials but Wort/must production are very different, is described. There is no must production as a step in ouzo manufacture but there is preparation of the raw Raw materials materials, especially the herbs and seeds for distillation. They are placed in water for 24 hours Ouzo: ethanol (agriculturally derived from prior to distillation. grapes, sugar cane and other sugary fruits), anise, In the case of raki, suma is prepared. The herbs and seeds (the herbs and seeds differ for raisins are soaked in water (solubilisation of each brand). raisins). The next step is grinding. The soaked raisins are passed through a hammer mill or Raki: is registered as a high proof spirit obtained roller crushers for complete disintegration by distilling distilled alcohol (suma) by itself or and pumped into tanks where they stay for 30 distilled alcohol (suma) mixed with agricultural- min at 22ºC. The whole process takes place in origin ethyl alcohol flavoured with Pimpinella stirred water in order to facilitate the extraction anisum (aniseed). For raki, the raw materials of sugars. The final amounts of the solubilised are thus: suma (distilled alcohol, grape-origin components increase with temperature and distillate). It is distilled up to maximum 94.5% stirring rate. Since high sugar concentration (v/v) alcohol in order to protect the taste or smell retards the fermentation, the final reducing of grapes (raisins). Selection of raw materials is sugar content of the must is diluted to give based on moisture and total sugar content (the approximately 17-21% (w/v) reducing sugars. 324 A. Varvagiannis et al.

Fermentation process distillery). The process is repeated the next day so the end product will be highly purified. This In the case of the raki, following the above product has an ethanol content from 75 to 80% process, the must is passed into yeast propagating (v/v). It will be diluted with water down to 42 to installations which have been sterilized by 48% (v/v) alcohol. heating with a steam coil and then cooled to 30°C by passing cold water through the coil. The must is heavily inoculated with a pure Raki yeast culture of Saccharomyces ellipsoideus and is left to incubate. During the fermentation In the case of raki distillation, the alcohol it is important to cool the must artificially and concentration increases by vaporizing the maintain the temperature below 30°C. Within alcohol of the must. The alcohol then passes to 64-72 h, the fermentation is generally completed the rectification section of the column where with a yield of 9-11% (v/v) of alcohol. its concentration reaches 94-96% (v/v) and unwanted compounds such as esters, aldehydes and fusel oils are removed. Suma is now ready Distillation for use, it is reduced to 30-40% (v/v) alcohol. Softened aniseed (6-10lt) is added to the suma Distillation takes place in traditional copper which is taken for distillation. During distillation, distilling alembics. In the case of ouzo there are 3 main fractions: ‘heads’, ‘heart (coeur)’ and two types of distillation where the end product ‘tails’ are separated. The aldehydes and esters will be either crude/cold ouzo (20% distillate) have lower boiling points than ethyl alcohol or 100% distillate. and are separated in the heads fraction, whereas the fusel oils and furfurals which have higher boiling points than ethyl alcohol are separated Ouzo in the tail fraction. The head fraction is produced with a temperature below 80°C, it is discarded The distillation process for the production of and distillation of the remaining suma continues crude/cold ouzo is as follows: a single distillation for a further 6 h. The head fraction containing (usually the beginning and the end fractions of about 82.4% (v/v) alcohol is thus separated. the distillation process (heads and tails of the After 31 h of further distillation at 85-91°C distillation) are rejected whereas the middle the main distillate heart averaging 80% (v/v) fractions, which will be diluted down with water alcohol is collected and stored in oak casks. to 40 degrees of ethanol, are kept. This will be The tail fraction is separated after a further 6-7 diluted to 20% (v/v) for the final package. The h of distillation at 92-101°C, above 101°C the ethanol percentage varies for every distillery alcohol content decreases below 76-78% (v/v) plant according to recipe. The distillation and the distillation is discontinued. process for the production of 100% distillate is as follows: the materials for the distillation (anise, herbs and seeds) after the water treatment Maturation and/or flavouring are placed with fresh water and ethanol in the still and the process of boiling begins. The first There is no maturation step for the 20% (v/v) liquified vapours are rejected, this point varies ethanol content ouzo whereas for the 100% from distillery to distillery. The middle fractions distillate the final product of the second are selected and the end fragments of the distillation (80% (v/v) ethanol) is placed in distillation process are rejected. The distillation stainless steel containers for 30 to 40 days in duration is around 12 h (depending on the order to get a homogenized final product. Then, Ouzo and raki 325 in a single day it is diluted down to 42-48% (v/v) aluminum bottles. The sealing materials are alcohol. After seven days quality control analysis usually metallic or cork (compressed cork) and takes place (a sensory evaluation is included). on very rare occasions, a plastic seal is used. Raki is not a matured spirit, but it is stored After filling the bottles with the liquid and in oak casks for 1-3 months to obtain a good sealing, they are packaged in boxes. The filling blend with anise. The final step before bottling of the boxes varies according to two parameters; is a filtration to polish the final product. Raki the bottles used and their volume, e.g. in the 0.7 contains a minimum 65% (v/v) of alcohol, which L packages the boxes contain 12 bottles and in is grape-origin distillate (suma), maximum 10 g/l the case of 0.2 L, 48 bottles. The Companies refined white sugar and anethole (amount must either have their own sales department or they be a minimum of 800 mg/l). use companies specialized in retailing. The product will reach the off-trade and on-trade, i.e. there is a separation between the supermarket, Packaging and marketing and kavas/restaurants/taverns.

The final product of ouzo is filled mainly in glass bottles and on rare occasions ceramic or 326 A. Varvagiannis et al. Rum and Cachaça 327 Chapter 47 Rum and cachaça

J.B. Faria UNESP, São Paulo State University, Departamento Alimentos e Nutrição, 14801-902, Araraquara, SP, Brazil

