THE ALCOHOL

TEXTBOOK 44TH
TH
E
EDITION
DITION
A reference for the beverage, fuel and industrial alcohol industries

Edited by KA Jacques, TP Lyons and DR Kelsall

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 Foreword iii

The Alcohol Textbook

4th Edition

A reference for the beverage, fuel and industrial alcohol industries

K.A. Jacques, PhD

T.P. Lyons, PhD
D.R. Kelsall

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iv T.P. Lyons

Nottingham University Press

Manor Farm, Main Street, Thrumpton
Nottingham, NG11 0AX, United Kingdom

NOTTINGHAM

Published by Nottingham University Press (2nd Edition) 1995

Third edition published 1999
Fourth edition published 2003
© Alltech Inc 2003

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.

ISBN 1-897676-13-1

Page layout and design by Nottingham University Press, Nottingham

Printed and bound by Bath Press, Bath, England

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 Foreword v

Contents

Foreword ix
T. Pearse Lyons
Presient, Alltech Inc., Nicholasville, Kentucky, USA

Ethanol industry today

1 Ethanol around the world: rapid growth in policies, technology and production 1
T. Pearse Lyons
Alltech Inc., Nicholasville, Kentucky, USA

Raw material handling and processing

2 Grain dry milling and cooking procedures: extracting sugars in preparation for
fermentation 9
Dave R. Kelsall and T. Pearse Lyons
Alltech Inc., Nicholasville, Kentucky, USA

3 Enzymatic conversion of starch to fermentable sugars 23

Ronan F. Power
North American Biosciences Center, Alltech Inc., Nicholasville, Kentucky, USA

4 Grain handling: a critical aspect of distillery operation 33

David J. Radzanowski
Alltech Inc., Nicholasville, Kentucky, USA

Substrates for ethanol production

5 Lignocellulosics to ethanol: meeting ethanol demand in the future 41

Charles A. Abbas
Archer Daniels Midland, Decatur, Illinois, USA

6 Ethanol production from cassava 59

Nguyen T.T. Vinh
The Research Institute of Brewing, Hanoi, Vietnam

7 Whey alcohol - a viable outlet for whey? 65

Jack O’Shea
Alltech Inc., Dunboyne, County Meath, Ireland

8 Treatment and fermentation of molasses when making rum-type spirits 75

Robert Piggot
Alltech Inc., Nicholasville, Kentucky, USA

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vi T.P. Lyons

Yeast and management of fermentation

9 Understanding yeast fundamentals 85

Inge Russell
International Centre for Brewing and Distilling, School of Life Sciences,
Heriot-Watt University, Edinburgh, UK

10 Practical management of yeast: conversion of sugars to ethanol 121

Dave R. Kelsall and T. Pearse Lyons
Alltech Inc., Nicholasville, Kentucky, USA

11 Continuous fermentation in the fuel alcohol industry:

How does the technology affect yeast? 135
W. M. Ingledew
University of Saskatchewan, Saskatoon, Saskatchewan, Canada

12 Understanding near infrared spectroscopy and its applications in the distillery 145
Don Livermore1, Qian Wang 2 and Richard S. Jackson2
1
Hiram Walker & Sons Ltd., Walkerville, Ontario, Canada
2
Bruker Optics Inc., Billerica, Massachusetts, USA

13 Emerging biorefineries and biotechnological applications of nonconventional

yeast: now and in the future 171
Charles A. Abbas
Archer Daniels Midland, Decatur, Illinois, USA

Beverage alcohol production

14 Production of Scotch and Irish whiskies: their history and evolution 193
T. Pearse Lyons
Alltech Inc., Nicholasville, Kentucky, USA

15 Tequila production from agave: historical influences and contemporary processes 223
Miguel Cedeño Cruz
Tequila Herradura, S.A. de C.V. Ex-Hda San Jose del Refugio Amatitán, Jalisco, México

16 Production of heavy and light rums: fermentation and maturation 247

Robert Piggot
Alltech Inc., Nicholasville, Kentucky, USA

17 From pot stills to continuous stills: flavor modification by distillation 255

Robert Piggot
Alltech Inc., Nicholasville, Kentucky, USA

18 From liqueurs to ‘malternatives’: the art of flavoring and compounding alcohol 265
Andy Head and Becky Timmons
North American Biosciences Center, Alltech Inc., Nicholasville, Kentucky, USA

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 Foreword vii

19 Production of American whiskies: bourbon, corn, rye and Tennessee 275

Ron Ralph
Ron Ralph & Associates Inc., Louisville, Kentucky, USA

Contamination and hygiene

20 Bacterial contamination and control in ethanol production 287

N.V. Narendranath
North American Biosciences Center, Alltech Inc., Nicholasville, Kentucky, USA

21 Managing the four Ts of cleaning and sanitizing: time, temperature, titration and
turbulence 299
Jim Larson and Joe Power
North American Biosciences Center, Alltech Inc., Nicholasville, Kentucky, USA

Recovery

22 Ethanol distillation: the fundamentals 319

P. W. Madson
KATZEN International, Inc., Cincinnati, Ohio, USA

23 Development and operation of the molecular sieve: an industry standard 337

R. L. Bibb Swain
Delta-T Corporation, Williamsburg, Virginia, USA

Engineering ethanol fermentations

24 Water reuse in fuel alcohol plants: effect on fermentation

Is a ‘zero discharge’ concept attainable? 343
W. M. Ingledew
University of Saskatchewan, Saskatoon, Saskatchewan, Canada

25 Understanding energy use and energy users in contemporary ethanol plants 355
John Meredith
Ro-Tech, Inc., Louisville, Kentucky, USA

The dryhouse, co-products and the future

26 Dryhouse design: focusing on reliability and return on investment 363

John Meredith
Ro-Tech, Inc., Louisville, Kentucky, USA

27 Ethanol production and the modern livestock feed industry: a relationship

continuing to grow 377
Kate A. Jacques
North American Biosciences Center, Alltech Inc., Nicholasville, Kentucky, USA

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viii T.P. Lyons

28 Biorefineries: the versatile fermentation plants of the future 387

Karl A. Dawson
North American Biosciences Center, Alltech Inc., Nicholasville, Kentucky, USA

The Alcohol Alphabet 399

A glossary of terms used in the ethanol-producing industries

Index 441

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 Foreword ix

Foreword
Alcohol production: a traditional process changing rapidly

T. PEARSE LYONS
President, Alltech Inc., Nicholasville, Kentucky, USA

In its simplest form, alcohol production is the liters/tonne. In fact, we predict yields of 3.2
process of preparing starch- or sugar-containing gallons in the near future. For molasses, the
raw materials for fermentation by yeast, which advent of detranases can ensure maximum
is currently the only microorganism used for yields. Every aspect of cooking and fermentation
converting sugar into alcohol. The ethanol is must be controlled, however.
then concentrated and recovered in a process
called distillation. Though essentially a simple
process, making it happen with maximum The cooking process
efficiency on a very large scale is a remarkable
combination of microbiology and engineering. Much debate exists at the moment regarding the
Over the past 25 years progress has been made cooking process. It is a sad reflection perhaps
in virtually all aspects of the process, but what on our industry that since the pioneering PhD
are the challenges facing alcohol in the future? work by Dr. John Murtagh in 1972 no further
Beginning with raw material processing, here scientific thesis has been published on cooking.
are the areas that we feel need to be addressed: Should grain be finely ground? Should cooking
involve high temperature α-amylase or should
an entirely different type of enzyme be used?
Raw material reception and processing Often the enzyme selected is one developed
based on experience in the corn wet milling
Despite many declarations to the contrary, it is sector, which may be far from ideal for dry mill
virtually impossible for a distillery to accurately alcohol production.
calculate true yields. A sampling of grains is Our industry needs a new enzyme, one tailored
either done in very cursory fashion or not at all. to our specific needs: converting grain to
The distiller must see himself first as a processor fermentable sugar. Raw materials contain not
of grain rather than a producer of alcohol. He only starch, but also hemicellulose (a polymer
must know accurately how much starch is of 5-carbon sugars) and cellulose. The cooking
present in his raw material and he must be able process must be designed to loosen these
to measure that immediately. NIR spectroscopy materials such that they release bound starch and
has much to offer in terms of raw material also become available in turn for fermentation.
applications; and this topic is detailed in this
volume. However, if we control the process and
use the appropriate enzymes from solid state Yeast and fermentation
fermentation, such as Rhizozyme™ and
Allzyme™ SSF, which are capable of releasing Despite all we know about it, yeast remains
not just bound starch but also hemicellulose and underrated and misunderstood. It is the
cellulose, we can and indeed some plants do, workhorse of the distillery, yet ‘economies’ on
get 2.9+ gallons per bushel (116 gallons or 420 quality and quantity of yeast are made; and we

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x T.P. Lyons

Corn Rye
STARCH
Wheat LIQUIFACTION Potatoes
Barley Milo (sorghum)
Enzymes

DEXTRINS Cellulose

Molasses Agave
Sugarcane SUGAR Sugarbeets
Whey
CO-PRODUCTS
Yeast CO2
DDGS BIOREFINERY
Wet cake
Value-added
Solubles
PRIMARY ALCOHOL co-products

POT STILL CONTINUOUS STILL

Irish Rum Grain Grain Fuel

Whiskey Whisky Alcohol Alcohol
Malt Tequila
Whisky
Bourbon Gin Vodka
Blended Whisky
Scotch Whisky
WHITE SPIRITS

MATURATION SPIRITS

often force it to grow and operate under uncontrolled, will prevent the yeast from
unsuitable conditions. As Professor Mike performing. Operating a fermentation at 23%
Ingledew has pointed out many times, the ethanol is totally different than operating a
fermentor is not a garbage can! We must make distillery at 8% ethanol. Any minor change,
sure that conditions in fermentation are optimum whether in mycotoxin level, salt level or nutrient
for yeast. level can have a major impact on performance.
Like our need for appropriate enzymes, ethanol
production also requires yeast with higher
ethanol and temperature tolerance if we are to Microbial contamination
keep progressing to higher levels of alcohol in
the fermentor. Today, 17-20% abv is becoming A distillery is never going to be a sterile
standard in our industry as we move to high fermentation and even those of us who have had
temperature Thermosacc TM -like yeast. When the good fortune to operate in sterile fermentation
used in combination with enzymes capable of conditions know how easily infection can take
extracting increasing amounts of sugar, such as hold. Since the yeast is a relatively slow-growing
those produced by surface culture fermentation, microorganism compared to most of the
yeast can bring us to a whole new level. infectious microorganisms (Lactobacillus, etc.),
While 23% ethanol by volume in the fermentor it is critical that we have an arsenal of
without loss of performance is possible given antimicrobial agents and effective cleaning
the right yeast and enzyme combination, it can products and programs ready to protect the
only be done by careful management of the so- fermentor environment.
called stress factors. Factors which, if left

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 Foreword xi

Recovery and utilization of co-products with PhD and masters degrees, our industry has
very few. Unlike the brewing industry, which
As we review our overall process, it is obviously has no less than five brewing schools around
critical that we maximize utilization of co- the world, the annual Alltech Alcohol School
products. Assuming the feedstock is corn, the remains the only venue for industry-wide
major cost factors are the raw material, energy, training.
enzymes, processing chemical cost (water The Alltech Institute of Brewing and Distilling,
treatment, etc.) and labor. Much of these costs established in 2000, set out to address this
can be offset with 17 lbs of distillers grains shortfall by working in conjunction with the
coming from each bushel of corn; but we must Heriot-Watt University in Scotland. Perhaps our
maximize the return on this essential product. industry should use the brewing industry as a
Distillers grains with solubles (DDGS) are model. Scholarships should be funded and
currently (July 2003) selling for around $80/ton perhaps a bioscience center concept established.
and corn for $2.20 per bushel. At $0.04 per lb Bioscience centers bridge the gap between
($80/ton), 17 lbs of DDGS gives us a credit of university and industry by creating an
$0.68. This is set against the $2.20 bushel of environment where students are encouraged to
corn or the $0.03-0.04 for enzymes. If on the complete higher degrees while doing industry-
other hand, new or novel products could be focused research.
made from distillers grains, (i.e. improved by-
pass proteins for cattle, pre-hydrolyzed DDGS
for human foods), the financial impact could be Conclusions
substantial. The future will see many new
products built around DDGS. This is the subject The alcohol industry, therefore, is alive and well,
of a separate chapter in this volume. and we have been given an opportunity to
As dry mills shift toward biorefining, instead achieve something that many of us thought
of simply grinding, corn, yield of ethanol would have been achieved by the mid-1980s.
becomes only one part of the economic We have a superb oxygenate that will help
equation. Perhaps in the future, further reduce global warming and at the same time
processing of distillers grains will create products enable us to add value to our grains. The world
that make spent grains the single most important is not short of starch and sugar, the world is short
raw material from a distillery. After all, they are of protein. The fuel alcohol industry and
rich and valuable protein sources, a commodity beverage alcohol industries therefore are in fact
in very short supply across the world. generators of protein; it only remains for us to
make sure the proteins we generate are the most
advantageous possible to man and beast.
Education The chapters in this book we hope will help
the reader realize the complexity and at the same
Perhaps the biggest surprise, as one surveys the time the simplicity of the process of converting
progress that has been made in the last 20 years sugars to ethanol. If we make your search for
is how little progress has been made in the area information just a little easier, we will have
of education. Unlike the brewing industry, which achieved our objective. If we can encourage
seems to have an abundance of professionals more research, then we will be well-rewarded.

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Ethanol industry today

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 Ethanol around the world: rapid growth in policies, technology and production 1

Chapter 1

Ethanol around the world: rapid growth in policies, technology and

production

T. Pearse Lyons
President, Alltech Inc., Nicholasville, Kentucky, USA

Fuel ethanol industry history: how did

today’s ethanol ‘boom’ happen?
The amazing expansion of the past few years is
happening in a wholly different industry than
when the first ‘boom’ occurred. In the late 1970s
and early 1980s an embryonic ethanol industry
was heralded as the renewable answer to
worldwide shortages of fossil fuels. Farmers in
America’s heartland had abundant raw materials
in the form of cereal grain; and it seemed at one
stage as if every farm would have its own
‘gasohol’ plant. Small-scale operations with a
myriad of new technologies emerged with
unique names like ‘OPEC Killer’ or ‘A Step to
Independence’.
Few of the early fuel ethanol plants were really
big, as it seemed the beverage plant size was
taken as an example. Many were only capable
of producing hundreds of gallons per day, but
others were well-engineered plants with
capacities up to 15-20 million liters per annum.
Competitions were held at state fairs in order to Figure 1. The alcohol industry reference books by
enable farmers to determine which design was Alltech Inc.
best. Enthusiasm, regardless of expertise, was
the order of the day. The industry was crying By the mid-1980s, the smaller plants were
out for training and technology; and during this beginning to run into typical start-up problems,
time Alltech Inc. emerged, with its philosophy many associated with poor design, little capital,
of marketing through education. Here also and relatively high labor costs. Perhaps they
emerged the concept of the ‘Alcohol Textbook’, were doomed to fail from the beginning, with
and the annual Alcohol School, which is now in or without government support. Production was
its 23rd year.

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2 T.P. Lyons

nowhere near the forecasted 7 billion liters (1.85 UNPRECEDENTED GROWTH IN THE US
billion gallons), and instead languished around
1.5 billion liters (0.4 billion gallons). Beverage In the United States, domestic events have given
ethanol remained the dominant ethanol product. the industry a big push. The US Clean Air Act
To most, it looked like the gasohol boom was mandated an average of 2% oxygen be present
over. in what was called ‘reformulated gasoline’
While smaller operations disappeared, other (RFG). Representing some 30% of the vast US
larger companies, well funded and with well- gasoline market (100 billion gallons), it created
designed plants, continued to grow. Corporations a clear demand for an oxygenate. MTBE (methyl
such as Cargill, Staleys and ADM became tertiary butyl ether), with approximately 18%
industry leaders. Today, one of these (ADM) is oxygen, and ethanol with 30% oxygen were
responsible for processing 45% of the corn that chosen. Both were excellent oxygen boosters.
is converted into ethanol in the US. This MTBE, a petroleum derivative, was the early
company and others of this size knew the ethanol choice, but a series of scares regarding its
industry had long term prospects and they possible carcinogenic nature and its detection
planned survival strategies accordingly. in groundwater led to its planned phaseout. The
Meanwhile in Brazil during the 1970s and alcohol industry, meanwhile, was beginning to
1980s, a similar revolution was occurring. In emerge, particularly in the Corn Belt (Nebraska,
1975 the so-called ‘Proalcohol’ program was Iowa, Minnesota and Illinois). Its capacity had
launched, with massive support by the reached 10 billion liters (2.7 billion gallons) by
government. Initially, the program focused on 2003 (Figure 2) and in anticipation of the
the production of anhydrous ethanol, which was phaseout of MTBE (not just in California, but
blended with gasoline to produce the Brazilian around the country) some 73 plants had been
gasohol. The emphasis quickly changed to built. At the time we are going to press, these 73
hydrous ethanol (95% ethanol/5% water), which plants, located in 20 states, have the capacity of
could be used in its pure form in cars with 11 billion liters (Figure 3). A further 13 plants
specially designed engines. By the mid-1980s, are under construction, which will add some 2
such cars represented the vast majority of the billion liters (500 million gallons) of capacity.
marketplace and it seemed the Proalcohol The ethanol industry also continues its streak
product was going to replace gasoline on a 1:1 of monthly production records. An all-time
basis. monthly production record of 181,000 barrels
The system, however, was very inflexible. per day (b/d) was set in June of 2003, according
When ethanol became difficult to get in the late to the US Energy Information Administration
1980s, consumers began to switch back to (Figure 4). The previous all-time record was
conventional cars in which gasoline (still 179,000 b/d, set in April of this year.
blended with a minimum level of ethanol) could
be used. Was the fuel ethanol program also Driving industry growth: Farmer-owned ethanol
going to falter in Brazil? plants

Unlike the earlier plants, which were dominated

by the large corporations, most of the new plants
Growing interest in fuel ethanol around are farmer-owned projects, as farmers seek to
the world add value to the commodities they grow and
participate in profit sharing when the plant
Against such a background of declining interest prospers. These plants have a typical size of 150
in ethanol in the early 1990s, what brought about million liters and a construction cost per liter of
the resurrection of the ethanol boom in the US $0.40-$0.50. The predominant raw material is
and elsewhere? The first driving factor was an locally-grown corn (maize); and ethanol
international agreement called the Kyoto production is the third largest and fastest growing
Protocol, in which most industrial nations agreed market for US corn (Figure 5). In 2002, over
to improve the environment and lessen the 800 million bushels of corn were processed into
greenhouse effect by burning less fossil fuel or ethanol and valuable feed co-products. The US
doing so more efficiently. It was agreed to roll Department of Agriculture estimates that more
back carbon dioxide emissions to a given year. than one billion bushels of corn will be
The impact was to be enormous.

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 Ethanol around the world: rapid growth in policies, technology and production 3

2500

2000

Millions of gallons
1500

1000

500

0
80

88

90

98

00
82

86

92

96

02
84

94
19

19

19

19

20
19

19

19

19

20
19

19
Figure 2. Historic US fuel ethanol production.

Ethanol production facility

Began productionn in 2002

Under constructioon

Source: Renewable Fueels Association

Figure 3. Fuel ethanol plants in the US (source: RFA).

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4 T.P. Lyons

190

Thousands of barrels/day
180

170

160

150

140

130

120

110
Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun

Figure 4. Monthly US ethanol production, 2002-2003 (1 barrel = 42 US gallons or 159 liters).

1000

800
Millions of bushels

600

400

200

0
80

88

90

98

00
82

86

92

96

02
84

94
19

19

19

19

20
19

19

19

19

20
19

19

Figure 5. Corn utilized in US ethanol production.

processed into ethanol in 2003, or about 10% to December 31, 2003, all major refiners
of the total US harvest. Since 1999, farmer- voluntarily switched to ethanol in early 2003,
owned ethanol plants have increased their resulting in a new market for more than 600
percentage of total production capacity from million gallons. In 2004, with the MTBE ban in
20% to nearly 35%. As a whole, farmer-owned place, California’s ethanol consumption will
ethanol plants are the largest ethanol producer increase to 750-800 million gallons annually.
in the US. MTBE bans in New York and Connecticut are
also scheduled to take effect on January 1, 2004.
New marketplace opportunities These two states combined represent a market
for more than 500 million gallons of ethanol. If
Following concerns regarding water all of the northeast were to replace current MTBE
contamination with MTBE, the state of California use with ethanol, a market for more than a billion
passed landmark legislation phasing out the use gallons of ethanol would be created. In total, 17
of MTBE in the state’s motor fuel supply. While states have passed legislation to phase out MTBE
the original deadline was delayed by one year use; and the future for ethanol looks bright.

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 Ethanol around the world: rapid growth in policies, technology and production 5

Comprehensive energy legislation offers higher than 2002, we can anticipate a parallel
opportunities for ethanol increase in production of feed co-products. A
waiting market exists in the world’s animal feed
The nation’s increasing reliance on imported industry (>600 million tonnes) where
energy, particularly from unstable regions of the traditionally used sources of protein such as
world, combined with erratic natural gas and animal by-products and fish meal have been
world oil markets has prompted the either eliminated due to concern surrounding
administration and US Congress to debate Mad Cow Disease (BSE) or reduced availability
comprehensive energy policy legislation. As part and higher prices. The combined protein and
of this legislation, an historic fuels agreement energy value of ethanol by-products gives them
supported by an unprecedented coalition of tremendous potential in animal feeds across the
agriculture, oil and environmental interests world.
would provide a growing market for renewable
fuels while addressing concerns regarding
MTBE water contamination and providing BRAZIL AND SOUTH AMERICA
needed flexibility in the fuels marketplace. This
agreement would establish a Renewable Fuel The US, however, is not the only country
Standard (RFS), which would set an annual and experiencing an increase in alcohol production.
increasing minimum level of renewable fuels In Brazil, new initiatives are underway and the
such as ethanol and biodiesel permitted in the inflexible Proalcohol program has been changed.
marketplace, growing to 5 billion gallons by Flex-fuel engines, which run equally well on
2012. either pure hydrous alcohol or gasoline, have
been launched. This technology can cope with
New uses for ethanol anything from 100% hydrous ethanol to a
gasohol mix of 80% gasoline and 20%
While ethanol today is largely used as a blending anhydrous ethanol, adjusting to any change
component with gasoline to add octane and within a fraction of a second. This compares very
oxygen, opportunities abound for ethanol to favorably to the engines in the US, which would
play a role in advanced vehicle technology and normally handle up to 85% anhydrous in a blend
alternative fuels markets. For example, as a and cannot use pure hydrous ethanol.
renewable fuel with an established fueling Furthermore, since ethanol sells in Brazil for less
infrastructure in the US, ethanol is an attractive than gasoline at the pumps (50-60% of the
fuel option for the fuel cell market in both power gasoline price) even taking into account that 30%
generation and transportation. Research is also more ethanol is required to travel the same
underway on E dieselTM, a mixture of ethanol, distance, the new cars are bound to be a success
diesel and a blending agent. And there is a and will drive up ethanol demand.
largely untapped market for E85, a blend of 85% It is expected that a million cars with Flex-fuel
ethanol and 15% gasoline, to power the growing engines could be sold each year. With an average
number of available flexible fuel vehicles. 240 liters (63 gallons) of ethanol per month
consumption, this could mean an additional 3
Valuable feed co-product production increasing billion liters would be needed over Brazil’s
current 12-13 billion liters of production.
Record ethanol production means record
production of valuable feed co-products for Table 1. Projected production of ethanol (billion liters)1.
livestock. In 2002, US dry mill ethanol facilities,
2003 2005
which account for approximately 65% of ethanol
production, produced about four million short USA 10 18
tons of distillers dried grains with solubles Brazil 13 16
(DDGS). Ethanol wet mills produced European Union Unknown 8
India 1.5 1.8
approximately 450,000 US tons of corn gluten
Thailand Unknown 0.7
meal, 2.5 million tons of corn gluten feed and Australia .04 .4
germ meal, and 530 million pounds of corn oil. China Unknown 2
With estimates for 2003 production nearly 30% 1
F.O. Licht, July 2003

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6 T.P. Lyons

While few people will say exactly how much it bioethanol as a way to reduce oil imports. It was
costs to produce a liter of fuel ethanol in Brazil, estimated that 0.7 billion liters of ethanol would
it is believed to be between $0.10 and $0.15. be required should they decide to introduce the
This puts the industry in a very favorable oxygenate at 10% of gasoline. Both cassava
position to export ethanol, since a similar cost (tapioca) and molasses were suggested as raw
in the US from corn would be more like $0.20. materials and eight projects were approved. At
Fuel ethanol emergence in South America is this time only one project is under construction.
not limited to Brazil. In Peru and Colombia Two distinct areas for ethanol production exist
interest is also growing. Colombia estimates an in China: the maize-rich north and the sugar-
annual need for almost one billion liters and 12 cane rich south. Both will feature in the country’s
production plants. ethanol future. The government sees ethanol as
part of its program to reduce pollution –
important with the looming 2004 Olympics –
THE EUROPEAN UNION and at the same time encourage rural
employment. China’s largest fuel ethanol
Driven by the Kyoto Agreement, the European facility, costing almost $300 million to construct,
Commission proposed in 2001 to increase the is in Jilin. With a capacity of some 600 million
use of crop-based, non-polluting renewable liters (165 million gallons), it will use 2 million
fuels. The target was for biofuels to account for tons of maize a year (13% of production in that
2% of all fuels in EU transport by 2005. It was province). By October 2003, all vehicles in the
mandated this would increase to 5.75% by 2010 Province of Jilin will use fuel containing at least
(from 0.3% in 2001). 10% ethanol.
Both ethanol and biodiesel (from canola) are
considered biofuels. In 2003 the targets were
made voluntary, but a direction on tax relief for AUSTRALIA
renewable energy was expected to be put into
place. These would augment the existing tax Uncertainty also exists in Australia, where a
relief schemes, which vary from country to target of 350 million liters was encouraged with
country. Spain, Germany, and Sweden, for tax exemptions and grants for up to six years.
example, have 100% tax relief on renewable Local governments have tried to boost ethanol
fuels, while France has 60% and Britain 40%. consumption. For example, in Cairns,
Across Europe, from Poland to Ireland, interest Queensland’s largest city, government-owned
is rising as the new legislation begins to take vehicles were mandated to use E10 (90%
effect. gasoline, 10% ethanol). Using some 20,000 liters
per day, the government hoped in this way to
avoid some of the negative publicity being
ASIA generated by the anti-ethanol lobby.
India’s transport sector accounts for over half
of the country’s oil consumption. It is not Conclusions
surprising, therefore, that interest is growing in
replacing dependence on fossil fuels. By January Ethanol, as a global product, is experiencing a
2003, nine states had mandated at least 5% significant jump in production and production
ethanol (E5) in transport fleets. Local distilleries capacity. Whether it be used domestically or
with a capacity of some 3.2 billion liters (900 exported to areas around the world (such as
million gallons) are currently only operating at Japan, Singapore and even California) where
50% capacity. The demand for an additional 400 production is not sufficient or not possible, it is
million liters under this new mandate is therefore clear it will become a global commodity. The
immediately being met. Although some political future, therefore, is very positive; and this future
maneuvering is still needed, the fuel ethanol will not be one dependent on subsidies. A 1997
program looks sound. study by the Kellogg School of Management at
In Southeast Asia, Thailand’s government, like Northwestern University concluded the net effect
many in the region, had an early interest in

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 Ethanol around the world: rapid growth in policies, technology and production 7

of ethanol production was a reduction in the US

federal budget deficit. This was brought about
by increased tax revenues due to the increases
in personal income from wages and salaries,
higher farm income, more corporate tax revenue,
and declining unemployment compensation
payments. Alcohol as a fuel is clearly with us to
stay.

