Blue Book on Yeast Updated Sept 2009 Final

An Introduction to

BREWING SCIENCE

& TECHNOLOGY

Series III

BREWER'S YEAST

THE INSTITUTE OF BREWING

An Introduction to

BREWING SCIENCE

& TECHNOLOGY

Series III

BREWER'S YEAST

G.G. Stewart1 and I. Russell2

('Heriot-Watt University, 2Labatt Brewing Company Limited)

THE INSTITUTE OF BREWING

ACKNOWLEDGEMENTS

The authors wish to thank their colleagues who have contributed to this book. To keep the

size small and easily readable, references to specific publications have not been used (with

the exception of where figures were adapted), but the list of source books used is included.

The authors also owe a special debt of gratitude for assistance with particular sections: Robert

Stewart, molecular biology; Heather Pilkington, biochemical pathways and viability;

Normand Mensour, immobilised cell technology; and Jadwiga Sobczak, light and electron

micrographs. Special thanks are due to Karen Ross for preparation of the figures and to

Dorothy Filsell and Janice Riddell for careful typing and editing of the manuscript.

ISBN No. 0900489 13 8

Copyright © 1998 The Institute of Brewing

All rights of reproduction are reserved in all countries in respect of all texts and illustrations.

No part may be reproduced or utilized in any form without written permission from the Institute of Brewing.

Published by: The Institute of Brewing. 33 Clarges Street, London W1Y 8EE, England.

BREWER'S YEAST

G.G. Stewart1 and I. Russell2

( Heriot-Watt University, 2Labatt Brewing Company Limited)

Contents

Introduction 3

Fundamentals 3

Characteristics of Brewing Yeasts 5Yeast Morphology 7

Yeast Cell Growth and Division 13

Genetic Characterisation of Yeast 14

Genetic Tests for Typing Yeast Strains 23Brewer's Yeast Performance 28

Uptake and Metabolism of Wort Nutrients 29

Wort Sugars and Carbohydrates 29

Control ofYeast Metabolism 35Pasteur effect 35

Crabtree effect (glucose repression, catabolite repression) 35Amino Acids, Peptides and Proteins 35

Oxygen ].".'.'."".'.'. 37Vitamins 40

Ions 41

Inorganic ions 41

Hydrogen ions 43

Potassium ions 43

Sodium ions 44

Divalent metal cations 44

Magnesium ions 44

Manganese ions 45

Calcium ions 45

Zinc ions 45

Copper and iron ions 45

Yeast Excretion Products 45

Organic and Fatty Acids 47

Higher Alcohols 47

Esters 49

Carbonyls 51

Sulphur Compounds 55

Flocculation 57

Yeast Management 62

Pure Yeast Cultures 62

Preservation of Stock Yeast Culture 64

Yeast Pitching and Cell Viability 64

Yeast Collection 65

Yeast Storage 66

Yeast Storage Conditions - Influence on Intracellular Glycogen

and Trehalose Levels 66

Yeast Washing 69

Contamination of Cultures with Bacteria 71

Contamination of Cultures with Wild Yeast 72

Yeast Cell Viability and Vitality 74

Use of Specific Dyes for Assessing Cell Viability and Vitality 74

Capacitance 74

The Power of Reproduction as a Viability Indicator 74

Viability and Vitality Methods Based on Cell Metabolic State 75

Adenosine triphosphate (ATP) 75

NADH fluorosensor 75

Specific oxygen uptake rate (BRF yeast vitality test) 77

Acidification power 77

Intracellular pH (ICP) method 77

Measurement of yeast vitality by stress response 77

Magnesium release test (MRT) 77

Electrokinetics 78

High Gravity Brewing 78

Continuous Fermentation 81

Immobilised Yeast Technology 84

Production of Alcohol-free and Low Alcohol Beers 86

Immobilised Lager Yeast to Reduce Maturation Times 87

Primary Fermentation with Immobilised Yeast 89

Distiller's Yeast 93

Malt and Grain Whisky 94

Ethyl Carbamate 97

Supplementary Readings 99

Internet Web Sites 100

Index 102Illustrations (Figures) 106

Tables 108

INTRODUCTION

The characteristic flavour and aroma of any beer is, in large part, determined by the yeast

strain employed. In addition, properties such as flocculation, fermentation ability (including

the uptake of wort sugars), ethanol tolerance, osmotolerancc and oxygen requirements have

a critical impact on fermentation performance. Thus, proprietary strains belonging to

individual breweries are usually (but not always) jealously guarded and conserved, however

this is not always the case. In Germany, most of the beer is produced with only four lager

strains and approximately 65% of the beer is produced with one strain!

FUNDAMENTALS

Yeasts arc non-photosynthetic, relatively sophisticated, living, unicellular fungi,

considerably larger in size than bacteria. Yeasts arc of benefit to mankind because they are

widely used for production of beer, wine, spirits, foods and a variety of biochcmicals.

Yeasts also cause spoilage of foods and beverages, and some species of yeast arc of medical

importance. At present, approximately 700 yeast species are recognised but only a few have

been adequately characterised. No satisfactory definition of yeasts exists, and commonly

encountered properties such as alcoholic fermentation and growth by budding arc not

universal in yeast [all brewer's yeast strains multiply by budding (Figure 1)]. There are

many definitions to describe the yeast domain, however, one that best describes the group

is: "Yeasts are unicellularfungi which reproduce by budding or fission". Yeasts are both

quantitatively and economically the most important group of microorganisms commercially

Figure 1. Electron micrograph of a budding yeast cell.

exploited on this planet. The total amount of yeast produced annually, including that

formed during brewing, wine-making, and in distilling practices, is of the order of a

million tonnes. Many microbiologists and fermentation technologists employ the term

"yeast" as synonymous with Saccharomyces cerevisiae. Although this yeast species is

of critical economic and biochemical importance, and most of the research on yeast has

been conducted on it, there are many exotic varieties of yeast species that offer

advantages for experimental studies. Nevertheless, the genus Saccharomyccs has often

been referred to as "the oldest plant cultivated by mankind". Indeed, the history of beer,

wine and breadmaking with the fortuitous use of yeast is as old as the history of

mankind itself. Many species of Saccharomyces are GRAS (Generally Regarded As Safe)

and produce two very important primary metabolites - ethanol and carbon dioxide.

The ethanol is used in both beverages and as a fuel, solvent and sterilant. The carbon

dioxide is employed for leavening in baked goods, for carbonation of beverages, as a

solvent in the liquid state (for example, for the production of hop extracts), and in

the culturing of vegetables and flowers in greenhouses under controlled environmental

conditions. In addition, there are a number of other important uses for yeast,

including cultures that have been genetically transformed to produce important non-yeast

proteins and peptides, such as the antiviral protein interferon, human serum albumin,

insulin and the acid protease chymosin used in the milk-clotting steps during cheese

production.

Brewer's yeast are of the genus Saccharomyces. In an acidic aqueous solution (wort), they

adsorb dissolved sugars, simple nitrogenous matter (amino acids and very simple peptides),

vitamins, ions, etc., through their outside cell membrane (the plasma membrane). Then

they employ a structured series of reactions known as metabolic pathways to use these

substances for growth and fermentation.

As a group of microorganisms, yeasts are capable of utilising a broad spectrum of

carbohydrates and sugars. Nevertheless, none of the yeast species isolated to date from

natural environments have been found capable of utilising all of the readily available sugar

carbohydrates. Saccharomyces cerevisiae has the ability to take up a wide range of sugars,

for example, glucose, fructose, mannose, galactose, sucrose, maltose, maltotriose and

raffinosc. In addition, as will be described in detail later, the closely related species

Saccharomyces diastaticus and Saccharomyces uvarum (carlsbergensis) (lager yeasts) are

able to utilise dextrins and melibiose respectively. However, Saccharomyces cerevisiae

and the related species are not able to metabolise all sugars. Examples of carbohydrates and

sugars in this category are pentose sugars (for example, ribose, xylose and arabinosc),

cellobiose (hydrolysis products of hemicellulose and cellulose), lactose (milk sugar), inulin

and cellulose.

Enzymatic hydrolysis of starch, as would occur during mashing, leads to a medium (wort)

consisting of a number of simple sugars. As a result, the fermentation of such a medium

requires that the yeast culture is able to metabolise several sugars either together or

sequentially. Further, as will be discussed in detail later, the repressive effects of one sugar

on the uptake of another have a profound influence on both the rate and extent of

fermentation.

Brewer's yeast strains are facultative anaerobes; that is, they are able to grow in the

presence or absence of oxygen. The formation of ethanol occurs via the Embden-

Meyerhof-Parnas Pathway (also called the Glycolytic Pathway) where, theoretically, I g of

glucose will yield 0.51 g of ethanol and 0.49 g of CO2. However, because some of the

glucose is used for cell growth (biomass production), it is more realistic to consider an

ethanol yield of 0.46 g of ethanol and 0.44 g of CO2 from 1 g of glucose. The glycolytic

pathway operates to convert glucose to pyruvic acid, energy and reduced nicotinamide

adenine dinucleotide (NADH - H+). The reaction can be summarised as:

glucose + 2 ADP + 2 Pi + 2 NAD+ = 2 Pyruvate + 2 ATP + 2 NADH + 2 H+

Heat is also produced during the reaction, although much of the energy liberated from the

biochemical steps is conserved by the yeast and stored as adenosine triphosphate (ATP) for

later use in biosynthctic reactions.

Brewer's yeast strains arc not very tolerant of high concentrations of acidic end products

such as pyruvic acid. Through evolution, they have developed a method to "detoxify" this

acidic end product by converting the pyruvic acid into CO2 and ethanol, both of which are

excreted out of the cell. As a result of this reaction, NADH formed during glycolysis is

reoxidised to NAD, which is then available to participate again in glycolysis. In this way,

the yeasts are able to continue to grow and metabolise sugar. The two-step reaction leading

to ethanol can be written:

NADH + H+

CH3 COCOOH » CO, + CHjCHO > CH3CH2OH

pyruvate pyruvate carbon dioxide + alcohol cthanol

decarboxylase acetaldchydc dehydrogenase

As will be discussed later pyruvate acts as precursor of many other key metabolites such as

esters, carbonyls and higher alcohols.

CHARACTERISTICS OF BREWING YEASTS

Identifying, naming and placing organisms in their proper evolutionary framework is of

importance to many areas of science that include agriculture, medicine, the biological

sciences, biotechnology and the food and beverage industries. Taxonomic concepts change

as a result of developments in science and philosophy. As a consequence, over the years,

several different species concepts have been applied to yeast systematics and taxonomy.

Microbiologists have studied yeast taxonomy for well over a century but, despite

considerable progress particularly as a result of developments in molecular biology, the

task of developing an accurate system of classification is far from complete. The need for

reliable identification is readily apparent for a number of reasons including the selection of

appropriate organisms for industrial fermentations such as brewing.

It is at the strain level that interest in brewing yeast centres. There are at least 1,000

separate strains of the species Saccharomyces cerevisiae. These strains include brewing.

baking, wine, distilling and laboratory cultures. There is a problem classifying such strains

in the brewing context; the minor differences between strains that the taxonomist dismisses

as inconsequential are of great technical importance to the brewer. The two main types of

beer, lager and ale, are fermented with strains belonging to the species Saccharomyces

uvarum (carlsbergensis) and Saccharomyces cerevisiae respectively. Currently, yeast

taxonomists have assigned to the species Saccharoinyces cerevisiae all strains employed in

brewing, indeed, increasingly they are referred to in the scientific/technical literature as

Saccharomyces cerevisiae (ale type) and Saccharomyces cerevisiae (lager type). However,

there arc several biochemical differences between these two types of yeast strains that

warrant maintaining them as separate entities. For example, they have been distinguished

on the basis of their ability to ferment the disaccharide melibiose (glucose-galactosc).

Strains of Saccharomyces tivarum (carlsbergensis) (lager type) possess the MEL gcne(s).

They produce the extracellular enzyme a-galactosidase (nielibiase) and are therefore able

to utilise melibiose. However, strains of Saccharomyces cerevisiae (ale type) do not possess

the MEL gene(s), consequently do not produce a-galactosidase, and are therefore unable to

utilise melibiose (Figure 2). Also, ale strains can grow at 37°C, whereas lager strains

cannot and this can be used as a distinguishing test.

Saccharomyces carlsbergensis (uvarum)

Raffinose

Galactose ■ ' Glucose' ' Fructose

Melibiase Invertase

Melibiose

Galactose ■ ■ Glucose

Melibiase

Saccharomyces cerevisiae

Raff/nose

Figure 2. Utilisation of the sugar

raffinosc and melibiosc by lager

ISaccharomyces uvarum

(carlsbergensis)] and ale

(Saccharomyces cerevisiae) yeast.

(Note: Saccharomyces cerevisiae does

not possess the enzyme melibiase.)

Galactose - ' Glucose - - Fructose

Invertase

Traditionally, lager is produced by bottom-fermenting yeasts at fermentation temperatures

between 7 and 15°C, and at the end of fermentation, these yeasts flocculate and collect at

the bottom of the fermenter. Top-fermenting yeasts, used for the production of ale at

fermentation temperatures between 18 and 22°C, at the end of fermentation form into loose

clumps of cells, which are adsorbed to carbon dioxide bubbles, and are carried to the

surface of the wort. Consequently, top yeasts are collected for reuse from the surface of the

fermenting wort (a process called skimming), whereas bottom yeasts are collected (or

cropped) from the fermenter bottom. As will be discussed later, the differentiation of lagers

and ales on the basis of bottom and top cropping has become less distinct with the advent

of cylindro-conical fermenters and centrifuges.

Novel methods of strain characterisation and identification will be discussed later, however,

a traditional method for this purpose that still has merit today is the Giant Colony Method.