Raw material of molasses is the total sugar (55-56% w/w) but other factors like sulphated ash, nitrogen, Rum and cachaça both originate from the sugar gums and unfermentable sugars also impact the cane (Saccharum officinarum) plant. The juice, molasses quality (Nicol, 2003). as extracted from the sugar cane stalks, may be directly used for cachaça production; or after Table 1. Molasses analyses. Results are expressed as industrial sugar processing, producing molasses, % (w/v). (From Nicol, D.A. (2003) Table 12.1 ©2003 used as raw material for rum production. The Kluwer Academic/Plenum Publishing, with kind permission). sugar cane juice, can be heat evaporated to obtain a concentrate similar to industrial molasses, to Typical analyses of make so called agricultural rum, or used in the molasses for rum fermenters as a source of fermentable sugar for production the production of lighter flavored rum. Thus rum Good Fair Bad and cachaça come from the same plant, but are obtained from distinct raw materials. Cachaça is Brix density 87.6 85.4 88.2 made from sugar cane juice, while rum production Total sugars as invert (TSAI) 57.97 52.91 49.93 is mainly based on sugar cane molasses. Sucros 36.44 31.30 34.61 Reducing sugars 19.61 19.96 13.50 Ash 7.31 9.35 11.57 Total nitrogen 1.10 0.60 0.45 Must production Total phosphate 0.19 0.09 0.21 Gums 5.5 5.7 6.3 Rum TSAI:ash ratio 7.93 5.65 4.61 Reducing sugars:sucrose ratio 0.54 0.64 0.39 As already mentioned, the main raw material Phosphate:total nitrogen ratio 0.17 0.15 0.47 for rum production is molasses provided by Gums:TSAI ratio 0.03 0.05 0.08 the sugar industry (see Table 1). For every one Aroma by steam distillation Good Fair Indiff- erent hundred tonnes of cane milled by the sugar industry, about 4-5 tonnes of molasses are Normally, molasses straight from the sugar mill, recovered. The most important constituent are delivery hot to the distillery (45-65°C) and 328 J.B. Faria considering the elevated temperature (>60°C), allowed to process naturally by yeasts and the molasses may degrade (Maillard reactions bacteria present in the air, contaminating the and eventual spontaneous combustion), it is dilution water or the sugar cane juice or lying recommended, when necessary to store molasses dormant in the molasses. Besides this old so that the temperature does not exceed 45°C traditional technique, the use of pure or mixed (Chen, 1985). In order to partially remove the cultures is being more and more employed in rum suspended solids, the molasses are first diluted to and cachaça fermentation. Rum fermentations are 45°Brix, and the temperature is raised to 70°C to mainly conducted by Saccharomyces cerevisiae, pasteurize the molasses. Sulphuric acid is added Saccharomyces bayanus, Schizosacharomyces at this stage (0.02–0.04% w/v), to lower the pH pombe and various strains of competing bacteria and to encourage sedimentation. from the dunder, the naturally fermented residue The diluted (45°Brix) and acidified molasses of wash distillation, that are used as a fermentation is then transferred into a large settling tank, inoculum. properly designed to decant the sludge. Finally, The sugar cane juice fermentation for cachaça the supernatant clarified molasses is diluted to production, is a very robust process that can occur the desired working specific gravity (16-20°Brix) even under technically adverse conditions, due and pumped to the fermenter, where it is dosed the higher adaptive capacity of the yeasts normally with nutrient and pitched with yeast (Nicol, 2003; used (Saccharomyces cerevisae mainly). The use Delavante, 2004). of selected yeasts is common in large distilleries, but many small-scale production units, still use bread-making yeasts or the so called ‘fermento Cachaça caipira’, natural or wild yeast mixtures, developed in the distilleries themselves (Faria, Loyola, Lopez The extracted sugar cane juice used to produce and Dufour, 2003a). cachaça, is itself a natural must, however to achieve better fermentation and more economic results, some procedures are performed. The juice The fermentation process is first left standing in a settling tank, to separate by gravity, the broad and heavy impurities usually Rum fermentation is usually carried out in present. There is dilution with water, in order to cylindro-conical fermenters with domed tops or adjust the sugar concentration to the fermentative in cylindrical open topped vessels with sloping yeast capacity. This dilution, avoids the inhibition bases. At the end of the process there is a high of the yeast action by high alcohol levels, reducing level of suspended solids from the molasses and sugar waste and preventing bacterial fermentation the yeast cells that need be separated to prevent of the remaining sugar. wash still fouling. The fermenters and others Correction of the juice temperature (30°C) equipments design, favour the separation of and addition of sulphuric acid to lower the these solids. The fermentation temperature, in pH (4.5), and nutritive materials to support the the Caribbean islands, are maintained around fermentation, are also usually performed. In some 30–33oC by using internal coil cooling or water cases, disinfectants and antibiotics are also used jackets. The molasses fermentation as described, in the must preparation, in order to eliminate takes around 24 hours and yields 5–7% (v/v) undesirable microorganisms. alcohol, depending on the original setting gravity. The cachaça fermentations are very similar Fermentation and depending on several factors that interfere with the process, can take from 20 to 30 hours to The yeasts be completed. In both cases, stainless steel is the material of choice but fermenters made of wood, Formerly, rum and cachaça fermentation were mild steel and other materials are still used. Rum and Cachaça 329

Distillation After that, the next development was the two retort still (see Figure 1), developed in Jamaica, From comparing the composition of the sugar where a single distillation can bring the strength cane juice and molasses, it is easy to understand up to 85% (v/v) alcohol (Delevante, 2004). In that after the fermentation, the resulting products this system, a pot still first concentrates the are quite distinct. The fermented molasses fermented molasses to about 30% (v/v) alcohol, certainly require a more carefully and controlled then the distillate, is forced to pass through the distillation in order to obtain a distillate free from liquids of the low wine and high wine retorts, to undesirable compounds present. That is why the be concentrated respectively to 60% (v/v) and distilling apparatus and procedures used for rum 85% (v/v) alcohol, and finally collected in the and cachaça distillation are not the same. condenser. After collecting the foreshots, the rum, in an alcohol range that gives its desired character, is collected as the first distillate. The Pot still distillation second and third fractions (high and low wines), are then collected and pumped back to the Pot distilled rum respective retorts, for the next distillation cycle. Finally, as also shown in Figure 2, it is possible To have a distillate with good sensory to distill light rum by using column distillation. characteristics, the first approach adopted was to The use of pot stills produces a more robust and distill the fermented molasses twice, in a single or heavier rum, that requires a more prolonged in a double pot still, in order to bring the distillate maturation time (Nicol, 2003). strength to about 65% (v/v) alcohol.

Water outlet

Vapour pipe

Temperature gauge Baffle Siphon

Water Rectifier outlet Condenser

Water Steam inlet Air valve inlet Wash Water inlet To test case Baffle

Retort Wash High wines Discharge Coopered wooden pot

Figure 1. Single rum pot still (from Nicol, D.A. (2003). Rum. In: Fermented Beverage Production. 2nd edition. Edited by Lea, A.G.H. and Piggott, J.R., Kluwer Academic/Plenum Publishers, New York, U.S.A., Figure 12.3 ©2003 Kluwer Academic/Plenum Publishers; with kind permission. 330 J.B. Faria

Sugar factory

Sugar cane Raw sugar 125 tonnes/hectare 5 tonnes sugar Molasses 60% sucrose 900 litres molasses

Yeast Dilution with H2O to 16% sugar

Fermentation 48 hours Wash 8% alc

Pot Rum - 87% alc Condenser

H2O Low wine High wine Rum at High Low Pot still - heavy rum 80% alc wine wine 75% 30%

Cond Light Heads Rum/vodka Rum 95% Heads 96%

Purifier Fusel Rectifier oil sh column Wa Fusel oil Steam Steam Steam Steam

Stillage H O Stillage 2

Coffey still - light rum 3 column still - molasses spirit

Figure 2. Heavy and light rum production in the Caribbean (from Delavante, M.P. (2004). Rum–the commercial and technical aspects. In: Distilled Spirits-Tradition and Innovation. Edited by Bryce J.H. and Stewart G.G., Nottingham University Press, Nottingham, UK, Figure 1 with kind permission. Rum and Cachaça 331

Pot still cachaça or ‘cachaça de alambique’ heart and tail fractions. The heart, that contains about 80-85% of the total distilling volume, and The use of only one simple pot still, also has from 40-48% (v/v) alcohol, can if desired be called alembic and made of copper or stainless bottled and consumed (Faria et al., 2003a). steel and copper, is enough to separate and The use of two or three alembics in series concentrate the desired distilled compounds, from improves the selectivity of the distilling process, the residual components present in the sugar cane representing a step towards the column distillation wine (these compounds are very low compared to (see Figure 5). A process of double distillation, using that found in fermented molasses). The alembics even the same alembic, was proposed in order to used for the cachaça distillation may have several obtain a spirit with a lighter aroma and taste. It can distinct shapes and sizes (see Figures 3 and 4), but be further aged, a practice adopted by several small in all cases is recommended to separate the head, producers (Novaes, 1999; Bizelli, 2000) which has the more volatile compounds, from the

4

3 5 2

1

(a) (b)

Figure 3. Some variations in cachaça alembic shapes. The “elephant head” type (a) and the “hot head” type (b) alembics. 1, kettle, 2, column; 3, head; 4, head cooler; 5, condenser tube; 6, coil cooler (Ribeiro, 1997, p.36 and 37).