Danisco Exhibit 1010, Page 18 of 85

 Raw material handling and processing

Danisco Exhibit 1010, Page 19 of 85

 Grain dry milling and cooking procedures 9

Chapter 2

Grain dry milling and cooking procedures: extracting sugars in preparation

for fermentation

DAVE R. KELSALL AND T. PEARSE LYONS

Alltech Inc., Nicholasville, Kentucky, USA

This chapter deals with the milling and cooking facilitate subsequent penetration of water in the
stages of alcohol production from whole cereals. cooking process. The optimum size of the
In brief, in this dry milling process the whole ground particle is the subject of disagreement.
cereal, normally corn (maize), is ground in a mill Some engineers believe the particle should be
to a fine particle size and mixed with liquid, as fine as possible to allow the water maximum
usually a mixture of water and backset stillage. access for hydrolysis of starch while others
This slurry is then treated with a liquefying believe a better yield is obtained if the particles
enzyme to hydrolyze the cereal to dextrins, which are larger and the jet cooker can act on the
are a mix of oligosaccharides. The hydrolysis particles. The key is simply to expose the starch
of starch with the liquefying enzyme, called α- without grinding so fine as to cause problems in
amylase, is done above the temperature of co-product recovery. A wide variety of milling
gelatinization of the cereal by cooking the mash equipment is available to grind the whole cereal
at an appropriate temperature to break down the to a meal. Normally, most distilleries use hammer
granular structure of the starch and cause it to mills, although some may use roller mills,
gelatinize. Finally the dextrins produced in the particularly for small cereal grains.
cooking process are further hydrolyzed to
glucose in a saccharification process using the
exoenzyme glucoamylase, and possibly another HAMMER MILLS
enzyme (Rhizozyme™). These enzymes may
also be added to the yeast propagation tank or In a hammer mill, the cereal grain is fed into a
the fermentor. These separate stages of milling, grinding chamber in which a number of
cooking and saccharification will be explained hammers rotate at high speed. The mill outlet
in more detail. This is called the dry milling contains a retention screen that holds back larger
process. Most of the new distilleries use this particles until they are broken down further so
process or a minor variation of it. that there will be a known maximum particle
size in the meal. The screens are normally in the
size range of 1/8-3/16 in. (2-4 mm).
Milling Sieve analysis (particle size distribution) shows
whether the hammer mill screens are in good
The purpose of milling is to break up the cereal order and whether the mill is correctly adjusted,
grains to appropriate particle size in order to and should be conducted regularly. Table 1

Danisco Exhibit 1010, Page 20 of 85

10 D.R. Kelsall and T.P. Lyons

shows a typical sieve analysis for corn. The two outside these specifications the mill should be
largest screens retain only 11% of the particles adjusted. Normally the hammers in a hammer
while the quantity passing through the 60-mesh mill are turned every 15 days, depending on
screen is also fairly low at 7%. For efficient usage; and every 60 days a decision should be
processing of the cereal starch into alcohol, the made as to whether or not to replace the hammers
particles should be as fine as possible. However and screens.
a compromise must be made such that the
Table 2. Typical alcohol yields from various cereals.
particles are not so fine that they cause balling
in the slurry tank or problems in the by-product Cereal Yielda
recovery process. Sometimes the sieve analysis (US gallons of anhydrous
will vary depending on the severity of the shaker alcohol/bushel)
and a consistent shaking should be done each Fine grind corn1, 3/16 in. 2.85
time the test is done. Coarse grind corn1, 5/16 in. 2.65
Milo 2.60
Barley 2.50
Table 1. Typical results of a sieve analysis of corn meal. Rye 2.40
a
Screen size Hole size (in) Corn on screen (%) Note that a distiller’s bushel is always a measure of weight.
12 0.0661 3.0 It is always 56 lb, regardless of the type of grain.
1
16 0.0469 8.0 Laboratory data using Rhizozyme™ as glucoamylase
20 0.0331 36.0 source.
30 0.0234 20.0
40 0.0165 14.0
60 0.0098 12.0 Since sieve analysis is critical, a case can also
Through 60 7.0 be made for recycling to the hammer mill any
grain not ground sufficiently fine. Fineness of
the grind also has an important bearing on
Fineness of the grind can be significant factor centrifugation of the stillage post-distillation. A
in the final alcohol yield. It is possible to obtain finer grind may yield more solubles and hence
a 5-10% difference in yield between a fine and place a greater load on the evaporator. However
a coarse meal. Table 2 shows the typical alcohol since the key is to maximize yield, dry house
yield from various cereals. It can be seen that considerations, while important, cannot override
the normal yield from corn is 2.85 gallons of yield considerations.
anhydrous ethanol per bushel (56 lbs). However,
the yield with coarsely ground corn may drop
to 2.65 gallons per bushel, a reduction in yield ROLLER MILLS
of 7.5%. This is a highly significant reduction
and would have serious economic consequences Some distillers use roller mills (e.g. malt whisky
for any distiller. Grinding is about expanding producers), particularly where cereals containing
the amount of surface area. When more surface substantial quantities of husk material are used.
area is exposed, water and enzymes are better In a roller mill, the cereal is nipped as it passes
able to penetrate the kernel. A particle 1 in. in through the rollers, thus exerting a compressive
diameter has a total surface area of 6 in2. If you force. In certain cases, the rollers operate at
grind the 1 in. particle into 1,000 particles, different speeds so that a shearing force can be
surface area becomes 6,000 in2. No amount of applied. The roller surfaces are usually grooved
heating or ‘exploding’ will overcome poor grind to aid in shearing and disintegration. Figure 1
as measured by yield. The key is to expose more shows the general configuration of a roller mill.
surface area, but to do so in a controlled manner. In Scotland the solids in whisky mash, made
Since grind is so important, it is recommended entirely from malted barley, are usually removed
that a sieve analysis of the meal be done at least by using a brewery-type lauter tun, which is a
once per shift. The distiller should set vessel with a perforated bottom like a large
specifications for the percentage of particles on colander. In this case, a roller mill should be
each sieve; and when the measured quantity falls used as the shear force allows the husk to be

Danisco Exhibit 1010, Page 21 of 85

 Grain dry milling and cooking procedures 11

Mash in Reduction in viscosity. Following gelatinization,

Feed roll viscosity is reduced allowing the slurry to be
moved through lines for subsequent processing.

1st pair
Cooking is the entire process beginning with
of rolls mixing the grain meal with water (and possibly
backset stillage) through to delivery of a mash
Anti-explosion
device
ready for fermentation. Figure 2 shows the
components that make up a typical milling and
Cylindrical
cooking system. This schematic diagram could
screen
represent the processes involved in beverage,
Revolving
beater
industrial or fuel alcohol production, except that
s
sk nowadays only the whisky distillers use malt as
Hu Coarse grits
Fine grits 2nd pair a source of liquefying and saccharifying
and flour of rolls enzymes. All other alcohol producers use
microbial enzyme preparations. The fastest
enzyme systems have low calcium requirements
(2-5 ppm), lower pH optima (less buffering
required) and high temperature stability. They
To meal bin are designed to reduce viscosity, with less
Figure 1. Schematic of a roller mill. emphasis on DE. The key to cooking is to liquefy
the starch so it can be pumped to the fermentor,
hence viscosity is critical.
separated with minimal damage. The husk then
acts as the filter bed in the lauter tun for the
efficient separation of solids and liquid.
HYDROLYSIS OF STARCH TO FERMENTABLE
SUGARS
Cooking Step 1: gelatinization
The purpose of cooking is 5-fold: The purpose of cooking and saccharification is
to achieve hydrolysis of starch into fermentable
Sterilization. The mash must be sterilized so that sugars, which is accomplished by the
harmful bacteria are minimized. A brewer endoenzyme α−amylase, followed by the
achieves this by boiling mash (wort) while the exoenzyme glucoamylase (amyloglucosidase) to
distiller achieves it by cooking. release glucose. However in order for the α-
Solubilization of sugars. Sugars are solubilized amylase to gain access to the starch molecules,
to a certain point, typically 2-3% free sugars. In the granular structure of the starch must first be
this way yeast growth can occur rapidly but not broken down in the process known as
excessively as would happen if too much sugar gelatinization. When the slurry of meal and
was released at once. water are cooked, the starch granules start to
adsorb water and swell. They gradually lose their
Release of all bound sugars and dextrins (chains crystalline structure until they become large, gel-
of sugar). Extraction must occur such that filled sacs that tend to fill all of the available
subsequent enzymatic hydrolysis can ensure that space and break with agitation and abrasion.
all sugars are utilized. Starch (precursor of The peak of gelatinization is also the point of
sugars) is in large bound to protein and fiber, maximum viscosity of a mash. Figures 3, 4 and
which is are freed during cooking. 5 show the progressive gelatinization of
Protein breakdown to amino acids should be cornstarch, as viewed on a microscopic hot stage.
minimized. Amino acids and peptides can In Figure 3 the granules are quite distinct and
become bound to sugars in Maillard reactions, separate from the surrounding liquid. In Figure
which leaves sugar unavailable.

Danisco Exhibit 1010, Page 22 of 85

12 D.R. Kelsall and T.P. Lyons

Raw material

α-amylase
α-amylase (0.02%)
(0.04 - 0.06%)
(High T L120™)
(High T L120™)

Hammer mill

Water Heat Cool Liquefaction

tank 90°C
Pre-mixing tank Cooker
40 - 60°C 90 - 120°C Glucoamylase
(0.06 - 0.12%)
(Allcoholase II L400™)
or
malt (up to 10%) (beverage alcohol only)
or
Yeast food Rhizozyme™ (0.05%)
Rhizozyme™
(0.01% supplement or
0.05% total replacement)
Saccharification tank (Rarely used)
60°C
Yeast
starter tank

Fermentors
Still Mash cooler
32°C

Figure 2. Components of a milling and cooking system.

4 these same granules have swollen in size and Table 3. Gelatinization temperature ranges of various
some of the liquid has entered the granules. feedstocks.
Figure 5 shows the granules as indistinct entities Gelatinization range (°C)
in which the liquid has entered to expand them
Corn
considerably. Standard 62-72
Gelatinization temperatures vary for the High amylose 1 67->80
different cereals (Table 3). Some distillers High amylose 2 67->80
consider it important for the slurrying Barley 52-59
temperature of the meal to be below the Rye 57-70
temperature of gelatinization. This avoids Rice (polished) 68-77
coating of grain particles with an impervious Sorghum (milo) 68-77
layer of gelatinized starch that prevents the Wheat 58-64
enzymes from penetrating to the starch granules
and leads to incomplete conversion. Many Step 2. Liquefaction: hydrolysis of starch to
distillers, however, go to the other extreme and dextrins
slurry at temperatures as high as 90°C (190°F).
At these temperatures starch gelatinizes almost Liquefaction is accomplished by the action of
immediately and with adequate agitation there α−amylase enzyme on the exposed starch
is no increase in viscosity and no loss of yield. molecules. Starch exists in two forms. One form

Danisco Exhibit 1010, Page 23 of 85

 Grain dry milling and cooking procedures 13

Figure 3. Gelatinization of cornstarch. Starch granules viewed on microscope hot stage at 67°C (under normal illumination).

Figure 4. Gelatinization of cornstarch. Same granules as in Figure 3 at 75°C (under normal illumination).

is the straight-chained amylose, where the Amylopectin has a branched structure (Figure
glucose units are linked by α−1,4 glucosidic 7). It has the same α−1,4 glucosidic linkages as
linkages (Figure 6). The amylose content of corn in amylose, but also has branches connected by
is about 10% of the total starch; and the amylose α−1,6 linkages. The number of glucose units in
chain length can be up to 1,000 glucose units. amylopectin can be as high as 10,000. Corn,
The other form of starch is called amylopectin, wheat and sorghum (milo), the three most
which represents about 90% of the starch in corn. common feedstocks for ethanol production, have

Danisco Exhibit 1010, Page 24 of 85

14 D.R. Kelsall and T.P. Lyons

Figure 5. Gelatinization of cornstarch. Same granules as in Figures 3 and 4 at 85°C (under normal illumination).

Amylose
α-1,4 linkage

CH2OH CH2OH
6 6

O O
H 5 H H 5 H
4 H 1 1 4 H 1
O OH H O OH H O
3 2 3 2

H OH H OH
n

Figure 6. Structure of amylose.

similar levels of starch, but percentages of Step 3. Saccharification: release of glucose from
amylose and amylopectin differ with grain and dextrins
with variety. Starch structures are reviewed in
greater detail in the chapter by R. Power in this Saccharification is the release of the individual
volume. glucose molecules from the liquefied mixture
The α−amylase enzyme acts randomly on the of dextrins. The dextrins will be of varying chain
α−1,4 glucosidic linkages in amylose and lengths. However, the shorter the chain length
amylopectin but will not break the α−1,6 the less work remaining for the exoenzyme
linkages of amylopectin. The resulting shorter glucoamylase, which releases single glucose
straight chains (oligosaccharides) are called molecules by hydrolyzing successive α−1,4
dextrins, while the shorter branched chains are linkages beginning at the non-reducing end of
called α-limit dextrins. The mixture of dextrins the dextrin chain. Glucoamylase also hydrolyzes
is much less viscous. α−1,6 branch linkages, but at a much slower

Danisco Exhibit 1010, Page 25 of 85

 Grain dry milling and cooking procedures 15

α-1,6 linkage
Amylopectin
CH2OH
6

O
H 5 H α-1,4 linkage
4
H 1

O OH H O CH2 CH2OH
3 2 6 6

O
H OH H 5 H H 5

4
H 1 4
H
O OH H O OH
3 2 3

H OH H

α-1,6 glucosidic linkages

α-1,4 glucosidic linkages = Glucosidic units

Figure 7. Structure of amylopectin.

rate. One real problem with HPLC analysis is that there are a variety of batch and continuous
that dextrins are given as total oligosaccharides cooking systems. For a batch system there is
with no differentiation between four glucose usually only one tank, which serves as slurrying,
units and 20 glucose units. The work of the cooking and liquefaction vessel. Live steam jets
glucoamylase is directly proportional to the are typically installed in the vessel to bring the
length of the dextrin chain; and there is currently mash to boiling temperature along with cooling
no way of knowing how well the α-amylase has coils to cool the mash for liquefaction. Figure 8
worked to produce small oligosaccharides. shows a typical batch cooking system.
In Scotch whisky production, malted barley is In the batch cooking system, a weighed
used as a source of both α−amylase and the quantity of meal is mixed into the vessel with a
exoenzyme ß-amylase. The fermentable sugar known quantity of water and backset stillage.
produced is maltose, a dimer made up of two These constituents of the mash are mixed in
glucose units. simultaneously to ensure thorough mixing. The
quantity of liquid mixed with the meal will
determine the eventual alcohol content of the
Cooking systems fermented mash. When a distiller refers to a ’25
gallon beer’, it means 25 gallons of liquid per
BATCH COOKING SYSTEMS bushel of cereal. For example, for a corn
distillery with an alcohol yield of 2.5 gallons of
In considering all the different processes that absolute alcohol per bushel, the 25 gallons of
make up cooking, it should first be explained

Danisco Exhibit 1010, Page 26 of 85

16 D.R. Kelsall and T.P. Lyons

Grain

Water

Backset
α-amylase
(High T L120™)

Cooling coils

Glucoamylase + distillers yeast

Steam
(Allcoholase II L400™ + Rhizozyme™ + Thermosacc™)

To fermentors
30°- 32°C

Heat exchanger

Figure 8. Batch cooking system.

liquid would contain 2.5 gallons of alcohol. The pH range for efficient α−amylase usage
Therefore it would contain 10% alcohol by is 6.0-6.5, although enzymes with good activity
volume (abv). Using the distillery alcohol yield, at pH 5.5-5.7 are now available. Therefore, mash
the distiller can determine the quantity of cereals pH should be controlled in this range from the
and liquid to use. Most distilleries traditionally first enzyme addition until the end of
operated with beers in the 10-15% abv range, liquefaction. The glucoamylase enzyme has a
although some beverage plants run at alcohol lower pH range (4.0-5.5), so after liquefaction
levels as low as 8%. More recently fuel alcohol the pH of the mash should be adjusted with either
plants run at alcohol levels as high as 19-20% sulfuric acid or backset stillage, or a combination
by volume, the average being nearer to16-17% of the two.
by volume. The quantity of backset stillage as a percentage
In the batch cooking system, a small quantity of the total liquid varies from 10 to 50%. On
of α−amylase is added at the beginning (0.02% one hand, the backset stillage supplies nutrients
w/w of cereal) to facilitate agitation in the high essential for yeast growth. However too much
viscosity stage at gelatinization. After boiling, backset stillage can result in the oversupply of
usually for 30-60 minutes, (sometimes under a certain minerals and ions that suppress good
slight pressure), the mash is cooled to 75-90°C fermentation. Especially noteworthy are the
and the second addition of α−amylase made sodium, lactate and acetate ions. Sodium
(0.04-0.06% w/w cereal). Liquefaction then concentrations above 0.03% or lactate above
takes place, usually over a holding period of 45- 0.8% or acetate above 0.03% can inhibit yeast
90 minutes. The mash should always be growth and can slow or possibly stop the
checked at this stage to make certain that no fermentation prematurely. Overuse of backset
starch remains. Starch produces a blue or purple (or even process condensate water) must be
color with iodine. Mash should not be transferred avoided to prevent serious fermentation
from the liquefaction hold until it is ‘starch- problems.
negative’.

Danisco Exhibit 1010, Page 27 of 85

 Grain dry milling and cooking procedures 17

CONTINUOUS COOKING SYSTEMS Modern systems have 29-33% solids. From the
liquefaction chamber, the mash is pumped
Few distilleries outside of beverage plants use through a heat exchanger to fermentation.
batch cooking. Most fuel ethanol distilleries use
a continuous cooking system. In the continuous
cooking process (Figure 9) meal, water and CONTINUOUS U-TUBE COOKING SYSTEM
backset stillage are continuously fed into a
premix tank. The mash is pumped continuously The continuous U-tube cooking system (Figure
through a jet cooker, where the temperature is 10) differs from the columnar cooking system
instantly raised to 120°C. It then passes into the in that the jet cooker heats the mash to 120-
top of a vertical column. With plug flow, the 140°C prior to being transferred through a
mash moves down the column in about 20 continuous U-tube. The retention time in the U-
minutes and passes into the flash chamber for tube is only three minutes, after which it is
liquefaction at 80-90°C. High temperature- flashed into the liquefaction vessel at 80-90°C
tolerant α−amylase is added at 0.04-0.08% w/w and the enzyme is added (high temperature-
cereal to bring about liquefaction. The retention tolerant α-amylase 0.05-0.08% w/w cereal). The
time in the liquefaction/flash chamber is a residence time in the liquefaction vessel is a
minimum of 30 minutes, but should be at least minimum of 30 minutes.
60 minutes. The pH from slurrying through to The main advantage of this system is the
the liquefaction vessel must be controlled within relatively short residence period in the U-tube.
the 6.0-6.5 range. The greatest advantage of this If properly designed there is no need to add any
system is that no enzyme is needed in the α−amylase enzyme in the slurrying stage.
slurrying stage, leading to significant savings in However, because of the relatively narrow
enzyme usage. It is critical to have plug flow diameter of the tubes, some distillers add a small
through the chamber along with good enzyme amount of enzyme to the slurry tank to guarantee
dispersion. The mash should have a relatively a free flow.
low viscosity and dextrose level of 2-3%.

α-amylase
(High T L120™)
Grain Steam Vacuum
0.05 - 0.8 w/w
ejector
Water
Pressure relief
Backset
(up to 40%)

Flash chamber 80-90°C

30 min retention time
Slurry tank Liquefaction
60°C max

Glucoamylase + yeast
(Allcoholase II L400™ +
Column
Rhizozyme™+ Thermosacc™)
20 min
120°C

To fermentor
30-32°C

Heat exchanger

Figure 9. Continuous columnar cooking system.

Danisco Exhibit 1010, Page 28 of 85

18 D.R. Kelsall and T.P. Lyons

Vacuum
Grain ejector

Water

Backset Pressure α-amylase (0.05%)

(up to 40%) control (High T L120™)

Flash chamber 80-90°C

30 min retention time
Slurry tank
60°C max
Glucoamylase + yeast
(Allcoholase II L400™ +
Rhizozyme™ + Thermosacc™)

Steam
To fermentor
Retention 'U' tube 30-32°C
3 min retention
140°C Heat exchanger

Figure 10. High temperature, short time, continuous U-tube cooking system.