This method involves inoculating a yeast culture onto solid media and examining the

colonial morphology that develops following incubation under standard conditions. It has

been found that gelatin, as the solidifying matrix with wort, tends to enhance the distinctive

features of the colonial morphology to a greater extent than does agar (Figure 3) and that

every strain of ale yeast has its own characteristic colonial morphology when cultured on

wort-gelatin. Lager yeast strain colonies however are not so distinctive and tend to have a

Figure 3. Giant colony morphology on wort gelatin plates of (A) a typical lager strain,

and (B) a typical ale yeast strain (Grown at 21°C for 21 days).

more uniform morphology. This method has two major shortcomings. Firstly in order to

obtain the characteristic colonial morphologies at least three weeks incubation at 21°C is

required. Secondly, it gives no information on the value of a particular strain for brewing

purposes. At a brewing congress nearly thirty years ago it was stated: "It is important to

realise that this procedure (the giant colony procedure) is rather like taking photographs of

those in this hall. The photographs would enable us to identify the individuals elsewhere

but would tell us nothing of their performance as maltsters, brewers and scientists".

YEAST MORPHOLOGY

Although brewing dates back to prehistory, it was not until 1841 that Mitcherlich

discovered that yeast was essential for fermentation. This was followed by Pasteur and

Buchner's fundamental studies that confirmed that yeast was responsible for the fermentation

of wort to beer. This research showed that alcohol and carbon dioxide are major by-

products of carbon metabolism and that the "non-living" zymase enzyme system is

responsible for the fermentation of sugar. Yeasts are quite small cells in size [5-10 microns

(1 micron = 1 n= 10"6 metres = 1CH centimetres)]. Individual cells are invisible to the

naked eye and require a microscope to be detected. Since Pasteur's time, it has become

clear that a most important part of the brewing process is the proper control of unwanted

micro-organisms (for example, bacteria and wild yeasts) and the careful management of the

brewing process.

Unstained cells exhibit little detail with the light microscope and even when inclusions in

the cytoplasm are recognisable, it is difficult to know whether they represent vacuoles,

granules or nuclei. Although more information can be obtained by using specific stains, it

is since the advent of the electron microscope that a clear picture of the yeast cell has

emerged. The cell is bounded by a thick cell wall. Inside it is impossible to recognise many

of the features of a typical cell: plasmalemma or plasma membrane, nucleus, mitochondria,

endoplasmic reticulum, vacuoles, vesicles and granules (Figure 4).

Mitochondrion

Bud vacuole

Nucleus

Golgi complex

Pore in nuclear

membrane

Vacuole

Endoplasmic reticulum

Vacuolar membrane

Lipid granule

Bud scar —i

Cell membrane

Cell wall -

Vacuolar

granules

Storage granule

Thread-like mitochondrion

Figure 4. Main features of a typical yeast cell.

manno-

protein Glucan

Glucan

Figure 5. Electron micrograph of a yeast cell with multiple

bud scars.

yeast strains (ale strains,

very rarely lager strains),

new rounds ofcell division

occur before cell

separation so clumps of

cells are produced - a

process known as chain

formation. The site of cell

separation is marked on

the mother cell by a

structure referred to as

the bud scar and on the

daughter cell by the birth

scar. These scars cannot

be seen under the light

microscope but can be

seen using fluorescence

microscopy after staining

with fluorescent stains

Cell

wall

The distinguishing feature

of a growing population

of yeast cells is the

presence ofdie buds which

are produced on the cell

wall when the cell

divides. The daughter cell

is initiated as a small

bud which increases in

size throughout most of

the cell cycle, until it is

the same size as the

mother cell. Most growth

in yeast occurs during

bud formation and the

bud is more or less the

same size as the mature

cell before it separates.

Cell separation usually

occurs soon after cell

division, however, in some

Fibrillar

layer

") Manno> protein

J layer

manno-

protein

Glucan

proteinmanno-

proteln

Glucan

Glucan

Glucan

Glucan

Glucan

Glucan

} Glucan

layer

plasma membrane

cytoplasm

Figure 6. Structure of the yeast cell wall.

such as calcafluor or primulin. Bud scars also show up as very distinct structures with the

electron microscope (Figure 5). No two buds arise at the same site on the yeast cell wall.

Each time a bud is produced, a new bud scar forms on the cell wall of the mother cell. By

counting the number of bud scars it is possible to establish the number of buds which have

been produced by a particular cell. This can be used as a measure of the age of the cell.

The cell wall is a rigid structure which is 25 nm thick and constitutes approximately 25% of

the dry weight of the cell. Chemical analysis of the cell wall indicates that the major

components are glucan and mannan, however chitin and protein are also present. Glucan is

a complex branched polymer of glucose units and although the structure is a matrix most of

the glucan is located in the inner layer of the wall adjacent to the plasmalemma (Figure 6).

It is the major structural component of the wall, since removal of the glucan results in total

disruption of the cell wall. Mannan, which is a complex polymer of mannose occurs mainly,

but not exclusively, in the outer layers of the cell wall. Since it is possible to remove the

mannan without altering the general shape of the cell, it appears that it is not essential to

the integrity of the cell wall. The third cell wall carbohydrate component is chitin which is

a polymer of N.acetyl-glucosamine, and is found in the cell wall associated with the bud

scars. Isolation of the bud scars by treating the cell wall with appropriate lytic enzymes has

shown that the chitin is arranged in a ring around the bud scar. Protein constitutes

approximately 10% of the dry weight of the cell wall. At least some of this protein is in the

form of wall bound enzymes. Several enzymes have been described as being associated

with the cell wall of yeast, including glucanase and mannanase, which are probably

involved in the "softening" of the cell wall to permit bud formation, invertase [which

hydrolyses sucrose (cane sugar)], alkaline phosphatase and lipase (which hydrolyses fatty

acids and lipids). Several of these enzymes, for example invertase, are mannoproteins and

contain up to 50% of mannan, as an integral part of the enzyme molecule. Much of the

remaining protein in the cell wall is also associated with mannan and this probably plays

a structural role as well as an enzymic role in the cell wall. In addition, the flocculation

properties of the cell are influenced by the mannoprotein structure of the cell wall; this will

be discussed in detail later.

The nucleus is the structure that contains most of the cell's deoxyribonucleic acid (DNA)

arranged into 16 chromosomes which contain over 6,000 genes and encode for all the

proteins synthesised in the cell. Recently the complete sequence of the chromosomes has

been published (the Yeast Genome Project). The compilation of this sequence of the

Sacchammyces genome was a considerable undertaking that required a high degree of

co-ordination but is, by itself, of little value in biological terms. Rather, it is the

information contained within the genes themselves that is more important so the first step

in the analysis of any sequence of DNA is to examine for individual genes. Once these

genes have been identified (and there arc clues in the DNA which reveal their location) the

amino acid sequences of the encoded proteins can be determined. What can the sequence

of the yeast genome tell us about brewer's yeast? The overall genetic picture will be very

similar for brewer's yeast, whether an ale or lager strain. New information on metabolic

pathways and cellular processes such as organelle biosynthesis will emerge from studies of

the yeast genome sequence. Also, the yeast strain chosen for genome sequencing was a

haploid (only one set of chromosomes) and was found to possess only one set of maltose

fermentation (MAL) genes. Brewing strains, which must ferment wort maltose as efficiently

as possible, are polyploid and may contain ten or more sets of MAL genes. There has

probably been selective pressure in brewing fermentations for yeast strains which possess

multiple sets of MAL genes and it comes as no surprise to find this reflected in the genetic

make-up of brewer's yeast.

Approximately 30% of the genes identified as part of the Yeast Genome Project encode

proteins with no clue to their function; this has led to them being called "orphan" genes.

10

Inner membrane

Matrix

The next phase of the Yeast Genome Project has already commenced with a European

Network of 144 laboratories carrying out a systematic analysis of 1,000 of the orphan

genes. New and existing molecular genetic methods will be applied to each of the genes in

an attempt to define the function of their encoded product. A similar, but complementary

research programme, will take place in the United States. The Genome Project is therefore

set to produce a flow of information on yeast, much of which will provide a better

understanding of industrial yeast strains.

Individual chromosomes arc very small and cannot be recognised as discrete structures by

light or electron microscopy. However, the advent of DNA fingerprinting (karyotyping) has

introduced an electrophoretic technique for separation of individual chromosomes and

this "fingerprint" can be

employed to type yeast

strains. (This will be

discussed in detail later.)

The membrane surrounding

the nucleus remains intact

throughout the cell cycle.

It is visible in electron

micrographs as a double

membrane which is

perforated at intervals with

pores. Associated with the

nuclear membrane is a

structure referred to as a

plaque. The characteristic

structure of a plaque is a

multilayered disc from

which microtubules extend

into both the nucleus and

the cytoplasm. These plaques

are the spindle apparatus

of the yeast nucleus and

they play an important part

in nuclear division. (More

of this later).

The mitochondria are readily

recognisable in electron

micrographs ofan aerobically

grown yeast cell as spherical

or rod-shaped structures

Figure 7. Structure of the mitochrondrion. surrounded by a double membrane. They

(A) A diagram showing the overall contain cristae which are formed by the

structure of the mitochondrion, and folding of the inner membrane (Figure 7).

(B) electron micrograph of A considerable amount of work has been

mitochondria. carried out on the structure of the

11

mitochondrion and the distribution of the many mitochondrial enzymes in the membranes

and the matrix of the mitochondrion. Most of the enzymes of the tricarboxylic acid cycle

are present in the matrix of the mitochondrion, whereas the enzymes involved in electron

transport and oxidative phosphorylation are associated with the inner membrane, including

the cristae.

At one time it was considered that mitochondria were absent from anaerobically grown (or

catabolite repressed) yeast since they could not be detected and also because such cells

lacked many of the enzymes associated with mitochondria. However, the use of freeze-

etching techniques has indicated that the apparent absence of mitochondria was due to

inadequate fixation techniques. Cells grown anaerobically in the absence of lipids have

very simple mitochondria, consisting of an outer double membrane but lacking cristae. The

addition of lipids such as oleic acid and ergosterol results in the development of the cristae.

The development of the mitochondrion is influenced by the lack of oxygen, the presence of

lipids and the level of glucose in the medium. Consequently, there is a change in the struc

ture of mitochondria upon transfer from anaerobic to aerobic conditions but no de novo

generation of mitochondria. The cytoplasm of the yeast cell contains a system of double

membranes known as the cndoplasmic reticulum. Some of these membranes are associated

with ribosomes, although as in other organisms, the endoplasmic reticulum appears to be

involved in many other cellular activities. The relationship between endoplasmic reticulum

and other organelles is unclear, however, there is continuity between the endoplasmic

reticulum, the outer membrane of the mitochondrion and the plasmalemma. The endoplasmic

reticulum is also involved in the formation of vesicles which are present in the cell. Mature

yeast cells contain a large vacuole. However, at the point in the cell cycle when bud

formation is initiated, the vacuole appears to fragment into smaller vacuoles which become

distributed between the mother cell and the bud. Later in the cell cycle, these small vacuoles

fuse to produce a single vacuole in the mother and daughter cell. The formation of the

vacuole is not completely established but it contains hydrolytic enzymes, polyphosphates,

lipids and low molecular weight cellular intermediates, and metal ions. In addition, it acts

• E ••

0 5 10 IS 20 25 30 35 40

Time (hours)

1x10*

1x10'-

1x10'I

1X105

Figure 8. Batch growth

curve for brewing yeast

culture in shake flasks at

20"C (A) log phase,

(B) accelerating,

(C) exponential phase,

(D) decelerating phase,

and (E) stationary phase

(adaptedfrom Priest and

Campbell, Brewing

Microbiology, 1996).

12

as a reservoir for nutrients and hydrolytic enzymes. Lipid granules are also present in the

cytoplasm and these are also probably derived from the endoplasmic rcticulum.

The technical problems of isolating and characterising the different membrane components

of yeast are considerable. Vesicles, vacuoles and other organdies are very fragile and easily

disrupted. Fragments of membrane from different organdies are a challenge to separate

but with the advent of differential centrifugal and electrophoretic separation techniques, this

is now possible. Nevertheless, considerable care must be exercised during the experimental

process.

Cell

separation Bud

initiation

YEAST CELL GROWTH

AND DIVISION

Late /nuclear A

division J

Figure 9. Cell cycle of Sacchammyces

cerevisiae.

Growth in brewer's yeast is associated

almost entirely with the growth of the

bud which reaches the size of the mature

cell by the time it separates from the

parent cell. Figure 8 illustrates the batch

growth curve of a brewing yeast culture

in shake flasks at 20°C. In rapidly growing

yeast cultures, all the cells can be seen

to have buds since bud formation occupies

the whole cell cycle. In fact both mother

and daughter cell can initiate bud

formation before cell separation has

occurred. In yeast cultures which are

growing more slowly, cells lacking

buds can be seen and bud formation

only occupies part of the cell cycle. The

cell cycle of yeast is normally defined

as the period between the end of one

cell division and the next cell division. In cells which are growing in an unrestricted

manner, all the contents of the cells double during this period. The cycle is divided into

four phases: Gl, S, G2 and M (Figure 9). The S period is the phase when DNA

synthesis occurs, the M phase is the period occupied by mitosis which is the mechanism

by which the chromosomes divide and separate. The phases Gl and G2 represent

the interval between mitosis and DNA synthesis (Gl), and DNA synthesis and

mitosis (G2).

The onset of bud formation coincides with the initiation of DNA synthesis. The initial steps

of bud formation involve the weakening of the cell wall caused by the action of lytic

enzymes which attack the polysaccharides of the cell wall. The bud is formed by new cell

material being laid down at the site of bud initiation, then as bud formation progresses and

it becomes larger, the deposition of new material becomes localised at the tip of the bud.

When the bud reaches full size, a complex septum is laid down in the neck of the bud which

contains chilin in addition to glucan and mannan. Cell separation is achieved when the layers

13

of (he septum separate leaving the bud scar on the mother cell and the birth scar on the

daughter cell.

During the S and G phases of the cell cycle, the nucleus moves towards the site of bud

formation, so that at onset of the M phase it is situated in the neck of the bud. Mitosis

occurs in the neck of the bud in such a manner that when it is completed, one of the nuclei

has moved into the bud whereas the other remains in the mother cell. As discussed

previously, it is not easy to recognise chromosomes in the nucleus of cells of brewer's yeast

strains because the nuclear membrane remains intact during mitosis. However, use of

electron microscopy has made it possible to identify different steps of the mitotic cycle by

studying the behaviour of the spindle plaques and the microtubules associated with them.