3

3' 4 4

2 2 5 Wine Wine 5 1 1 Distillate

Head products (a) (b) Distillate

Figure 4. Some modified alembics. a, alembic with modified head cooler and coil cooler; b, alembic with head cooler; wine preheater and coil cooler. 1, kettle; 2, column; 3, head cooler 3’, modified head cooler; 4, condenser tubes; 5, coil cooler; 5’, heating wine coil (Ribeiro, 1997, p.41 and 42). 332 J.B. Faria

characteristics very similar to the pot still rums Wine (Delavante, 2004). A two column patent still of French Savalle design, can also be employed to produce at least six distinct products, from light 1 5 2 to medium flavored rums to rectified spirits for 3 c vodka and gin production (Nicol, 2003). 2 14

6 b Column cachaça or ‘cachaça de coluna Distillate Head products 1 As mentioned, column distillation has greater d Vapor a distilling selectivity than the single alembic units, making it possible to separate the undesirable Figure 5. A semicontinuous distillation system with three alembics. 1, kettle; 2, column; 3, condenser compounds (produced by defective fermentations) tubes; 4, bored coil; 5, wine heating coil; 6, coil and resulting in lighter, standardised and cheaper cooler (Ribeiro, 1997, p.44) products. The sensory characteristics of cachaça distilled in alembics are distinct from that produced in The cachaça distilled in alembics, like the pot still colunns, and together with other regional and rum, usually has a more complex composition, technical practices, adopted all over the country, mainly due to the reactions among volatiles make possible the great range of variations, compounds that occur during the distillation easily perceived among the distillates produced process, catalysed by the copper of the stills in the 50,000 big, medium and small units, that (Cole and Nobel, 1995). make cachaça in Brazil.

Column distillation Maturation After the original Coffey still, the use of column Aging distilled spirits in small oak casks favours distillation, became an option for every kind of the development of good characteristics from drinking spirit distillation. the oak and the loss of the harsh characteristics The main advantages of column distillation are of the new distillates. In some cases, the aging or great distilling selectivity, increase in production, maturation steps are part of the overall production energy savings, decrease in undesirable sensory process, as adopted for whisky and brandy. defects and standardization of the distillate. Compared with pot stills or alembics, column distillation can produce lighter rums and cachaças, as well as neutral spirits. Rum aging

The aging process is associated to rum processing Rum column distillation and definition, being part of the organoleptic characteristics normally associated with rum. The two columns (beer/rectifier) as proposed by The rum distillates, normally collected at Coffey, is still used to produce lighter flavored between 80-94% (v/v) alcohol, are aged in rums up to 95% (v/v) alcohol, as well as a new remade American Bourbon casks, at strengths three column still, used to produce an even of 83-85% (v/v) alcohol, in temperatures varying lighter spirit. It is possible too, using a single between 27-32ºC and relative humidity from column distillation, to produce distillates with 75-90%. Rum and Cachaça 333

Distillates with different congener known as a very versatile beverage and is defined complements, for example light column and by at least 20 different international standards of heavy pot distilled rums, can be pre-blended identity. Some countries insist that rum must be before maturation. Also, it is very common after aged in small oak casks or in large oak vats, but blending to marry mature rums in casks for six the majority of the rums sold all over the world months. There are premium rums that are aged are not aged (Delavante, 2004). for 21 years or more (Nicol, 2003).

Cachaça Cachaça aging The estimated volume of Brazilian cachaça Very few spirits have as good a sensory quality production is around 1.3 billion litres a year, just after being distilled as cachaça. Perhaps representing the third biggest production that is why aging cachaça was not in the past a of distilled drinking spirit in the world. normal practice, although this process produces Unfortunately, the great part of it is consumed a significative improvement in its flavour quality in Brazil. During recent years, the volume of (Faria, Cardello, Boscolo, Isique, Odello and exported cachaça has increased, as well the Franco, 2003b). number of countries that are importing this Besides the use of remade oak casks from beverage (Faria et al., 2003a). America and Scotland, a great number of The excellence of several cachaça brands, Brazilian wood species have been tested for and the growing tendency to age cachaça, were aging cachaça. Some good results have already already responsible for the consumption of been obtained with ‘amendoin’, ‘cabreuva’, good cachaça brands, not only as ‘caipirinha’, ‘freijó’ and ‘pereiro’ (Faria et al., 2003b). For another great invention of the Brazilian people, very good reasons, it has been concluded, that but also pure, like the best known traditional the maturation of cachaça will represent the aged spirits. next great improvement in the sensory quality Improvements in the chemistry and sensory of this beverage. control of cachaça quality, the Government’s efforts to promote cachaça export, the association of smaller producers interested in exporting Packaging and marketing their products, and the growing acceptance of cachaça, all over the world, are showing the Rum great potential of this beverage, as an important Brazilian item for export. Since the beginning of rum commercial production, this beverage has been bottled or traded in casks or larger containers to be matured References in the USA, Canada or Europe. It has reached all the world markets and become a very well Bizelli L.C (2000). Influência da condução da known product. Nowadays, several islands dupla destilação nas características físico- and countries, having favorable latitudes for químicas e sensoriais da aguardente de cana. planting sugar cane and making sugar, produce Piracicaba- SP, (Dissertação de Mestrado) more than 230 bottled rum products, so varied, Escola Superior de Agricultura ‘Luiz de that they can be used for making long drinks or Queiroz’ – USP mixed beverages, as well as other special drinks Chen, J.C.P. (1995). Cane Sugar Handbook. to savour after a good meal (Nicol, 2003). Rum Wiley Interscience, New York, U.S.A does not have a geographical heritage. It is well Cole, V.C. and Nobel, A.C. (1995). Flavor 334 J.B. Faria