COMPARING COOKING SYSTEMS brought about instantaneously by a jet cooker

or ‘hydroheater’.
The relative heat requirements of the three One purpose of the cooking process is to
cooking systems can be seen in Table 4. cleave the hydrogen bonds that link the starch
Surprisingly, the batch system is the most molecules, thus breaking the granular structure
energy-efficient. Batch systems also generally and converting it to a colloidal suspension.
use less enzyme than the other systems, possibly Another factor in the breakdown of starch is the
due to the difficulty of accurate dosing and good mechanical energy put into the mash via
mixing with the continuous systems. The main agitation of the different vessels in which the
disadvantage of the batch system compared to cooking process takes place. Well-designed
the continuous system is the poor utilization or agitation is very important in a cooking system;
productivity per unit of time. The temperature- and the problem is intensified when plug flow
time sequences for the three systems shown in is also desired.
Figure 11 demonstrate how much more Mash viscosities give an indication of the
efficiently time is used in continuous systems relative ease or difficulty with which some
compared to the batch system. cereals are liquefied. Figure 12 compares
viscosity against temperature for corn and waxy
Table 4. Relative heat requirements of cooking systems.
maize (amioca) and illustrates the difference in
Batch 1 viscosity profiles.
Continuous columnar 1.18 All of the cooking systems described require
Continuous U-tube 1.37 the addition of enzymes at least for the
liquefaction stage where most of the hydrolysis
takes place. Many distilleries now use a high
In the continuous systems, the flow diagrams temperature-tolerant α−amylase. The optimum
show steam addition to raise the mash pH range for this enzyme is between 5.8 and
temperature. This temperature increase is 6.5, although it shows good stability up to pH

Danisco Exhibit 1010, Page 29 of 85

 Grain dry milling and cooking procedures 19

160 Continuous U-tube cooker

140 Continuous columnar cooker

120 Batch cooker

100
Temperature °C

Holding time

80

60

40

20

0
0 25 50 75 100 125
Time (minutes)

Figure 11. Temperature/time sequences in various types of cooking systems.

8.5 (Figure 13) while the optimum temperature

range is 88°C-93°C (Figure 14). Typically, this
100
type of enzyme would be used at between 0.04%
and 0.08% by weight of cereal. Where it is
necessary to add some α−amylase enzyme to
the slurrying vessel, the dosage rate may be
75
slightly higher.
% activity remaining

50
18
Corn
Viscosity in poises

14

10 25

Treatment time: 20 minutes

6 Calcium: 15 ppm
Waxy maize
2 (Amioca) 0
4 5 6 7 8 9 10 11 12
Held at
65 75 85 85 75 65 55 45 35 pH
95°C
Temperature (°C)
Figure 13. Effect of pH on activity of α-amylase.
Figure 12. Increasing viscosity with cooking temperature.

Danisco Exhibit 1010, Page 30 of 85

20 D.R. Kelsall and T.P. Lyons

100 to a distillery fermentor (Table 5). Rhizozyme™

has particularly found favor in SSF situations
because the enzyme can work at near its
optimum in the fermentor. Because the organism
75
used to produce Rhizozyme TM is grown on a
solid substrate (eg wheat bran), this enzyme
% activity remaining

source also contains side activities that assist in

50
releasing more carbohydrate and protein (Table
Substrate: 4% w/v soluble starch 5). These side activities work to release sugars
+ 10 ppm calcium from other structural carbohydrates besides
Treatment time: 15 minutes starch, which also releases bound starch that is
25 then available for conversion. This approach to
enzyme addition is able to increase yield to
higher levels than normally seen with single
activity liquid glucoamylase. Levels in excess
0 of 2.9 gallons/bushel have been reported; and
49 54 60 65 71 77 82 88 93 100 the target is 3.1 gallons/bushel (122 gallons or
Temperature (°C) 456 liters per tonne).

Figure 14. Activity of high temperature-tolerant α-amylase Table 5. Advantages of Rhizozyme™ over conventional
in relation to temperature. glucoamylase.

Conventional Rhizozyme™
The reaction time for enzyme-catalyzed glucoamylase
reactions is directly proportional to the
Temperature optimum, °C 60+ 30+
concentration of enzyme. Consequently, distillers pH optimum 4.0-5.5 3.5-5.0
wishing to minimize the quantity of enzyme Amylase activity,
used should design equipment to have long SKB units None 50,000
residence times to allow the reactions to be Cellulase activity
completed with minimal dosage of enzyme. CMC-ase units None 2500
Amylopectinase, AP units None 5000

Saccharification: should we have high

IF A SACCHARIFICATION STEP IS USED
levels of sugar going into the fermentor?
Mash from the liquefaction vessel is cooled,
Saccharification of distillery mashes is a usually to 60-65°C, and transferred to a
somewhat controversial subject. Over the last saccharification vessel where the glucoamylase
10 years many distillers have changed from (amyloglucosidase) enzyme is added. This
saccharifying mash in a dedicated sacchari- exoenzyme starts hydrolyzing the dextrins from
fication vessel (or ‘sacc’ tank) to adding the the non-reducing end of the molecule and
saccharifying enzyme directly to the fermentor progressively, though slowly compared to
in a process referred to as Simultaneous endoenzymes, releases glucose. The saccharifying
Saccharification and Fermentation (SSF). process is usually carried out with a residence
Saccharification in a separate vessel is still the time of between 45 and 90 minutes, but can be
practice in some distilleries, particularly for as long as 6 hrs, and the glucoamylase is added
beverage production and continuous at 0.06-0.08% by weight of cereal used. Some
fermentation. distillers measure the quantity of glucose
Another factor is use of Rhizozyme™, an produced by measuring the dextrose equivalent
enzyme produced by surface culture (solid state (DE) of the mash. A DE of 100 represents pure
fermentation). Rhizozyme™ has an optimum pH glucose, while zero represents the absence of
of 3.5-5.0 and an optimum temperature of 30- glucose. This test is rarely used nowadays, as
35°C. As such, it is a glucoamylase more suited many distillers have high performance liquid

Danisco Exhibit 1010, Page 31 of 85

 Grain dry milling and cooking procedures 21

chromatography systems (HPLC) that can demonstrates the diversity of approach possible,
measure sugars directly. Recent experience, yet points out many similarities (Table 6). It
however, shows that DE, provided it is above would appear that there are three schools of
10, is of no concern. In focusing on 23% ethanol, thought:
the key in any cooking and liquefaction process
is to liquefy, i.e., lower viscosity, so that mash a) A long liquefaction period (1-2 hrs at 90+ °C)
can be pumped through the heat exchanger to after the high temperature jet cooker and low
the fermentor (for SSF) or to the saccharification temperature slurry (no enzymes).
tank.
The functional characteristics of liquid b) A short high temperature slurry prior to a
glucoamylase prepared from the microorganism long liquefaction period (1-2 hrs at 90+ °C)
Aspergillus niger, can be seen in Figures 15 and after the high temperature jet cooker.
16. Two parameters, temperature and pH, dictate Enzymes used in slurry.
how enzymes can be used. While liquefaction c) Choice of saccharification stage or SSF.
is carried out at a pH of 6.0-6.5 and a
temperature of 90°C, this is not at all acceptable From the experience of the authors,
for saccharification. The pH must be in the 4-5 recommendations are for a short slurry at 140-
range for saccharification; and the optimum 150°F followed by jet cooking (10-15 min.) and
temperature for the glucoamylase activity is a long (1.5 hrs) liquefaction step. The mash
60°C. The mash, therefore, must be acidified should have a sugar profile similar to Table 7. It
with either sulfuric acid or backset stillage or should then be cooled to fermentation
both before addition of the glucoamylase. temperature on the way to the fermentor. This
Temperature must also be adjusted. As mentioned profile will ensure a rapid start by yeast with no
previously, normal mash saccharification sugar overload. In the presence of Rhizozyme™
temperature is 60-65°C; although for or glucoamylase, dextrins will continue to be
microbiological reasons 70-75°C would be ‘spoon-fed’ to the yeast during fermentation.
preferable. Lactobacillus can survive at 60°C; and Enzyme additions should include all the amylase
frequent infection of saccharification systems has during liquefaction and Rhizozyme™ during
caused many distillers to change to fermentation.
saccharifying in the fermentor.
Table 7. The target sugar profile following liquefaction.

IF NO SACCHARIFICATION STEP IS USED pH 4.2

Solids 29-35
If no saccharification step is planned, the Glucose, % 1.8-2.5
liquefied mash is simply cooled from 90°C Maltose, % 0.7-0.9
through a heat exchanger and transferred to the Maltotriose, % 0.2-0.3
fermentor. A portion of the liquefied mash is Lactic, % 0.07-0.08
diverted to a yeast starter tank where yeast, Glycerol, % 0.2-0.3
glucoamylase and the Rhizozyme TM is added.
Conventional glucoamylase (L400) is added at
0.06% with 0.01% Rhizozyme™ recommended The future
as a supplement. Rhizozyme™ alone can be
added at 0.05%, in which case no glucoamylase Many new enzyme systems including xylanases,
is required. hemicellulases, ligninases (esterases) and others
are being developed and the cooking system
must be flexible enough to handle them. The
Recommendations objective is to maximize the biochemical activity
of the enzyme so as to maximize alcohol yield,
Given the goals and the variables involved, which not to fit an enzyme to an existing engineering
cooking system should be chosen? A design.
comparison of systems used in four distilleries

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 Production of Scotch and Irish whiskies: their history and evolution 193

Chapter 14

Production of Scotch and Irish whiskies: their history and evolution

T. PEARSE LYONS
President, Alltech Inc., Nicholasville, Kentucky, USA

Introduction
Whisky is the potable spirit obtained by Within the blend, there may be as many as 20-
distillation of an aqueous extract of an infusion 30 individual malt whiskies and grain whiskies.
of malted barley and other cereals that has been These blends are, by law, matured for at least
fermented with strains of Saccharomyces three years but in practice this period is much
cerevisiae yeast. Various types of whisky are longer. Unblended Scotch malt whiskies are
produced in a number of different countries in usually matured for a minimum of eight years.
the world. They differ principally in the nature The cereals used in the manufacture of Scotch
and proportion of the cereals used as raw grain whisky are malted barley, together with a
materials along with malted barley, and also in high proportion (up to 90%) of wheat or corn
the type of still used for distillation. (maize). Currently wheat is the main cereal,
The principal types of whisky are also chosen on the basis of cost and the attraction of
characteristic of particular geographical regions using a Scottish-grown cereal. All whiskies are
of the world. In Scotland, the characteristic legally protected and defined, mainly because
product is manufactured using only malted of the huge revenues that governments obtain
barley as the raw material; and the fermented from their sale. The Scotch Whisky Order (1990)
malt mash is distilled in relatively small pot stills. and the Scotch Whisky Act (1988), in defining
The product, known as Scotch malt whisky, is Scotch Whisky, state that to be called Scotch
produced in small distilleries, of which there are Whisky spirits must be:
over 100 in Scotland. Scotch malt whisky is
marketed both as a straight malt whisky, many 1. produced at a distillery in Scotland
brands of which have recently become
extremely popular throughout the world, and 2. produced from water and malted barley to
also as a blend with another type of whisky which only whole grains or other cereals may
produced in Scotland known as ‘Scotch grain be added
whisky’ or (because it is distilled continuously 3. processed at that distillery into a mash
in Coffey-type patent stills) as ‘patent-still
4. converted to fermentable carbohydrate only
whisky’. Most Scotch whiskies available on the
by endogenous enzymes
international market consist of blends with 20-
30% malt whisky and 70-80% grain whisky. 5. fermented only by the addition of yeast

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194 T.P. Lyons

6. distilled to an alcohol strength less than industries producing a sufficiently acceptable

94.8% so that the distillate has the aroma and product for them to venture into the export
taste derived from the raw materials industry. Other countries, including the
Netherlands and Spain, have whisky-distilling
7. matured in Scotland in oak casks of less than
industries that cater mainly, if not exclusively,
700 litres for a minimum of three years
for home consumption. The measures that some
8. be a product that retains the color, aroma and of these industries have taken to imitate Scotch
taste derived from the raw materials become apparent when the spirits are sampled.
9. produced with no substance other than water One of the two whisky distilleries in Spain
and spirit caramel added located northwest of Madrid in the Guadarrama
Hills near Segovia produces a very acceptable
Scotch-type whisky. Its quality is attributed in
The word ‘Scotch’ in this definition is of part to the fact that the water used, which comes
geographical and not generic significance. from the surrounding hills, closely resembles that
Irish whiskey (spelled with an e) is a distinctive used in highland Scotch whisky distilleries.
product of either the Republic of Ireland or of Recommended descriptions of Scotch whisky
Northern Ireland. In the Republic of Ireland, manufacture have come from Brander (1975),
definitions were enacted by the Parliament in Daiches (1969), Dunnett (1953), Gunn (1935),
the Irish Whiskey Acts of 1950 and 1980. The Robb (1950), Ross (1970), Simpson (1968),
1950 Act distinguished pot still whiskey from Simpson et al. (1974), Ross Wilson (1959, 1970
blends and stated that the title ‘Irish Pot-Still and 1973) and Philip (1989). There are a few
Whiskey’ was reserved solely for spirits distilled smaller books describing individual distilleries
in pot stills in the Republic from a mash of cereal in Scotland. Recommended are those by
grains normally grown in that country and McDowell (1975) and Wilson (1973; 1975).
saccharified by a diastase of malted barley. The Recommended reading on Irish whiskey
1980 legislation specified that the term ‘Irish includes an account by McGuire (1973) and
Whiskey’ only applied to ‘spirits distilled in the short review articles by Court and Bowers (1970)
Republic or Northern Ireland from a mash of and Lyons (1974).
cereals saccharified by the diastase of malt
contained therein, with or without other natural
diastases’. This meant that unlike Scotch, Irish History of whisky production
whiskey may be produced with the use of
microbial enzyme preparations in addition to The origins of the art of distilling potable spirits
malt. The 1980 Act also specified that the are obscure, and probably date back to ancient
whiskey must be aged for at least three years in China. However, the first treatise on distilling
Ireland in wooden barrels. While not possessing was written by the French chemist Arnold de
the ‘smoky’ taste and aroma of Scotch, Irish Villeneuve around 1310. The potency of distilled
whiskey is usually more flavorful and has a spirits caused many to be known as the ‘water
heavier body than Scotch. Moreover, the of life’, a description that survives today in such
whiskey is distilled not twice, as in Scotland, names as eau de vie for French brandy and
but three times to give a very strong spirit of 86o akvavit and aquavit for spirits in Northern
GL compared with the 71o GL whisky distilled Europe. The name ‘whisky’ is a corruption of
in Scotland. ‘uisgebaugh’, the Gaelic word for water of life.
The tremendous popularity of whiskies Uisge was corrupted first into ‘usky’, which
manufactured in Scotland, Ireland, the US and finally became whisky after several centuries.
Canada has prompted several other countries to Dr. Johnson sang the praises of this potable spirit,
try manufacturing whiskies, usually ones although in his Dictionary of 1755 it is listed
designed to resemble Scotch. Indeed, the number under ‘u’ and not ‘w’.
of countries with minor but nevertheless Much to the chagrin of the Scotsman, it is
significant whisky-distilling industries must now likely that the first whisky was distilled not in
be well over a dozen. Some countries, notably Scotland but in Ireland. The spirit was known in
Australia and Japan, have whisky-distilling Ireland when that land was invaded by the

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 Production of Scotch and Irish whiskies: their history and evolution 195

English in 1170. In all likelihood the art of riots occurred. The English, meanwhile, had
distillation was imported into Scotland by cultivated a taste for French brandy, there being
missionary monks from Ireland. Two of today’s very little whisky consumed at that time outside
main centers of Scotch distilling, namely the of Scotland. However around 1690 William III
island of Islay and the Speyside town of began to wage commercial war against the
Dufftown, were the sites of early monastic French, and imposed punitive taxes on imports
communities. of French brandy into England. The English
Whisky, principally Scotch whisky, has for reacted by acquiring a taste for gin, which was
many years been one of the most popular distilled locally. The scale of drunkenness that
distilled beverages in the world; and it was in developed with the popularity of gin had to be
Scotland rather than in Ireland that its qualities controlled by law; and the Acts of 1736 and 1713
came to be extensively appreciated. This has levied high taxes on gin manufacturers. Both of
continued to the present day and in the these acts contained clauses exempting Scotland,
intervening period many Scotsmen have felt but not for long. The Parliament in London saw
compelled to record for posterity their thoughts the prospect of a rich harvest of taxes in the
and inspirations on the potable spirit. There are distilleries of Scotland; and in a series of acts
numerous histories of whisky distilling in starting in 1751, production of whisky in
Scotland, some more comprehensive than others. Scotland was increasingly subjected to taxation.
For good general accounts, the reader is referred The outcome of these punitive measures was
to the texts by Brander (1975), Daiches (1969), not surprising. An extensive and thriving
Ross (1970) and Ross Wilson (1970). business in illicit distillation of whisky grew up
Whisky distilling flourished in Scotland not in Scotland as described by Sillett (1965).
least because consuming the spirit helped the Curiously, illicit production of Scotch hardly
inhabitants to withstand the climatic rigors of extended over the border into England, although
this northern region of Britain. The first recorded there are a few records of the operation of illicit
evidence of whisky production in Scotland is stills in the Cheviot Hills west of Newcastle-
an entry in the Exchequeur Rolls for the year upon-Tyne. Following the Act of 1823, which
1494. It reads “To Friar John Cor, by order of introduced much stiffer penalties for illicit
the King, to make aquavitae, eight bolls of malt”. distillation, and to some extent because of the
Production of whisky was therefore being increased standards of living in northern
controlled; and an Act of 1597 decreed that only Scotland, illicit manufacture of whisky declined.
earls, lords, barons and gentlemen could distill Indeed, many erstwhile illicit distillers emerged
for their own use. To many Scots of this era to become legal and registered distillers of
whisky was a medicine, and in 1506 King James Scotch whisky.
IV of Scotland had granted a monopoly for In 1826, Robert Stein of the Kilbagie distillery
manufacture of ‘aqua vitae’ to the Guild of in Clackmannanshire, Scotland, patented a
Barber Surgeons in the City of Edinburgh. continuously operating still for whisky
Taxation on whisky production first appeared production. However, this invention was
in the 17th century. Breaches of the monopoly superseded in 1830 with the introduction by
regulations and the need to raise money to send Aeneas Coffey of an improved version of this
an army into England to help the English type of still. The appearance of continuous stills
Parliament in its war against Charles I led to the sparked off a period of turmoil in the Scotch
Act of 1644, which fixed a duty of two shillings whisky industry, it being claimed that the product
and eight pence Scot’s on a pint of whisky (the from the continuous distillation of a mash that
Scot’s pint was then about 1.5 litres). But the contained unmalted grain (described as neutral
tax was short-lived and was replaced by a malt or ‘silent spirit’) could not be called whisky,
tax that later was also repealed. since it had not been distilled in the traditional
At the time of the Treaty of Union between pot still. The battle was waged for about three
Scotland and England in 1707 there was a tax quarters of a century; and in 1908 a Royal
on malt in England, but not in Scotland. The Commission decided that malt whisky and grain
English were irate; and in 1725 when Lord neutral spirit, when blended, could be labeled
Walpole’s administration decided to enforce the whisky.
tax in Scotland, the first of a series of Malt Tax

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196 T.P. Lyons

The major factors which have affected the is entitled to be called a highland malt whisky,
development of the whisky distilling industry while whiskies distilled in areas south of the line
in Scotland in this century have been economic. are designated as lowland whiskies. Of the 104
The industry has had to endure the privations of malt whisky distilleries in Scotland, 95 are
two world wars, the economic depression in highland malt whisky distilleries. Of these, no
Great Britain during the 1920s and prohibition fewer than 49 are situated in an area measuring
in the United States from 1920 to 1933, which 50 miles east to west and 20 miles southwards
greatly affected export of Scotch to North from the Moray Firth. This area of Speyside has
America. Since 1945, however, the industry in been called the ‘Kingdom of Malt Whisky’
Scotland has consolidated and expanded. The (Cameron Taylor, 1970). Classification of the
20th century has also witnessed a considerable four whiskies distilled on the islands of Jura,
improvement in the quality of whisky distilled Orkney and Skye is disputed. Some authorities
and blended in Scotland as a result of the list them along with the Islay whiskies as ‘island’
acceptance of blending malt whiskies with grain whiskies, others as highland whiskies, which is
whisky and the amalgamation of several smaller geographically correct. There are also eight grain
distilleries into combines. whisky distilleries in Scotland.
Scotch malt whiskies can be divided into Whiskey distilling in Ireland was, as has been
‘highland’, ‘lowland’, ‘Islay’ and ‘Campbeltown noted, first recorded in the 12th century. By
whiskies’ (Simpson et al., 1974). The ‘highland 1556, it had become sufficiently widespread to
line’, which separates the areas in Scotland in warrant legislation to control it. A statute
which the first two types of spirit are distilled, is proclaimed that year stated that a license was
a straight line which runs from Dundee in the required to manufacture the spirit, but that peers,
east to Greenock in the west (Figure 1). It then gentlemen owning property worth £10 or more
extends southwards, below the Isle of Arran. and borough freemen were exempt (McGuire,
Any whisky produced north of this line, 1973). Taxation of whisky distilling gradually
including those from Campbeltown and Islay, became more excessive and collection of taxes

Highland whisky region

D
Lowland whisky region

Northh Sea

ENGLA
LAND
AND

Figure 1. Highland and lowland whisky-producing regions of Scotland.

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 Production of Scotch and Irish whiskies: their history and evolution 197

became increasingly efficient. However, in 1779 Outline of whisky production processes

there was an important change in the distillery
laws. An attempt was made to limit the extent of Whiskies differ basically in the nature and
evasion of spirit duty by prescribing a minimum proportion of the cereals used as raw materials
revenue to be exacted from the owner of each and on the type of still used in the distillation
still. The effect of this legislation was dramatic. process. These differences in the production
In 1779, there was said to be 1,152 registered process are illustrated in the flow diagram in
stills in Ireland. By 1790, this number had fallen Figure 2 for production of Scotch malt whisky
to 216 and this inevitably fostered widespread (production of Irish whiskey is very similar).
illicit distilling (McGuire, 1973). This legislation Detailed accounts of each of the unit processes
lasted until 1823 when it was replaced by laws in whisky production are given in subsequent
that taxed Irish whiskey on the volume of sections of this chapter.
production, legislation that is essentially still in A characteristic of Scotch malt whisky is that
force today. Development of the Irish whiskey the only cereal used in its manufacture is malted
distilling industry in the present century has barley (Table 1). After milling, the meal is
inevitably been influenced by economic mashed in a mash tun (Figure 2) similar to that
circumstances and by the political division of used in breweries for beer production. During
Ireland into the Republic and Northern Ireland mashing or conversion, enzymes in the malt
that occurred in 1922. Barnard (1887) described catalyze the hydrolysis of starch into fermentable
visits to 28 distilleries in Ireland, but closures sugars. In the manufacture of Scotch grain
and amalgamations followed such that when whisky and Irish whiskey, other cereals are used
McGuire (1973) prepared his account, there along with malted barley to provide additional
were only two whiskey-distilling companies in starch in the mash tun (Table 1). Owing to the
Ireland, one with distilleries in Dublin and Cork high gelatinization temperature of their starches,
in the Republic and the other with plants in unmalted cereals must be precooked before they
Bushmills and Coleraine in Northern Ireland. are incorporated into the mash.
These two companies have since amalgamated The wort, or clear mash, leaving the mash tun
and have concentrated their distillery operations is cooled and fed into a vessel where it is mixed
in Cork and Bushmills. There has also been a with yeast. In Scotland and Ireland these
move towards production of a lighter Scotch- fermentation vessels have a relatively small
type whisky in Ireland to replace the heavier capacity and are known as ‘washbacks’ (Figure
traditional Irish whiskey. 2).

Table 1. Raw materials and unit processes used in the production of Scotch and Irish whiskies.