Growth of the yeast cell wall occurs during growth of the bud resulting in progressive

increase in the size of a rigid spherical structure. As has been discussed previously, the yeast

cell wall is very complex and knowledge of its structure and biosynthesis is still increasing. Its

biosynthesis must involve the formation of the major components: glucan, mannan, chitin and

protein, and their assembly into a three dimensional structure in a precise manner outside

the plasma membrane. The formation of the cell wall poses several interesting questions:

• What is the nature of the precursors from which the wall is synthesised?

• Which enzymes are involved in its biosynthesis?

• How do these enzymes control the three dimensional structure of the cell wall?

• Where does cell wall biosynthesis occur?

• At what stage in the biosynthesis are cell wall components transported across the cell

membranes?

The cell wall polysaccharides glucan, mannan and chitin are produced from mannose,

glucose and N-acetyl-glucosamine respectively. However, the immediate precursors of the

polysaccharides are not the free sugars but uridine diphosphate (UDP) or guanosine

diphosphate (GDP), derivatives of the sugars. The cell wall proteins are produced from

amino acids by the normal process of protein biosynthesis. There arc differences between

the mechanisms of glucan and mannan synthesis. Glucan synthesis can occur in the absence

of protein synthesis and microfibrils of glucan can be seen on the cell surface. Mannan

synthesis, on the other hand, cannot proceed in the absence of protein synthesis. Inhibitors of

protein synthesis such as cycloheximide block mannan synthesis and mannan microfibrils do

not accumulate during mannan biosynthesis. This dependence on protein synthesis has

been interpreted as indicating that mannan synthesis can only be initiated by the attachment

of mannose units to amino acids such as serinc, thrconine and asparagine in wall proteins.

GENETIC CHARACTERISATION OF YEAST

The behaviour, performance and quality of a yeast strain is influenced by three sets of

determining factors, collectively called nature-nurture effects. The nurture effects are all

the environmental factors, (i.e. the phenotypes), to which the yeast is subjected from

inoculation (pitching) onwards. On the other hand, the nature influence is the genetic

make-up (i.e. the genotype) of a particular yeast strain.

14

There are a number of methods that are employed in the genetic research and developmentof brewer's yeast strains. Classical approaches to strain improvement include mutation andselection, screening and selection, and cross-breeding (hybridisation). Mutation is anychange that alters the structure of the DNA molecule, thus modifying the genetic material.The mutagenised strains often no longer exhibit many desirable properties of the parent

Diploid Phase (2n)

p/a diploid

Haploid Phase (n)

2 mating type a

and

2 mating type a

4 spored ascus

meiosis and

sporulation

Figure 10.

Haploid/diploid life

cycle of

Saccharomyces

cerevisiae.

s v, 20(im

Figure 11. Sporulating yeast cell (A) wet mount preparation, and (B) stained preparation.

15

strain and in addition may exhibit a slower growth rate and produce a number of undesirable

taste and aroma compounds during fermentation. Mutagenesis is seldom employed with

brewing strains due to their polyploid/ancuploid nature.

Screening of cultures to obtain spontaneous mutants or variants has proved to be a more

successful technique as it avoids the use of destructive mutagens. To select for brewery

yeast variants with improved maltose utilisation rates, 2-deoxy-glucose, a glucose analogue,

was employed and spontaneous mutants selected which were resistant to 2-dcoxy-glucose.

These isolates were also found to be dcrcprcsscd for glucose repression of maltose uptake.

This resulted in faster wort fermentation rates and no alteration in the final flavour of

the beer.

The study of yeast genetics was pioneered in the Carlsberg laboratory in Denmark. In 1935

they established the haploid - diploid life cycle in yeast (Figure 10). Sacchammyces

species can alternate between the haploid (a single set of chromosomes in the nucleus) and

diploid (two sets of chromosomes) states. Yeast can display two mating types (sexes),

designated "a" and "a", which arc manifested by the extracellular production of an "a" or

an "a" mating factor (pheromonc). When "a" haploids are mixed with "a" haploids,

mating takes place and diploid zygotes are formed. Under conditions of nutritional

deprivation, diploids undergo reduction division by mciosis and differentiate into

tctranuclcate asci, containing four uninucleate haploid ascospores, two of which arc "a"

mating type and two of which are "a" mating type (Figure 11). Ascus walls can be removed

with a specific lytic enzyme preparation (glucanase). The four spores from each ascus can be

isolated by use of a micro-manipulator, induced to germinate, tested for their fermentation

ability, and subsequently employed for further hybridisation work. Both haploid and

diploid organisms can exist stably and undergo cell division via mitosis and budding.

Brewing yeast strains are not immediately amenable to hybridisation techniques because

they are usually not haploid or diploid, but rather aneuploid or polyploid. Consequently,

such strains possess little or no mating ability, poor sporulation and the spores that do

form have low spore viability. In recent years it has been shown that it is possible to

increase sporulation ability of brewer's yeast strains by manipulation of the medium and

the incubation conditions.

Although the technique of hybridisation (cross-breeding) fell into disfavour for a number

of years, when new biotechnological methods such as recombinant DNA were thought to

be the complete solution to the development of novel brewer's yeast strains, it has again

come to be accepted as a very valuable technique. For example, using traditional genetic

techniques, a yeast that produced beer with only 10% of the normal diacctyl level at the

end of fermentation has been produced. Also, hybrids with crosses between ale and lager

segregants exhibited faster attenuation rates and produced beers of good palate which

lacked the sulphury character of a lager but retained the estery aroma of the ale. One of

the major advantages to cross-breeding is that this technique carries none of the burden of

ethical questions and fears that can accompany the use of recombinant DNA technology.

Rare mating, also called forced mating, is a technique that disregards ploidy and mating

type and thus is ideal for the manipulation of polyploid/aneuploid strains where normal

hybridisation procedures cannot be utilised. When non-mating strains are mixed at a

16

high density, a few hybrids with fused nuclei form and these can usually be isolated using

appropriate selection markers. A possible disadvantage to this method is that while

incorporating the nuclear genes from the brewing strain, the rare mating product can also

inherit undesirable properties from the other partner, which is often a non-brewing strain.

A good example of this is the successful construction of a dextrin-fermenting brewing

strain using this technique which unfortunately introduced the POF gene (Phenolic-Off-

Flavour) which imparts the ability to decarboxylale wort ferulic acid to 4-vinyl guaiacol,

giving beer a phenolic or clove-like off-flavour (the characteristic flavour of "weissbier").

This made the hybrid product unsuitable for the production of lagers and ales from a taste

perspective but acceptable from a dextrin utilisation standpoint.

Figure 12.

Saccharvmyces

brewing yeast with and

without

zymocidal "killer"

activity.

"Killer" Yeast Lawn of

Sensitive Yeast

"Non-Killer'

Yeast

Laboratory Haploid

-Killer" Strain

brewing Lager

Strain

Rare Mating

(Forced Mating)

S\ Hybrid(Heterokaryon)

Segregation under

Influence of Kargone

(Kar = Karyogamy defective)

Brewing Lager

Strain

Laboratory Haploid

-Killer Strain

True Hybrid

Figure 13.

Rare mating

protocol to produce

brewing strains with

zymocidal

"killer" activity.

Heteroplasmon

17

Cytoduction is a specialised form of rare mating in which only the cytoplasmic components

of the donor strain are transferred into a brewing strain. The process of cytoduction requires

the presence of a specific nuclear gene mutation designated Kar, for karyogamy defective. This

mutation impairs nuclear fusion. Cytoduction can be used in three ways: substitution of the

niitochondrial genome; introduction of DNA plasmids; or, transfer of double-stranded

RNA species. When used in the substitution of the mitochondria! genome, it is possible to

study the effects of these genetic elements on various cell functions such as respiratory

activity, cell surface activities and various other yeast strain characteristics. Also, as will be

discussed below, rare mating has been employed to transfer "zymocidal" or "killer" factor

from laboratory haploid strains to brewing strains without altering the primary fermentation

characteristics of the brewer's yeast strain.

Some strains of Saccharomyces species secrete a proteinaceous toxin called a zymocidc or

killer toxin that is lethal to certain other strains of Saccharomyces. Toxin-producing strains

are termed killer yeasts and susceptible strains arc termed sensitive yeasts. There are strains

that do not kill and are not themselves killed, and these arc called resistant (Figure 12).

The "killer" character of Saccharomyces is determined by the presence of two species of

cytoplasmically located dsRNA plasmids (termed M and L). The M-dsRNA "killer" plasmid

is "killer" strain specific and codes for "killer" toxin (an extracellular protein) and also for a

protein or proteins that make the host immune to the toxin. The L-dsRNA codes for the production

of a protein that encapsulates both forms of dsRNA, thereby yielding virus-like particles.

These virus-like particles are not naturally transmitted from cell to cell by any infection process.

Brewing strains can be modified such that they are both resistant to killing by a zymociclal

yeast and so that they themselves have zymocidal activity, thereby eliminating contaminating

yeasts (which must be sensitive). Rare mating has been successfully employed to

produce brewing "killer" yeast strains by crossing a brewing lager yeast with a Kar "killer"

strain (Figure 13). Wort fermentations have been conducted with this strain, the finished

beer packaged and subject to tasie assessment. The beer brewed with the "killer"

Figure 14.

Triphenyl tetrazolium

overlay of yeast colonies (A)

Respiratory Deficient (RD)

mutants - petite white

colonies, (B) Respiratory

Sufficient (RS) colonies

(red),

and (C) mix of RS and RD

colonies.

18

strain was acceptable but contained an ester note that was not present in the control. Aquestion often asked is whether the toxin is still active in the finished beer. The toxin isextremely heat-sensitive, and a brewery pasteurisation cycle of 8 PU's has been shown tocompletely inactivate it.

The introduction of a "killer" strain into a brewery where several yeasts are employed for theproduction of different beers can present logistical problems. An error on an operator's part inkegging lines and yeast tank lines could have serious consequences, since accidental mixingwould prove fatal for the normal brewer's yeast. In a brewery with only one yeast strain,this would not be a cause for concern. It is worthy of note that a number of commerciallyavailable wine yeasts contain the "killer" characteristic, the purpose being to eliminate

some of the yeasts that occur in the must that originates from the natural flora of the grapes.

Yeast mutations arc a common occurrence throughout the growth and fermentation cycle, butthey are usually recessive mutations, due to functional loss of a single gene. Since brewer'syeast strains are usually aneuploid or polyploid, the dormant gene will function adequately

in the strain and it will be phcnotypically normal. Only if the mutation takes place in bothcomplementary genes will the recessive character be expressed. If the mutation weakens theyeast, the mutated strain will be unable to compete and soon be outgrown by the non-mutatedyeast population. The accepted view until recently was that brewer's yeast strains arc

genetically very stable, however, with the advent of DNA fingerprinting (karyotyping) it

has been found that instability in many production brewer's yeast strains is not uncommon.

This finding has reinforced the view that there should be strict adherence to yeast generation

production specifications. This topic will be discussed in greater detail when yeastmanagement techniques are considered.

Only three characteristics are routinely encountered resulting from yeast mutation that archarmful to a fermentation. These are:

• The tendency of yeast strains to mutate from flocculent to non-flocculent;

• The loss of ability to ferment maltotriose; and

• The presence of respiratory deficient mutants.

The respiratory deficient (RD) or "petite" mutation is the most frequently identified mutant

found in brewing yeast strains. The mutant arises spontaneously when a segment of the DNA

in the mitochondrion becomes defective to form a flawed mitochondrial genome. Themitochondria are then unable to synthesise certain proteins. This type of mutation is alsocalled the "petite" mutation because colonies of such a mutant are usually much smaller

than the normal respiratory sufficient (RS) culture (also called "grande") (Figure 14). Therespiratory deficient mutation normally occurs at frequencies of between 0.5% and 5% of

the population but in some strains, figures as high as 50% have been reported. Deficiencies

in mitochondrial function result in a diminished ability to function aerobically and as aresult these yeasts are unable to metabolise non-fermentable carbon sources such as lactatc,

glycerol or ethanol. Many phenotypic effects (actual expressed properties, such as theyeast's ability to perform a particular chemical reaction) occur due to this mutation andthese include alterations in sugar uptake, metabolic by-product formation, and toleranceto stress factors such as ethanol and temperature. Flocculation, cell wall and plasmamembrane structure, and cellular morphology are affected by this mutation.

19

Beer produced with a yeast that is respiratory deficient or that produces a high number of

respiratory deficient mutants is likely to have flavour defects and fermentation problems.

For example, beer produced from these mutants contained elevated levels of diacetyl and

higher alcohols. Wort fermentation rates were slower, higher dead cell counts were

observed, and biomass production and flocculation ability were reduced.

A significant reduction in diacetyl production has been achieved by the selection of

spontaneous mutants from brewer's yeast cultures using resistance to the herbicide

sulphometuron methyl (SMM). The SMM resistant strains produce 50% less diacetyl than

the parent strain due to partial inactivation of the enzyme that produces the diacetyl

precursor, a-acetolactatc (a-acetolactate synthetase).

Saccharomycesdiastaticus

Saccharomyces

uvarum

(carlsbergensis)

Whole cells

Spheroplasts IDEX

Fusing spheroplasts ( DEX • FLO

t/

Fused spheroplasts

spheroplasting enzymes

fusing agent

(polyethylene glycol)

Regenerated fused cell

cell wall regeneration in

complete growth medium

DEX- Dextrin fermentation

FLO - Flocculation

Fusion

product

Figure 15. Spheroplast fusion of two yeast strains.

The advent of the new biotechnology has been stimulated by the development of novel

methods of genetic manipulation - spheroplast (protoplast) fusion and recombinant DNA.