Chemistry and Assessment. In: Fermented Faria, J.B., Franco D. W. and Piggott J.R. Beverage Production. Edited by Lea, A.G.H. (2004). The quality challenge: cachaça and Piggott, J.R., Blackie Academic and for export in the 21st century. In: Distilled Professional, Glasgow, UK, pp. 361-381 Spirits-Tradition and Innovation. Edited by Delavante, M.P. (2004). Rum–the commercial Bryce J.H. and Stewart G.G., Nottingham and technical aspects. In: Distilled Spirits- University Press, Nottingham, UK, pp. Tradition and Innovation. Edited by Bryce 215-221 J.H. and Stewart G.G., Nottingham University Nicol, D.A. (2003). Rum. In: Fermented Press, Nottingham, UK, pp. 209-221 Beverage Production. 2nd edition. Edited Faria, J.B.; Loyola E.; Lopez, M.G. and Dufour by Lea, A.G.H. and Piggott, J.R., Kluwer J.P. (2003a) Cachaça, Pisco and Tequila. In: Academic/Plenum Publishers, New York, Fermented Beverage Production. Second U.S.A., pp. 263-287 Edition, Edited by Lea, A.G.H. and Piggott, Novaes, F.V. (1999). Processo de dupla J.R., Kluwer Academic/Plenum Publishers, destilação. In Programa de fortalecimento New York, U.S.A., pp. 335-363 do setor de aguardente de cana-de-açúcar e Faria, J.B., Cardello H.M.A.B., Boscolo, M., seus derivados do Estado do Rio de Janeiro. Isique W.D., Odello, L. and Franco, D. Sebrae, Rio de Janeiro-RJ, p. 118. (2003b). Evaluation of Brazilian woods as Ribeiro, J.C.G.M. (1997). Fabricação Artesenal an alternative to oak for cachaça aging. da Cachaça Mineira. Editora Perform, Belo European Food Research and Technology Horizonte, MG. 218: 83-87, 2003 Pálinka - Hungary's national drink 335 Chapter 48 Pálinka - Hungary’s national drink

L. Szabó Agárdi Distillery, Sreiner Tanya, Hungary

Introduction for medicinal reasons. A century later crematura was served in the days of King Matthias Corvinus. Pálinka is recognised as Hungary’s national The word pálinka, used to denote liquor distilled drink - a Hungaricum is a term used for products from fruit, wine or corn, became current in the originating from - and usually produced only in 1500s, and has been used ever since. Pálinka was - Hungary. Similar to Scotch whisky, Hungary’s served at royal banquets, genteel dinner parties national drink may only be called pálinka if it and peasant celebrations. has been distilled in Hungary. There is, however, For various reasons, pálinka has remained one more criterion for calling a distillate pálinka: famous only within the territory of the Carpathian it must be made from 100% fruit. No flavouring, basin, although its quality and unique flavour is colouring or preservatives may be added. This recognised widely: the future Edward VIII, then is widely recognised in law in accordance with heir to the English throne, is said to have claimed the regulations of the European Union. - when visiting Hungary in 1935 - that “the apricot The main characteristics of these drinks are pálinka of Kecskemét drunk with soda is better the smooth, airy and rounded tastes, with their than whisky, and with tea better than rum.” flavours and aroma harmoniously integrated so Recent decades saw a decline in quality, the that they inimitably resemble the original fruit. reasons lying largely in mass production and the lack of quality standards and strict regulations. A few years after Hungary’s transition to the free The past market, new distilleries were founded by private entrepreneurs dedicated to traditions amassed Pálinka has played an important role in Hungary’s during centuries and with the latest technology gastronomic tradition for centuries. The first to assure the highest quality. mention of ‘aqua vitae’ appeared in the 14th century at the court of King Caroberto. It should be noted that this expressive name for the fiery liquid Present has the same meaning as that of ‘uisge beatha’ - the water of life - used by Ancient Celts for the Following the regulation of the European distillate they produced. Originally it was applied Union recognizing pálinka as a true Hungarian 336 L. Szabó product in July 2002, a brand new “pálinka law” Distillation has been enacted recently by the Hungarian Government. The decree defines various types There are two methods for distilling pálinka. In of pálinka, minimum time of maturation, etc. the traditional way the crude alcohol is distilled Simultaneously with work of the official bodies, twice in large copper pot stills. Employing the both private and national institutions emerged most advanced technology, the liquid wash to unify those operating in the spirits industry. is distilled in a multi-step operation system. Among the main goals of these organizations The raw material, the geographical factors and are the implementation of quality assurance climatic conditions are all decisive factors in the systems with clear norms and rules, consumer process of pálinka making as are the skill and education, restoration of the prestige of pálinka experience of the master distiller supervising the - in short, to stand up for the mutual interests of whole process of distillation. Needless to say, the industry. it is the precision of the master distiller that is required to preserve the distinct characteristics and quality of the distillate. The main steps of pálinka manufacture

Fermentation Maturation

One of the key factors determining the quality Unlike in the case of whisky - where the Scottish of the distillate is the fruit, whose delicious and climate is extremely important, particularly distinctive floral scent and light spicy bouquet when the whisky is maturing and the soft air are transmitted to fruit-specific noble spirits. As permeates the casks and works on the whisky mentioned above, the raw material may only eliminating harsher constituents to produce be fruit fermented by the addition of yeast. The a mellow distillate - maturation is not vital in living yeast attacks the sugar in the wort and determining the quality of pálinka. However, converts it into crude alcohol. This means that many distillers mature their products in wooden the making of pálinka requires only materials barrels and/or stainless steel tanks. The barrels that are provided naturally. are kept at constant temperature and medium The fruits most widely used for pálinka air humidity. Minimum maturation time in a making are: 1,000-litre wooden barrel is 3 months, in bigger barrels 6 months. The colour of these drinks • Pome fruits like (apple, pear, quince, rowan will be derived from the casks in which they and service tree) have been matured. The alcoholic strength of • Stone fruits (apricot, cherry, plum and the mature distillate, which originates solely peach) from the alcohol obtained from the distillation • Berries (blackberry, raspberry, blueberry, of the fruit, is adjusted by adding soft water. currants) Pálinka must be bottled at a minimum 37.5% • Others (strawberry and grape). (v/v) alcohol.

Geographic and climatic factors - Hungary being situated in the temperate zone with a high Challenges number of sunshine hours and low humidity - influence flavour and bouquet to a considerable As a responsible industry the following are degree: they may generate distinctive flavours important: each year depending on the vintage. • Ensure the authenticity of distilled spirits Pálinka - Hungary's national drink 337

• Balance between tradition and innovation Notwithstanding the uniform customs regulations • The continuity of supply of high quality raw within the EU, the whole system is still far too materials complicated (and bewildering even for customs • Commitment to quality officers). The product is also heavily taxed. The • A Code of Good Practice largest organization - and the one with the most • Consumer education; commitment to social significant influence - is the National Trade responsibility Association unifying the biggest companies • Moderation messages in the industry. There are, however, several • Avoidance of drinking and driving societies of private distilleries that realised the • Measures to combat alcohol abuse need for a joint effort to preserve traditions, ensure quality assurance methods and to gain ground in the field of marketing this top quality alcoholic beverage worldwide. 338 L. Szabó Education and training in the Scotch whisky industry 343 Chapter 50 Education and training in the Scotch whisky industry

J.B. Eaton International Centre for Brewing and Distilling, Heriot-Watt University, Edinburgh, UK