Scotch malt Scotch grain Irish

Raw materials Peated and unpeated Wheat or corn and a small Unmalted barley and unpeated,
malted barley proportion of malted barley malted barley
Conversion Infusion mash Mash cook followed by Infusion mash
conversion stand
Fermentation Distillers yeast and Distillers yeast Distillers yeast
brewers yeast
Distillation Two pot stills Continuous still Three pot stills
o o
Maturation At 62 GL in used charred Up to 67 GL in used charred At 70o GL in used charred oak
oak bourbon whiskey oak bourbon whisky barrels bourbon whisky barrels or sherry
barrels or sherry casks or sherry casks for at least casks for at least three years
for at least three years three years

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198 T.P. Lyons

Fermentation is conducted with strains of the species Hordeum vulgare L. and Hordeum
yeast Saccharomyces cerevisiae that are usually distichon (Hough et al., 1971). Malted barley is
specially propagated for the purpose, although employed as a source of enzymes (principally
Scotch malt whisky distillers may use some amylolytic) that catalyze the hydrolysis of
surplus brewers yeast (Table 1). The process is starches and in some instances serves as a source
allowed to proceed to a point at which the of starch that is converted ultimately into ethanol.
specific gravity of the fermented mash has These two demands must be finely balanced. In
usually dropped to below 1.000. In pot still the manufacture of Scotch malt whisky, where
distilleries, the fermented mash or ‘wash’ (beer) only malted barley is used, care must be taken
is fed directly to a still known as the wash still, when the grain is sprouting during the malting
from which the distillates are redistilled in the process to ensure that only a limited amount of
second or low wines still. In Ireland, and in one enzyme activity is produced. This is because
Scotch malt whisky distillery, a third distillation enzyme is produced at the expense of the
is carried out. Finally, the freshly distilled whisky fermentable materials in the grain (referred to
is stored in charred oak barrels for minimum as ‘extract’). However in the manufacture of
periods of time that depend on the legislation in other types of whisky, malted barley is often
the producing country (Table 1). Scotch malt and used as the only source of amylolytic enzyme
Irish whiskies are customarily matured for much in a mash bill that contains a high proportion of
longer than the legal minimum period. unmalted grain. In this type of whisky production
the enzyme activity of the malted barley must
be greater than that used in Scotch malt whisky
Individual operations manufacture.
Traditionally, barley used in the production of
RAW MATERIALS
Scotch malt whisky was malted on the distillery
Malted cereals premises using a floor malting system and dried
over fires of coke and peat in the pagoda-shaped
Malted barley is the principal malted cereal used kilns which are still a feature of these distilleries.
in whisky production. Like the brewer, the To a large extent, this system has been
whisky distiller uses barley cultivars of the superseded by mechanical maltings, which
Yeast

Malt
storage Hot
bins water Condensers

Washback
(fermenter)
Spirit or middle run
Foreshot and feints
(Head & tails)

Malt
mill

Mashing
machine
(mixer)

Maturing warehouse
Wash still Low wines
Heat still
exchanger (pot still)

Low
wines Oak casks
(barrels)

Draft Mash tub

(grain (cooker)
solids) Spirit receiver
Feints and low
wines
Wort

Figure 2. Flow diagram showing the principal operations during production of Scotch malt whisky.

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 Production of Scotch and Irish whiskies: their history and evolution 199

produce malt for groups of distilleries. In order have a nitrogen content of 1.8% or higher
not to destroy the enzyme activity developed (compared with a brewer’s malted barley with a
during malting, a balance must be achieved in nitrogen content in the region of 1.5%) (Hough
the kiln between drying the green malt to a et al., 1971). Malting aids, such as gibberellic
suitably low moisture level for storage, curing acid and bromate, are not normally used in
to give it the appropriate flavor and retaining production of malted barley for whisky
sufficient enzyme activity (Simpson, 1968). In manufacture.
maltings attached to the distillery, the kiln Because of the high cost of malted cereals,
temperature is increased slowly over a 48 hr considerable effort has been expended by the
period to achieve an even rate of drying and the whisky distiller in attempting to devise methods
desired flavor. The latter character is achieved that allow prediction of the yield of alcohol
by fuelling the furnace with peat during the early expected using different proportions of malted
part of the kilning period when the green malt is barley in the mash bill. Unfortunately, the
moist and readily absorbs the peat smoke or methods customarily used by the brewer, such
‘reek’. In mechanical maltings the green malt is as those recommended in Great Britain by the
dried at a faster rate with a forced-air draught, Institute of Brewing Analysis Committee (1975),
but a supplementary peat-fired kiln is often used have proved of limited value. The brewer has
to produce flavored malts. The amount of peat used measurements of diastatic power,
used varies with different maltings. Some of the expressed as the Lintner value, which is a
distilleries on Islay in Scotland specialize in measure of the extent of saccharification of
producing a whisky with a very pronounced peat soluble starch present in a cold water extract of
flavor, and they therefore use heavily-peated the malt (Lloyd Hind, 1948) as an indicator of
malts. Malted barleys used in the manufacture malt quality. Diastatic activity measured in this
of Scotch grain and Irish whiskies are not dried way includes contributions from both α- and ß-
over a peat fire. They generally have a greater amylases. However, Preece and his colleague
enzyme activity, so the relatively small (Preece, 1947; 1948; Preece and Shadak-
proportion of malted barley used in the mash sharaswamy, 1949 a,b,c) have shown that high
contains sufficient enzyme activity to convert ß-amylase activity, as determined by the Lintner
all of the starch in the mash (principally supplied value, is not always accompanied by high α-
by unmalted cereals) into fermentable sugars. amylase activity. Pyke (1965) showed that the
The greater enzyme activity in these malted Lintner value of a malt is only useful for
barleys is reflected in their nitrogen content. predicting the spirit yield in manufacture of
Malts used in production of Scotch grain whisky Scotch grain whisky when the proportion of

375
Spirit yield (Liters absolute
per tonne of grain)

350

325

300
0 10 20 30
Malt enzyme activity (°Lintner)
Figure 3. Relationship between the diastatic activity of a Scotch grain whisky mash bill and spirit yield. Laboratory fermentations
were conducted using mash bills containing different proportions of malt mixed with corn, and therefore with different Lintner
values. It can be seen that only when the proportion of malt is rate-limiting can spirit yield be correlated with the Lintner value
(Pyke, 1965).

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200 T.P. Lyons

malted barley in the mashbill is low (Figure 3). chromatography (GLC). The peat smoke oil was
Determination of α-amylase activities of the malt more complex, and no fewer than 30 peaks,
gave a less satisfactory correlation than the some of them created by more than one
Lintner value, an observation which agrees with compound, were obtained by GLC. The
that made earlier by Thorne et al. (1945). Further compounds included hydrocarbons, furfural
evidence for the unsuitability of employing derivatives, benzene derivatives and phenols.
traditional malt specifications for predicting The authors stressed that using their GLC
performance in whisky manufacture has come techniques, 3,5-xylenol is masked by the peaks
from Griffin (1972). of m-ethylphenol and ρ-ethylphenol, two
Although the degree to which a malted barley compounds thought to make an important
has been peated can to some extent be assessed contribution to peat aroma and taste. Figure 4
by smell, such is the importance of this character shows gas liquid chromatograms of extracts of
in malt that it must be determined in a more a peated and a unpeated malt.
rigorous fashion. Peat smoke or ‘reek’ contains
a wide range of compounds, but it is generally Unmalted cereals
held that the peaty character is imparted to the
malt largely as a result of absorption of phenols. Fewer problems are encountered in arriving at
For some years, Scotch distillers used a method specifications for the unmalted cereals used in
based on a reaction of phenols with diazotized whisky manufacture, namely wheat, corn, rye
sulphanilic acid. A lack of specificity in this and barley. The corn (varieties of Zea mays) used
method, coupled with the instability of the in mash bills for manufacturing Scotch grain
diazonium salt, prompted MacFarlane (1968) to whiskies is usually of the yellow dent type,
recommend an alternative method involving generally obtained from France. Occasionally
extraction of phenols from malt with diethylether white corn is used, and it is reputed to give a
under acid conditions with absorptiometric higher alcohol yield. Corn is a popular grain
measurement of the color developed when the because it has a high content of starch that is
phenols are reacted with 4-aminophenazone. readily gelatinized and converted into
MacFarlane applied the method to a wide variety fermentable sugars. The US has imposed controls
of malts, both peated and unpeated, and reported on the quality of corn used for whisky
values ranging from zero (for an unpeated grain) manufacture. In the US there are three grades of
to as high as 9.4 ppm for a malt produced on corn, with only grades 1 and 2 being approved
Islay in Scotland. (It has been calculated that to for spirit manufacture. In Great Britain, on the
obtain a malt with 10 ppm phenols, one tonne other hand, the corn used is normally grade 3
of peat must be used for drying each tonne of on the US scale.
malted barley). Unmalted barley used in manufacture of Irish
Scotch whisky distillers have been concerned whiskey has a quality intermediate to that used
by the possibility that colorimetric methods, such for malting and that used for cattle feed. In this
as those recommended by MacFarlane (1968) way, the maltster can select the best barley
and Kleber and Hurns (1972), may not assay all available on the market at the time of purchase.
of the organoleptically-important compounds For many years, a small percentage (about 5%
that malt acquires as a result of peating. To of the total) of unmalted oats (Avena spp.) was
examine this possibility, MacFarlane et al. included in mash bills for manufacturing Irish
(1973) produced peat smoke condensate on a whiskey. It was contended that these grains, with
laboratory scale and separated the oil and their large husks, improved the texture of the
aqueous phases from the wax fraction, as only grain bed in the mash tun (to assist in straining
the two former would contain components that off the clear wort from the mash solids) and that
might appear in the distilled whisky. Six oats influenced the flavor of Irish whiskey.
compounds, namely furfural, 5-methylfurfural, Whether either or both of these effects were
guiacol, phenol, ρ-cresol and 5-xylenol, were important will probably never be known, for oats
detected in the aqueous fraction by gas liquid are no longer used in the production of Irish
whiskey (Court and Bowers, 1970).

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 Production of Scotch and Irish whiskies: their history and evolution 201

Unpeated malt

ρ-Cresol + Phenol + Guaicaol

ρ-Ethylphenol

Phenol + ρ-Cresol + Guaicaol

Peated malt
ρ-Ethylphenol

ρ-Cresol

Figure 4. Gas-liquid chromatograms of extracts of unpeated and peated Scoth barley malts showing the contribution peating
makes to the content of phenols in the malt. Peating leads to an increase in the size of the peak corresponding to phenol, ρ-cresol
and guiacol, and of the peaks which lie to the right of the mixed phenol peak (attributable to furfurals and hydrocarbons). ρ-cresol
is also detectable on the chromatogram of extracts of peated malt. The ratio of the area of the total phenol peaks to that of ρ-
ethylphenol is used as an indication of the peatiness of the malt.

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202 T.P. Lyons

MASHING AND COOKING The meal from overhead bins is mixed with hot
water at 60-65oC in the proportion of one part
Mash production in Scotch and Irish distilleries meal to four parts water, and the mash is
involves a process not unlike that used in homogenized by action of revolving rakes. In
breweries to prepare wort for beer manufacture. the traditional mashing process, the mash is
However, where cereals other than malted barley usually loaded into the tun to a depth of about
are used, the malt mashing is preceded by a high one meter and allowed to stand for about one
temperature cooking process. hour, after which the wort is drained off from
under the grain bed. This liquid extract, which
Mashing has a specific gravity (SG) of 1.070-1.060
Regardless of whether the mash bill contains (Figure 5) is collected in an intermediate vessel
cereals other than malted barley, the main known as an ‘underback’. After being cooled to
biochemical changes that take place during around 25oC in a heat exchanger, the wort is
mashing are hydrolysis of starch, protein and pumped into the fermentation vessel. The bed
other biopolymers in the meal to produce water of grains in the tun is then re-suspended in water
soluble low molecular weight compounds that at 75oC and a second batch of wort is drawn off
form a fermentable substrate (or wort). The major at a specific gravity of around 1.030 and passed
starch-liquefying and saccharifying enzymes are into the underback. This process is known as
α- and ß-amylases, while the limit dextrins the first aftermash and is repeated twice more;
formed by action of amylases on amylopectin except that the dilute worts drawn off are not
are further hydrolyzed by limit dextrinases. passed to the underback, but are returned to the
Barley malt used in the manufacture of Scotch hot water tank to be used in the next mash. The
malt whisky is coarsely ground in a roller mill wort in the underback has a pH value of about
adjusted to give a grind no finer than the malt 5.5, a specific gravity of 1.045-1.065, and an
warrants. Too fine a grind can give rise to a ‘set amino nitrogen content of about 150-180 mg/
mash’ that settles on the bottom of the mash tun L. The spent grain residue or ‘draff’ is removed
to block and impede drainage of the liquid from the mash tun and sold as animal feed. Many
(Simpson, 1968). The mash tun is usually distilleries have now reduced this process to just
preheated with water and the perforated bottom three water additions. Larger distilleries are now
plates are flooded before mashing commences. installing semi-lauter tuns in place of the
traditional mash tuns. These have vertical knives,

Malt meal

First Second Third

Mash
aftermash aftermash aftermash

Wort Weak wort Weak wort Weak wort

(SG 1.060-1.070) (SG about 1.030) (SG about 1.010) (SG about 1.006)

Fermentation Weak wort for next mash

Figure 5. Flow diagram showing the mashing cycle in a Scotch malt whisky distillery.

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 Production of Scotch and Irish whiskies: their history and evolution 203

enabling the structure of the mash bed to be loss of sugar and decreased spirit yield. Pyke
maintained whilst speeding wort draining, and (1965) has provided an account of the cooking
sparging rings to allow simultaneous draining of corn in its production of Scotch grain whisky
and sparging. The result is a faster more efficient and a similar process is still used today. The
extraction without loss of clarity or changing the conventional cooker is a horizontal cylindrical
composition of the wort. vessel capable of working at pressures up to 90
Mash preparation in the manufacture of Irish psi (63 x 104 Pa), and fitted with stirring gear. A
whiskey is very similar to that used in Scotch typical cycle in cooking corn might consist of
production, but there are certain differences. Use 1-1.5 hrs of heating with live steam injection to
of a high percentage of raw barley in the mash reach a temperature of 120oC and a pressure of
bill (up to 60%) has necessitated the use of stone 15 x 104 Pa. The mash is held at this temperature
mills or hammer mills to achieve the required and pressure for a further 1.5 hrs. The liquid
grind. The unmalted barley is sprayed with water used for the mash is often that from the third
to give a moisture content of about 14% and aftermash mentioned earlier. At the end of the
then dried to around 4.5% moisture before cooking time, the pressure is released and the
grinding. The malted barley used is roller-milled. hot cooked corn mash is blown directly into a
In recent years a plant has been installed in saccharification vessel containing the malted
Ireland which uses a wet milling process that barley suspended in hot water. Cold water is then
eliminates the need for watering and drying of added to bring the temperature in the mash tun
the unmalted barley. Mash tuns used in Irish to around 63oC. Good mixing is also essential
whiskey distilleries differ from those used to at this stage; and failure to achieve it can lead to
make Scotch in that they are larger, simply the entire mash solidifying. The need for rapid
because these distilleries have stills with a greater cooling of the corn mash was emphasized by
capacity than those in Scotland. (This difference Murtagh (1970), who showed that with slow
dates back to when there was a flat rate of tax cooling or holding at a temperature around 82oC
per still in Ireland, rather than a tax per gallon lipid-carbohydrate complexes could be formed
of product.) Moreover, the mashing cycle differs that are not fermentable and can lead to a loss
in that the weak wort from the first aftermash is of 1-2% in spirit yield.
not passed to the underback, but is mixed with After a suitable mashing period, the mash may
worts from the second and third aftermashes to be filtered as described previously. More
be used in the subsequent mash (Lyons, 1974). commonly, the entire mash may be pumped via
a heat exchanger into a fermentor. Pyke (1965)
has published the composition of a typical
Cooking followed by malt mashing Scotch grain whisky mash (Table 2).
In the manufacture of Scotch grain whisky, the
The wheat, corn and rye used in production of proportion of malted barley in the mash bill is
Scotch grain whisky must be cooked before usually around 10-15%, a proportion far greater
being added to the mash containing malted than that required merely to provide a source of
barley in order that the starch in these grains amylolytic enzymes. It is a practice of long
can become gelatinized and accessible to the standing, and is done to obtain the required malt
malt enzymes. Traditionally, cooking was carried flavor in the grain whisky. This was noted as
out as a batch process, but it has to some extent early as 1902 by Schidrowitz and prompted
been superseded by continuous processes. further comment from Valaer in 1940.
For batch cooking, the grain is freed from Continuous cooking has been adopted in
extraneous material by passage through screens recent years in Scotland. Stark (1954) listed the
and then ground either in a pin-type mill or a advantages of using continuous cooking. While
hammer mill. It is then conveyed to the cooker practices vary to some extent in different
and subjected to a cooking cycle involving high distilleries, essentially the cereal is slurried at
temperatures and pressures designed to bring around 50oC and then pumped through a steam-
about complete gelatinization of the starch. jet heater into the cooking tubes where the
Inadequate cooking will sometimes leave starch residence time is normally 5-10 minutes. The
granules intact in the mash, while excessive continuous cooker has a narrow tube (6-16 cm
heating can cause caramelization and therefore

Danisco Exhibit 1010, Page 43 of 85

204 T.P. Lyons

in diameter) which reduces the incidence of fermentors were made of wood, usually of larch
charring and carbon deposition. The mash or Oregon pine, but in recent years they have
passes through the tube at about 20 meters/ been constructed of steel or aluminum. In some
minute, with the temperature reaching near 65oC Scottish distilleries, timber is still used as a
and the pressure 65 psi (40 x 104 Pa). covering material.
When the fermentor is partly filled, the mash
Table 2. Chemical composition of a typical scotch grain is inoculated with a suspension of
mash*. Saccharomyces cerevisiae. The source of yeast
varies with the location and size of the distillery.
% Distilleries in Scotland and Ireland, particularly
Total soluble carbohydrate (as glucose) 9.00
the pot whisky distilleries, rarely have their own
Insoluble solids 2.20 yeast propagation equipment. They rely on
Fructose 0.13 specially-propagated pressed yeast for use in
Glucose 0.29 fermentations. In Scotch malt whisky distilleries,
Sucrose 0.28 this pressed yeast is augmented, usually on an
Maltose 4.65 equal weight basis, with surplus brewery yeast
Maltotriose 0.96 typically supplied in compressed form from
Maltotetraose 0.45 breweries that may be as far as 500 km from the
Dextrin 2.54 distillery.
Amino nitrogen (as leucine) 0.09
Ash 0.27
The requirements for a strain of Saccharo-
containing P2O5 0.09 myces cerevisiae yeast used in distillery practice
K 2O 0.09 have been described by Harrison and Graham
MgO 0.02 (1970). Apart from the obvious need to select a
strain that maintains very high viability in the
µg/ml
Thiamin 0.46
pressed state (containing 25% dry weight), other
Pyridoxine 0.61 very important properties of these strains are
Biotin 0.01 ability to tolerate concentrations of ethanol of
Inositol 236 the order of 12-15% (v/v) and the capacity to
Niacin 11.1 metabolize oligosaccharides such as maltotriose
Pantothenate 0.71 and maltotetraose in order to maximize the
conversion of starch into ethanol and carbon
*Pyke, 1965. dioxide.
Whisky worts usually have a specific gravity
in the range 1.050-1.080, a pH value of around
Fermentation 5.0, a total acid content of 0.1% and an optical
rotation of +30 o. After inoculation, the yeast
The objectives in fermenting a whisky distillery content is 5-20 million cells/ml. The bacterial
mash with strains of Saccharomyces cerevisiae count varies with the cleanliness of the plant and
are to convert the mash sugars to ethanol and the extent to which the raw materials were
carbon dioxide while producing quantitatively endowed with a microbial flora. Scotch grain
minor amounts of other organic compounds whisky fermentations create little if any foam
which contribute to the organoleptic qualities of because of their large content of suspended
the final distilled product. Fermenting vessels solids. However, in most Scotch malt whisky
vary considerably in volume, depending on the fermentations only a small proportion of the
distillery. The small Scotch whisky distillery at suspended solids in the mash is retained in the
Edradour near Pitlochry, for example, has fermentation vessel. These fermentations tend
fermentors with a capacity of only 4,500 liters. to foam and the distillers have resorted to the
In contrast, other Scotch and Irish pot whiskey use of various types of antifoams.
distillers have fermentors with a capacity in the Changes over the time course of a typical
range 50,000 to 150,000 liters. Much larger fermentation in a Scotch malt whisky distillery
fermentors are found in Scotch grain whisky are depicted in Figure 6. Fermentation proceeds
distilleries. Traditionally, the smaller pot distillery vigorously for the first 30 hrs, during which time

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 Production of Scotch and Irish whiskies: their history and evolution 205

1.060
5.0 +30 0.30
1.050 pH
4.5 +25 0.25

Optical rotation (a °D)

Specific gravity

Total acid (%)

1.040
4.0 +20 0.20
1.030 pH 3.5 +15 0.15
Acidity
1.020
3.0 +10 0.10
1.010
2.5 Specific Optical rotation +5 0.05
0 gravity
2.0 0 0
0.995
0 10 20 30 40 50

Fermentation time (h)

Figure 6. Changes in specific gravity, optical rotation, pH value and acidity during fermentation of a mash
in the production of Scotch malt whisky (Dolan, 1976).

the specific gravity falls to 1.000 or below and (Pyke, 1965). Over the first 30 hrs the pH value,
the optical rotation to around zero. The sugars after declining to around 4.2, rises to about 4.5.
in the wort are utilized in a particular sequence During the first 30 hrs the specific gravity drops
with glucose and fructose being fermented first, at a rate of about 0.5o per hour accompanied by
followed by maltose and then maltotriose. The a massive evolution of heat. While many of the
removal of sugars during fermentation of a larger grain whisky distilleries have fermentors
Scotch grain whisky mash is shown in Figure 7 fitted with cooling coils, these are absent, or if

50
Concentration of fermentable sugar (g/l)

Maltose

30

Maltotriose

10
Glucose

0
10 30 50
Fermentation time (hrs)

Figure 7. Time course removal of fermentable sugars from a Scotch grain whisky mash (Pyke, 1965).

Danisco Exhibit 1010, Page 45 of 85

206 T.P. Lyons

fitted are relatively inefficient in most malt During the first 30 hrs or so of malt whisky
whisky distilleries where temperature can rise fermentation there is vigorous fermentation and
by the end of the fermentation to as high as 35- the majority of the aerobic bacteria die. This,
37 o C. The distiller is concerned about the however, provides ideal conditions for growth
temperature rise during fermentation since this of anaerobic or microaerophilic bacteria,
can cause the fermentation to stop or become principally lactic acid bacteria (mainly strains
‘stuck’. Temperature rise can be controlled by of Lactobacillus brevis, L. fermenti and
using a lower starting temperature or, because Streptococcus lactis) with the result that the
glycolysis of sugar is a heat-producing process, concentration of lactic acid in the fermented mash
by using a lower initial concentration. Strains of can be as high as 30 mg/L (MacKenzie and
Saccharomyces cerevisiae are well suited for Kenny, 1965). A wide range of lactobacillus
malt whisky distillery fermentations since they species have been identified in Scotch whisky
can ferment efficiently over a wide temperature fermentations including L. fermentum, L. brevis,
range. Fermentation is usually continued for at L. delbrueckii, L. plantarum, L. casei and a
least 36 hrs and frequently longer, at which time bacterium resembling L. collinoides, in addition
the ethanol content of the wash is 7-11% (v/v). to Leuconostoc spp., Streptococcus lactis and
In larger distilleries, particularly those in the US, Pediococcus cerevisiae (Bryan-Jones, 1976).
the carbon dioxide evolved is collected, liquefied More recently Barbour (1983) isolated many
and sold. Smaller distilleries, particularly the malt species that did not conform to recognized
whisky distilleries in Scotland, usually do not species of lactic acid bacteria, a point
have this facility. emphasized by Walker et al. (1990) who used
It should be noted that mashes in malt whisky DNA hybridization techniques to classify
distilleries are not boiled, so any enzyme activity distillery bacteria. Growth of lactic acid bacteria
manifested at the temperature of the mash and is probably enhanced by yeast excretion of
any microorganisms that can survive at that nitrogenous nutrients at the end of a vigorous
temperature will continue to be active during the fermentation. Kulka (1953) demonstrated the
fermentation. The continued activity of limit ideal nature of yeast autolysate for growth of
dextrinases in unboiled distillery mashes lactobacilli. Bacterial activity in the fermenting
increases the concentration of sugars available wort also leads to removal of some acids.
for fermentation by the yeast. Hopkins and Actively growing yeasts secrete citric and malic
Wiener (1955) calculated that with amylases acids, but MacKenzie and Kenny (1965)
alone the yeast cannot metabolize the equivalent attribute the lower concentrations of these acids
of the final 12-16% of the starch. in malt distillery worts (as compared to brewery
Another important consequence of using non- worts) to their partial removal by bacteria.
sterile conditions in distillery fermentations is Occasionally, the extent of the bacterial flora
the activity of bacteria that pass through in the in the fermenting wort can become too large.
mash, which are encouraged to some extent by This causes problems due to sugar utilization
the relatively high temperatures to which the by the bacteria that lead to an overall decrease
fermentations can rise. In addition to lactic acid in spirit yield. In addition, the bacteria may
bacteria, the flora can include other Gram- produce organoleptically-undesirable
positive as well as Gram-negative strains. The compounds and also release hydrogen ions
concentration of the flora depends on a number causing the pH value of the wort to fall too low,
of factors including the extent to which the lactic thereby providing suboptimal conditions for
acid bacteria grew during yeast propagation, the action of certain enzymes. Examples of
extent of the flora on the cereal raw materials undesirable compounds that may be excreted
and on the standard of hygiene in the distillery. by bacteria are hydrogen sulfide and other
There is no doubt, however, that the controlled sulfur-containing compounds (Anderson et al.,
activity of this bacterial flora, and particularly 1972). Lactobacilli can also metabolize glycerol
of the lactic acid bacteria, is accompanied by (excreted by the yeast during fermentation) to
excretion of compounds that contribute to the produce ß-hydroxypropionaldehyde, which
organoleptic qualities of the final whisky subsequently breaks down on distillation to give
(Geddes and Riffkin, 1989). acrolein (Harrison and Graham, 1970). Acrolein

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 Production of Scotch and Irish whiskies: their history and evolution 207

imparts a pungent, burnt and often peppery odor hrs and a lower optical rotation of the mash after
to the whisky (Lyons, 1974). In a later paper, about 40 hrs. In the fermentations there is often
Dolan (1976) concentrated on the problems a difference of up to 4 hrs from the time a rise in
arising in malt whisky distilleries when there is the acid content is detected to the point when
an unacceptably high concentration of bacteria the pH value of the fermentation begins to fall.
in the mash. Table 3 shows changes in the Dolan (1976) attributes this to the buffering
concentrations of Gram-negative and Gram- capacity of the mash. The data in Table 4 show
positive bacteria and (separately) of lactobacilli the effect of different levels of infection after 30
during fermentation of a minimally-infected hrs fermentation of a malt distillery mash on
mash and of a heavily-infected mash. The time spirit yield and the associated financial losses to
course of fermentation of an unacceptably- the distiller. Dolan (1976) recommends upper
infected malt distillery mash (Figure 8) shows, limits of 1,500 bacteria, 50 Gram-positive and
in comparison with similar data for fermentation 10 lactic acid-producing bacteria per million
of an acceptable mash (Figure 6), a greater rise yeast cells in the mash at the start of fermentation.
in the acid content of the mash after about 35

Table 3. Changes in the concentration of bacteria during fermentation of a minimally infected and a heavily infected
Scotch malt whisky wort.*

Minimally-infected wort Bacteria/ml Heavily-infected wort

Age Gram- Gram-negative Lactobacilli Gram-positive Gram-positive Lactobacilli
(hours) negative rods cocci rods cocci

At setting 2,000 3,700 n.d. 52,000 60 0.6 x 106

10 150 n.d. n.d. n.d. n.d. 2.3 x 106
20 n.d.** n.d. 1.53 x 106 n.d. n.d. 18.8 x 106
30 n.d. n.d. 10.2 x 106 n.d. n.d. 96 x 106
40 n.d. n.d. 10.2 x 106 n.d. n.d. 502 x 106
50 n.d. n.d. 50 x 106 n.d. n.d. 1 x 109

*Dolan, 1976.
**None detected.