Spheroplast fusion is a technique that can be employed in the genetic manipulation of

brewer's yeast strains. The method does not depend on ploidy and mating type and

consequently has great applicability to such strains because of their polyploid nature

20

and absence of mating type characteristic. The yeast cell wall is removed with lytic

enzymes such as extracts of snail gut or enzymes from various microorganisms. Removal

of yeast cell walls results in osmotically fragile spheroplasts, which must be maintained in

an osmotically stabilised medium such as 1 M sorbitol. The spheroplasting enzyme is

removed by thorough washing, and the sphcroplasts are then mixed and suspended in a

fusion agent consisting of polyethylene glycol (PEG) and calcium ions in buffer.

Subsequently, the fused spheroplasts must be induced to regenerate their cell walls and

recommence division. This is achieved in solid media containing 3% agar and sorbitol. The

action of PEG as a fusing agent is not fully understood, but it is believed to act as a

polycation inducing the formation of small aggregates of spheroplasts (Figure 15).

Some examples of fusions with commercial brewing strains arc:

• The construction of a brewing yeast with amylolytic activity by the fusion of

Saccharvmyces cerevisiae and Saccharumycei' diaslaticus;

• A polyploid strain capable ofhigh ethanol production by fusion of a flocculcnt strain with

Sake yeasts; and

• Construction of strains with improved osmotolerance by fusion of Sacchammyces

diastaticus and Saccharomyces rvuxii (an osmotolerant yeast species).

Although spheroplast fusion is an extremely efficient technique, it relies mainly on trial

and error and is not specific enough to modify strains in a predictable manner. The fusion

product is nearly always very different from both original fusion partners because the

genome of both strains become integrated. Consequently, it is difficult to selectively

introduce a single trait such as flocculation into a strain using this technique. Spheroplast

fusion has been found to be a viable technique when flavour of the final product is not

critical, for example, fusion products that could survive high osmotic pressure, elevated

fermentation temperatures (ca. >40°C) and increased ethanol tolerance. Such strains

are successfully being used in the industrial alcohol industry but produce beer with

unsatisfactory beer flavour/taste profiles.

Although the techniques of hybridisation, rare mating and spheroplast fusion have met with

success, they have their limitations, the principal one being the lack of specificity in genetic

exchange. It is only since 1978 that a DNA transformation system for yeast has been

available and great strides have been made in the past two decades. It is now possible to

modify the genetic composition of a brewer's yeast strain without disrupting the many other

desirable traits of the strain and it is also possible to introduce genes from other sources.

This technology employs a set of methods called recombinant DNA which had its origins

in two related fields. The first, microbial genetics, studies the mechanisms by which micro

organisms inherit traits. The second, molecular biology, specifically studies how genetic

information is carried in molecules of DNA and how DNA directs the synthesis of proteins.

During the 1970's and 1980's, the practical application of microorganisms expanded almost

beyond imagination with the development of new, artificial techniques for making

recombinant DNA. Although natural recombination makes it possible for closely related

organisms to exchange genes, the new techniques make it possible to transfer genes

between completely unrelated species. These techniques are so powerful that the term

21

recombinant DNA is now widely understood to mean any artificial manipulation of genes,

whether within a particular species or between different species.

A gene from a vertebrate animal, including a human, can be inserted into the DNA of a

bacterium, or a gene from a virus into a yeast. In many cases, the recipient can then be made

to express the gene, which may code for a commercially useful product. Thus, yeast, with

genes for human insulin, arc being used to produce insulin for treating diabetics or a

vaccine for hepatitis B is being made from a gene for part of the hepatitis virus (the yeast

produces a viral protein).

oPlasmid DNA

spheroplasting

enzymes

Donor DNA

Cut DNA

Pieces

(JCut Plasmid

Pieces

anneal and ligate

oRecombinant

Plasmid

cell wall regeneration

Transformed Yeast Cell

Figure 16. Production of a recombinant DNA brewer's yeast.

Recombinant DNA techniques can also be used to make thousands of copies of the same

DNA molecule - to amplify DNA, thus generating sufficient DNA for various kinds of

experimentation or analysis. Artificial gene manipulation is popularly known as genetic

manipulation. In fact, the term biotechnology, which correctly has been defined to include

all industrial applications of biological systems and processes, has increasingly become

erroneously identified in the public mind as only the industrial application of genetic

engineering. Genetic engineering has been made possible by the discovery and development

of a number of tools and techniques. The most important was the discovery of restriction

22

enzymes, bacterial enzymes that can be used to cut DNA from different sources into pieces

that are easy to recombine in vitro (in vitro means "in glass" - that is, a test tube rather than

inside a living organism). Genetic manipulation required the development of methods for

inserting recombinant DNA molecules into cells by using so-called vectors. If a mosquito,

carrying the virus for yellow fever, bites and infects a human, the mosquito is considered

a "disease vector" because it can transmit the virus from one host to another. The term

vector, or cloning vector, has generally been adopted to describe a self-replicating DNA

molecule that is used as a carrier to transmit a gene from one organism to another.

Recombinant DNA technology has been used for improving brewer's yeast strains, and

some successful examples that can be cited are:

• Glucoamylase activity from the fungus Aspergillus niger;

• Glucanase activity from the bacterium Bacillus subtilis, the fungus Trichoderma reesii

and barley;

• a-Acetolactate decarboxylase activity from the bacteria Enterobacter aerogenes and

Acetobacter spp.;

• Extracellular protease for chill-proofing beer; and

• Modification of the yeast's flocculation properties.

What are the future prospects for the use of recombinant DNA with brewer's yeast and their

use in the brewing industry? At this time this is a difficult question to answer. It is quite

surprising that there are not a number of recombinant brewer's yeasts commercially in use

today. Permission has already been granted in the U.K. from the Ministry of Agriculture

Foods and Fisheries Advisory Committee on Novel Foods and Processes for the use of

a baker's yeast strain that is genetically manipulated to enhance baking properties and for

a brewing strain, cloned with DNA from Saccharomyces diastaticus, that secretes

glucoamylase to produce low caloric beer (Figure 16).

Perhaps the availability of alternative inexpensive traditional solutions for many of the

problems that it was hoped a cloned yeast could solve, such as inexpensive sources of

glucanase and gluco- and a-amylase, has retarded implementation. Also in some cases

recombinant DNA technology is ahead of the knowledge base in yeast biochemistry. There

is also still concern over consumer acceptance. Although this is a difficult hurdle, it is

thought that as people become accustomed to Pharmaceuticals produced by recombinant

DNA, and more plants with improved characteristics for farming/food gain regulatory

approval and customer acceptance, the current reluctance to use the products of this

technology in the brewing industry will slowly disappear.

GENETIC TESTS FOR TYPING YEAST STRAINS

Traditional methods for differentiating brewing strains of yeast are relatively simple

biochemical or microbiological tests. Typically the tests are designed to detect differences

in such properties as colony morphology, flocculence and sensitivity to antibodies and

other chemicals. Such tests have a number of drawbacks:

• Lack of objectivity - the results may be open to misinterpretation;

• Poor sensitivity - it is often difficult to detect differences between closely related strains;

23

1234 1234

Figure 17.

Restriction

patterns of

(A) yeast

DNA, and (B)

DNA

hybridisation

map.

• Lengthy response time - this may be a week or

more for some growth tests; and

• Poor reproducibility and lack of "robustness"

- minor changes in the way the yeast is

prepared for the test, or the way the test is

carried out, may have a profound effect on its

outcome.

As previously discussed, yeast strains vary from

one another because of differences in their genetic

make-up, so it follows that the most direct

approach to distinguish yeast strains should

involve some method of DNA analysis. There are

essentially three such methods, each of which has

its advantages but also its disadvantages. They are:

• DNA fingerprinting by hybridisation with a

DNA probe;

• Karyotyping, the analysis of whole chromosomes;

and

• Polymerase chain reaction (PCR) for

amplification of DNA in vitrv.

DNA fingerprinting using hybridisation with a

DNA probe is a technique which allows the

identification of specific DNA fragments in an otherwise complex mixture. The result

is a pattern or profile (resembling a bar code) which is characteristic for each strain. The

technique is perhaps best explained by considering just how a sample of DNA must beprepared from a yeast strain. If a sample of DNA from a strain of brewer's yeast is subjected

to agarose gel electrophoresis, all that can be seen is a broad band (not shown). Although

the DNA sample actually consists of many large molecules of various sizes, conventional

agarose gel electrophoresis cannot resolve them and instead they appear as one band.

If the same DNA sample is digested with a nuclease (a restriction endonuclease or

restriction enzyme) before agarose gel electrophoresis then many smaller fragments can be

seen. This is illustrated in lanes 1-4 of Figure 17A. Generally, different restriction enzymes

will cut at specific sequences in a DNA molecule; typically the recognition site for a

given restriction enzyme is 4 to 6 base-pairs (bp) in length. The DNA in lane 1 ofFigure 17A, for example, has been cut with the enzyme EcoRl which has the recognition

sequence GAATTC, whereas the DNA in lanes 2 and 3 were cut by the enzymes Hindlll

and Pstl, respectively.

Restriction enzymes are produced by bacteria as a defence against incoming foreign DNA

(in effect to "restrict" the entry of DNA especially from viruses); EcoRl is the name givento the enzyme produced by the Escherchia coli (E. coli) bacteria; likewise Hindlll is

derived from the bacterium Haemophilus influenzae and Pstl is derived from the bacteriumProvidencia stuartii. However, in molecular biology, the real value of restriction enzymes

lies in their use as tools for the dissection of DNA and over 100 different restriction

enzymes are now commercially available.

24

Figure 18. DNA-DNA

hybridisation test.

Digesting a sample of yeast

DNA with a restriction

enzyme such as EcoRl

should generate a

characteristic pattern of

fragments but this is not

obvious from lane 1 of

Figure 17A because of the

number fragments which

have been produced. The

patterns in lanes 2 and 3

also have many bands.

What is needed is a

method of detecting

specific DNA fragments

such that a clearer pattern

of fewer fragments can

be resolved. Hybridisation

of the digested DNA with

<£>

Step 1 -

Collection of

organisms on a

filter matrix

Step 2 -

Cell lysis and

DNA strand

separation

Step 3 -

Binding of DNA

to filler matrix

Step 4 -

Addition of labeled

DNA Probes

Step 5 -

Hybridisation of labeled

probes to complementary

DNA from organisms

a DNA probe enables this to be achieved. Before hybridisation with a probe can be carried

out, the restriction enzyme-digested DNA sample (or samples) must be transferred from

the agarose gel to a membrane of nitro-cellulose or, because of its greater strength and

DNA binding capacity, nylon. Transferring the DNA to the surface of a suitable membrane

makes it accessible to the probe and provides a much more solid support than agarose gel.

This process of transferring the DNA from an agarose gel to a membrane is often referred

to as "Southern blotting" after its inventor, Edwin Southern who invented the techniquein 1975.

The choice of which type (i.e. sequence) of DNA is used for the probe is important.

Multi-locus probes, are so-called because they can bind to more than one site in a sample

of DNA, are the ones most likely to succeed in detecting differences between closely related

strains of yeast. The hybridisation of a multi-locus probe to a restriction enzyme-digested

DNA sample on a nylon membrane will, as discussed earlier, be detected as a pattern ofbands resembling a bar code.

The probe must be labelled or tagged in some way that allows its detection by hybridisation

on the membrane. Before use, the double-stranded DNA probe is denatured (i.e. made single-stranded) and this is usually achieved by boiling it for a few minutes. The single-stranded

probe can now hybridise with complementary, single-stranded DNA in the membrane to

form stable, double-stranded hybrids. A typical protocol would allow this step to take placeovernight. The membrane is then washed to remove excess or loosely bound probe, and the

label is detected by the appropriate method (discussed below). The whole transfer anddetection process is summarised in Figure 18.

25

When DNA fingerprinting was first developed, radioactive probes were used. Specifically,

they were labelled with phosphorus-32 which could be readily detected by autoradiography

with X-ray film. Radioactive probes of this sort are hazardous and very unstable (they have

to be used more or less immediately after they arc made) and these problems limit the use of

radioactive probes outside the specialised laboratory. Probes with non-radioactive labels

have been developed which are stable, sensitive and safe to handle. They also give sharper

bands in the final fingerprint. Recently, one label which

has been widely employed is the plant steroid digoxigenin

(DIG). The probe is labelled with DIG in a reaction

catalysed by DNA polymcrase and using the unlabelled

DNA as a template. This leads to the synthesis of new

copies of the probe which are labelled with DIG. The

DIG-labelled probe is then detected (after hybridisation

to the DNA on a membrane) by a colourimetric reaction.

Figure 17B shows the DNA fingerprints obtained for four

production lager strains of yeast following hybridisation

with a DIG-labelled probe. In Figure 17A, the DNA was

digested with the restriction enzyme prior to agarose gel

electrophoresis. Digesting the DNA with Hindlll instead

of EcoRl shows a clear difference in the hybridisation

pattern (Figure 17A, lane 2), as does the pattern from

Pstl digest in lane 3. Together the patterns produce a

fingerprint which is unique to individual lager strains.

How can DNA fingerprinting be of value to the brewer?

It offers the opportunity to "catalogue" yeast strains; this

could provide a reference point for regular checks on the

yeast strains as they are freshly propagated. The introduction

of new strains into brewing operations may call for them to

be properly typed so that they can be clearly differentiated

from strains already in use, and DNA fingerprinting

addresses this need. In cases where a change in the

properties of a yeast strain is suspected (perhaps by

altered fermentation behaviour), then it would be possible

to investigate this further by DNA fingerprinting. The

technology of DNA fingerprinting requires further

development, specifically, to simplify it and make it

more rapid. Nevertheless, as it presently stands it can be

a useful tool in the quality control of yeast supply. Figure 19. Chromosomal

fingerprints of three brewing

lager yeast strains.Karyotyping is an electrophoretic technique that separates

whole chromosomes based on their different sizes. Asdiscussed above, the haploid yeast genome is contained in 16 distinct, linear chromosomes,

each of which is of a different size. Yeast chromosomes are readily separated from one

another by the technique of pulsed field electrophoresis using commercially available

equipment. The chromosomes are resolved into a bar code-like pattern which can be

made visible by staining with cthidium bromide and viewing under UV light (Figure 19).

26

A haploid strain may appear to have less than the 16 expected bands as similarly sized

chromosomes may co-migrate. Diploid strains will often display a somewhat larger number.