Introduction • The COMAH (Control of Major Accident Hazards) Regulations for Category 1 high The Scotch whisky industry is a blend of the risk sites is forcing companies, as part traditional - with pot still distillations carried of their agreed procedures, to train their out in very much the same way as 100 or more operators and to be able to demonstrate their years ago - to the modern, with highly automated competence. large scale grain whisky distilleries and high • The downsizing of centralised functions has speed packaging plants. The training needs put a greater responsibility onto distillery of the industry are therefore diverse. Through managers for both the technical as well the Training Committee of the Scotch Whisky as the operational management of their Association, a detailed evaluation of these distilleries. requirements has been carried out and presented • The global marketplace, including the as a qualifications tree or route map covering all international ownership of previously local levels of the operation from technical expert to companies, has driven the need for greater operator. efficiency and maximised yields and the need Analysis of this tree and research of the for managers who can deliver. training marketplace has identified gaps in provision which are being filled by bespoke This paper looks at the historical background to training packages or by the training industry. distilling training, outlines how technical training One such example is the vocational training has been provided and the opportunities to package for operators introduced in April 2001 extend this to a greater worldwide audience. It by the Distilling Industry at Scottish Vocational will cover some of the historical factors affecting Qualification (SVQ) levels 1 and 2. education and training in the distilling industry, Since the Scotch whisky industry is steeped in and will also look at the recent history and what tradition, one might surmise that training would is available today. It will be mainly based on the be conducted to ensure that operations do not practice of the Scotch whisky industry but will deviate from the procedures of many centuries. conclude with a discussion on how the training So why is this not the case and why has technical is developing in order to address an international training increased in recent years? There are market. several factors driving change: 344 J.B. Eaton

Historical background required would have been manual dexterity in quickly dismantling the still, a good local The illicit distillers of the past are romanticised, knowledge and a fast pair of legs – or a four- but of course they were not illicit (illegal) until the legged accomplice – a horse. The art of distilling government, around the time of the Act of Union would still have been passed from father to with England in 1707, decided to tax excessively son, mother to daughter, but not with so much the production of spirits and to criminalise sharing for security and commercial reasons. those who chose to continue as before and A few malt distilleries in Scotland trace their not pay the unwarranted tax. Distilling must foundation to before the Excise Act of 1823, have been as commonplace and as accepted as which legalised distilling on payment of a bread making. The expertise would have been small fee - £10. There was still a lot of illicit passed from parents to children and it is open distilling at that time, with some 14,000 stills to debate whether that was more likely to have being confiscated each year but the legalisation been within the female or male line. A culture of led to a surge in legal distilleries (Figure 1). shared knowledge would have prevailed since it This was not surpassed until the 1880s when was in everybody’s interest that the best possible the Phylloxera beetle destroyed the grape vines spirit was made. If invited to dram with your of France, causing a virtual cessation of wine neighbours, it would be a harrowing experience and brandy production, and creating sudden if the dram was not up to scratch! demand for Scotch whisky (Figure 1). A further As long as it remained a cottage industry, production surge in the 1970s was due to competition was not an issue and sharing optimistic sales predictions – which proved to experience and knowledge was the way that be over-optimistic! improvement could be made. Inevitably, not Whilst the illicit distilleries were isolated everyone wanted to carry out all domestic for tax evasion, the legal distilleries, probably tasks, so the formation of “businesses” would founded at the locations of the previous illegal have evolved with payment by barter. The distilleries, were equally isolated. Father to son criminalisation of distilling led to a new set heritage transfer still prevailed and the route to of skills for the “illicit distiller”. Evasion of the promotion would have been dead man’s shoes Exciseman would have been one of the key - internal promotion from mill man to mash performance indices and a sub-set of skills man to stillman to brewer to distillery manager.

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Figure 1. Founding of Scotch malt whisky distilleries in each decade from 1740 to 2000 (partly based on data from Craig, 1994). Education and training in the Scotch whisky industry 345

Technical training would have been rare but this Drivers for training did not mean that education did not happen. For example, in the still house at William Grants, the In the distilleries, until the last ten years, taking children did their schoolwork whilst at the same examinations was not a major focus. A few time watching the stills and learning about the distillers would take the brewing examinations distilling process. of the Institute of Brewing, maltsters would The larger companies set up teams of take the Maltsters Association of Great Britain specialists and experts who would visit the Diploma and those in packaging the Institute distilleries when problems arose, for example, of Packaging Diploma, but numbers taking the technical teams at Glenochil - of whom formal examinations were low. But matters it was said – they had more doctors working have changed, so what have been the drivers there than in the whole of the National Health for change? Service! The local distillery was not expected to Competition between companies has no have technical people – they were expected just doubt played a part – with a drive for lower costs, to run the plant as set up and directed by these fewer people are now employed and the very “expert” teams. visible and costly technical centres have been There were also large distances between downsized or closed completely. the different parts of the process. Distilling, for Mobility of personnel both within companies, example, in one of the major areas on Speyside and from without, has increased and companies was separated by 200 km from the packaging have become national and international. Some plants (Figure 2). Combined with the many companies embrace both brewing and distilling years of spirit maturation, this led to little or no and opportunities for cross-fertilisation between transfer of people or knowledge between these brewing and distilling have grown. Personnel two aspects of the industry. Departments have become Human Resource

Distilling on Speyside

200 km MaturationMaturation andand packagingpackaging in in centralcentral ScotlandScotland

Figure 2. The geographical separation of distilling and packaging/maturation activities in Scotland. 346 J.B. Eaton

Departments and with it the concept of people as There are two levels but both are concerned a resource has developed- sometimes too often with distilling industry operations. Over 190 just as a resource and not as people! This has candidates have so far enrolled at Level 1 and led to a much greater “specification” of people. over 140 at level 2, and these are recognised The need has arisen to define the purpose of qualifications – they give “status” and are much employees, to set them targets against which they appreciated by operators. are appraised and to formulate training plans to fill the gaps in their abilities. On the other side of the coin, there are also greater individual General Certificate in Distilling (GCD) expectations of what a company will provide in terms of training. The General Certificate in Distilling was launched Downsizing and delivering targets has led to by the Institute of Brewing and Distilling (IBD) the discovery of the “middle manager” as a key in 2002 and was for the cereal based spirits – person for successful operation. The growing malt whisky and grain whisky. It has since been and development of their own people to better expanded to cover gin, vodka and neutral spirit, fill these roles within companies has started all spirits made from cereal substrates. These and at operator level there is the expectation examinations are aimed at plant operators and that cross-skilling will happen. Legislation are backed up by a comprehensive study book. has also played a role in driving training. The There are elective sections so that an operator COMAH legislation has required companies to in a malt distillery can answer questions related demonstrate operator competence and this has particularly to malt distillery operation and been used by companies to entice operators out similarly for grain distilling. Numbers taking of a rut and start training initiatives on the back these examinations have increased year on year, of a legal requirement. and there are currently over one hundred per year taking the examinations (Figure 3). The cereal-based examinations, however, SWA Training Committee could not address the needs of distillers using molasses and grape as substrates and hence in The Scotch Whisky Association (SWA), the response to interest, mainly from South Africa, trade body representing the majority of whisky the General Certificate in Distilling International producers in Scotland, has put in place a forum was born. Its first examination was held in for the sharing of best practice, the development November (2002) in South Africa and it was of industry specific training with links to training taken in English and Afrikaans. A great deal of providers and benchmarking against other help, initiative and support was received from industries. There is a wealth of training taking the IBD Africa Section and from the distilling place within companies and it is beyond the industry in South Africa in establishing this scope of this paper to look in detail at all this examination. training. These are examinations for plant operators predominantly and it cannot be expected that operators can gain experience outside Scottish Vocational Qualifications their workplace or country. However, it was (SVQs) cumbersome to manage two examinations and so a single examination has been developed At the operator level, the SWA and the industry to incorporate key aspects of distilling in have developed the Distilling Industry SVQs. general with options to study cereal, grape or These are recognised within both the Scottish molasses based spirits. From next year, on-line and National Qualifications frameworks. examinations could be offered. Whilst computer Education and training in the Scotch whisky industry 347