1.060 0.30
5.0 +30
Optical rotation (a °D)

1.050 0.25
4.5 +25
Specific gravity

pH
Total acid (%)

1.040 0.20
4.0 +20
1.030 0.15
pH

3.5 Acidity +15

1.020 0.10
3.0 +10
1.010 Specific Optical rotation 0.05
2.5 +5
gravity
0 2.0 0
0
0.995 -2.5
0 10 20 30 40 50

Fermentation time (h)

Figure 8. Changes in specific gravity, optical rotation, pH value and acidity during fermentation of a Scotch malt whisky mash
containing an unacceptably high concentration of bacterial infection (Dolan, 1976).

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208 T.P. Lyons

Table 4. Effect of the level of bacterial infection on the loss of spirit incurred following fermentation of a Scotch malt
whisky mash.*

Infection Bacteria/ml in 30 hr old Approximate loss in Approximate financial loss in

rating mash (million) spirit yield (%) US dollars (thousands)

At filling Duty paid

a 0-1 <1 <26 <369
b 1 - 10 1- 3 26 - 79 369 - 1106
c 10 - 100 3- 5 79 - 131 1106 - 1844
d >100 >5 >131 >1844

*Dolan, 1976.

Much less has been published on the effect of major higher alcohols was increased in the
retaining solid material in the fermenting mash. presence of solids, the effect being particularly
However, marine microbiologists have long noticeable with isobutanol and 2-methylbutanol.
known that the presence of solid particles in a The effect of low insoluble solids content is a
liquid medium can affect bacterial growth, factor relevant to congener levels in malt whisky
probably because of the concentration of fermentations. In grain whisky production,
nutrients at the solid-liquid interface (Heukele- where ‘all-grains-in’ fermentations are generally
kian and Heller, 1940; Zobell, 1943). Moreover, used, the degree of rectification during
Cromwell and Guymon (1963) found that form- distillation is the principle determinant of higher
ation of higher alcohols during fermentation of alcohol levels in the spirit.
grape juice is stimulated by the presence of grape
skins or inert solids. Beech (1972) made similar
observations on cider fermentations. Merritt DISTILLATION
(1967), in the only detailed report on the role of
solids in whisky distillery fermentations, states Whether the fermented beer is distilled in a pot
that a dry solid concentration of 50 mg/100 ml still, as in production of Scotch malt and Irish
might typically be expected, although much will whiskies, or in a continuous still based on the
clearly depend on the design of the mash tuns Coffey design as in the manufacture of Scotch
used in individual distilleries. Merritt went on to grain whiskies, the objectives are the same:
report that a concentration of dry solids as low selective removal of the volatile compounds
as 5 mg/100 ml causes an increase in yeast (particularly the flavor-producing congeners)
growth, and that solids also enhance the rate of from the non-volatile compounds and to create
production of ethanol and glycerol. There was additional flavor-producing compounds as a
also an effect on production of higher alcohols result of chemical reactions that take place in
by the yeast (Table 5). With the possible the still. Nevertheless, it is still most convenient
exception of n-propanol, production of all of the to discuss whisky distillation under the separate
headings of pot still and continuous distillation.

Table 5. Production of higher alcohols (mg/100 ml).

Insoluble solids
content 2-methyl butanol 3-Methyl butanol
Mash (mg/100 ml) n-Propanol Isobutanol (amyl alcohol) (isoamyl alcohol) Total

1 0 1.5 4.2 3.0 8.0 16.7

25 1.5 6.5 4.4 8.8 21.2
2 0 2.8 4.5 2.7 9.5 19.5
35 3.3 6.6 3.7 10.1 23.7
3 0 1.7 4.8 3.1 8.0 17.6
50 1.7 6.8 4.6 9.5 22.6

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 Production of Scotch and Irish whiskies: their history and evolution 209

Pot still distillation condenser. In Irish pot stills this purifier function
is effected by a trough fitted around the lyne
The copper pot still, which is the feature arm through which running water is circulated.
dominating any Scotch malt or Irish whiskey Pot stills in Scotch and Irish distilleries are
distillery, has changed hardly at all over the traditionally constructed of copper. The reason
centuries, except of course in size. Traditionally, for this adherence to copper is more than
the onion-shaped stills were fired from beneath tradition. It has been established that copper
and had the vapor pipe or ‘lyne arm’ from the fixes some volatile sulfur-containing
still projecting through the distillery wall to compounds that are produced during
connect with a condenser in the form of a coil fermentation but undesirable in the distilled
immersed in a water tank fed from a local stream spirit.
(Figure 9). Internal steam-heated calandria are Early whisky distillers realized that although
now preferred to direct firing because this the objective of distilling was to separate volatile
decreases the extent of pyrolysis of the still constituents from the beer, collecting the
contents and results, for example, in a lower distillate not as a whole but in several fractions
concentration of furfural in the whisky. Variations and combining certain of these fractions gave a
in still design include expansion of the surface much more acceptable product. Pot still
area of the column into a bulbous shape, water distillation in Scotland and Ireland differ not only
jacketing and return loops from the first stage in the size of the stills (25,000-50,000 liters in
of the condensation. (Nettleton (1913) provided Scotland vs 100,000-150,000 liters in Ireland),
a valuable account of early still design). In many but also in the different ways in which fractions
distilleries, condenser coils have been replaced are collected from the stills.
by tubular condensers that have an advantage In Scotland, the beer is subjected to two
in that they are designed to conserve the heat distillations. In the first, carried out in the beer
extracted from the distillates. Yet other pot stills still, the beer is brought to a boil over a period
are fitted with ‘purifiers,’ which consist of a of 5-6 hrs and the distillate is referred to as low
circular vessel cooled by running water wines. This distillation effects a three-fold
interposed between the neck of the still and the

Lyne arm

Lyne arm tank

Condensate
Rousing return pipe
gear Worm
rub

Heating
coils

Product
Pot still

Figure 9. Diagram of an Irish distillery pot still. Designs used in Scotch malt whisky distilleries are similar except that the pot is
onion-shaped and the still usually has a shorter lyne arm not surrounded by a lyne-arm tank (Lyons, 1974).

Danisco Exhibit 1010, Page 49 of 85

210 T.P. Lyons

concentration of the alcohol in the beer (Figure whisky distilled over in the middle fraction has
10). The residue in the wash still, known as ‘pot an alcohol content of 63-70o GL.
ale’, is either discharged to waste or evaporated The manufacture of Irish whiskey involves
to produce an animal feed (Rae, 1967). three rather than two distillations (Figure 11).
Distillation of the low wines in the spirit is still The fermented beer is heated in the wash still,
more selective. The first fraction, which contains and the first distillation (‘strong low wines’) is
low boiling point compounds, is rejected as ‘fore- collected until the distillate reaches a
shot heads’. At a stage determined by continued predetermined specific gravity. The distillate,
hydrometric monitoring, which usually occurs then known as ‘weak low wines’, is switched
when the distillate has an alcohol content of into a separate vessel. The weak low wines are
approximately 70-73 o GL, the distillate is pumped into the low wines still and are re-
switched from the fore shots tank to the whisky distilled to produce two fractions termed ‘strong
receiver tank. This switch has traditionally been feints’ and ‘weak feints’. Strong feints are mixed
made at the discretion of the distiller; and he with the strong low wines in a spirit still.
has been aided by the disappearance of a bluish Distillates from this are collected in the same
tinge when water is added to the distillate. The fashion as in production of Scotch. The whiskey
monitoring process takes place in a spirit safe collected usually is about 89-92o GL.
secured with a Government Excise lock.
Collection of the distillate is terminated when Continuous distillation
the alcohol content has fallen to a specified value,
although distillation of the feints or tails is No fundamental changes have been introduced
continued until all of the alcohol has been into the design of the patent or Coffey still over
removed from the low wines. The residue the past century. Automation, particularly of the
remaining in the spirit still is known as ‘spent beer feed, is now commonplace, as is continuous
lees’, and like pot ale is either run to waste or monitoring of other stages in the distillation
evaporated to manufacture animal feed. The process. Nevertheless, many Scotch and Irish

Fermented beer
(about 8°GL)

Pot Ale Wash Still

Low Wines Still

(about 24°GL)

Spent Lees Spirit Still

Recycle

Recycle

Foreshots Whiskey Feints

(heads) (about 63-70°GL) (tails)

Figure 10. Flow diagram showing the stages in distillation of Scotch malt whisky.

Danisco Exhibit 1010, Page 50 of 85

 Production of Scotch and Irish whiskies: their history and evolution 211

Fermented beer
(about 8°GL)

Wash Still

Strong low wines Weak low wines

(tails)

Low Wines Still

Strong Feints Weak Feints

(tails)
Spirit Still

Heads Whiskey Feints

(about 89-92°GL) (tails)

Figure 11. Flow diagram showing the stages in distillation of Irish whiskey.

producers of grain whiskies continue to use a about by storing the whisky in oak barrels for
still which, like the original Coffey still, has just periods of time that depend on traditional
two columns: a beer stripper (or analyzer) and a practice and legal requirements. In general,
rectifier. whiskies are matured for far longer than the
A description of the operation of two column legally-required period of time. The raw spirit is
continuous stills in the manufacture of Scotch taken by pipeline from the distillation plant to
grain whisky has come from Pyke (1965). In the tank house where it is diluted with water to
order to obtain whisky of high quality from these the required strength and then transferred into
stills, they must be operated such that the alcohol barrels.
concentration of the spirit at the spirit draw tray Maturation in barrels is accompanied by a loss
in the rectifier is not less than 94.17o GL. The of liquid by evaporation, and the relative rates
manner in which the precise control of still of loss of water and of alcohol determine
operation can affect the composition of the whether the aged whisky has a higher or lower
whisky is shown in Figure 12. As illustrated, if alcoholic strength than that at filling. In Scotland,
conditions are changed in either direction on the where the barrels of whisky are stored in cool,
abscissa, the concentration of congeners will unheated, but humid warehouses, the alcoholic
alter with a possible adverse effect on final strength decreases (Valaer, 1940). In contrast
product quality. Valaer and Frazier (1936) reported that in the
US storage conditions cause an increase in
alcoholic strength. Maturation in barrels is also
MATURATION AND AGING accompanied by changes in the chemical
composition of the whisky. These changes are
Freshly distilled whisky of any type is very attributable to extraction of wood constituents
different from the spirit that is later bottled, either from the barrel, oxidation of components present
singly or blended. The transformation is brought in the original whisky as well as those extracted

Danisco Exhibit 1010, Page 51 of 85

212 T.P. Lyons

102

Amyl alcohol 100

Isobutanol

10 90

Ethanol 80

Ethanol concentration (%w/w)

70

1 60

50
Composition (% w/w)

n-Butanol 40

10-1 30

n-Propanol 20
Total esters
10
Total aldehydes
0
10-2

Total acids

10-3 Furfural

10-4
0 5 10 15 20 25 30

Tray number

Figure 12. Changes in composition of the vapor at different trays in a Coffey still rectifier used in the manufacture
of Scotch grain whisky (Pyke, 1965).

Danisco Exhibit 1010, Page 52 of 85

 Production of Scotch and Irish whiskies: their history and evolution 213

from the wood, reactions between components desired value by adding caramel. Some
in the whisky and removal and oxidation of brownish pigment is extracted from the casks,
highly volatile sulfur components by the carbon but this may not be sufficient to provide the
char on the inner surface of the barrel. desired color. Finally, the whisky is clarified for
Some of the earlier investigators reported on bottling by filtration through sheets of cellulose
changes in the composition of the major classes (Simpson, 1968). Chill filtration may also be
of organoleptically-important compounds practiced, since it removes tannin material from
during maturation in barrels. Thus, Schidrowitz the whisky and prevents subsequent appearance
and Kaye (1905) found increased concentrations of haze.
of volatile acids in aged whiskies, a trend also
described in the report of the Royal Commission
on Whisky and Other Potable Spirits (1909). EFFLUENT DISPOSAL AND SPENT GRAINS
Several reports followed. Liebmann and Scherl RECOVERY
(1949), for example, reported increased
concentrations of acids, furfural, tannins and Traditionally effluents from whisky distilleries
color with maturation in barrels. The arrival of were disposed of in the most convenient manner.
the gas liquid chromatograph and HPLC greatly Spent grains were retailed, often quite cheaply,
accelerated research on this topic; and more to local farmers as animal fodder while pot ale
recent data on chemical changes that take place and spent lees were simply discharged into the
during maturation are described later in this local sewer, stream or river. This is no longer
chapter. the case, mainly because of the distiller’s
Maturation of whisky in oak barrels is an awareness of the nutritional value (largely the
expensive process; and it is hardly surprising protein content) of some of these effluents, and
that consideration has been given to methods public awareness of environmental problems
for acceleration. Jacobs (1947) described several arising from uncontrolled disposal of effluents
of these methods including pretreatment of the into waterways. Simpson has described the
beer with activated carbon, chemical treatment production of ‘distillers dark grains’. These
of the whisky to convert aldehydes into esters processes are widely used to dispose of effluents
and use of oxidation treatments. Such techniques from whisky distilleries, especially where the
are not used in the industry, which adheres to traditional methods of disposal are forbidden or
the traditional nature of the whisky-producing uneconomic.
process.

Organoleptically important components

BLENDING AND COLORING of whisky
Whisky manufacturers take great pride in the Modern analytical techniques have enabled
quality of their straight or blended whiskies and major advances in the understanding of the
in their ability to maintain the quality of a compounds responsible for the organoleptic
particular product over the years. Blending is properties of whiskies. However, the first reports
conducted by experts who advise manufacturers on the nature of flavor-producing compounds
on mixing or blending large volumes of whisky in whiskies antedate the era of gas liquid
after appropriate sniffing and tasting sessions on chromatography by nearly half a century. Two
experimental blends. Blended whiskies are filled publications by Schidrowitz (1902) and
into re-used barrels after dilution with water (or Schidrowitz and Kaye (1905), dealing
not) and stored for a further period of time exclusively with Scotch whiskies, reported on
referred to as ‘marrying’. The water used for the higher alcohol, acid and ester contents of
dilution is softened or demineralized, since water some 50 different brands. They reported
containing an appreciable concentration of salts analyses of several Campbeltown Scotch malt
can cause hazes in the whisky (Warricker, 1960). whiskies. A report by Mann (1911) published a
After marrying, and dilution if necessary, the few years later also quoted values for acidity
color of the whisky may be adjusted to the and levels of furfural, aldehydes, esters and

Danisco Exhibit 1010, Page 53 of 85

214 T.P. Lyons

alcohols in Scotch whiskies imported into sensitivity and discrimination, a major problem
Australia. in analyzing whisky by GLC is the
overwhelming preponderance of ethanol and
water. Only one volatile compound, namely
CONCENTRATIONS OF ORGANOLEPTICALLY isoamyl alcohol, is likely to be present in a
IMPORTANT COMPOUNDS concentration exceeding 0.01%; while most of
the others are present in concentrations that
Since the introduction of GLC into distillery rarely exceed 50 ppm. Indeed many compounds
laboratories, several reviews have been now understood to have an important impact on
published on the composition of the major whisky flavor are present at ppb levels.
flavor-producing compounds in whiskies Carter-Tijmstra (1986) described a technique for
(namely higher alcohols, esters, carbonyl measuring dimethyl trisulfide, a compound with
compounds, organic acids, aromatic a threshold of only 0.1 ppb present in whisky at
compounds) and on the identification of concentrations below 50 ppb. Analyses are most
individual compounds that make up these conveniently conducted on extracted fractions
fractions. Higher alcohols, which are still of the different classes of compounds. When
routinely determined with GLC using a polar direct analysis of whiskies has been employed,
stationary phase (Aylott et al., 1994), are only a limited number of components have been
quantitatively the most important. Scotch malt determined (Morrison, 1962; Bober and
whiskies are richest in higher alcohols, with Haddaway, 1963; Singer and Stiles, 1965).
contents often well over 2 g/L. Free fatty acids Recent developments in headspace analysis
are relatively volatile and make a major using trapping and thermal desorption
contribution to the organoleptic qualities of techniques (Pert and Woolfendon, 1986) have
whiskies. Concentrations of acids in some Scotch enabled analysis of the more volatile
malt whiskies can be as high as 0.4-1.0 g/L components of whisky flavor whilst avoiding
absolute alcohol (Duncan and Philip, 1966). interference from high concentrations of other
Roughly comparable concentrations of esters are congeners. Headspace analysis using trap and
found in whiskies, although those produced with purge techniques would now be the method of
pot stills generally have higher concentrations choice for measuring highly volatile sulfur
than those from continuous stills. Ester compounds such as dimethyl trisulfide.
concentrations in Scotch and Irish pot still Some idea of the variety of compounds
whiskies have been reported in the range of 0.27- detected in whiskies came from a compilation
0.87 g/L absolute alcohol (Valaer, 1940). Lighter of both published and unpublished sources
whiskies contain lower concentrations of (Kahn 1969, Kahn et al., 1969). Of the some
carbonyl compounds, although the 200 compounds listed there are 25 higher
concentration varies with the brand. Grain alcohols, 32 acids, 69 esters and 22 phenolic
whisky may have as little as 20 mg/l of compounds. Undoubtedly, this list could now
aldehydes, while in a mature Scotch malt whisky be extended quite considerably. Of the higher
the concentration may be as high as 80 mg/L alcohols, isoamyl alcohol and optically-active
(Duncan and Philip, 1966). amyl alcohol predominate, accompanied by
lower concentrations of isobutanol and n-
propanol. Characteristically, there are usually
CHEMICAL NATURE OF only low concentrations of n-butanol and
ORGANOLEPTICALLY-IMPORTANT secbutanol. The principal organic acid in
COMPOUNDS whiskies is acetic acid, which can account for
Duncan and Philip (1966) reviewed chromat- between 50 and 95% of the total content of
ographic and other methods used to separate the volatile acids determined by titration. Of the
various organoleptically important compounds remaining acids, caprylic, capric and lauric are
from whiskies. Even though analytical methods quantitatively the most important (Suomalainen
such as capillary column gas chromatography and Nykänen 1970a). Some of the characteristic
linked to a mass selective detector (GC-MS) flavor and aroma of Irish whiskies may be
(Aylott et al., 1994) have developed in terms of attributed to somewhat higher concentrations of

Danisco Exhibit 1010, Page 54 of 85

 Production of Scotch and Irish whiskies: their history and evolution 215

the odoriferous butyric acid. Compared with CONTRIBUTION OF COMPOUNDS TO

other types of whisky, Scotch whisky ORGANOLEPTIC PROPERTIES
characteristically contains more palmitoleic acid
(and its ethyl ester) than palmitic acid. Published information on compounds
Suomalainen (1971) has suggested that the responsible for the organoleptic qualities of
typical stearin-like smell of Scotch malt whisky whiskies is meagre and very largely confined to
may be attributed to long chain fatty acid ethyl reports by Suomalainen and his colleagues from
esters. It is not surprising to find that ethyl acetate the State Alcohol Monopoly in Helsinki, Finland.
is the major whisky ester in view of the In order to assess the contributions made by
prevalence of acetic acid in the distillate. whisky components to the odor of these spirits,
Concentrations of 95 mg/L have been detected Salo et al. (1972) concocted a synthetic whisky
in a blended Scotch whisky (de Becze, 1967). with components that chromatographic analysis
Other esters such as ethyl caprate are present in had revealed were present in a light-flavored
much lower concentrations, on the order of 2- Scotch whisky. The synthetic whisky was made
10 mg/L. Of the carbonyl compounds, using 576 g of a mixture of higher alcohols, 90
acetaldehyde is the principal component mg of acids, 129 mg esters and 17.4 mg
together with a range of other short chain carbonyl compounds in highly-rectified grain
aldehydes. Furfural, with an aroma resembling spirit diluted to 34o GL in water that had been
that of grain, also occurs with as much as 20-30 ion-exchanged and treated with activated
mg/L in Scotch malt whiskies (Valaer, 1940). charcoal. This imitation whisky contained 13
Acrolein, a pungent and lachrymatory alcohols in addition to ethanol, 21 acids, 24
compound, is also present; and it has been esters and 9 carbonyl compounds. Caramel
suggested that it may contribute to the ‘peppery’ coloring was used to give it the color of a distilled
smell of whisky. and matured whisky. Odor thresholds of the
A variety of other organoleptically important individual compounds and groups of
compounds has also been detected in different compounds were determined as described by
whiskies, many of which result from maturing Salo (1970). Experienced taste panel participants
the whisky in charred oak barrels. Scopoletin were easily able to distinguish the imitation
and other aromatic aldehydes, including vanillin, whisky from a blended Scotch whisky; but when
were detected in bourbon by Baldwin and his the concoction was mixed with an equal amount
colleagues (1967). These compounds were of the Scotch, only 6% correct judgments above
previously identified by Black et al. (1953) in chance were made. This suggested that the
ethanolic extracts of plain and charred American concentrations of, and interactions between,
white oak from which the bourbon whiskey components of the synthetic whisky were not
barrels subsequently used for the maturation of greatly dissimilar from that in the Scotch used
Scotch and Irish whiskies are constructed. (It for comparison.
should be noted that the US regulations for Odor threshold determinations of individual
bourbon whiskey production require that new components in the imitation whisky revealed that
charred oak barrels be used for maturation; so the contributions made by the mixture of
there is a continuous supply of once-used alcohols and acids accounted for only about 10%
bourbon barrels that are shipped to Ireland and of the total odor intensity, despite the fact that
Scotland for re-use.) A lactone dubbed ‘whisky the alcohols themselves accounted for over 70%
lactone’, also appears in whisky following of the total concentration of organoleptically-
storage in oak barrels. This compound, ß-methyl- important compounds in the concoction. Esters
octalactone, was first isolated from Scotch and carbonyl compounds had a much greater
whisky by Suomalainen and Nykänen (1970b). influence, particularly butyraldehyde,
It has since been reported that both cis and trans isobutyraldehyde, isovaleraldehyde, diacetyl,
diastereomers of the compound occur in whisky and the ethyl esters of acetic, caproic, caprylic,
(Nishimura and Masuda, 1971). Other capric and lauric acids. Since just three of the
compounds detected in whisky include phenols most important carbonyl compounds could
(Salo et al., 1976), glycerol and erthritol (Black substitute for the whole carbonyl fraction, there
and Andreasen, 1974), pyridine, α-picoline and would seem to be considerable homogeneity in
various pyrazines (Wobben et al., 1971). the odor contributions made by these

Danisco Exhibit 1010, Page 55 of 85

216 T.P. Lyons

compounds. Interestingly, the relative used to ferment the wort and the charred oak
contributions made by the different classes of barrels in which the whisky is matured.
compounds are not very different from the Suomalainen and Nykänen (1966) fermented a
contributions Harrison (1970) reported that they nitrogen-free medium containing sucrose with
make towards the taste of beers. a strain of Saccharomyces cerevisiae and distilled
Threshold values can be assessed not only for the fermented medium either after removing the
individual components in whisky, but for the yeast by centrifugation or with the yeast
total aroma of the beverage. Salo (1975) remaining in the medium. Gas chromatographic
examined this by diluting different whiskies with analyses of these distillates are reproduced in
water until the characteristic whisky aroma could Figure 14, which also shows a chromatogram
only just be recognized. Values for the threshold of Scotch whisky for comparison. There is
dilution of several commercial whiskies shown clearly a similarity among the three analyses;
in Table 6 reflect the differences in aroma although differences, such as the higher
strength for several different commercial proportion of isoamyl alcohol in the distillate
whiskies. from the fermented medium, can be detected.
An excellent review of how these Also worth noting is the greater concentration
characteristics can be evaluated from an of ethyl caprate in the distillate from the spent
organoleptic point of view comes from Jack
(2003). Sensory data are only as good as the Pungent
2.5
sample preparation and guidelines were set as Stale Phenolic
applicable to the Scotch whisky industry. It is 2

important that the line between sample Sulfury 1.5 Feinty

preparation and assessment should be minimized 1
since time and the temperature of nosing have a 0.5
major impact on sensory character. As Soapy Cereal
0
temperature increases, a greater amount of the
flavor volatiles are released, thus altering the
sensory profile. Effects of changes in room Sour Aldehydic
temperature on flavor scores determined by a
sensory panel for a blended Scotch whisky are
Oily Estery
illustrated in the form of a spider diagram in
Figure 13. Woody Sweet

ORIGIN OF ORGANOLEPTICALLY IMPORTANT 5°C Room temperature 30°C

COMPOUNDS

The two main sources of the organoleptically Figure 13. Impact of temperature on flavor profile of Scotch
important compounds in whisky are the yeast whisky (adapted from Jack, 2003).