The fingerprints ofcommon brewing strains and laboratory strains arc generally distinguishable.

This technique is relatively simple and economical. The gel apparatus can be purchased for

£5,000-£6,000 and reagents for a set often chromosome preparations cost approx. £25. The

chromosomal isolation procedure takes 2-3 days though for many strains a procedure

Figure 20. Polymerase

chain reaction. Target DNA

(A) is heat denatured, (B)

at 94°C. Primers are

annealed (C) at 55°C and

then primer extension (D)

proceeds at 72CC. The cycle

(A-D) is then repeated (E)

until 25-40 cycles have

been completed. (F) time-

temperature representation

of a typical PCR cycle, and

(G) quantitation of

amplified DNA product.

Copies of amplified DNA

increase exponentially as

number of cycles increases.

B

51.

31-

Target DNA

B Cycle 1Denature!ion

5, 04-C

3'

51-

3'-

Primer Annealing

55"C

D Primer Extension„ 72*C

3'-

5'-

•5'

-3'

E Cycle 25'

-5'

•3'

25~«> cycles

3'

5'

3'

51

3'

5'

9a

94

72

60

30

Oonalurolion

1—1 l~~\/ 1 Primer / \/ 1 Extension / 1

/ \ 1 \/ ' ' «/ Primer/ Anneatng

F

1 min 1 min 2 min 1 min

■4— Cycle 1 —►

Number of cycles

►

Figure 21. Fingerprint patterns using

polymerase chain reaction (PCR)

technology to differentiate yeast strains.

Lane A - ale yeast, Lane B - wild yeast,

Lane C - lager yeast, and

Lane D - DNA size standards.

—222249

27

taking only 6 hours is effective. The electrophoresis needs to be run for a minimum of 16 hours

for a full fingerprint, although a "snapshot" can be obtained much faster. Gel to gel

reproducibility is generally good, but a new batch of a particular reagent (even water) can

sometimes introduce quite startling changes. It is, therefore, important to have good

control samples on every gel. Clone to clone reproducibility is good for most chromosomes.

Karyotypes are generally reproducible though variation is very common in chromosome

XII and it is better not to read any significance into its wanderings.

Polymerase chain reaction (PCR) is an in vitro method for amplifying very small amounts

of selected nucleic acids (DNA or RNA) by several orders of magnitude over a short period

of time (hours). This technique permits the detection of specified DNA fragments by making

multiple copies. The process requires a thermostable DNA polymerase, the four

deoxyribonucleoside triphosphates (dNTPs) and two short pieces of DNA (primers) which

are complementary to the 3' ends of the double-stranded fragment to be amplified. A small

sample of chromosomal DNA (less than a picogram) is heat denatured, then cooled in

the presence of excess primer molecules, enzymes and dNTPs. The primers anneal to

their complementary targets and the polymerase extends them at their 3' ends, copying

chromosomal DNA. As a result, the DNA flanked by the primers is duplicated. If the

sample is heated and cooled again, the primers can anneal again to the chromosomal target

as well as to the new copies, and following primer extension the target sequence is

duplicated. After 20 replication cycles, the target DNA is amplified over a million-fold

(Figure 20).

Employing this technique, a specific fragment of DNA (or RNA) from a particular

micro-organism (for example a contaminating bacteria or yeast) can be isolated and

amplified with PCR, or it can also be used to produce a fingerprint of different yeast strain,

as shown in Figure 21. This technique can theoretically be used to identify a contaminant in

any part of the brewing process, provided that the DNA sequence of one or more of the

target organisms' genes is known. However, the exceptional degree of specificity of the PCR

technique means that only the target organism will be detected, and every target organism

must, therefore, have its own PCR test, i.e. a PCR test for wild yeast will not detect any

lactic acid bacteria. The advantages of this method for recognising contaminants is that it

is sensitive, specific, versatile, affordable and fast. Besides the limitation discussed above,

PCR requires the operator to possess an advanced level of technical laboratory skills, and

the laboratory must also take the precautions needed to avoid the possibility of cross-

contamination and false negatives.

BREWER'S YEAST PERFORMANCE

The objectives of wort fermentation are to consistently metabolise wort constituents into

ethanol and other fermentation products in order to produce beer with satisfactory quality

and stability. Another objective is to produce yeast crops that can be confidently re-pitched

into subsequent brews. During the brewing process overall yeast performance is controlled

by a plethora of factors. These factors include:

• The yeast strains employed and their condition at pitching and throughout fermentation;

• The concentration and category of assimilable nitrogen;

28

The concentration of ions;

The fermentation temperature;

The pitching rate;

The tolerance of yeast cells to stress factors such as osmotic pressure and ethanol;

The wort gravity;

The oxygen level at pitching;

The wort sugar spectrum; and

Yeast flocculation characteristics.

These factors influence yeast performance either individually or in combination with others

and also together permit the definition of the requirements of an acceptable brewer's yeast

strain: "In order to achieve a beer of high quality, it is axiomatic that not only must the

yeast be effective in removing the required nutrientsfrom the growtli/fermentation medium

(wort), able to tolerate the prevailing environmental conditions (for example, ethanol

tolerance) and impart the desiredflavour to the beer, but the microorganisms themselves

must be effectively removedfrom the wort byflocculation, centrifugation and/orfiltration

after they havefulfilled their metabolic role".

It is worthy of note that brewing is the only major alcoholic beverage process that recycles

its yeast. It is, therefore, important to jealously protect the quality of the cropped yeast

because it will be used to pitch a later fermentation and will, therefore, have a profound

effect on the quality of the beer resulting from it.

Over the years, considerable effort has been devoted in many research laboratories to the

study of the biochemistry and genetics of brewer's yeast (and industrial yeast strains in

general). The objectives of the studies have been two-fold:

• To learn more about the biochemical and genetic makeup of brewing yeast strains; and

• To improve the overall performance of such strains, with particular emphasis being

placed on broader substrate utilisation capabilities, increased ethanol production, and

improved tolerance to environmental conditions such as temperature, high osmotic

pressure and ethanol, and finally to understand the mechanism(s) of flocculation.

UPTAKE AND METABOLISM OF WORT NUTRIENTS

When yeast is pitched into wort, it is introduced into an extremely complex environment

due to the fact that wort is a medium consisting of simple sugars, dextrins, amino acids,

peptides, proteins, vitamins, ions, nucleic acids and other constituents too numerous to

mention. One of the major advances in brewing science during the past 25 years has been

the elucidation of the mechanisms by which the yeast cell, under normal circumstances,

utilises in a very orderly manner, the plethora of wort nutrients.

Wort Sugars and Carbohydrates

Wort contains the sugars sucrose, fructose, glucose, maltose and maltotriose togetherwith dextrin material. In the normal situation brewing yeast strains (ale and lager strains) are

capable of utilising sucrose, glucose, fructose, maltose and maltotriose in this approximate

29

Glucose

Fructose

Maltose

Maltotriose

Dextrins

24 12048 72 96

Fermentation time (hours)

Figure 22. Order of uptake of sugars by yeast from wort.

144

sequence (or priority)

(Figure 22), although

some degree of overlap

does occur. The majority

of brewing strains leave

the malto-tetraose and other

dextrins unfermented,

but Saccharomyces

diastaticus is able to

utilise dextrin material.

The initial step in the

utilisation of any sugar

by yeast is usually

either its passage

intact across the cell

membrane or its

hydrolysis outside the

cell membrane followed

by entry into the cell by some or all of the hydrolysis products (Figure 23). Maltose and

maltotriose are examples of sugars that pass intact across the cell membrane whereas

sucrose (and dextrin with Saccharomyces diastaticus) is hydrolysed by an extracellular

enzyme, and the hydrolysis products are taken up into the cell. Maltose and maltotriose are

the major sugars in brewer's wort and as a consequence, a brewer's yeast's ability to use

these two sugars is vital and depends upon the correct genetic complement. It is probable

that brewer's yeast possess independent uptake mechanisms (maltose and maltotriose

permease), to transport the two sugars across the cell membrane into the cell (Figure 24).

Once inside the cell, both sugars are hydrolysed to glucose units by the a-glucosidase

system. It is important to re-emphasise that the transport, hydrolysis and fermentation of

maltose is particularly

important in brewing, since Maltose Maltotriose

maltose usually accounts for p^mease50-60% of the fermentable

sugar in wort. Maltose

fermentation in Saccharomyces

yeasts requires at least one

of five unlinked (each

independent) MAL loci each

consisting of three genes

encoding the structural gene

for a-glucosidase (maltase)

(MAL S), maltose permease

(MAL T) and an activator

(MAL R) whose product

co-ordinately regulates the

expression of the a-

glucosidase and permease

genes. The expression of

MAL S and MAL T is

Maltose Maltotriose

a-glucosidase a-glucosidase

GLUCOSE

permease permease

glucose

tglucoamylase

IStarch/Dextrin

glu

\permease

I+ fructrose

invertase

ISucrose

Figure 23. Uptake of sugars by the yeast cell.

30

Maltose

IMaltotriose

I

icell / "Carrier Protein

membrane % (maltose permease)"Carrier Protein \

(maltotriose permease) /

t tMaltose Maltotriose

a-Glucosidase

Glucose

Figure 24 (left). Uptake and

metabolism of maltose and

maltotriose by the yeast cell.

| glucose!

£ADPglucose 6-phosphate

6-p

I

fructose 6-phosphate

ATP

ADP

fructose 1,6-diphosphate

glyceraldehyde

3-phosphate

L

JNADHj,1,3-diphosphoglycerate

ADP

atp

3-phosphoglycerate

[ dihydroxyacetone

phosphate

12-phosphoglycerate

phosphoenol pyruvate

J{ ADP

55-

5-

45

J 4-

1 3.5-

i '■

1":2 1.5-

1

05-

t Ale brewing 3train

O—O Oorepressed variants

12 24 38 48 60 72 84 86 106 120

Time (hours)

B

Ipyruvate]

0 12 2-1 36 48 60 72 84 9B 108 120

Time (hours)

Figure 25 (above). (A) Degree plato

reduction, and (B) ethanol production

by an ale brewing strain and its

depressed variants.

Figure 26 (left). Embden-Meyerhof-Parnas (EMP,

glycolysis, glycolytic) pathway.

31

regulated by induction by maltose and repression by glucose. When glucose concentrations

are high [greater than 1% (w/v)] the MAL genes are repressed and only when 40-50% of

the glucose has been taken up from the wort will the uptake of maltose and maltotriose

commence. Thus, the presence of glucose in the fermenting wort exerts a major repressing

influence on wort fermentation rate. Using the glucose analogue 2-deoxy-glucose (2-DOG),

which is not metabolised by Saccharvmyces strains, spontaneous variants of brewing

GLUCOSE

Citrate

Isocitrate

I— NADP4/—►NADPH,

Biosynthesis

excretion

Succinyl CoA

GTp QDp Biosynthesis

excretion

Figure 27. Kreb's Cycle (adaptedfrom Priest and Campbell, Brewing Microbiology, 1996).

strains have been selected in which the maltose uptake is not repressed by glucose, and as

a consequence these variants (called derepressed) have increased wort fermentation rates

(Figure 25).

Once the sugars are inside the cell, they are converted via the glycolytic (also known as

Embden-Meyerhof-Pamas, EMP, glycolysis) pathway into pyruvate. Figure 26 shows the

basic steps in the glycolytic pathway and where ATP is broken down and created. This

conversion to pyruvate generates a net total of 2 ATP molecules which supply the yeast cell

with energy.

32

dihydroxyacetone

phosphate

aldehydes

oxaloacetate

fumarate

diacetyl

dimethyl sulfoxide

NADH

dehydrogenases

NAD+

gtycerol

8thanol|

and fusel alcohols

• malate

■ succinate

2,3-butanedbl

dimethyl sulfide

GLYCOLYSIS

Figure 28. Regenerating NAD+ by fermenting yeast (adaptedfrom Lewis and Young,

Brewing, 1995).

In Figure 26 the enzyme cofactor called NAD+ (nicotinamide adenine dinucleotide), acofactor for dehydrogenase enzymes controlling oxidative reactions in catabolism, is

observed. Reduced NAD+ (or NADH2) is formed when electrons are transferred to NAD+as hydride ions [H]:

NAD* + [2H] NADH + H+ (or NADH2)

When yeast are respiring in an aerobic environment, the Kreb's cycle [aJso known as thetricarboxylic acid cycle (TCA)] and oxidative phosphorylation (also called the electron

transfer chain) occurs. This massive electron transfer system produces large amounts of

energy in the form of ATP. The synthesis of citrate, isocitratc and 2-oxoglutarate for nucleic

acid and amino acid synthesis also occurs during the Kreb's cycle and these organic acidswill spill over into the fermented beer. The additional substrates that are generated from theKreb's cycle may be used to supply additional substrates for biosynthesis (Figure 27). Inrespiring cells, molecular oxygen is used as the final H+ acceptor and glucose is completelyoxidised. By the end of oxidative phosphorylation, one glucose molecule yields 2 ATPfrom the glycolytic pathway, 2 ATP from the Kreb's cycle, and 24 ATP from oxidativephosphorylation. Thus, respiration of 1 glucose molecule yields 28 molecules of ATPoverall. In respiring yeast, NAD+ is regenerated using oxidative phosphorylation and theKieb's cycle.

Under anaerobic conditions, the Kreb's cycle may operate partially, but the extent ofoperation has yet to be determined. When yeast are in the fermentative state, NAD+ isregenerated using a range of hydrogen acceptors (Figure 28). For example, yeast are

33

glycogen

mannan

glucan

trehalose

hexose

monophosphate

pathway

glucose-6-phosphate

triose phosphates

phosphoenol pyruvate

nucleotides

inoaciKrebs'

Cycle

succinate < u-oxoglutarate

Figure 29. The contribution of carbohydrate catabolism to intermediate compounds for

biosynthetic reactions (adaptedfrom Hough el al.. Malting & Brewing Science, Vol. 2,

Hopped Wort and Beer, 1982).

not tolerant of highly acidic environments, and therefore pyruvic acid is converted to

carbon dioxide and acetaldehyde and finally into ethanol:

pyruvate

CHjCOCOOH

co2

acetaldehydc

■*- CH3CHO -

ethanol

CH3CH2OH

NADH? NAD*

This serves two purposes, the cofactor NAD* molecules are regenerated that were consumed

during glycolysis, and the yeast cell is detoxified by the conversion of pyruvic acid into

carbon dioxide and ethanol. These are the key reasons that ethanol is produced during

fermentation. Other hydrogen acceptors used to restore the redox ratio of the cell include:

diacetyl, fumarate, oxaloacetate, aldehydes, etc.