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Figure 3. Number of candidates taking the General Certificate in Distilling. marking and an instantaneous result is an option, needs to have a breadth of knowledge in line the IBD continues with the more personal with his/her status and therefore requires a approach and will mark examinations and advise working knowledge of all three substrates. candidates of their results. The Diploma in Distilling is a professional qualification and the IBD is the examining and awarding body for this qualification. There will Diploma in Distilling therefore be only one Diploma in Distilling, encompassing all three substrates which need The Diploma in Distilling is aimed at the to be mapped and accredited to the relevant Technician /Manager level and numbers taking qualifications frameworks. the examination have been increasing steadily This situation for the distilling candidate is not in recent years. The distilling examination was dissimilar to the requirement for the Diploma in based on the equivalent brewing examinations brewing candidate - they need to know about (which have been in existence since 1933) by ale production if they are a lager brewer and deleting brewing topics from the syllabus and vice versa, and a keg producer needs to know adding distilling topics. Whilst this approach about small pack production. A Diploma in successfully launched the examinations, there distilling candidate also needs a similar breadth was a residue of brewing remaining in the of knowledge. This will not diminish the distilling syllabus and the structure was not ideal achievement of present Diploma holders of the for distilling. This has recently been addressed cereal examinations and parity will be achieved with a new syllabus for distilling. However, the by maintaining a balance of breadth and depth. present Diploma in Distilling only considers Furthermore, the IBD has now developed a cereal substrates. Diploma qualification in Packaging which will The IBD is now extending these examinations be of relevance to many in the spirits industry. into the international market which requires molasses and grape substrates to be included. However, the question arose as to whether Degree level qualifications in Distilling there should be optional sections as with the GCD? The GCD examinations are vocational For the technician and manager, the International and workplace based and need to be industry Centre for Brewing and Distilling (ICBD) at specific. However, the professional distiller Heriot-Watt University offers undergraduate 348 J.B. Eaton and postgraduate degrees in distilling, as well Conclusions as brewing. Although Heriot-Watt University celebrated a centenary of teaching brewing just There has been major investment in education a few years ago (2003), a formal distilling focus and training in distilling over the last 10 years has been relatively recent (1989). However, that and this is most welcome. The IBD examinations is not to say that the University did not contribute have a lot to offer the distilling industry in terms in major ways in the past to the understanding of qualifications for operators and managers of cereals, malting, mashing and fermentation alike. However, it will need the input and which are as important in distilling as in brewing. the views of the industry in order to structure Courses are also offered by distance learning these examinations to deliver exactly what is with about 140 students presently enrolled. required and to make the examinations truly The ICBD Brewing and Distilling Degrees are international. unique, but there are several Universities around the world who offer distilling as part of other courses. Listed below are a few examples: Acknowledgements

• Stellenbosch University, South Africa Acknowledgement is given to the ICBD, and – BScAgric in Wine Production Systems. in particular Professor Graham Stewart, for his • Corvinus University of Budapest support, and also to many from the distilling – BSc Food Technology – brewing and industry for their help in putting topics in this distilling specialism. paper into context. • Technische Universitat Munchen - Weinstephan – BSc Brewing and Beverage Technology. Reference • Islamabad Higher Education Commission Craig, H.C. (1994). The Scotch Whisky Industry – BSc/MSc Food Science and Technology. Record: An Industry history Reference Book. Index Publishing, UK. Distilling is also a major part of alcohol technology courses that are available at a number of centres, and listed below are just a couple of courses available:

• Vasantdada Sugar Institute, Manjari, Pune – Diploma in Industrial Fermentation and Alcohol Technology. • National Sugar Institute, Kalyanpur, Kanpur, Uttar Pradesh – Diploma in Industrial Fermentation and Alcohol Technology. Index 349 Index

2D- Polyacrylamide Gel Electrophoresis (2D-PAGE), 94-97 Aroma wheel Acetic acid bacteria, 198, 316 Brandy, 22 Acidifying bacteria, 260 Aromatic aldehydes, 220 Acids, 220 Associação Brasileira de Bebidas (ABRABE), 170 Aging ATP synthase, 102 Rum, 165 ATPase, 102, 106 Aguardiente, 283 Authenticity indicators, 283, 284, 287 Alcohol Yield Potential (PSY), 68, 70, 71 Autophagy, 115 Alembic, 317 Cachaça production, 329 - 332 Backset, 296 Copper, 17,18 Effect on limit dextrinase, 84 Stainless steel, 17,18 Bacteria, 133, 257, 258, 316, 328 Alien, 269 Action on molasses, 176 Allergens, 277-9 Contamination, 160 Cereal, 279, 280 Spoilage, 317 Milk, 279, 280 Barley (Hordeum vulgare), 37, 81 Nuts, 279, 280 Chariot, 83 Whey, 279, 280 Decanter, 3,4, 83 Alpha-lactoglobulin, 278 Malting, 3, 47-50, 295 American bourbon, 219 Optic, 4, 78, 82 Amino acids, 111, 209 Raw material in spirit production, 299 Ammonium sulphate, 148 Transgenic, 81 Anaerobic degradation, 257 Barley koji Angelica root, 300 In Shochu production, 305 - 308 Animal feed, 265 Batch distillation, 169 Aniseed (Pimpinella anisum), 315, 323, 324 Bentonite, 248 Anisoles Beta glucanase, 83 2,3,4,6-tetrachloroanisole, 269 Biodiesel, 4 2,4,6-tribromoanisole, 269, 271, 272 Bioethanol, 4, 88 2,4,6-trichloroanisole, 269 Biofuel, 4, 89, 252, 266 Bromo-, 270-273 Biogas, 260-2 Chloro-, 270-273 Biopetrol, 266 Analysis of Variance (ANOVA), 23, 76, 171, 195 Bleaching, 271 Arabinoxylan, 64 Boiling point congeners, 220 Aroma, 205 Wood extractives, 220 350 Index