Table 6. Threshold dilution levels for nine different types of whisky*.

Whisky Threshold dilution (x 10-4) One standard deviation range (x 10-4)

Scotch malt 0.56 0.2 - 1.3

Scotch, old blend 0.87 0.5 - 1.5
Scotch, blend 1.20 0.3 - 4.2
Irish 1.30 0.4 - 2.0
Bourbon 2.40 0.6 - 11.5
Irish 4.50 2.7 - 7.5
Canadian 10.40 3.0 - 37.0

*Salo, 1975.

Danisco Exhibit 1010, Page 56 of 85

 Production of Scotch and Irish whiskies: their history and evolution 217

medium containing yeast as compared with that Extensive research in Scotland revealed the
obtained by distilling the medium from which mechanism of ethyl carbamate production and
yeast had been removed. It would be interesting facilitated the introduction of very effective
to learn of the importance of yeast strain in control measures. These measures maintain
production of organoleptically-important levels close to zero and always less than 30 ppb
compounds in whisky. Unfortunately, there is a in distilled whisky spirit.
lack of published data on this matter. Cook (1990) reviewed the outcome of this
research and the resulting control procedures.
Scotch whisky When barley sprouts during malting, a
Isoamyl Alcohol

Ethyl Caprylate
Ethyl Caprate
Ethyl Laurate

glycoside, epiheterodendrin (EPH) is present in

Ethyl Palmitate

the acrospire. This glycoside, which survives

kilning and mashing, is extracted into the wort
and converted by yeast enzymes to glucose and
isobutyraldehyde cyanohydrin (IBAC). The
IBAC is stable during fermentation, but when
Sugar fermentation heated above 50oC at distillation it breaks down
Isoamyl Alcohol

to form volatile nitriles. There are a multiplicity

Ethyl Caprylate
Ethyl Caprate
Ethyl Laurate

Ethyl Palmitate

Yeast separated
before distillation of potential routes nitriles can follow including
reaction with other beer components or complex
reactions often mediated indirectly by copper.
Some of the volatile nitriles may escape these
reactions and pass into the distillate. A number
Sugar fermentation of reactions can remove the nitriles from the
Isoamyl Alcohol

Ethyl Caprylate
Ethyl Caprate
Ethyl Laurate

spirit, one being the reaction with ethanol to form

Ethyl Palmitate

Distilled with yeast

ethyl carbamate.
Two control strategies may be used to prevent
ethyl carbamate reaching the final spirit. Firstly,
some varieties of barley produce low levels of
the glycoside EPH (Cook, 1990), and plant
80 120 160 200 210
65 breeders are now concentrating on incorporating
Temperature (°C) this character in all varieties for the distilling
industry. The second strategy relies on the low
volatility of ethyl carbamate. Control of
Figure 14. Gas liquid chromatograms of aroma compounds distillation conditions eliminates all the volatile
produced by yeast in a nitrogen-free sugar fermentation, precursor formed by copper mediated reactions.
with a trace for comparison of the aroma compounds Non-volatile substances are formed prior to the
detected in a sample of Scotch whisky (Suomalainen final distillation in malt distillation or before
and Nykänen, 1966). rectification in grain distillation. Thus, ethyl
carbamate can be minimized in the final spirit.
The complexity of the processes for determining A considerable number of studies have been
the presence and concentration of compounds published on those organoleptically important
in whisky may be illustrated by looking at one compounds arising either directly or indirectly
compound for which this process has been from the oak barrels in which whisky is matured.
elucidated in detail. In the 1980s ethyl carbamate, The increase in coloring, tannin, dissolved solids
a naturally-occurring component of many and acid concentrations are not observed when
alcoholic drinks, was identified as being whisky is stored in glass, which is proof of the
undesirable. Canada specified by regulation a importance of the oak barrels in the maturation
maximum limit for ethyl carbamate in whisky process. An analysis of heartwood of the
spirit of 150 ppb and the US set guidelines of American oak (Quercus albus) gave cellulose
120 ppb. Control of this compound was only (49-52%), lignin (31-33%), pentosans (or
possible after the processes resulting in its hemicelluloses, 22%) and compounds extracted
presence in spirit were fully understood. with hot water and ether (7-11%; Ritter and Fleck,

Danisco Exhibit 1010, Page 57 of 85

218 T.P. Lyons

1923). However, when charred oak sawdust was evaporation and adsorption on the charred
directly extracted with water or 96o GL ethanol, surface of the barrel (Perry, 1986). Lastly there
the extracts obtained differed markedly in odor are reaction processes including establishment
from aged whisky. Moreover, none of the of equilibria among acetaldehyde, ethanol and
various fractions of ethanol-soluble oak acetal (Perry, 1986), polymerization reactions
extractives contained flavors that resemble (Nishimura and Matsuyama, 1989) and
mature whisky (Baldwin et al., 1967). As a oxidation-reduction reactions (Perry, 1986;
result, it is now generally held that the Connor et al., 1990). Many of the reactions
maturation process involves not only extraction involve, and are indeed dependent on, the
of compounds from the oak but also chemical components extracted from the wood.
modifications of at least some of the compounds Looking at several of these reactions in more
extracted from the wood. detail will serve to illustrate the complexity of
For a long time, most of the work reported on the maturation process. The work at the Seagram
this aspect of maturation of whisky came from laboratories, whilst focused on bourbon, is
the laboratories of Joseph E. Seagram and Sons directly relevant to Scotch and Irish whiskies for
in the US. More recently, accounts of the which once-used bourbon barrels are extensively
mechanisms of Scotch whisky maturation have utilized for maturation. Changes in the
been given by Philip (1989) and Perry (1986) concentrations of organoleptically-important
and on the maturation of whisky generally, by compounds during a 12 year storage of a 109o
Nishimura and Matsuyama (1989). A theme from US proof (54.5o GL) bourbon, on a 100o proof
all this work has been the identification of a (50 o GL) basis, are shown in Figure 15. The
number of mechanisms of maturation common nature and origin of some of these compounds
to all whiskies. These divide into addition, have been examined in some detail. Among the
subtraction and modification by reaction. There aldehydes, scopoletin and the aromatic
is addition of components from the oak wood, aldehydes syringaldehyde, sinapaldehyde,
including those derived from lignin, tannins and coniferaldehyde and vanillin are important.
oak lactones. There is the subtraction of volatile According to Baldwin et al. (1967), these
compounds from the maturing whisky by compounds could be formed by ethanol reacting

260
240
Concentration (g/100L) at 100° proof

Absorbance at 430 nm at 100° proof

220
Total solids
200
180 1.8
160 1.6

140 1.4
Color
120 1.2

100 1.0
80 ids
0.8
Total ac
60 0.6
40 Tannins 0.4
Esters Aldehydes
20 0.2
Furfural
0 0
1 2 3 4 5 6 7 8 9 10 11 12
Age (years)

Figure 15. Changes in composition of the vapor at different trays in a Coffey still rectifier used in the manufacture
of Scotch grain whisky (from Pyke, 1965).

Danisco Exhibit 1010, Page 58 of 85

 Production of Scotch and Irish whiskies: their history and evolution 219

with lignin in the oak wood to produce coniferyl compounds extracted from charred oak wood
alcohol and sinapic alcohol. Under mildly depend on the ethanol concentration of the
oxidizing conditions, these alcohols could be whisky. It is to some extent an advantage to
converted into coniferaldehyde and mature whisky at a high proof, since this requires
sinapaldehyde, respectively. Vanillin could then fewer barrels and saves on storage space. Until
arise from coniferaldehyde, and syringaldehyde 1962 the US Treasury Department limited the
from sinapaldehyde. The increase in aldehyde barrelling proof of whisky to a maximum of 110o
content during maturation is also attributable in US proof (55o GL). In anticipation of this limit
part to formation of acetaldehyde by oxidation being raised to 125 o US proof (62.5 o GL),
of ethanol. Formation of ethyl acetate probably Baldwin and Andreasen initiated a series of
accounts for the steady rise in the ester content experiments in 1962 to establish the importance
of whisky during maturation. of barrelling proof on changes in color and
Several other groups of compounds not concentrations of organoleptically-important
described in Figure 14 are also important in the compounds during maturation of bourbon
maturation process. Monosaccharide sugars are whiskies. Their report in 1973 indicated that
found in mature whisky, and probably arise from color intensity and congener concentration of
the pentosans and other polysaccharides in the whiskies matured for 12 years decreased as the
oak wood. Otsuka et al. (1963) reported that a barrelling proof was raised from 109o US proof
mature Japanese whisky contained xylose, (54.5o GL) to 155 o US proof (77.5 o GL). The
arabinose, glucose and fructose, while Black and one exception was the higher alcohol content,
Andreasen (1974) added rhamnose to this list which remained approximately constant.
when they analyzed a mature bourbon. The latter
workers found that the concentrations of
arabinose and glucose increased at a faster rate Is it really Scotch?
than those of xylose and rhamnose over a 12
year maturation period. Salo et al. (1976) also Analysis of the variety of compounds in spirit is
detected low concentrations of mannose and also of interest for reasons of identifying or
galactose in a matured Scotch malt whisky in authenticating product origins or types. Adam
addition to the sugars already noted. The et al. (2002) investigated whether malt, blended
concentrations of sugars in mature whiskies (of and grain whiskies could be differentiated based
the order of 100 mg/L) are too low to suggest on content of various metals. While it was not
any sweetening effect on the beverage. Phenols possible to define a ‘metal fingerprint’ that would
are also detectable in mature whisky, although identify a whisky as to origin, malt whiskies had
some of these probably arise during mashing markedly higher concentrations of copper than
(Steinke and Paulson, 1964) or from malt blended or pure grain whiskies. Differences were
produced using peat-fired kilns (MacFarlane, significant with malt whiskies containing 385-
1968). However, Salo et al. (1976) reported an 480 ng Cu/mL and grain and blended whiskies
increase during a one year maturation of a contained 131-242 ng/mL. Since malt Scotch is
Scotch malt whisky in the concentration of produced in traditional copper pot stills while
eugenol, which is a major phenol extracted from grain whisky used in blending is made in
oak chips by ethanol (Suomalainen and continuous patent stills, copper content was
Lehtonen, 1976). Also present in mature suggested as a means of distinguishing a malt
whiskies are sterols, which may precipitate in Scotch from a blended product.
bottled whisky stored at room temperature. Another approach to distinguishing between
Black and Andreasen (1973) found campesterol, malt and blended products is based on the
stigmasterol and sitosterol in mature bourbon, differences between barley, which is used to
in addition to sitosterol-D-glucoside, although produce malt Scotch and the corn (maize) that
the possibility that some of these were formed is typically used in production of grain whisky.
during mashing cannot be excluded. Finally, Parker et al. (1998) found that the ratio of 12C
reference has already been made to the whisky and 13 C isotopes in volatiles such as
lactone, ß-methyl-octalactone, and its origin. acetaldehyde, ethyl acetate, n-propanol and
Not surprisingly, the nature and amounts of others differed among whiskies. This was a

Danisco Exhibit 1010, Page 59 of 85

220 T.P. Lyons

reflection of the different types of photosynthetic biology I I:178.

metabolism of barley, a cool season plant with Black, R.A. and A.A. Andreasen. 1973. Journal
C3 metabolism, versus maize, which being of the Association of Official Analytical Che-
adapted to hotter climates has C4 metabolism. mists 56:1357.
There is no doubt value in being able to Black, R.A. and A.A. Andreasen. 1974. Journal
chemically define the special characteristics that of the Association of Official Analytical Che-
distinguish an ordinary whisky from malt Scotch. mists 57:111.
Authentication is often needed; and a thorough Black, R.A., A.A. Rosen. and S.L. Adams. 1953.
understanding of the chemical basis of Journal of the American Chemical Society
organoleptic properties might pave the way to 75:5344.
production of even finer whiskies than those we Bober, A. and S.W. Haddaway. 1963. Journal
enjoy most today. For the moment, however, the
of Gas Chromatography 1 12: 8.
magic remains. The distiller’s art continues to
Brander, M. 1975. A Guide to Scotch Whisky.
elude the chemist.
Johnson and Bacon, Edinburgh. p. 96.
Bryan-Jones, G. 1976. Lactic Acid Bacteria in
Beverages and Foods. Proceedings of the
Acknowledgments
Fourth Long Ashton Symposium. (J.G. Carr,
The author wishes to thank his friends and C.V. Cutting and G.C. Whiting, eds). Acad-
colleagues in whisky production in many emic Press, London. pp. 165-175.
countries of the world for their cooperation and Cameron Taylor, I.B. 1970. Whisky Export, 2nd
willingness to supply information on the issue, June-Sept. Aldiffe Publishing Co. Ltd.,
processes of whisky production. Particular Wilmslow, Cheshire.
thanks are due to Dr. Gareth Bryan-Jones of Carter-Tijmstra, J. 1986. Proceedings of Second
United Distillers, Menstrie, Scotland, for his Aviemore Conference on Malting, Brewing
assistance in providing research reports. and Distilling. pp. 413-416.
Connor, J.M., A. Paterson and J.R. Piggot. 1990.
Proceedings of Third Aveimore Conference on
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Convention, Vienna. p 253. Court, R.E. and V.H. Bowers. 1970. Process
Aylott, R.I., A.H. Clyne, A.P. Fox and D.A. Biochemistry 5:17.
Walker. 1994. Analyst 119:1741-1746. Cromwell, E.A. and J.F. Guymon. 1963.
Baldwin, S. and A.A. Andreasen. 1973. Journal American Journal of Enology and Viniculture
of the Association of Official Analytical 12:214.
Chemists 57:940. Daiches, D. 1969. Scotch whisky, its past and
Baldwin, S., R.A. Black, A.A. Andreasen and present. Andre Deutsch, London. p. 168.
S.L. Adams. 1967. Journal of Agriculture and Dolan, T.C.S. 1976. Journal of the Institute of
Food Chemistry 15:381. Brewing. 82:177.
Barbour, E.A. 1983. Taxonomy of some lactic Duncan, R.E.B. and J.M. Philp. 1966. Journal
acid bacteria from scotch whisky distilleries. of the Science of Food and Agriculture 17:208.
PhD Thesis, Heriot-Watt University. Dunnett, A. 1953. The land of scotch. Scotch
Barnard, A. 1887. The whisky distilleries of the Whisky Association, Edinburgh. p. 170.
United Kingdom. Proprietors of Harpers Geddes, P.A. and H.L. Riffkin. 1989. Distilled
Weekly Gazette, London, pp. 457. Beverage Flavour (ed. Piggot, J.R. Ellis,
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Beech, F.W. 1972. Progress in Industrial Micro- ledge, London. p 198.

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222 T.P. Lyons

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46:39.

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 Tequila production from agave 223

Chapter 15

Tequila production from agave: historical influences and contemporary

processes

MIGUEL CEDEÑO CRUZ

Tequila Herradura, S.A. de C.V. Ex-Hda San Jose del Refugio Amatitán, Jalisco, México

Introduction: agave beverage alcohol

products
Tequila is classically associated with Mexico and 159 gallons or barrels, and extra-aged (Añejo)
has been long identified as more than just the matured at least 12 months in white oak barrels.
national drink, but a symbol of our culture and More than 300 brands (labels) of tequila are
environment. As the reach of the global trade produced in the 104 distilleries registered in
has extended, tequila is now consumed around Mexico by the Tequila Regulatory Council (CRT
the world. by its acronym from the Spanish: Consejo
This alcoholic beverage is obtained from the Regulador del Tequila), which verifies
distillation of fermented juice of the agave plant, compliance with the Mexican official standard
Agave tequilana Weber var. azul. A. tequilana (Secofi, 1994) through routine on-site
is often confused with various cacti growing in inspections.
same desert environment, but they belong to Tequila is differentiated from the beverage
different botanical families: agaves to Agavaceae known as ‘mezcal’ by the type of agave used in
and cacti to Cactaceae families, respectively. its production. Mezcal is made from Agave
There are two classifications of tequila based potatorum, which grows in the states of Oaxaca,
on agave content: 100% tequila, produced using San Luis Potosi, Guerrero and Zacatecas. Most
only agave, and 51% tequila, which contains mezcal producers use a rudimentary
sugars from other sources such as cane and corn fermentation and distillation process (Sánchez,
added in the fermentation step, at up to 49% by 1991). There is no technical reason for, or any
weight. It is important to note that only tequila improvement in the organoleptic characteristics
51% can be exported in bulk outside Mexico from, the worm inside the bottles of some mezcal
since 100% tequila must be state-bottled. brands. The worm is primarily a commercial
Based on maturation, there are four ploy. Worms are grown in agave plants and
classifications of tequila: silver, without introduced manually in the bottling line.
maturation, gold, containing permitted additives ‘Pulque’ is another beverage obtained by
and colors (generally caramel color), aged fermentation of the juice obtained from several
tequila (Reposado) matured at least two months species of agave, A. atrovirens and A. salmiana
in white oak tanks with a maximum capacity of among others, by a complex succession of yeast

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224 M. Cedeño Cruz

and bacteria that produce ethanol, a diversity of codex, which notes that certain tribes had learned
chemical compounds, and some polymers that to cook agave plants and used them as food and
give a sticky consistency to the final product to compensate for the lack of water in desert
(Rzedowski, 1978; Sánchez Marroquin and lands. Also, these tribes discovered that cooked
Hope, 1953). Pulque is sometimes mixed with agave when soaked in water would ferment,
fruits or vegetables, but has poor stability as it is producing a much appreciated beverage. In fact,
neither distilled nor pasteurized. this primitive and rudimentary method was used
Agave plants still serve as food in some states for centuries to produce beverages from agave,
of Mexico; and other fermented regional considered a sacred plant possessing divine
beverages are produced (e.g., ‘Sotol’ in the state properties. In other codices, such as Nutall,
of Chihuahua and ‘Bacanora’ in the state of Laud, Borgia and Florentine, there are many
Sonora), but only tequila and more recently references to uses of the agave plant for soap
mezcal have reached international recognition. manufacture, a source of fiber, footwear,
Another difference between tequila and mezcal medicine and sewing needles as well as thread,
or other regional drinks is that both are subject paper and rope. In fact, Indians distinguished
to an official standard that for tequila is NOM- the different species of agave by color, size, stem,
006-SCFI-1994 (Secofi, 1994), and production leaf width and the different uses given each plant
is supervised by the Mexican government. Table (Muria, 1990). The great religious importance
1 shows the main differences among the regional of agave was apparent in those codices, as only
beverages. warriors and priests used fermented drinks in
ritual ceremonies.
In pre-hispanic Mexico the general name for
Origin and history of tequila all species of agave (or mezcal as it is also
known) was Metl, which is a representation of
The word ‘tequila’ is believed to originate from the goddess Mayahuel (Figure 1). The alcoholic
the tribe of ticuilas who long ago inhabited the drink produced was called Iztac octli (white
hillside of a volcano bearing the same name wine). The first Spaniards to arrive in Mexico
located near the city of Tequila. Another possible referred to the plant as ‘maguey’, a name used
origin is the Nahuatl word tequitl, which means for an identical plant they had seen in the
work or employment, and the word tlan, which Caribbean Islands, where they first encountered
means place. Therefore ‘tequila’ would mean new world plants and animal life (Bottorff,
place in which labor or work is done. 1971). It was not until the arrival of the
The oldest reference to the existence of agave Spaniards, who brought knowledge of
and its different uses is from the era before the distillation techniques, that tequila took its
Spaniards in several codices preserved to the present form (Goncalves, 1956). Tequila is
present time (a codex, from the Latin codex considered the first distilled beverage in North
meaning board or writing tablet, is a manuscript America and has been given several different
volume, especially of a classic work or scripture). names including agave wine, mezcal wine and
The most important is the Tonalmatlnahuatl finally tequila.

Table 1. Agave alcoholic beverages produced in Mexico.

Beverage Agave Distillation Producing Fermentation:

species process region main microorganisms

Tequila tequilana Yes Jalisco Yeast

Mezcal potatorum, salmiana, angustifolia Yes Oaxaca, San Luis Potosi Yeast
Bacanora pacifica, palmieri, angustifolia Yes Sonora Yeast
Sotol angustifolia, dasylirion Yes Chihuahua Yeast
Pulque atrovirens, salmiana No Central Yeast, bacteria

Danisco Exhibit 1010, Page 64 of 85

 Tequila production from agave 225

of the following states: Jalisco (all), Nayarit (8),

Michoacan (30), Guanajuato (7) and Tamaulipas
(11) (Figure 2). The oldest, the Tequila-Amatitán
region that comprises the Amatitán village,
developed at the end of the 17th century. The
second region, the Jalisco Highlands, appeared
in the last decade of the 19th century (Luna,
1991).
The first tequila production process with a
commercial purpose was established in the city
of Tequila around the end of the 18th century.
The main consumers were in the mining zones
located in the state of Jalisco. The Spaniards tried
to suppress consumption of tequila in order to
reduce competition for brandy and other wines
imported from Spain with a decree signed by
Carlos III forbidding sale and production of
tequila under the pretext that its consumption
was the cause of several illnesses. The results
were negative. The governor of the region later
issued a decree imposing a tax on tequila in order
Figure 1. Representation of the goddess Mayahuel. to enrich the royal coffers, thus permitting its
sale in all of New Spain. By the end of the 19th
There are specific regions in Mexico where century, expansion of the tequila industry, helped
agave should be grown, where tequila must be by the railway, was evident. However it was not
produced and where 100% tequila must be state- until the first casks were exported to the US that
bottled. This area comprises the municipalities tequila was known beyond Mexico’s borders.

Tamaulipas (11)

Guanajuato (7)
Nayarit (8)

Jalisco (all)

Michoacán (30)

Figure 2. States and municipalities for agave cultivation and tequila production in Mexico.