34

Control of Yeast Metabolism

Pasteur effect

If oxygen is introduced during fermentation, the yeast cell will revert to respiration.

This means that pyruvate from glycolysis will move directly into the Kreb's cycle and

oxidative phosphorylation in the presence of oxygen. In this case glucose is oxidised

completely into carbon dioxide and water. A key observation of Pasteur was that the uptake

of glucose is slower in respiring cells than in non-respiring (fermenting) cells. This is

due to the fact that aerobic respiration produces more energy to the cell for each

glucose molecule (or other carbon source) compared to fermentation, and therefore less

substrate is needed to supply the yeast cell with a given amount of energy.

Crabtree effect (glucose repression, catabolite repression)

Respiration is inhibited and fermentation occurs. Even if oxygen is present, if glucose

levels are high, the fermentative pathway is used rather than the Kreb's cycle. In

Saccharomyces cerevisiae, a glucose sensitive yeast, respiration is repressed in the presence

of a small (0.4% w/v) concentration of glucose in the medium. This is regardless of the

presence or absence of molecular oxygen. During a typical brewery fermentation, wort

contains about 1% glucose, so it would be assumed that the yeast cells are repressed.

In the absence of repressive amounts of glucose and in the presence of molecular oxygen,

glucose is completely oxidised to carbon dioxide and water through to the glycolytic

pathway and the Kreb's cycle. Neither maltose nor maltotriose exhibits a repressive action

on respiration.

This phenomenon may be explained somewhat by the model of Sols. ATP has been shown

to inhibit the enzyme 6-phosphofructokinase in the glycolytic pathway, whereas ADP and

AMP cause activation. Thus in high energy situations (i.e. during respiration), the flux of

glucose through the EMP pathway is lowered. Also, as ATP levels increase, the intracellular

reserve of inorganic phosphate decreases and the operation of the glycolytic pathway also

decreases, resulting in a lowered glucose flux.

Figure 29 shows how the glycolytic pathway and the Kreb's cycle provide intermediates for

biosynthetic reactions.

Amino Acids, Peptides and Proteins

Active yeast growth involves the uptake of nitrogen, mainly in the form of amino acids,

for the synthesis of proteins and other nitrogenous compounds of the cell. Later in the

fermentation as yeast multiplication stops, nitrogen uptake slows or ceases. In wort, the

main nitrogen source for synthesis of proteins, nucleic acids and other nitrogenous cell

components is the variety of amino acids formed from the proteolysis of barley proteins.

Brewer's wort contains 19 amino acids and as with wort sugars the assimilation of amino

acids is ordered. Four groups of amino acids have been identified on the basis of

assimilation patterns (Table 1). Those in group A are utilised immediately following yeast

pitching, whereas those in group B are assimilated more slowly. Utilisation of group C

amino acids commences when group A types are fully assimilated. Proline, the most

plentiful amino acid in wort and the sole group D amino acid, is utilised poorly or not

at all. Proline is usually still present in beer at 200-300 mg/L, however, under aerobic

35

Table 1. Classification Of Amino Acids According To Their Speed Of Absorption From

Wort By Ale Yeast Under Brewery Conditions [Pierce, JIB, 1982, 88(4), 232].

A - Fast

Absorption

Glutamic acid

Aspartic acid

Asparaginc

Glutamine

Serinc

Threonine

Lysine

Arginine

B - Intermediate

Absorption

Valine

Methionine

Leucine

Isolcucine

Histidine

C - Slow

Absorption

Glycine

Phenylalanine

Tyrosine

Tryptophan

Alaninc

Ammonia

D - Little or No

Absorption

Proline

conditions proline is assimilated after exhaustion of the other amino acids since its uptake

requires the presence of a mitochondrial oxidase.

The regulation of amino acid uptake by brewer's and related yeast strains is complex,

involving carriers specific to certain amino acids and a general amino acid permease of

broad substrate specificity. The utilisation pattern of wort nitrogen is due to a combination

of the range of permeases present, their specificity, and feedback inhibition effects resulting

from the composition of the yeast intracellular amino acids.

The metabolism of assimilated amino nitrogen is dependent on the phase of the fermentation

and on the total quantity provided in the wort. The majority of amino nitrogen is ultimately

utilised in protein synthesis and, as such, is vital for yeast growth. It would appear that

amino acids are not usually incorporated directly into proteins but are involved in

transamination reactions, a significant proportion of the amino acid skeletons of yeast

protein being derived via the catabolism of wort sugars. This explains why the total amino

content of wort is important in determining the extent of yeast growth, the amino acid

spectrum being somewhat secondary.

Table 2. Classification Of Amino Acids According To The "Essential" Nature Of Their

Keto-Acid Analogues In Yeast Metabolism [Jones & Pierce, 1969, JIB, 75(6), 520J.

Class 1

Glutamic acid

Asparaginc

Glutamic acid

Glutamine

Threonine

Serinc

Methionine

Proline

Class 2

Isolcucine

Valine

Phenylalanine

Glycine

Alanine

Tyrosine

Class 3

Lysine

Histidine

Arginine

Leucine

36

The amino acid spectrum of wort does influence beer flavour. In this respect wort amino

acids can be further subdivided on the basis of their "essential" nature (Table 2). The initial

concentration of Class 1 amino acids is considered relatively unimportant since they may be

incorporated directly from the wort when available, or synthesiscd from sugar metabolism

and transamination in later fermentation. Deficiencies in Class 2 and Class 3 amino acids

have considerable effects on beer quality. Thus, in the later stages of fermentation when the

supply of exogenous amino acids is exhausted, the keto-acid moiety of Class 2 amino acidsmust be synthesised solely from sugars.

The nitrogen obtained from the amino acids in wort is used to synthesise amino acids and

ultimately proteins intracellularly. The yeast assimilates the wort amino acids and a

transaminase system removes the amino group and the carbon skeleton is anabolised,

creating an intracellular oxo-acid pool. The oxo-acid pool generated by the transaminases

and anabolic reactions is a precursor of aldehydes and higher alcohols which contribute to

beer flavour. Thus the formation of higher alcohols (i.e. higher in number of carbon atoms

than ethanol) is tied in with nitrogen metabolism.

Normally only the necessary amount of (a keto-acid (2-oxo-acid) is produced for the

synthesis of required amounts of amino acid. The production is controlled by feedback

inhibition of the required amino acid. However, as nitrogen shortage develops later in the

fermentation (e.g., by slow transfer of the remaining amino acids or by using wort with high

level of nitrogen free adjunct) feedback control deteriorates. Larger quantities of various

keto- (or oxo-) acids are produced in attempt to guarantee synthesis of missing amino acids

(see Figure 29). When the necessary nitrogen is not available, synthesis of missing amino

acids is not possible and since accumulation of keto-acids is not tolerated by yeast,

compounds are reduced to corresponding alcohols. Therefore higher alcohols of beer have

structural similarity to amino acids. The reduction of a keto-acid to alcohol is the same as

the mechanism of conversion of pyruvic acid to cthanol.

Carbonyl by-products (for example, diacetyl) of the syntheses of certain of these keto-acids

impart deleterious flavours to beer if present in excess. A major aim of fermentation

management is to ensure that these carbonyls are present at an appropriate concentration

in the finished beer (details will be discussed later). This will be facilitated if the wort

contains a suitable proportion of Class 2 amino acids. In the case of Class 3 amino acids,

the contribution made by the sugar synthetic route is small and the yeast is dependent on

an adequate exogenous supply. Therefore, a deficiency in Class 3 amino acids results in

major perturbations in nitrogen metabolism, yeast growth and, by inference, beer flavour.

It is apparent that the amino nitrogen composition of wort has far-reaching effects upon

fermentation performance and on beer flavour. Where malt is used as the principal source

of extract, the quantity and composition of amino acids are such that these problems are not

encountered. However, care must be exercised when using adjuncts, many of which are

relatively deficient in amino nitrogen.

Oxygen

Wort fermentation in beer production is largely anaerobic, but when the yeast is first

pitched into wort, some oxygen must be made available to the yeast. Indeed, it is now

37

evident that this is the only point in the brewing process where oxygen is beneficial.

Oxygen must be excluded, as far as it is possible, from all other parts of the process

because it will have a negative effect on beer quality. Specifically, it will promote beer

flavour instability. The widespread adoption of high gravity brewing procedures (which

will be discussed in detail later) has increased our awareness of the importance of oxygen

during wort fermentation and has stimulated basic and applied research on the

mechanisms of oxygen interactions during cell growth and the application of this

knowledge in the process.

Oxygen has a profound influence on the activity of yeasts and particularly on yeast growth.

Certain yeast enzymes only react with oxygen and it cannot be replaced by other hydrogen

acceptors. This applies to the oxygenases involved in the synthesis of unsaturated fatty

acids and sterols, which are vital components of cell membranes. Quantitative studies on

the effect of aeration on yeast growth and fermentation have been given little serious

consideration until the last 25 years. The traditional concept of beer fermentation was that

growth occurred prior to the fermentation of most wort sugars and that fermentation was

carried out by non-growing, stationary phase cells. It is now known that yeast growth, sugar

utilisation and ethanol production are coupled phenomena. For example, the rate of

fermentation by growing, exponential phase cells of an ale yeast strain is 33-fold higher

than that of non-growing cells.

For a brewery fermentation to proceed rapidly there must be sufficient amounts of yeast

synthesised. Inadequate growth of a brewer's yeast culture will result in poor attenuation,

altered beer flavour, inconsistent fermentation times and recovered pitching yeasts which

arc undesirable for subsequent fermentations. It has been discussed already the effect

that spontaneous respiratory deficient (RD) mutants of brewer's yeast strains (mutants

with impaired yeast aerobic metabolism) have on wort fermentation characteristics and

beer flavour.

Trace amounts of oxygen have profound stimulatory effects on yeast fermentation and

particularly on yeast growth. Pasteur demonstrated that oxygen was necessary for normal

yeast reproduction, although excessive wort aeration caused undesirable flavour effects on

the finished beer. Oxygen requirements were confirmed by such early notable brewing

researchers as Adrian Brown, Horace Brown and Frans Windisch. Windisch concluded

that over-vigorous aeration of fermenting worts led to yeast "weakness", illustrated by

increasingly sluggish fermentations characterised by longer lag phases, a slower specific

rate of fermentation and/or residual sugar remaining in the final beer. The critical importance

of oxygen was confirmed when in 1954 it was shown that under anaerobic conditions

Saccharomyces yeast strains require both prc-formed sterols and unsaturatcd fatty acids

as growth factors. These two lipids are both found in membranes and are critical for

membrane function and integrity. Both of these lipid classes require molecular oxygen

for their biosynthesis.

Lipids in beer quantitatively form an almost negligible component, but can influence its

organoleptic and physio-chemical properties. Malt is the main source of unsaturated fatty

acids in wort. Wort concentrations of these acids are sub-optimal and can be growth-limiting.

During fermentation, yeast can take up free fatty acids from wort, most of which are

incorporated as structural lipids.

38

Table 3. Effect Of Linoleic Acid And Oxygen On Ester

Lentini el «/., 1994, Proc. Com hist. Brew. (Asia

Linoleic Acid

(Hg/gDW yeast)

Wort oxygen

concentration (mg/L)

Total esters (mg/L)

High Trub

Wort

6180

8

18.3

High Trub

Wort

5510

4

26.5

Production /adaptedjmm

Pacific Sect.), Sydney, 23, 89-95}.

Low Trub

Wort

880

8

24.2

Low Trub

Wort

510

4

34.6

A typical lipid composition of brewing yeast would consist of70-90% fatty acids. The fatty acid

composition of the yeast lipids shows a preponderance of C16 and C|8 acids. Yeast usually

contains a high content of unsaturated fatty acids if they are grown aerobically. Under these

conditions oleic acid (C 18:1) is a major component. Fatty acid composition is an extremely

important variable in determining membrane structure, morphology and function. Although

Sacchammyces cerevisiae and related species require unsaturated fatty acids during aerobicand anaerobic growth, respiratory growth requires four times as much unsaturated fatty acids

due to their function as co-factors coupling oxidative phosphorylation to ATP synthesis.

Yeast cultures synthesise fatty acids throughout fermentation but the ratio of the acids

varies with time. Unsaturated fatty acid [for example, palmitoleic (C|6.|) and oleic (C|8:))

acids] synthesis only occurs in the presence of dissolved oxygen. Oxygen is present in

aerated/oxygenated pitched wort for a relatively short period (3-9 hours) and during this

period there is a large increase in the percentage of unsaturated fatty acids. When oxygen

is depleted there is an increase in the production of short-chain fatty acids (C6 - C]2).

The sterol component of brewing yeast ranges from 0.05-0.45% of the cellular dry weight

(depending on the prevailing environmental conditions) and accounts for less than 10%

of the total cell lipid. Ergosterol is the major sterol in brewing yeast strains and can

account for over 90% of the total sterol. The biosynthetic pathway for sterol formation is

complex. The important fact for this dissertation is that the precursor sequences can be

synthesiscd anaerobically, but the final reaction that produces ergosterol requires molecular

oxygen. The major function of sterols in yeast is to contribute to the structure and dynamic-

state of the membranes. The primary role is to modulate membrane fluidity under fluctuating

environmental conditions. For example, ergosterol confers increased resistance to ethanol

and multiple freeze-thawing effects. A decrease in the ergosterol level of membranes has

been directly related to a reduction in cell viability in the presence of ethanol.