Botrytis cinerea, 311 Congeners, 222, 299 Bourbon, 297 Continuous distillation, 169, 299, 317 Barrels, 169 Controlled Activities Directive, 254 Brand equity, 282 Copper Brand protection, 281 - 288 Contact, 206, 207 Brandy, 21-28, 229-236, 319 Distillation, 22 Aroma wheel, 22 Copy DNA (cDNA), 93 Maturation, 332 Coriander seeds, 300 Brazilian wood, 179-188 Corn, 88 Amendoim, 179, 182, 185, 186 Fuel ethanol production, 145 Balsamo, 179, 182 Raw material in spirit production, 299 Canela-sassafras, 179, 182, 186, 187 Corn size Castanheira, 179, 182, 185-7 Effect on distillation potential of wheat, 67-74 Ipe, 179, 182 Counterfeiting, 281 - 288 Jatoba, 179, 182, 185, 186 Crop Evaluation Ltd (CEL), 52 Louro-canela, 179, 182, 187 Curie point pyrolysis-gas chromatography-mass spectrometry Brewers yeast, 296 (Py-GC-MS), 213 - 215 Brown spirit, 299 British Society of Plant Breeders (BSPB), 52 Department of Environment, Food and Rural Affairs Bubbing, 133 (DEFRA), 67, 73 Bureau National Interprofessionel du Cognac (BNIC), Demethylising column, 299 310, 313 Diatomaceous earth, 248, 249 By-products and effluent treatment, 255 Dielectric permittivity monitoring, 119 Differential Scanning Calorimetry (DSC), 152 Cachaça, 15,16,19, 169-179, 189-192, 197, 198, 283, Diploma in Distilling, 347 327-334 Direct fired stills, 206 Carbon Dioxide, 87-89, 123-126 Directive 2003/89/EC, 277 Indicator of fermentation performance, 213 Directive 2005/26/EC, 279 Carbonyls, 297 Distillation Cask Brandy, 235 American bourbon, 332 Cognac , 311 American oak, 298 Grappa, 317 Brazilian wood, 332 Palinka , 336 Castanheira , 16,17,18 Raki, Ouzo, 324 Charred, 219, 220, 225-227, 298 Rum, 163 Curing, 219, 224 - 227 Distilling Ex-bourbon, 219 Grain, 59 Ex-sherry, 219 Wheat, 59, 62, 64, 65 Oak, 16-18, 219, 297, 333 Dimethyl Sulphide (DMS), 220 Regeneration, 219, 224 DNA Fingerprinting, 33 Toasting, 235 Double distillation, 22 Cassia bark, 284 Dry yeast Cellulose, 90, 219, 220 Fuel ethanol production, 147 Cereals Dual purpose barley, 3 Sustainability, 1-6 Charentais pot still, 311 ELISA, 278 Cognac production, 237, 238, 240, 242 Energy recovery, 257 - 264 Charente distillation, 311 Environmental issues, 205, 207 Chlorophenols, 271 Enzymes, 90, 91 Chrysonilia sitophila, 271 Esters, 111, 119, 120, 151, 153, 155, 162, 189, 198, Climate change, 87, 251, 254 243-250, 324 Climate Change Levy, 254 Beta-phenyl acetate, 243 Chemical oxygen demand (COD), 257, 258, 260-2 Ethyl acetate, 243, 249, 250 Cognac, 151, 179, 233, 234, 237, 282, 309-314, 319 Ethyl caproate, 243 Common Agricultural Policy, 1 Isoamyl acetate, 243, 249, 250 Compounded gin, 301 Isobutyl acetate, 243 Confederation of European Spirits Producers (CESP), 287 Ethanol Production, 88 Index 351

Ethylbenzene, 216 Expended, 319 EU Green Fuels Initiative, 4 Grapes, 21 European Carbon Trading Scheme, 254 Cognac production, 309 - 311 European Confederation of Spirit Producers (CEPS), 277 Ouzo and Raki production, 323, 324 European Food Safety Authority (EFSA), 277, 278 Raw material in spirit production, 299 Extractive distillation, 299 Grappa, 315 - 322 GREEN grain project, 67, 72, 73 Feed wheat, 2 Fermentation Headspace solid phase microextraction (SPME), 221, 222 Brandy, 235 Heat shock proteins, 103, 105 Cognac, 311 Hemicellulose, 90, 151, 219 Efficiency, 123, 145 Home Grown Cereals Authority (HGCA), 52, 67, 73 Grappa, 316 Hierarchical Cluster Analysis, 169 Palinka , 336 High Gravity Performance, 123-126 Brewing, 245 Raki, Ouzo, 323 Fermentation, 129 Rum, 162 Wort, 127, 129, 131 Flavour, 205, 208, 209, 211 High performance anion exchange (HPAE), 94 Flavour congeners, 207, 209, 211 Hogshead, 225, 226 Flavour wheel, 208 Hot Water Extract (HWE), 47 Food Safety Act 1990, 281 Hydrolysable tannins, 220 Fourier-transform infrared (FT-IR) spectroscopy, 213, 214 Hydroselection, 299 Free amino nitrogen (FAN), 94, 96, 104 Hypochlorite, 272 French oak Barrels, 22 Indian herbs and spices Freshwater Fisheries Directive, 253, 256 In alcoholic beverage production, 7,9 Fruits Indirect heating, 206 Palinka production, 336 Institute of Brewing and Distilling (IBD), 346, 347 Raw material in spirit production, 299 International Centre for Brewing and Distilling Fuel ethanol, 145-150 (ICBD), 347 Fungal alpha-amylase (termamyl), 53 Irish Distillers Group, 29 Fungi, 269 - 272 Irish distilling industry, 29 to 36 Lignin-degrading, 216 Irish malt, 295 Fungicides, 271 Furaldehydes, 220 Juniper, 299, 300 Furans, 213 Berries, 299 Fusel alcohols, 111 Kilning, 81, 213 Gas chromatography (GC), 170, 283 Kjeldahl, 53 GC-MS, 220 - 222 K-nearest neighbour analysis, 169 GC-MS olfactometry, 208, 200-223 General Certificate in Distilling (GCD), 346 Labelling information, 277 Gin, 282-284, 299-304, 332 Lactic acid bacteria, 96, 111, 147 Gilbeys, 284 Lactobacilli, 148 Gordons, 284 Lactose, 278, 279 Tanquery, 284 Least Significant Difference, 195 Gin and Vodka Association of Great Britain (GVA), 287 Lignin, 219 Gluten, 278 Degradation, 219 Glycerol, 146 Limit dextrinase, 81-86 Grain, 1 London Gin, 301 Grain spirit, 51 Grain whisky, 47, 295, 297 Maillard Reaction, 176, 209, 220, 229 Production of, 75, 78 Maize, 1,2, 51, 75, 78, 295, 296 Grape Raw material for grain distillation, 59-66 Brandy, 229, 235 MALDI-TOF, 94, 95 Grape marcs, 315 Malolactic fermentation, 311 Storage, 316 Malt enzymes, 82 352 Index