Danisco Exhibit 1010, Page 65 of 85

226 M. Cedeño Cruz

The agave plant

According to Granados (1985), the genus Agave, Agave has commercial importance as a fiber
which means ‘noble’ in Greek, was defined by source; and this industry developed in the 19th
Linneaus in 1753 when he described the plant century in Mexico mainly in the Yucatan state
A. americana as the first agave species known with the cultivation of henequen, A. fourcroydes,
to science. Agave plants, which are often to export fiber to Europe. However, the
confused with cacti, belong to the family Philippines, Indonesia and Tanzania in Africa
Agavaceae and are succulent plants with competed in this market with fiber produced by
spirally-arranged leaves forming a rosette. Some A. sisalana. The use of agave as a forage for
have definite trunks, but more often they are cattle is a recent development for milk
nearly stemless. The leaves are bluish green in production, but must be enriched with a protein
color, over 1 m long in mature plants, and end source. Other agaves such as A. americana, A.
in a sharpened brown thorn. As Backman (1944) victoria regina, A. filifera and A. stricta among
pointed out, the widespread distribution of some others are used as ornamentals.
300 species, combined with the fact that the In the beverage industry, A. salmiana and A.
plants require approximately 8-12 years to atrovirens are valued for pulque production, A.
mature and hybridize very easily, make the potatorum, A. salmiana and A. angustifolia for
taxonomic and phylogenetic study of the genus mezcal production, A. pacifica, A. palmieri and
Agave extremely complicated and very difficult. A. angustifolia for the elaboration of bacanora
The family Agavaceae includes 20 genera and and A. dasylirion and A. angustifolia for sotol
nearly 300 species. Of these, around 200 are production. Finally, A. tequilana Weber var.
found in Mexico. In Figure 3, a diagram of an azul, named around 1900 by the German
agave plant in soil shows the main structural botanist Weber (Diguet, 1902), is used to produce
features. tequila.

CULTIVATION AND HARVEST

The blue agave, as it is known, is the only species

out of hundreds of Agavaceae with the
appropriate characteristics for tequila
production. These include a high inulin
concentration, low fiber content and the
chemical compounds present in the plant that
contribute to the final taste and flavor of tequila
to give the beverage its particular character.
Some have attempted to produce tequila in other
states with other agave species, but without
success. The blue agave is mainly cultivated in
the state of Jalisco in the two regions with the
right climate and soil composition for its growth,
namely the Highlands and the Tequila-Amatitán
region. The temperature conditions for good
agave yields are a minimum of 3°C, an optimum
of 26°C and maximum of 47°C. Soil should be
fertile but not very deep, 30 to 40 cm. Good
drainage is required to avoid effects of flooding,
which are very harmful for development of the
agave. Agave are planted at 800 to 2500 m
above sea level where the annual rainfall is about
Figure 3. Structure of Agave tequilana Weber var. azul. 800 to 900 mm. The correct planting time is

Danisco Exhibit 1010, Page 66 of 85

 Tequila production from agave 227

immediately before the rainy season, from June theobromae and Colletotrichum agavae may
to September, so that the plants do not suffer cause serious damage to agave leaves (Halffter,
from water stress during the first year of growth. 1975; Agricultural Research Service, 1972).
Propagation is accomplished by vegetative Agave plants, adapted to hot and very arid
means in agave. Sexual reproduction via seeds conditions, employ crassulacean acid
is not usual. Asexual bulbils develop in the metabolism (CAM). Crassulacean acid
inflorescence at the base of the flowers, metabolism is similar to photosynthetic processes
producing small plants that after some time in other hot temperature-adapted plants in that
detach themselves from the floral peduncle and the enzyme phosphoenolpyruvate (PEP)
fall to the soil where they root. Another mode carboxylase is used to fix CO2 into a 4-carbon
of asexual propagation is by suckers, which are compound (C 4 metabolism). However in most
a characteristic type of lateral bud or branch hot season plants the light-dependent reactions
developing at the base of the main stem. Plants and CO2 fixation are separated in different cells
developing near each mother plant are separated of the leaf. In contrast, in plants with crassulacean
at the age of 3-4 years. These baby plants are acid metabolism such as cacti and agave these
called ‘first-class seed’ because they are better reactions occur at different times of the day.
and healthier (Sánchez, 1991). In practice, people Carbon dioxide (CO 2) enters the leaf through
use the word ‘seed’ to refer to such young stomata with the simultaneous loss of water. By
plants, but from a botanical point of view these only requiring the stomata to be open during the
are rhizome shoots or suckers (Valenzuela, cooler night for CO 2 uptake and keeping them
1992). Plant cell culture is used by some tequila closed during the hotter day, the agave plants
companies in order to maintain plants of lose much less water (Ehrler, 1967). Malic acid
consistent quality, reduce the cost of labor for formed by CO2 fixation using PEP carboxylase
sowing the plants, eliminate use of fungicides accumulates during the night, and is broken down
and antibiotics during sowing, and have bigger during the day (Figure 4). The CO 2 that is
plants with higher inulin content at the end of released can be re-fixed by the normal
the cultivation period. photosynthetic Calvin cycle (C3 metabolism) to
Land for agave cultivation must be cleared and produce sugars and then inulin.
deep-ploughed, sometimes twice. Agave is The thick cuticle overlying the epidermis,
planted approximately 2-4 m apart in straight which is quite evident in tequila agave,
lines called ruts. Sowing is done by hand in holes apparently prevents damage to the leaf from high
15 cm in depth. Plant density is around 2000- temperatures (Casado and Heredia, 2001). This
4000 plants per hectare, depending on the waxy cuticle produces turbidity in tequila
plantation system used; and yields can be because it dissolves in the distillation step and
between 30,000 and 200,000 kg/ha, assuming produces a haze in the final product when it is
that the weight of a harvested plant varies from diluted or cooled. One way to avoid this is to
15 to 50 kg. This variation is caused by treat tequila with activated charcoal and to filter
differences in soil conditions, quality of plants it through pure cellulose filter pads. This,
sown, rainfall, pests and fertilization. Sometimes unfortunately, results in the loss of some aromas.
agave is interseeded with nitrogen-fixing crops Agave fertilization is determined by soil
such as peanuts, beans, chickpeas or soybeans. composition, plant age, and the type of chemical
After the agave has been in the soil for a year, compound used. The normal procedure is to use
visual inspection is conducted and sick or dead urea as a nitrogen source in amounts of 30-70 g
plants are replaced. This operation is called re- per plant incorporated directly into the soil. In
seeding. The percentage of dead plants depends some areas, phosphorus and potassium
on soil and plant characteristics, but is generally fertilization is also required. As some agave
from 8 to 15% (Pérez, 1990). regions are also involved in cattle, swine, and
Agave plants regularly host borer insects that chicken production, manure is sometimes
live in the stems, leaves and fruits during the employed for fertilization (GEA, 1992).
larval stage. These include butterflies of the The average maturity time for agave is seven
family Megathymidae and moths of the family years; and at this time the content of inulin is at
Prodoxidae. Also, the fungi Diplodia its highest level (Mendez, 1999). Every year

Danisco Exhibit 1010, Page 67 of 85

228 M. Cedeño Cruz

Day Night

Air
CO2
H2O

Leaf Stomata (open)

n
Inuli

CO2
Calvin PEP
Cycle
Glycerol CO2 fixation

Growth

C4 acids

Figure 4. Schematic representation of carbon dioxide fixation by agave (PEP: Phosphoenolpyruvate).

following planting, fields are cultivated to loosen are harvested; but on the 12th year, all plants
the soil and weed and pest control is carried out. remaining (the weakest plants) are cut. These
Each plant matures individually. Harvest begins are called ‘drag’ and are generally discarded.
in the seventh year. The leaves are cut from the Agave composition varies seasonally, but an
base and left in the field to recycle nutrients. average would be (wet basis) 27% (w/w)
The harvested plants free of leaves look like large reducing sugars, a juice content of 0.572 ml/g
pineapples and weigh from 20 to 90 kg. They and a pH of 5.2.
are transported to the distillery. Only the better In Figure 5 the amount of agave harvested for
plants, meaning those of good size and high tequila production is presented for the period
inulin content (measured as reducing sugars), 1995-2002. These figures show, first of all, a

800 Tequila100%

700 Tequila
Tons of agave ('000s)

Total
600

500

400

300

200

100

0
1995 1996 1997 1998 1999 2000 2001 2002

Figure 5. Agave harvested for tequila production (CRT, 2002).

Danisco Exhibit 1010, Page 68 of 85

 Tequila production from agave 229

decrease in total agave production. This reflects advances that improve process efficiency and
the shortage caused by disease in agave consistency; and some have implemented a ISO-
plantations in the year 2000, which was mainly 9000 standard. Whatever the process used,
due to a bacteria Erwinia carotovora and a tequila manufacture comprises four main steps:
fungus Fusarium oxysporum, which destroyed cooking, milling, fermentation and distillation.
more than 25% of the total agave plants for An extra step, maturation, is required for aged
tequila. From this graph, it is evident that the products. Figure 6 shows a flow diagram for the
agave utilization for 100% tequila is growing, tequila production process, which is explained
since that beverage is being recognized as a in detail in the following paragraphs.
premium product not only in Mexico but also
overseas.
Processing harvested agave
Tequila production Agave is transported from the fields to the
factories as soon as possible to avoid weight
Tequilas differ greatly depending on agave losses, because today most distilleries pay by
quality and origin (Highlands or Tequila- weight and not by inulin content. The heads are
Amatitán regions). The production process also unloaded from the truck in the receiving area of
strongly influences quality of the final product. the factory and must be protected from the sun
Some distilleries still employ rudimentary and rain in order to avoid withering and fungal
production methods, just as they did several growth. Although the agave has already been
decades ago (Pla and Tapia, 1990). Most inspected during growth and at harvest, it is
companies, however, employ technological examined again to reject visually unacceptable

Autoclave

Oven

AGAVE COOKING MILLING BAGASSE

Yeast

MATURATION 2nd 1st

FERMENTATION
DISTILLATION DISTILLATION
DILUTION

STILLAGE STILLAGE

DISSOLUTION OF
BOTTLING OTHER SUGARS

Figure 6. Flow diagram for the tequila production process.

Danisco Exhibit 1010, Page 69 of 85

230 M. Cedeño Cruz

plants or those damaged by pests. At this point, hydrolyzed corn syrup may be used to formulate
a representative sample of agave is taken for the wort. All sugars used in wort formulation
laboratory analysis. Modified AOAC (1990) are routinely analyzed by measuring solids
procedures are used to determine reducing sugar content and reducing and fermentable sugar
content (after acid hydrolysis of inulin) along content.
with pH, moisture, dry weight, juice and ash
content. CH2OH
Agave heads usually weigh between 20 and H
O
H
60 kg, although some can reach 100 kg. They H Glucose
are cut to sizes that facilitate uniform cooking OH H
and handling. Different agave cutting systems OH
exist, but the use of axes and a specialized tool
H OH O
called ‘coa’ are the most popular. The heads are
cut in halves or quarters, depending on the CH2OH O
Fructose
weight, and the pieces are arranged manually in
an oven or autoclave. Band saws can also be H OH
used to cut the agave heads, since they are faster H
CH2
n
and less labor-intensive than the manual
procedure, but the belts break frequently because OH H
of the resinous consistency of agave. Some O O
CH2OH
factories tear uncooked agave first with a knife
and place the resulting pieces mixed with water H OH
into autoclaves to be cooked. H
CH2OH
OH H
OTHER RAW MATERIALS FOR TEQUILA
PRODUCTION Figure 7. Structure of inulin, in which chain length, n, varies
from 3 to 19, depending on the maturity of the agave plant
When producing 100% agave tequila, the only
(Mendez, 1999).
source of carbohydrate is the inulin hydrolyzed
from agave in the cooking step. Inulin, a polymer
of fructose with a terminal glucose, belongs to
the family of fructans (Figure 7). After starch,
fructans are the most abundant non-structural The cooking step: hydrolysis of inulin
polysaccharide found in nature.
Cooking the agave serves three purposes. First,
For other kinds of tequila the law permits use
the low pH (4.5) together with the high
of other sugars in amounts up to 49% by weight
temperature hydrolyzes inulin and other
in wort formulation. There are no legal
components of the plant. A recent study (Pinal,
specifications regarding the type of adjunct sugar
2001) showed that the concentrations of furfural,
sources to be used in tequila manufacture; and
5-hydroxymethyl furfural, 5-methylfurfural and
theoretically any kind of fermentable sugar is
2-acetylfuran increase with length of cooking
allowed in wort formulation. In practice and from
time. These components are important as they
an economic point of view, only cane sugar,
may influence fermentation rate as well as the
Piloncillo, cane molasses and acid- or enzyme-
organoleptic characteristics of the final tequila.
hydrolyzed corn syrup are employed. Cane
For this reason, time control during cooking is
sugar is received in 50 kg bags and stored in a
very important. In addition, cooked agave has a
dry, cool place for subsequent utilization.
soft consistency that facilitates the milling
Piloncillo consists of brown cones of crystallized
operation.
complete cane juice, sometimes individually
In the pre-Hispanic era, agave was cooked in
wrapped in corn or cane leaves and packed in
holes filled with stones heated using wood for
sacks. Cane molasses is also used but it is difficult
fuel. The stones retained the heat for the time
to handle and there is risk of spoilage over a
needed to cook the agave. Nowadays some
long storage period. Acid- or enzyme-

Danisco Exhibit 1010, Page 70 of 85

 Tequila production from agave 231

distillers have replaced the heated stone holes additional steam, cooking slowly in the
with brick ovens and heating is accomplished remaining heat. This step produces a syrup with
by steam injection after the chopped raw agave a high sugar concentration (>10% by weight)
has been introduced into the oven. Oven cooking that is later used to formulate the initial wort. To
is slow, and steam injection lasts around 36-48 calculate the yield and efficiency of this step,
hrs to obtain temperatures of 100°C. After that the amount of cooking honey and its reducing
period, the steam is shut off and the agave is left sugar content as well as the cooked agave are
in the oven for a further two days to complete measured. Figure 8 illustrates the temperature
the cooking process. During this step, a sweet profile over time for cooking agave in an
liquid called ‘cooking honey’ is collected and autoclave compared with an oven.
used later as a source of free sugars, mainly The main difference between autoclaved and
fructose. Also during this step some of the sugars oven-baked agave is that careful control of
are caramelized; and some of the compounds cooking time, temperature and steam pressure
that contribute significantly to the aroma and must be maintained in autoclaves to prevent
flavor in wort formulation are due to its high overcooking or burning the agave. Overcooking
content of fermentable sugars (>10% w/v). gives a smoky taste to the tequila, increases the
Finally, the oven door is opened to allow the concentration of furfural in the final product and
cooked agave to cool. The agave is then ready reduces ethanol yield due to the caramelization
for milling. of some of the fermentable agave sugars. This
In most distilleries brick ovens have been is why some factories with both cooking systems
replaced by steel autoclaves. Autoclaves have reserve the ovens for their better-quality
superior efficiency and allow good pressure and products. Although it is easier to obtain well-
temperature control, enabling homogeneous and cooked agave in an oven than in an autoclave,
economic cooking. In a typical autoclave there is no difference in terms of flavor and
cooking operation, steam is injected for 1 hr so fermentability between agave cooked in
that steam washes the agave. This condensed autoclaves or in ovens if both are correctly
liquid is called ‘bitter honey’ and is discarded controlled.
because it contains waxes from the agave cuticle
and has a low sugar content (<1 % w/w). Steam
is injected for an additional 6 hrs to obtain a Extraction of agave juice: milling
pressure of 1.2 kg/cm 2 and a temperature of
121°C. At the end of that time the agave remains Milling has gone through three historical stages.
in the autoclave for another 6 hrs without In ancient days cooked agave was crushed with

140
Autoclave
120
Oven
Temperature (°C)

100

80

60

40

20

0
0 20 40 60 80
Time (hours)

Figure 8. Temperature profile for agave cooking in oven and autoclave.

Danisco Exhibit 1010, Page 71 of 85

232 M. Cedeño Cruz

wood or steel mallets to extract the juice. Later, board, or enzymes (cellulase and pectinase).
a rudimentary mill consisting of a large circular Many of these projects are at the laboratory stage,
stone 1.3 m in diameter and 50 cm thick was and there is not enough information to evaluate
used. Driven by animals, the stone turned in a feasibility.
circular pit containing cooked agave and In milling, as in all steps of the tequila process,
extracted the juice. The resulting juice was a sugar balance is computed to determine the
collected by hand in wooden basins and carried yield. If the yield decreases, the extraction
to fermentation tanks. By the 1950s modern pressure in the mill and the water/agave ratio are
systems were implemented in which cooked increased to improve efficiency.
agave was passed through a cutter to be
shredded (except in factories that did this
operation before cooking); and with a Fermentation
combination of milling and water extraction,
sugars were extracted. The mills used for agave WORT FORMULATION
are similar to those used in the sugarcane
industry but are smaller in size (normally 50 cm To produce 100% agave tequila, only agave may
wide). This system is still employed in most be used and the initial sugar concentration ranges
distilleries. from 4 to 10% w/v, depending on the amount of
In recent years the tequila industry has been water added in milling. When other sugars are
using a technology to extract fructose in cooked employed, they are previously dissolved and
agave or inulin in raw agave based on mixed with agave juice to obtain an initial sugar
countercurrent extraction by means of a piece concentration of 8-16%, depending on sugar
of equipment called a diffusion band. With the tolerance of the yeast strain. Wort formulation
use of this technology, used for many years in in most distilleries is based solely on previous
the extraction of sugar and alcohol in grapes in experience. A few distilleries base wort
Spain, the process has been improved and the formulation on composition of raw materials and
bagasse at the end of the extraction has nearly nutritional requirements for yeast growth and
no residual sugar. fermentation. In these distilleries response
Juice obtained in milling is mixed with the surface methodology is the preferred method to
syrup obtained in the cooking step and with a optimize nutrient concentrations, using
solution of sugars, normally from sugarcane (if fermentation efficiencies and taste of the
the tequila to be produced is not 100% agave), resulting tequila as responses (Montgomery,
and finally pumped into a fermentor. Although 1984). To correct nutritional deficiencies of
the amount of sugar employed as an adjunct is agave juice and sugars employed in the growth
regulated by law and must be less than 49% by and fermentation steps, urea, ammonium sulfate,
weight at the beginning of the fermentation, each ammonium phosphate or magnesium sulfate can
factory has its own formulation. be added. Because pH of the agave juice is
The milling step generates a by-product called around 4.5, there is no need for adjustment and
bagasse, which represents about 40% of the total the same wort composition is used for both
wet weight of the milled agave. Bagasse inoculum growth and fermentation.
composition (dry weight basis) is 43% cellulose,
19% hemicellulose, 15% lignin, 3% total
nitrogen, 1% pectin, 10% residual sugars and YEASTS
9% other compounds. The bagasse, mixed with Some companies do not inoculate a specific
clay, is used to make bricks; but it is also the strain of S. cerevisiae and instead allow natural
subject of research to find alternative uses. fermentation to proceed. Others inoculate the
Examples are use of bagasse as an animal feed wort with fresh packages of baker’s yeast or a
or as a substrate on which to grow edible fungi commercial dried yeast to obtain initial
(Iñiguez et al., 1990; 2001). Attempts are also populations of 20-50 x 106 cells/ml. The dried
underway to recover bagasse components yeasts were originally prepared for wine, beer,
(cellulose, hemicellulose and pectin) using high- whisky or bread production; and sometimes the
efficiency thermochemical reactors (Alonso et quality of the tequila obtained using these yeasts
al., 1993), to obtain furfurals, make particle is not satisfactory, with large variations in flavor

Danisco Exhibit 1010, Page 72 of 85

 Tequila production from agave 233

and aroma. To achieve high yields and maintain a pleasant aroma to the final product. The
a constant quality in tequila, some companies difference in alcohol yield in tequila production
use yeast strains isolated from a natural using a selected commercial strain of S.
fermentation of cooked agave juice. Nutrients cerevisiae compared with wild yeast is presented
are added and special conditions such as a high in Figure 9. The higher alcohol concentration at
sugar concentration or temperature are the end of the fermentation process clearly
maintained. These isolated and selected yeast means better productivity and a lower
strains have been deposited in national production cost.
microbial culture collections, the most important Fermentation temperature is important in
being the Biotechnology and Bioengineering tequila production since most of the distilleries
Department Culture Collection of CINVESTAV- are located in regions where ambient
IPN, located In México City. temperatures are normally warm, especially
Toward the goal of improving fermentation during the summer, and may be as high as 37ºC.
productivity, one alternative is to use a strain of Not all strains of S. cerevisiae are resistant to
yeast capable of both efficiently converting wort such high temperatures; and the selection of a
sugars into alcohol and of producing the commercial strain is very important as it can
appropriate organoleptic compounds that impart represent an advantage in process yield. Figure
4. 00
Wild yeast 26°C
3. 50
Alltech Thermosacc 37°C
Alcoholvolume (%)

3. 00

2. 50

2. 00

1. 50

1. 00

0. 50

0. 00
0 20 40 60 80 10 0
Fermentation time (hours)
Figure 9. Alcohol concentration in a fermentation for tequila production using wild yeast compared with
a commercial strain of yeast.
5. 00
Alltech Thermosacc 37°C
4. 50
4. 00 Alltech Thermosacc 26°C
Alcoholvolume (%)

3. 50
3. 00
2. 50
2. 00
1. 50
1. 00
0. 50
0. 00
0 20 40 60 80 10 0
Fermentation time (hours)
Figure 10. Alcohol concentration in tequila fermentation using a thermotolerant commercial strain of yeast
at temperatures of 26 and 37ºC.