Pitching yeasts arc propagated under weakly aerated conditions or recovered from previous

fermentations. In both cases, the cells are lipid-depleted and to promote normal growth and

attenuation cither pre-formed lipids must be added to the wort or oxygen must be made

available for their synthesis. In commercial brewing, only the second alternative is feasible.

Wort is cooled and aerated/oxygenated to 8-16 mg/L dissolved oxygen (DO). Within a few

hours of pitching, most of this oxygen is removed from the wort. During this time there is

intensive synthesis of lipid (stcrol and fatty acid) and a decrease in cellular glycogen (the

39

role of glycogen in yeast will be discussed later). In practice, sterol synthesis by brewing

yeasts in the presence of oxygen appears to be of greater significance than unsaturated fatty

acid synthesis. This may be due to the contribution of wort to the fatty acid pool. Wort does

not contribute exogenous sterol to the fermentation.

There is a wide range of oxygen requirements amongst ale and lager yeast strains. In ale

strains, oxygen requirements have been assessed by comparative fermentations of worts

pitched with anaerobically-grown and aerobically-grown yeast. It has been found that ale

yeasts are divisible into four classes based on their oxygen requirements:

Class 01 requiring 4 mg/L DO

Class 02 requiring 8 mg/L DO

Class 03 requiring 40 mg/L DO

Class 04 requiring over 40 mg/L DO

The different oxygen requirements amongst ale strains disappear if the pattern of

oxygen supply is modified. For example, the differences between Class 01 and Class 04

strains disappear when 4 mg/L DO was supplied in four increments over a period of

12 hours. Differences in oxygen requirements may simply reflect the fact that some

strains require oxygen at a later stage in growth than others. This may be due to unequal

partitioning of unsaturated lipids from mother to daughter cells during cell division.

Lager strains have also been divided into four groupings with respect to their oxygen

requirements. Group I yeasts are the least sensitive to anaerobic propagation and the

sensitivity increases from Group I to IV, indicating that the yeasts that already have high

oxygen requirements more easily develop an additional requirement.

There is a relationship between wort trub levels and wort DO at pitching. Trub contains

high concentrations of unsaturated fatty acids, particularly linoleic acid. This linoleic acid

is absorbed by yeast and has a negative effect on ester production. In a similar manner, high

concentrations of oxygen have a similar negative effect on ester production (Table 3). The

role of linoleic acid in ester biosynthesis is not fully understood but it has been suggested

that it plays a role in modifying membrane structure and affects ester synthesising enzymes,

some of which are membrane bound.

Vitamins

Yeast vary widely in their need for vitamins for metabolism and, in a given strain, this need

may also vary between active respiration and growth on the one hand, and alcoholic

fermentation on the other. Almost all vitamins (except mesoinositol) required by yeast

function as a part of a coenzyme, serving a catalytic function in yeast metabolism.

Brewer's wort is a rich source of vitamins and contains biotin, thiamine (B,), calcium

pantothenate, nicotinic acid, riboflavin, inositol and pyridoxine, pyridoxal and

pyridoxamine (Table 4). Most brewer's yeast have an absolute requirement for biotin and

many require pantothenate. Inositol is sometimes required and pyridoxine and thiamine

appear to be needed only by ale yeast. Although brewer's wort is a rich source of most of

these growth factors and deficiencies are rare, there have been reports of

fermentation problems due to lack of biotin and inositol in wort.

40

Vitamin

Biotin

Thiamin(Bl)

Calcium pantothenate

Nicotinic acid

Riboflavin

Inositol

Pyridoxine,

pyridoxal, &

pyridoxaminc

Level in Wort

/100 ml

0.56 u.g

60 ug

45-65 ug

1000-1200 Ug

20-50 ug

9.3 mg (free) and

18.9 mg (total)

85 Hg

Some Metabolic Functions

Carboxylation reactions, protein, nucleic

acid, carbohydrate, fatty acid

Decarboxylation of pyruvate

rearrangements in pentose cycle,

transketolase reactions, isoleucinc and

valine biosynthesis

Coenzyme A, acetylation reactions

(Under anacrobiosis) as coenzymes in

oxidation/reduction reactions

(Under anacrobiosis) as coenzymes in

oxidation/reduction reactions

Membrane phospholipids (structural)

Amino acid metabolism

Table 4. Vitamins In Sweet Wort And Functions Of Certain Essential Vitamins In Yeast

Metabolism [adaptedfrom Reed & Nagodawithana, 1991, Yeast Technology).

Ions

Inorganic ions

Yeast requires a number of inorganic ions for optimum growth and fermentation.

Appropriate concentrations of these elements allow for accelerated growth and increased

biomass yield, enhanced ethanol production, or both with a higher final substrate to

product yield. An imbalance in inorganic nutrition is reflected in complex, and often

subtle, alterations of metabolic patterns and growth characteristics (for example, cellular

morphology, tolerance to the environment and by-product formation). The role played by

these ionic species is both enzymatic and structural. A number of ions function as the

catalytic centre of an enzyme, as an activator or stabiliser of enzyme function, or to maintain

physiological control by antagonism between activators and deactivators. Zn2+, Co2+, Mn2+

and Cu2+ are common catalytic centres whilst Mg2+ acts as one of the most common

activators of enzyme activity and K* commonly functions in the role of metal coenzyme.

In the structural role, ionic species act to neutralise electrostatic forces present in the

various cellular anionic units. For polyphosphate, DNA, RNA and proteins, K+ and Mg2+

are most commonly encountered in this role. The charged structural membrane

phospholipids are shielded principally by Ca2+ and Mg2*. Cell wall phosphate ions are

typically complexed to Ca2+ (as will be described later it has a critical role in flocculation)

although other cations can replace this ion.

Inorganic ions are divided into anions (negatively charged) and cations (positively

charged). Anions that will be considered are: phosphate, sulphate, chloride and nitrate; and

monovalent cations considered are: hydrogen, potassium and sodium; and divalent cations

are: magnesium, manganese, calcium, zinc, copper and iron.

41

Phosphorus is essential to yeast cells for incorporation into structural molecules

(for example, phosphomannan and phospholipids), nucleic acids (DNA and RNA) and

phosphorylated metabolites (for example, ATP and glucose-6-phosphate). Phosphorus

is commonly available to yeasts in the form of inorganic orthophosphate (H2PO4)

which is rapidly metabolised to nuclcoside triphosphate (for example, ATP)

on entry into yeast cells. Orthophosphate transport in yeast occurs against a

concentration gradient and is, therefore, active (requires the expenditure of

metabolic energy). Generally, transport is highly dependent on both the intra- and

extracellular pH, and on Mg2+, K+ and phosphate concentrations in the growth medium

(wort). It is also dependent on the presence of fermentable sugars such as maltose

and glucose.

In brewer's and related yeast strains, at least three systems are thought to translocate

orthophosphate into the cell: high affinity system, low affinity system, and sodium-

phosphate transporter. It is conceivable that both low and high affinity systems may

operate simultaneously depending on phosphate availability. In fact, phosphate uptake may

be controlled by the concentration of intracellular orthophosphate. When this is high, no net

phosphate uptake occurs, but when it declines (as during yeast growth and fermentation)

the rate of phosphate uptake increases. In addition to yeast cell membrane transport of

phosphate, other membrane transporters are known to operate. For example, in yeast,

mitochondria exhibit both high and low affinity transport, and in vacuolcs the formation of

insoluble polyphosphate may contribute to the way in which yeast controls cytosolic

phosphate levels.

Inorganic sulphur in the form of sulphate anions, is transported by yeast for assimilation

into sulphur-containing amino acids such as methionine (Figure 30) and the tripeptide

glutathione (glutamic acid-cysteine-glycine). Sulphate uptake by yeast is an active process.

The mechanism involves an inducible anion which is energised by proton motive force. This

sulphate-proton symport is counterbalanced by K+ efflux. The existence of two (high and

low affinity) independent sulphate transporter proteins in brewer's yeast has been confirmed.

In the presence of excess sulphate, yeast can store sulphur intracellularly in the form of

glutathione, which can account for as much as 1 % of the cellular dry weight. Therefore,

under conditions of sulphate limitation or starvation, glutathione may act as an endogenous

sulphur source for the biosynthesis of sulphur amino acids. It should be noted that

glutathione also plays a number of other significant roles in the physiology of the yeast

cell including functioning as a primary scavenger of oxygen free radicals and in conferring

protection from oxidative stress (Figure 31).

There is no evidence of an active chloride uptake system in the yeast plasma membrane.

It has been suggested that chloride transport occurs via a proton-chloride or sodium-

chloride symport mechanism which may be involved in regulation of yeast cell

water content.

Yeast cells take up inorganic cations for several reasons. These may involve regulation of

intracellular pH homeostatis and generation of proton motive force (in the case of H+

transport); osmoregulation and charge balancing (in the case of K+); enzyme cofactor functions

(in the case of Mg2+ and Mn2+); metallo-enzyme structural functions (in the case of

42

micronutrient divalent cations such as Fe2+, Zn2* and Ni2+) and single transduction second

messenger functions (in the case of Ca2+). Although advances arc being made into

elucidation of cation transport mechanisms in yeast using molecular biological approaches,

relatively few cell physiological studies have been reported in recent years.

Hydrogen ions

Yeast cells are not freely permeable to hydrogen ions and trans-membrane proton gradients

are established by active proton pumping mechanisms. The electrochemical trans-membrane

proton gradient is generated by H+-translocating ATPase enzymes which provide the driving

force for the transport of many yeast nutrients. The yeast cell membrane H+-ATPase is a

major constituent of the plasma membrane (comprising as much as 50% of total membrane

protein in Succharomyces cerevisiae) and has been described as the "master enzyme" in

many yeasts and mycelial fungi. This is because it controls cell pH, nutrient and ion

transport, and overall cell growth. Also, the activity of the enzyme declines significantly as

yeast cells enter the stationary growth phase.

The H+-ATPase is instrumental in modulating both the intra- and extracellular pH.

Intracellular pH of brewing yeast strains remains relatively constant (to within 0.4 pH

units) at about pH 5.2, even when the extracellular pH fluctuates. This constancy is

maintained primarily through the activities of the cell membrane H+-ATPase.

Extracellular acidification and concomitant intracellular alkalinization are important yeast

growth responses. The plasma membrane H+-ATPase activity is, therefore, inextricably

linked with yeast growth and has the capability of generating a 10,000-fold difference

between the concentration of protons on either side of the membrane. The magnitude of the

gradient in yeast depends on the presence of other cations, notably K+ which is exchanged

for H+ in a 1:1 stoichiometry. Control of proton exchanges in growing yeast cells is directly

relevant to wort fermentation. The acidification response of yeasts to addition of a carbon

substrate can be exploited in order to assess the metabolic competence of brewer's yeast

cultures. The so-called "acidification power" test (also called the vitality test) for yeast

membrane proton efflux capacity is useful in distinguishing vitality from viability, where

in broad terms viability is a cell's ability to divide and vitality is a cell's ability to take up

and ferment appropriate substrates.

Potassium ions

Active K+ transport in yeast requires a fermentable or respirable substrate. It occurs against

a considerable concentration gradient (5000:1) and exhibits substrate saturation kinetics.

The K+ carriers also transport other monovalent (Rb+, Cs+, Li+, NH4+) and divalent

(Ca2\ Mg2+) cations, albeit with much lower affinity than K+. Several components of the

monovalent cation transport system have been identified. The site is a transporter for

alkali metal cations and also possibly Mg2+, when present in high concentrations.

Another (modifier) site exists where other ions (both monovalent and divalent) bind in a

non-competitive fashion. The third site, referred to as the activation site, may be

equivalent to the high affinity K+ carrier which is only expressed in cells grown in low K+

ion concentrations. The multi-component K+ transporter is also implicated in potassium

efflux from yeast and net translocation into cells is dependent upon the balance between

uptake and efflux. In fermenting yeast cells, the net K+ uptake is rapid. Resting cells leak

K+ slowly in the absence of an energy source.

43

Sodium ions

Yeast cells do not accumulate Na+ ions under normal growth conditions. Conversely, yeast

continuously excretes Na+ in order to maintain very low cytoplasmic concentrations of this

cation. This is accomplished via a Na+-H+ antiport mechanism. In the presence of high

salt concentrations, yeast cells osmoregulate by producing intracellular compatible solutes

such as glycerol and arabinitol. Cells can be artificially "loaded" with Na+ which, under

such non-physiological conditions, probably enters via a low-affinity K+ transporter and

perhaps also Na+-substrate symportcrs. Na+ toxicity in brewing and related strains may be

due to antagonism of essential K+-functions.

Divalent metal cations

There is still much to learn about divalent (and trivalent) cation uptake but several general

statements concerning transport mechanisms can be made. Uptake is biphasic, involving

firstly non-specific cell surface binding of cations followed by a more regulated, carrier-

mediated translocation across the plasma membrane. This secondary phase involves energy-

dependent transport driven by the electrochemical membrane gradients generated by proton

and potassium ion pumps. However, it is the trans-membrane potential which is the primary

driving force for divalent cation uptake. The extracellular concentrations of glucose,

phosphate and potassium greatly influence divalent cation uptake. Once transported, certain

cations are subject to intracellular compartmentalisation most notably in the yeast vacuole.

Some cation carriers may have a very high affinity and be singularly specific for certain

ions, whereas others may possess broader specification and be capable of transporting a

multitude of divalent ions. Controlled efflux of certain cations (for example, Ca2+ and Cu2+)

also exists and is important to maintain intracellular levels at very low, sub-toxic levels.

Magnesium ions

Magnesium is the most abundant intracellular divalent cation in yeast cells and it acts

primarily as an enzyme cofactor. Although still far from being fully understood, uptake of

Mg2+ ions in yeast is thought to be driven by both the proton and potassium ion trans-

membrane gradients. Mg2+ uptake through the low-affinity K+ transporter is thought to be

of major significance in yeast. It is not known how many Mg2+ carriers exist but a general

divalent cation transport was described 30 years ago.

Mg2+ transport occurs with simultaneous uptake of phosphate and reserves of Mg-

orthophosphate have been found in the yeast vacuole. This intracellular segmentation of

Mg2+ indicates that vacuolar transport mechanisms are involved in regulating free Mg2+ ion

concentrations in the yeast cytoplasm.