Malt whisky, 47, 297 Palinka, 335-337 Definitions, 289 - 291 Palinka law, 336 Counterfeit, 292 Pearl barley, 306 Maltase (alpha-D-glucosidase), 101, 105 Peat , 213-218 Malted barley, 47-50, 295 Pentosans Malting Wheat, 59 Whisky production, 295 Phenol, 270 Malting barley, 3 Cresol, 213 Maltol, 220 Guaiacol, 213 Mash rheology, 29 Pilot scale distillery, 237, 242 Mashing Pisco, 230, 236 Whisky production, 295 Plymouth Gin, 301 Maturation Polyphenols, 24, 27 Brandy, 26, 235 Pot still, 169, 170 Cognac, 312 Potatoes Grappa, 320 Raw material in spirit production, 299 Orange liquor, 15,19,20 Potential Spirit Yield (PSY), 29, 31, 35, 47 Palinka, 336 Principal Component Analysis (PCA), 31, 33, 35, 169 Raki, Ouzo, 324 Principal component discriminant function analysis Whisky, 332 (PC-DFA), 214, 215 Messenger RNA (mRNA), 93 Product Dimension Analysis (PDA), 14 Metallised polypropylene laminates, 270 Proteinase A, 106 Methanogenic bacteria, 258, 260 Pyrolysis, 214 Methanol, 283 Microarrays, 93 Quantitative Descriptive Analysis (QDA), 140, 207, 221 Microbial contamination, 22, 141 Quality Function Deployment (QFD), 14 Microbiota, 271 Micro-organisms, 269, 271, 272 Raki , 323-5 Milk, 277-279 Rapid Visco Analyser (RVA), 33, 37-46, 60-62, 65 Millet, 59-65 Recommended Level Trials, 54 Minitab, 171 Recycling, 257 Mixed fermentation, 111 Red wine, 151 Molasses, 159, 327, 328, 330 Residual Viscosity Analysis (RVA), 60, 61, 62, 64 Fuel ethanol production, 146, 148 Rheology, 37 Raw material in spirit production, 299 Rice Mouthfeel, 205 Raw material in spirit production, 299 Rum, 8, 23, 159-168, 169-178, 327-334 NABIM, 52 Rye, 88, 295 Near Infrared (NIR), 47 Fuel ethanol production, 146 Near-infrared Reflectance (NIR) Spectroscopy, Gene, 52, 55, 56 29,30,31,35 Whiskies, 219 Near-infrared Transmittance (NIT) Spectroscopy, 30 Neutral spirit, 299 Saccharomyces cerevisiae, 161, 198, 199, 248, 249, New product development (NPD), 14 296 Nitrogen, 60-65, 67-74, 147, 213 Cachaça production, 189 Nitrogen FC9, FA1, SM102, 311 Barley, 47 S228C, 105 Non-cooked granular starch, 146 Saccharomyces ellipsoideus Nuclear Magnetic Resonance (NMR) spectroscopy, 152 Ouzo and Raki production, 324 Scotch Whisky, 127, 131, 151, 282, 295 Oak Barrel, 165, 169, 170, 312 Scotch Whisky Act, 138 Oak Cask, 151, 152, 155, 324 Scotch Whisky Association (SWA), 343, 346 Oak Lactones, 220 Scotch Whisky Association Code, 292 Oak Maturation of brandy, 21 Scotch Whisky Research Institute (SWRI), 52, 76, 277 Orange liquor, 15-19 Scottish Agricultural College (SAC), 52 Orange peel, 300 Scottish Agronomy, 52 Ouzo, 323-5 Scottish Crop Research Institute (SCRI), 76 Oxidative stress, 103 Index 353

Scottish Executive Environment and Rural Affairs Volatiles Department (SEERAD), 67 Furanol, 220 Scottish Vocational Qualifications (SVQs), 346 Guaiacol, 220 Sensory descriptive analysis (SDA), 22, 25, 26, 118 Sotolon, 220 Shochu, 305 - 308 Honkaku, 305 Wastewater treatment, 257 - 264 Korui, 305 Water Framework Directive, 254 Soil Directive, 254 Water supply, 251 Solid Phase Extraction (SPE), 171 Wheat, 3, 75, 78, 88, 295, 296 Solid State Fermentation (SSF), 90 Ambrosia, 77 Sorghum, 88 Atlanta, 77 Red, 60, 61, 65 Bread-making, 52 White, 60, 61, 65 Buchan, 54, 55 South African Brandy Board, 23 Claire, 4, 51, 54, 72 Spearman’s rank correlation, 190 Consort, 4, 51, 54, 70, 76-78 Sphagnum moss, 214 Cultivars, 67 Spirit character Deben, 70, 72, 76, 77 Green/grassy notes, 205 Dickson, 55, 77 Oily, 205 Eclipse, 54, 55 Spirit congeners, 151 Endosperm, 51 Spirit quality, 205 Ethanol production, 67 Spirit Yield Performance, 2 Fuel ethanol production, 146 Stainless steel Genetic markers, 67 Column still, 170 Glasgow, 55, 72 Strathclyde Simulated Mouth, 153, 155 Grain whisky production, 59 Stress response proteins, 103 Istabraq, 55, 70, 77 Styrene, 216 Madrigal, 54, 55 Submerged Fermentation (SMF), 90 New varieties, 67 Sugar beet, 88 Nijinsky, 55 Sugar cane Nitrogen, 51 Saccharum officinarum, 15, 27, 88, 169 Non-ruminant feeding, 67 Spirit, 189-196, 197-204 Raw material in spirit production, 299 Sulphur compounds, 110, 111, 209-211 Riband, 51, 54, 55, 76 Suma Robigus, 51,54,55 Grape origin distillate, 323, 324 Winter, 51 to 58 Wizard, 76, 78 Taints, 269 - 276 Whiskey, 37 Cork, 269, 271-3 Whisky, 23, 179, 319 Wood/paper, 269 Maturation, 332 Taste, 205 White koji fungus TCA, 272 Aspergillus kawachii, 306 Terpenes, 230-232, 236 White spirit, 299 Geraniol, 230, 231 Wild yeast, 147, 148 Linalool, 230, 231 Wood casks/ ageing, 179 Nerol, 230, 231 Oak, 179, 182 Toasting Wood maturation, 21,26,27 Barrels, 21 Wort Toluene, 216 Clarity, 243, 248 Trade Descriptions Act 1966, 281 Composition, 243 Original gravity, 243 Urban Waste Water Treatment Directive (UWWTD), 253 Sugar spectrum, 243, 244 Urea, 148 UV/Vis Spectroscopy, 283 Yeast, 88, 109-116, 117-122, 127, 133-138, 169, 243, 249, 250, 316 Vanillin, 220 Ale, 93, 120 Very High Gravity fermentation, 145, 146 Brazilian fuel ethanol, 139, 141, 142 Vodka, 8, 282-4, 299-304, 332 Brewers, 94, 109-111, 114, 115, 117, 121, 133, 135, Smirnoff, 283 139, 296 354 Index

Compressed, 134, 137 Orange liquor fermentation, 16 Cognac production, 311 Palinka production, 336 Cream, 134, 135 Pitching rate, 129, 130 Distillers’, 109, 110, 111, 112, 114, 117 Propagation, 110, 148 Dry, 119, 121, 137, 147, 235, 311 Saccharomyces bayanus, 328 Kerry, 118 Saccharomyces cerevisiae, 104, 105, 161, 198, 199, Kerry M-type, 128 , 129, 130 248, 249, 296, 328 Kerry MX, 128 , 129, 130 Safwhisky M1, 136 Lager, 93 Schizosaccharomyces pombe, 328 Mauri (M), 53, 93, 94, 98, 104, 105, 118, 141, 143, Scotch whisky fermentation, 139 248, 249 Stress factors, 128 Management, 133, 145 Wild, 147, 148 Mauri Pinnacle, 128 , 129, 130 Wine, 93 MX, 141