Danisco Exhibit 1010, Page 73 of 85

234 M. Cedeño Cruz

10 illustrates the fact that alcohol volume in the Otherwise, microorganisms present in the wort
fermentation process for a thermotolerant strain carry out the fermentation. If an inoculum is not
is better at 37ºC than at 26ºC. added, the fermentation could last as long as
seven days. With an inoculum the fermentation
time ranges from 20 hrs in the faster process to
INOCULUM GROWTH three days in the slower one.
Production of ethyl alcohol by yeast is
When an inoculum is used, it is grown in the associated with formation of many fermentation
laboratory from a pure culture of S. cerevisiae compounds that contribute to the final flavor of
maintained on agar slants in lyophilized form the tequila. These are organoleptic compounds
or frozen in liquid nitrogen. All laboratory or their precursors produced either in subsequent
propagation is carried out under aseptic maturation of the wort before it goes to the
conditions using a culture medium with the same distillation step, in the distillation process, or in
ingredients used in the normal process but the barrels if tequila is aged. The factors
enriched to promote cellular growth. The influencing formation of the organoleptic
inoculum is scaled-up with continuous aeration compounds in alcoholic beverages have been
to produce enough volume to inoculate reviewed by many authors (Engan, 1981; Berry,
fermentation tanks at 10% of the final volume. 1984; MacDonald et al., 1984; Ramsay and
Populations of 200-300 x 10 6 cells/ml are Berry, 1984(a); Geiger and Piendl, 1976).
normally achieved. Strict cleanliness is Experience in the tequila industry is that the
maintained in this step as bacterial contamination amount of organoleptic compounds produced
is highly undesirable. When contamination is is lower in fast fermentations than in slow
detected, antibiotics or ammonium bifluoride are fermentations. As a consequence, the flavor and
used as antimicrobial agents. Once an inoculum general quality of tequila obtained from worts
is grown, it is maintained by mixing 10% of the fermented slowly is best. The rate of
volume of an active culture with fresh agave fermentation depends mainly on the yeast strain
juice and nutrients. used, medium composition and operating
Although inoculation with commercial yeast conditions, as previously explained. The wort
greatly improves yield and turnover time, some sugar content decreases from an initial value of
companies prefer a more complex (in terms of 4-11% to 0.4% (w/v) reducing sugars if an
the microbial diversity) fermentation. While efficient yeast strain is employed. Otherwise, the
yields might be lower and turnover time higher, residual sugar content could be higher,
the range of microorganisms produces more increasing production costs.
compounds contributing to a more highly Fermentation vessels vary considerably in
flavored tequila. It is also important to recognize volume, depending on the distillery. Capacity
that a change in taste and flavor could negatively ranges from 12,000 liters for small tanks to
affect the market for a particular brand of tequila. 150,000 liters for the largest ones; and they are
For this reason it is very important that when a constructed of stainless steel in order to resist
commercial strain of yeast is used in the the acidity of the wort. Ethanol production can
fermentation process, the final aroma profile in be detected almost from the onset; and a pH drop
tequila must be evaluated using a sensory from 4.5 to 3.9 is characteristic of the
evaluation panel together with gas chromat- fermentation. The alcohol content at the end of
ography analysis. fermentation is between 4 and 9% v/v,
depending on the initial sugar concentration. In
order to increase the fermentation yield, in
FERMENTATION OF AGAVE WORT
addition to selection of a good yeast strain,
another option is the use of enzymes or enzyme
Once a wort is formulated with the required complexes, to convert residual polymers from
nutrients and the temperature is around 30°C, it agave into fermentable sugars, which are
may be inoculated with 5 to 10% (volume) of a converted mainly into alcohol improving the
previously grown S. cerevisiae culture with a productivity of tequila production. Figure 11
population of 100-200 million cells/ml. shows the results of an experiment using

Danisco Exhibit 1010, Page 74 of 85

 Tequila production from agave 235

Alcoholvolume (%)
3
Control 35°

2 0.25% Rhizozyme 35°

0.5% Rhizozyme 35°

1
1% Rhizozyme 35°

0
0 20 40 60 80 10 0 12 0 14 0
Time (hours)

Figure 11. Alcohol volume in the wort at different concentrations of an enzyme complex (RhizozymeTM)
for tequila production at 35ºC.

different doses of a commercial enzyme complex compounds used in the propagation step may
in the alcohol concentration of the wort. be used here to decrease common bacterial
Alcohol losses may be significant because contaminants found in tequila worts.
many fermentation tanks are open, allowing Lactobacillus, Streptococcus, Leuconostoc, and
evaporation of alcohol with carbon dioxide. Pediococcus are the most common
Some of the largest distilleries have a cooling contaminants, but Acetobacter may be found in
system that keeps fermentation temperature fermented worts that are left inactive for a long
within a tolerable range for yeast, but small time prior to distillation.
producers do not have these systems. The In contrast to other distilled beverages, the
fermentation temperature can exceed 40°C, organoleptic characteristics of tequila come from
causing the fermentation to stop with an the raw material (cooked agave) as well as from
accompanying loss of ethanol and flavors that the fermentation process. In most of the processes
consequently decreases yields and affects the used in the tequila industry, fermentation is
quality of the tequila. Fermentations carried out spontaneous with the participation of
with pure agave juice tend to foam, sometimes microorganisms from the environment, mostly
requiring the addition of silica. In worts with yeasts and a few bacteria. This peculiarity brings
added sugars, foaming is usually not a problem. about a wide variety of compositions and
Non-aseptic conditions are employed in organoleptic properties. However, the special
fermentation, and in consequence bacterial characteristics of the wort make it a selective
activity may increase. The size of the bacterial medium for the growth of certain yeasts such as
flora depends on a number of factors including Saccharomyces and to a lesser extent, acetic and
the extent to which bacteria grow during yeast lactic bacteria. In experiments isolating microbial
propagation (if used), the abundance of bacteria flora from musts of various origins, different
on the raw materials and hygiene standards in microorganisms were isolated. In most cases,
the distillery. There is no doubt that the activity this difference in flora is responsible for the wide
of these bacteria contributes to the organoleptic variety of organoleptic characteristics of tequila
characteristics of the final product. Occasionally, brands (Pinal, 1999). Where a single purified
the size of the bacterial population in fermenting strain is used, the final flavor and aroma are
wort may become too large (>20 x 10 6 cells/ more neutral since the bouquet created by the
ml), in which case the bacteria use the sugars, contribution of several stains is richer than that
decreasing ethanol yields and sometimes obtained from only one type of yeast. Moreover,
excreting undesirable compounds. The same when yeast produced for bakery applications is

Danisco Exhibit 1010, Page 75 of 85

236 M. Cedeño Cruz

used, the final product is also more neutral. It is whisky (Ramsay and Berry, 1984(b)) and beer
also recognized that when non-100% agave (García et al., 1994).
tequila is made, a poorer bouquet is obtained The carbon:nitrogen ratio also has a significant
because a more defined medium yields a more influence on higher alcohol production. In
defined product. tequila musts, which contain mainly fructose
(≈95%) as a carbon source and an inorganic
nitrogen source (ammonium sulfate), it was
ORGANOLEPTIC COMPOUNDS GENERATED found for both native and bakery yeast strains
DURING FERMENTATION that low carbon:nitrogen ratios result in low
amounts of isoamyl alcohol: 19 mg/L in bakery
Fusel oil strains and 30 mg/L for native strains vs 27 and
As in many other alcoholic fermentation 64 mg/L, respectively, for high carbon:nitrogen
processes, higher alcohols are the most abundant ratios. A similar relationship exists for isobutyl
compounds produced along with ethanol. We alcohol production (Figures 12 and 13).
have found (in decreasing order of abundance) Temperature is a third factor affecting isobutyl
isoamyl alcohol, isobutanol, active isoamyl and isoamyl alcohol production with higher
alcohol and phenylethanol. It has been temperatures (e.g. 38 vs 32°C) yielding higher
established that production of isoamyl and concentrations of those alcohols. On statistical
isobutyl alcohols begins after the sugar level is analysis it was found that in addition to the direct
lowered substantially and continues for several effects of yeast strain, carbon:nitrogen ratio and
hours after the alcoholic fermentation ends. In temperature, the interaction of these three factors
contrast, ethanol production begins in the first also had an impact on higher alcohol
hours of the fermentation and ends with concentration in tequila (Pinal et al., 1997).
logarithmic yeast growth (Pinal et al., 1997). These results are consistent with the fact that with
The most important factor influencing the high carbon:nitrogen ratios there is a tendency
amount of isoamyl alcohol and isobutanol is the to use amino acids as a nitrogen source, which
yeast strain. It was found that a native strain implies the production of fusel oil as a by-
isolated from tequila must produces a higher product (by the Erlich pathway). On the other
amount of such compounds when compared hand, variables such as the type of nitrogen
with a strain usually employed in bakeries. These source (urea or ammonium sulfate) or the
results agree with those reported for Scotch amount of inoculum used for fermentation had

8.4 140

120
Log cells/ml
8.0 100
Reducing sugars, g/L
Ethanol, g/L 80

60

40

20
Isoamyl + isobutyl alcohols, mg/L
7.2 0
0 5 10 15 20 25 30 35
Time (hrs)

Figure 12. Higher alcohol production in tequila wort by a baker’s yeast strain.

Danisco Exhibit 1010, Page 76 of 85

 Tequila production from agave 237

8.4 140

120
Log cells/ml
8.0 100
Reducing sugars, g/L

80

60

40
Ethanol, g/L
Isoamyl + isobutyl 20
alcohols, mg/L
7.2 0
0 5 10 15 20 25 30 35
Time (hrs)

Figure 13. Higher alcohol production in tequila wort by a native yeast strain.

little or no effect on the production of higher amounts of methanol in musts with the same
alcohols. Pareto diagrams involving all the composition but fermented with different strains
variables tested are depicted in Figures 14 and (Téllez, 1999).
15.
Aldehydes
Methanol
Along with the production of ethyl acetate, the
Another characteristic compound present in oxidation of ethanol also generates acetal-
tequila is methanol. It is generally thought that dehyde, an intermediate in the production of
methanol is generated through hydrolysis of acetic acid. It is well known in commercial
methylated pectins present in the agave plant. practice that an oxidation process instead of
Nevertheless, it is also believed that some yeast fermentation begins after the sugar concentration
strains, natural or inoculated, have pectin methyl declines, thus provoking the increase in
esterases. Some authors describe various acetaldehyde levels. However, there is no formal

A -26.68
21.61
AD -15.56
10.32
BD 8.34
-7.38
ABD -6.71
Factors

-6.41
ABE 5.61 A: Strain
-5.10
B: Temperature
BE -4.65
3.70 C: Nitrogen source
CDE -3.09 D: Carbon:nitrogen ratio
-2.53 E: Inoculum amount
ACE 2.50
-1.93
E 1.00

0 10 20 30 40 50
Effect level

Figure 14. Pareto diagram for isoamyl alcohol production in tequila.

Danisco Exhibit 1010, Page 77 of 85

238 M. Cedeño Cruz

A -41.85
16.33
AD -13.41
12.94
BD -12.73
-8.47
ABD 8.13
Factors

7.35
ABE 6.99 A: Strain
-6.56
B: Temperature
BE 6.56
C: Nitrogen source
-6.12
CDE -6.03 D: Carbon:nitrogen ratio
-6.01 E: Inoculum amount
ACE 5.66
3.84
E 1.55

0 10 20 30 40 50
Effect level

Figure 15. Pareto diagram for isobutyl alcohol production in tequila.

report of such phenomena in tequila, as is ethanol. Acetic acid can also be produced by
described in beer (Hammond, 1991). the oxidation of ethanol when the fermentation
has ceased and an oxidative process starts on
Organic acids the surface of the fermentation tank by
Saccharomyces and many other yeasts such as
Small organic acids (up to six carbons) and larger Brettanomyces. Therefore, long fermentation
molecules (fatty acids) are produced during periods (a current practice in the tequila
fermentation. The smaller molecules can be industry) yield high ethanol oxidation. In
products of intermediate metabolism of the addition, in open fermentation tanks with worts
normal microbial flora; and their production at low pH containing alcohol, ethanol is also
depends on the presence of oxygen. The larger transformed to acetic acid (itself a precursor of
fatty acids are synthesized for membrane ethyl acetate) by bacteria of the genera
structures during cell growth and can also Acetobacter. Besides ethyl acetate, the presence
appear at the end of the fermentation when lysis of several other esters has been described
takes place. The presence of octanoic and including ethyl and isoamyl esters. Some of the
decanoic acids in the final product has been most important esters found in silver tequila are
described particularly for tequila. listed in Table 2.

Esters Table 2. Most abundant esters found in silver tequila.1

Esters are very important compounds in their Ester %
particular contribution to flavor and aroma, since Ethyl acetate 17.77
they have the lowest organoleptic threshold Ethyl decanoate 2.78
values (Ramsay and Berry, 1984a). In particular, Ethyl lactate 2.74
ethyl acetate is the most abundant and important Ethyl octanoate 1.92
compound of this family. Ethyl acetate has been Ethyl dodecanoate 0.95
reported to be the second most abundant Ethyl butanoate 0.63
Isoamyl acetate 0.58
compound in tequila after isoamyl alcohol. The Ethyl propanoate 0.57
quantity of this compound present in the final Ethyl hexanoate 0.48
product can vary widely, since it is synthesized Ethyl hexadecanoate 0.48
from acetic acid (in the form of acetyl CoA) and 1
Estarrón et al., 1999.

Danisco Exhibit 1010, Page 78 of 85

 Tequila production from agave 239

Distilling
Distillation involves the separation and of trays. Steam is injected from the bottom in a
concentration of the alcohol from the fermented coil and strips the wort of its volatile
wort. In addition to ethanol and other desirable components. Vapors condense higher in the
secondary products, wort contains solid agave column, depending on component volatility,
particles consisting mainly of cellulose, pectin, allowing liquids to be drawn off or recycled at
and yeast cells in addition to proteins, mineral the various plates as appropriate. Sometimes
salts and some organic acids. Although many tequila obtained in this way is mixed with tequila
types and degrees of distillation are possible, the from pot stills to balance the amount of
most common systems used in the tequila organoleptic compounds because in general,
industry are pot stills and rectification columns. tequila obtained through continuous columns
The pot still is considered the earliest form of has less aroma and taste than tequila obtained
distilling equipment. It is of the simplest design, from pot stills.
consisting of a kettle to hold the fermented wort, The presence of methanol in tequila is still a
a steam coil, and a condenser or a plate heat subject of discussion because whether methanol
exchanger. Pot stills are often made of copper, is produced only by a chemical reaction or in
which ‘fixes’, according to Thorne et al. (1971), combination with a microbial hydrolysis has not
malodorous volatile sulfur compounds produced been satisfactorily demonstrated. The chemical
during fermentation. Batch distillation using pot reaction is demethylation of agave pectin by the
stills is carried out in two steps. First the high pH during cooking and the first distillation
fermented wort is distilled to increase the alcohol steps. The microbial reaction could be the
concentration to 20-30% by volume, separating hydrolysis of agave pectin by the enzyme pectin
out the first fraction called ‘heads’, and the last methyl esterase produced by some
fraction, called ‘tails’. Composition of these microorganisms during the fermentation step, but
fractions varies depending on many factors this has not yet been demonstrated. Preliminary
including the yeast strain employed, wort results favor the first theory, but research is still
nutrient composition, fermentation time and needed on this matter because it is important to
distillation technique; but in general, heads are maintain the methanol concentration within the
rich in low boiling point compounds such as limits established by the official standard, which
acetaldehyde, ethyl acetate, methanol, 1- is 300 mg per 100 ml of anhydrous ethyl alcohol.
propanol, 2-propanol, 1-butanol, and 2-methyl Table 3 shows the specifications for tequila
propanol, which give a very pleasant flavor and according to the standard of quality from the
taste to tequila. Heads are normally mixed with Mexican Official Norm (Secofi, 1994).
the wort being distilled. The tails contain high
boiling point components such as isoamyl
alcohol, amyl alcohol, 2-furaldehyde, acetic acid Effluent disposal
and ethylactate, giving a strong taste and flavor
to the tequila; and when the concentration is The discharge from pot stills or distillation
above 0.5 mg/ml, the final product becomes columns is known as stillage, slops or vinasse;
unpleasant. This fraction is not used. and in a typical tequila distillery 7 to 10 liters of
In the second step, the liquid obtained from effluent are produced per liter of tequila at 110º
the first stage is re-distilled in a similar pot still proof. Tequila stillage has a biological oxygen
in order to obtain a final product that is 110° demand (BOD) of 25 to 60 g/L. In addition to
proof if it is sold in bulk (reducing transport the dissolved salts (mainly potassium, calcium,
costs) or 80° proof if it is to be bottled. Some and sulfate ions) and the low pH (<3.9) of the
companies obtain high proof tequila and dilute stillage, there are significant disposal or treatment
it with demineralized water or water purified by problems. A general solution to the disposal
reverse osmosis. problem does not exist because every factory
In continuous distillation systems, the has its own production process and is located
fermented wort enters the feed plate of the either in a city or near agave fields. As a result
column and flows downward, crossing a series of the difficulties of treating vinasses and due to

Danisco Exhibit 1010, Page 79 of 85

240 M. Cedeño Cruz

Table 3. Tequila specifications according to NOM.

Tequila blanco Tequila joven u oro Tequila reposado Tequila añejo

(White tequila) (Gold tequila) (Aged) (Extra-aged)
min max min max min max min max

Alcohol, % bv at 20ºC 38.0 55.0 38.0 55.0 38.0 55.0 38.0 55.0
Dry extract, g/L 0 0.20 0 5.0 0 5.0 0 5.0

Values expressed as mg/100 ml pure ethanol

Higher alcohols 20 400 20 400 20 400 20 400

(as amyl alcohol1)
Methanol2 30 300 30 300 30 300 30 300
Aldehydes 0 40 0 40 0 40 0 40
Esters 2 270 2 350 2 360 2 360
Furfural3 0 1 0 1 0 1 0 1
1
Using a Gas Chromatograph the value could be up to 500 mg/100 ml.
2
Minimum could be less than this value if the distillery could demonstrate the use of a technology to reduce methanol
concentration.
3
Using chemical analysis the maximum value could be up to 4 mg/100 ml.

their high concentrations of dissolved matter, a and Marchant, 1980). Stillage may be used as a
host of utilization schemes have been proposed. food supplement for cattle, but it has an
Some of the methods indicated below are under undesirable laxative effect on animals.
investigation and others are in use. Biological, aerobic, or anaerobic treatment offers
Recycling reduces the volume of waste to be a real means of disposal, but the cost is likely to
treated. Stillage can be recirculated, mixing 5 to be as high as the fermentation costs themselves
10% of the total volume of the waste obtained (Speece, 1983; Maiorella, 1983). Ultimately,
with clean water to substitute for the dilution tequila vinasses should be viewed as a raw
water used to prepare the initial wort. This can material rather than a waste, and a strategy
be carried out for a number of cycles, usually should be devised that maximizes economic and
no more than five, because the concentration of social benefits and reduces recovery costs.
dissolved salts increases and could affect the
fermentation process. Also, great care must be
taken with the final taste and flavor of the tequila Maturation
because some components present in stillage
could affect the organoleptic characteristics of Distillation is the final stage of tequila production
the final product. Currently, only one tequila if silver or white tequila is the desired product.
company uses this system. For rested (reposado) or aged (añejo) tequila,
Direct land application as irrigation water and maturation is carried out in 200 liter white oak
fertilizer in agave fields is under careful casks or in larger wood tanks. The time legally
evaluation to determine the optimum loading required is two months for rested tequila and 12
rates and the effects on the agave over the long months for aged tequila. Tequila is generally
time needed to reach maturity. Evaporation or matured for longer periods, depending on the
combustion of stillage could provide fertilizer characteristics each company desires for its
or potash, but the high cost of such a process is particular brand.
a serious limitation (Sheenan and Greenfield, As tequila ages in barrels, it is subject to
1980). The production of biomass and changes that will determine its final quality.
biochemicals including fodder yeast is a Thickness and quality of the stave, depth of the
possibility (Iñiguez et al., 1996), but the char, temperature and humidity in the barrelling
remaining liquor still has a high BOD (Quinn area, entry proof (40-110° proof), length of

Danisco Exhibit 1010, Page 80 of 85

 Tequila production from agave 241

storage and number of cycles for the barrel (in million liters of tequila (at 40% alcohol by
Mexico barrels may be re-used several times) volume) in 1999 (Figure 16). After that, a
all affect the final taste and aroma of the tequila. shortage of agave occurred due to a combination
Fusel oil content decreases during maturation of environmental and market factors. A bacterial
owing to the adsorbant nature of the char, infection (Erwinia carotovora) and a fungus
smoothing the final product. Complex wood (Fusarium oxysporum), affected agave harvests,
constituents are extracted by the tequila, while the growing number of distilleries,
providing color and the particular taste. development of many new brands in the market,
Reactions among certain tequila compounds and an increase in tequila consumption in the
yield new components; and oxidation reactions domestic and export market affected demand.
change some of the original components in The drop in production began in 2000 with a
tequila and those extracted from the wood. As a reduction to 181.6 million liters and in 2002
result of all of these changes, the concentration declined to 141 million liters.
of acids, esters and aldehydes is increased, while Production of 100% agave tequila had the most
the concentration of fusel oils decreases as pronounced decrease due to the agave shortage,
tequila reposes in barrels. because 6.0 kg of agave are required to produce
After aging and dilution with demineralized 1 liter of tequila (at 55% abv) whereas other
water (if necessary) the color of the tequila may tequilas require only 3.0 kg of agave per liter.
be adjusted to the desired value by the addition Most tequila producers moved into production
of caramel. Alternatively, some companies blend of other tequila types and reduced amounts of
different batches of tequila to obtain a 100% agave tequila.
standardized final product. The outlook for the future appears promising
Government inspectors supervise the entire for tequila production, and a return to previous
aging process. Prior to bottling, tequila is filtered industry growth rates is expected by the end of
through cellulose filter pads or polypropylene 2003.
cartridges. Sometimes afterwards a pretreatment In the export market the scenario was very
with charcoal is used to eliminate turbidity. similar but the effect of the agave shortage lasted
through 2002 since the main export companies
sell tequila in bulk (Figure 17). Total export
Production statistics volume was at its lowest in 2001 (75.6 million
liters of tequila at 40% abv) with the beginning
Tequila production has grown since 1995 at an of recovery noted in 2002. Exports of 100%
average rate of more than 10% annually and agave tequila became almost stable at a volume
reached its maximum production of 190.6

200 Tequila100%
Tequila
Total
150

100

50

0
1995 1996 1997 1998 1999 2000 2001 2002

Figure 16. Tequila production in Mexico in millions of liters at 40% abv (CRT, 2002).

Danisco Exhibit 1010, Page 81 of 85

242 M. Cedeño Cruz

Tequila100%
10 0
Tequila
Total
80

60

40

20

0
1995 1996 1997 1998 1999 2000 2001 2002

Figure 17. Exports of tequila by type in million liters at 40% abv (CRT, 2002).

near 8 million liters. Again, it should be noted million liters of tequila at 40% abv), this has been
that this type of tequila can only be exported growing since 1995 when 6.5 million liters were
bottled and not in bulk. state-bottled (Figure 19).
Although the European and other markets are
growing, nearly 80% of all tequila exported
continues to go to the US market with volumes Future developments
that in 2002 represented over 69 million liters
of tequila at 40% abv, which is more than the 53 Research and future developments in the
million liters consumed in the domestic market production of tequila and agave cultivation can
(Figure 18). Finally, even though only 25% of take many directions, but the implementation of
the tequila is exported bottled during 2002 (22 any change must allow quality of the final

10 0 USA Europe Other Total

80

60

40

20

0
1995 1996 1997 1998 1999 2000 2001 2002

Figure 18. Exports of tequila by destination in million of liters at 40% alcohol in volume (CRT 2002).

Danisco Exhibit 1010, Page 82 of 85

 Tequila production from agave 243

10 0
Bulk
Bottled
80
Total

60

40

20

0
1995 1996 1997 1998 1999 2000 2001 2002

Figure 19. Exports of tequila in millions of liters at 40% abv (CRT, 2002).

product to be maintained and provide substantial knowledge of distillation techniques, that tequila
improvement in the process. There are several acquired its present form. The agave plant, which
key areas where important developments could is often confused with cacti, is propagated
take place. Development of new varieties of through traditional methods or by means of plant
agave endowed with resistance to pests or cell culture. It is grown for 7 to 8 years before it
extremely dry environments, use of can be harvested and sent to the distillery.
micropropagation techniques, satellite remote The tequila production process comprises five
inspection to evaluate health status of agave stages. In the first step, agave is cooked to
plantations, higher inulin content, low wax hydrolyze the polymers present in the plant,
content in the leaf cuticle and superior growth mainly inulin, into fermentable sugars. In some
rates would all be of benefit. Mechanization factories this step is accomplished using stone
would improve aspects of cultivation and harvest ovens and in others it is carried out in autoclaves.
of agave. In addition, optimization of the The second stage is sugar extraction from cooked
cooking and fermentation steps would improve agave through milling; and the agave juice
yields and reduce the amount of waste, while obtained in this step can be mixed with sugars
yeast strain selection could improve ability to from other sources, normally sugar cane, if 100%
ferment musts with high concentrations of sugars. agave tequila is not desired. The third and most
Another area of opportunity is the use of isotope important stage is fermentation in which sugars
(H2 and O18) measurement in tequila to authenticate are transformed into ethanol and other
the beverage. Finally, low cost alternatives for compounds such as esters and organic acids.
waste (bagasse and stillage) treatment are These, along with other substances derived from
needed. the cooked agave, give the characteristic flavor
and taste to tequila. It is of great importance to
have a good yeast strain and nutritionally
Summary balanced wort for tequila production, as losses
can be as high as 35% of the total production if
Tequila is a beverage obtained from the inefficient yeast is used or nutrients are not
distillation of fermented juice from the agave present in the right proportions. In the fourth
plant (Agave tequilana. Weber var. azul). The stage, fermented wort is distilled, normally using
use of agave to produce drinks in Mexico dates pot stills or in some cases rectification columns,
from long ago when certain tribes used them to obtain the final product. At the end of the
for religious ceremonies. It was not until the distillation process white tequila is obtained.
arrival of the Spaniards, who brought Maturation, the last stage, in white oak barrels

Danisco Exhibit 1010, Page 83 of 85

244 M. Cedeño Cruz

is required for rested or aged tequila. The Diguet. L. 1902. Estudio sobre el maguey de
minimum maturation times are 2 and 12 months, tequila. Generalidades e historia. El Prog. Mex.
respectively, for rested and aged tequila as 9:424.
required by government regulations. At every Ehrler, W.H. 1967. Agave plant, efficient water
step of the production process, most companies user. USDA Agr. Res. 16(4):11.
employ several quality control analyses in order Engan, S. 1981. Beer composition: volatile
to ensure the quality of the product and the substances. In: Brewing Sciences, Vol. 2
efficiency of the process. Some major producers (J.R.A. Pollock, ed.) Academic Press, London,
of tequila are certified through an ISO-9000 p. 98.
standard. Tequila production is governed by the Estarrón, M., T. Martín del Campo and R. Cosío.
official norm NOM-006-SCFI-1994, which must 1999. Identificación de los componentes
be followed by all tequila producers to guarantee
volátiles que caracterizan la huella
a good quality final product.
cromatográfica distintiva de tequilas. Technical
Report for Tequila Herradura S.A.
García, A.I., L.A. García and M. Díaz. 1994.
Acknowledgement
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to Tequila Herradura, S.A. de C.V., for its support GEA. 1992. La fertilización, La Jima. Boletin
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