The brewing significance of Mg2+ transport in yeast lies in the central importance of this

metal cation in governing several aspects of yeast growth and metabolism. With regard

to growth, cell Mg2+ has been shown to fluctuate during the cell cycle and it has been

postulated to play a role in co-ordinating cell growth and division by regulating key events

during mitosis. With regard to yeast fermentative metabolism, there has been found to be

correlation between cellular Mg2+ uptake and alcoholic fermentation in industrial strains of

Saccharomyces cerevisiae including brewing strains. Also Mg2+ may exert a protective

effect on yeast cultures subjected to a variety of physical and chemical stresses and as will

be discussed later, stimulates fermentation during the metabolism of high gravity worts.

44

Manganese ions

Manganese is essential for yeast growth and metabolism in trace levels and may also

act as an intracellular regulator of key enzymes. Mn2+ ions accumulate to a greater

extent than Ca2+ in yeast cells, but to a much lesser extent than Mg2+. AlthoughMn2+ can be substituted for Mg2+ as an enzyme cofactor in vitro, this is unlikely tobe of any physiological significance due to the different transport magnitudes and

resulting intracellular concentration differences between Mn2+ and Mg2+ in yeast

cells (|iM vs. mM respectively). The possibility that Mn2* may substitute for Ca2+ ions

in regulating the yeast cell division cycle will be discussed later. Mn2+ uptake in yeast,

which is strongly inhibited by Mg2+, is maximal during exponential growth anddecreases on entry into stationary phase. Like Mg2+, Mn2+ is accumulated in the yeast

vacuole. Energy-dependent transport of Mn2+, which is optimal at pH 5, is counter

balanced by K+ efflux to maintain electroneutrality. Mn2+ uptake and toxicity isstrongly influenced by the intracellular levels of Mg2+ ions.

Calcium ions

Calcium stimulates yeast growth but it is not a growth requirement. It is involvedin the membrane structure and function. Yeast cells maintain cytosolic Ca2+ at very lowlevels. This is accomplished by means of the efflux and compartmentalisation via plasma

membrane and tonoplast (scmipermeable membrane surrounding the cell vacuole) Ca2+

transporters and by means of sequestering with specific Ca2+ binding proteins like

calmodulin. The presence of Ca2+-H+ antiporter activity in the yeast cell membrane has

been demonstrated. Such a carrier exists in the vacuolar membrane indicating the energy-

dependent uptake of Ca2+ into the vacuole may be involved in regulating Ca2+ metabolismin yeast.

The physiological and biotechnological significance of Ca2+ uptake in yeast lies in the

multifunctional role of this cation as a modulator of growth and metabolic responses.In relation to yeast cell division, Ca2+ ions have been linked to cell cycle regulationand have been implicated in the transition from log to exponential phase in batchcultures of yeast. Also, culture media requirements for Ca2+ in yeast growth and

division have been highlighted recently by findings that indicate Mn2+ caneffectively replace Ca2+ in modulating events leading to cell cycle progression in

Saccharomyces cerevisiae. Calcium also plays an important role in flocculation.

Zinc ions

Trace levels of Zn2+ arc essential for yeast growth. For example, Zn2* deprivation inSaccharomyces cerevisiae prevents budding and arrests cells in the G, cycle of thecell cycle. Zn2+ requirements for the growth of yeast cannot be met by other metalions. Metabolic roles for Zn2+ indicate that it is essential for the structure and function

of many enzymes. For example, the important terminal step enzyme in yeastalcoholic fermentation, namely, alcohol dehydrogenase is a zinc-metalloenzyme.The brewing significance of this lies in the phenomenon of "stuck" fermentationswhich may be ameliorated following appropriate supplementation with zinc salts(Figure 32).

45

180-1

160-

140-

120-

100

Strain A

Strain B

02 0.3

Zinc addition mg/L

Figure 30. The effect of

zinc levels in wort on

primary fermentation time

(adaptedfrom Skands et

ai, Proc. EBC Cong.,

Maastricht, 1997, p. 413).

Copper and iron ions

Both copper and iron are

essential nutrients for yeast

which act as cofactors in

several enzymes including

the redox pigments of

the respiratory chain. The

assimilation of these two metals and their subsequent metabolism is closely interconnected

in yeast, as in other organisms. Copper is an essential micronutrient at low concentrations,

but is toxic at high concentrations. Copper toxicity towards yeast cells involves intracellular

interaction between copper, nucleic acid and enzymes. However, the major mode of action

is disruption of plasma membrane integrity. Copper ion homeostasis in yeast is controlled

by several uptake, efflux and chelation strategies depending on the external bioavailability

of copper. One mechanism relates to sequestration of copper by a copper-metallothionein

protein. Such low molecular weight proteins are generally synthesised as a protective

response to high levels of potentially toxic metal ions. Up to 60% of cellular copper in

Saccharomyces cerevisiae can be in the form of copper-metallothionein and this protein

plays an important role in copper resistance in this yeast.

Yeasts have adopted a number of strategies for converting insoluble (Fe3+) into biologically

active and soluble ferrous (Fe2+) ions. In Saccharomyces cerevisiae this is accomplished

by extracellular reduction by plasma membrane ferri reductase activity. Also in

Saccharomyces cerevisiae it is now recognised that several transporters exist in the cell

membrane. Some of these systems are non-specific for iron and also code for cobalt,

cadmium and nickel transport.

YEAST EXCRETION PRODUCTS

Although ethanol is the major excretion product produced by yeast during wort fermentation,

this primary alcohol has little impact on the flavour of the final beer. It is the type and

concentration of the many other yeast excretion products produced during wort fermentation

that primarily determine the flavour of the beer. The formation of these excretion products

depends on the overall metabolic balance of the yeast culture, and there are many

factors that can alter this balance and consequently beer flavour. Yeast strain, fermentation

temperature, adjunct type and level, fermenter design, wort pH, buffering capacity, wort

gravity, etc., are all influencing factors.

Some volatiles are of great importance and contribute significantly to beer flavour, whereas

others are important in building background flavour. The following groups of substances

46

arc found in beer: organic and fatly acids, alcohols, esters, carbonyls, sulphur compounds,amines, phenols and a number of miscellaneous compounds.

Organic and Fatty Acids

Some 110 acids, both organic and short-to mcdium-chain-lcngth fatty acids occur in beer.In part these are derived from malt or other wort constituents, but a proportion arise during

fermentation as a result of yeast metabolism. Organic acids contribute to the decrease in pH

observed during fermentation and many are flavour-active. They arise from carbohydrate

metabolism and include pyruvate, succinate, citrate, and acetate. It is presumed that mostof these arise as a consequence of the incomplete tricarboxylic acid cycle which occurs

under anaerobic conditions. It has been observed that pyruvate is secreted into the wortduring the active fermentation phase and that in later stages, when yeast growth has ceased,

it is re-utilised and the accumulation of acetate occurs. This observation provides evidence

for the overspill model of ethanol formation, already discussed. Thus, for pyruvate secretion

to occur it would suggest that under conditions of high glycolytic flux the pathways devolvingfrom pyruvate are rate-determining.

Medium-chain-length fatty acids (C6-CUI) arise via the activity of fatty acid synthetase asintermediates in the formation of longcr-chain-length fatty acids, which are incorporatedinto the various classes of yeast lipids. In addition, a proportion are derived from theassimilation and further metabolism of wort lipids. The release of medium- and long-

chain-length fatty acids during fermentation is probably associated with some loss of yeast

viability and subsequent cell lysis. This may occur during beer maturation.

The concentration of fatty acids formed as a result of yeast metabolism is inversely related to

fermentation rate. Thus, those parameters that increase fermentation rate, such as elevated

temperature and pitching rate, result in decreased accumulation of fatty acids. However, as

previously discussed, increased levels of wort oxygen favour yeast growth, with a concomitant

requirement for increased synthesis of membrane lipids. This depletes the acetyl CoA pool

such that less is available for the formation of medium-chain-length fatty acids. This may

be due to a general effect of fermentation rate. Also, the nitrogen content is important sinceacids such as isocaproic and isovaleric may be excreted as intermediates in the formation

of the corresponding ammo acids (leucinc and valine respectively).

Higher Alcohols

In flavour terms, the higher alcohols (also called fusel oils) that occur in beer and many

spirits are: n-propanol, isobutanol, 2-methyl-l-butanol and 3-methyl-l-butanol. However,more than 40 other alcohols have been identified. Regulation of the biosynthesis of higher

alcohols is complex since they may be produced as by-products of amino acid catabolismor via pyruvate derived from carbohydrate metabolism (Figure 33).

The catabolic route (a biochemical process in which organic compounds are digested,usually an energy-liberating process) involves a pathway in which the keto-acid produced

from an amino acid transamination is decarboxylated to the corresponding aldehyde, thenreduced to the alcohol via an NAD-linked dehydrogenase. In this way, for example,isobutanol may be produced from valine, 3-methyl-l-butanol from leucine and 2-methyI-1-butanol from isoleucine.

47

sugar

carbohydrate

metabolism

aldehydes

ethanol and

fusel alcohols

oxoacids

ammo

acids

Figure 31. Production of higher alcohols

(adaptedfrom Lewis and Young, Brewing, 1995).

The anabolic route (a biochemical process involving the synthesis of organic compounds,

usually an energy-utilising process) utilises the same pathways as those involved in the

biosynthesis of amino acids. As in the catabolic route, the keto-acid intermediate is

decarboxylated and the resultant aldehyde reduced to the alcohol.

The relative contribution made by the two routes varies with individual higher alcohols.

Since there is no corresponding amino acid, the anabolic route would seem to be the sole

mechanism for the formation of n-propanol. In general, the catabolic route would seem to

predominate during the early growth phase when exogenous amino nitrogen is plentiful. In

the later stages when the wort becomes deficient in assimilable nitrogen, the anabolic route

is probably the major source of higher alcohols.

The total concentration of higher alcohols produced during fermentation is linearly related

to the extent of yeast growth. Thus, conditions that promote growth, such as an increased

provision of oxygen, will result in increased production of higher alcohols. Similarly,

supplementation of worts with additional amino nitrogen also results in stimulation of

higher alcohol synthesis. In this case the nature of the amino acids present is reflected in

48

the spectrum of higher alcohols produced. Application of pressure during fermentation,

which may be accomplished by restricting the release of evolved carbon dioxide, results in

reduced yeast growth and is accompanied by a similar reduction in the extent of higher

alcohol formation.

Esters

Esters are important flavour components which impart flowery and fruit-like flavours and

aromas to beers, wines and spirits. Their presence is desirable at appropriate concentrations

but failure to properly control fermentation can result in unacceptable beer ester levels.

Organoleptically important esters include ethyl acetate, isoamyl acetate, isobutyl acetate,

ethyl caproate and 2-phcnylethyl acetate. In total, over 90 distinct esters have been

detected in beer.

unsaturated fats

and phospholipids

saturated fats

and phospholipids

sterols

cunsaturated

acyl CoAs

!■squalene

Imevalonic

acid

/C acyl CoAs

r.e\i , _

membrane

/

T

(oxoacidsV-*-

talcohols

\

M

/di and tri- -4—pyru-( carboxylicV acids

nitrogen

metabolism

V.

1i

^\

vate

J<s

i

ESTERS amino acids fermentable

carbohydrate

Figure 32. Metabolic interrelationships leading to ester formation

(adaptedfrom Hough el ai, Mailing & Brewing Science, Vol 2,

Hopped Wort and Beer, 1982).

49

Many factors, in addition to the yeast strain employed, have been found to influence the

amount of esters formed during a wort fermentation. These include: fermentation temperature,

where an increase in temperature from 10 to 25°C has been found to increase the

concentration ofethyl acetate from 12.5 to 21.5 mg/L; fermentation method, where continuous

fermentation results in higher levels of esters than conventional batch fermentation; pitching

rate, where higher rates have been reported to result in lower levels of ethyl acetate; and

wort aeration, where low levels of oxygen appear to enhance ester formation.

Esters arise as a result of a reaction between an alcohol, which may be ethanol or of longer-

chain-length, and a fatty acyl-CoA ester. The reaction is catalysed by an alcohol acetyl

transferase. The acyl component of the activated fatty acid may be acetate, produced by the

action of pyruvate dehydrogenase. Alternatively, acetate and longer-chain-length acids may

be activated directly by an acyl-CoA synthetasc.

The spectrum of esters produced is largely strain-specific. This may reflect the presence of

a family of alcohol acetyltransferases with different substrate specificities. The relative

activities of these enzymes will depend, to some extent on the availability of the respective

substrates. The rate of formation and type of ethyl ester produced are influenced by the

availability of the respective fatty acids which will be synthesised de novo or assimilated

from the wort. In the case of the synthesis of acetate esters the availability of the

corresponding higher alcohol is important.

The total quantities of esters produced during fermentation are influenced by the wort gravity,

the oxygen availability and the temperature (which should not be a process variable).

An increase in the concentration ofoxygen supplied at pitching is associated with a progressive

decline in the ester content of the final beer. It is assumed that since increased oxygen availability

promotes greater yeast growth more of the acetyl-CoA pool is utilised in biosynthetic

reactions, as seen in Figure 34, thereby restricting that available for ester synthesis.

The effect of wort gravity is particularly relevant to modern practice since in some

circumstances an increase in this parameter is associated with elevated ester levels. Many

other factors are pertinent (which will be discussed later), and this phenomenon defines an

upper limit that can be used in high gravity brewing. The explanation for the relationship

between wort gravity and ester levels would appear to reside in the use of sugar adjuncts

in concentrated worts. This increases the C:N ratio of the wort such that growth becomes

limited by nitrogen depletion, thereby allowing the excess carbon to be metabolised to

acetyl-CoA and hence provides a supply of substrate for ester synthesis. In addition, the

concentration of unsaturated fatty acid may be diluted, which would tend to promote ester

synthesis by relieving repression of the alcohol acetyltransfcrase.

Practical measures which can be taken to control ester levels (particularly in high gravity

worts) include wort with a suitably low C:N ratio and an adequate supply of oxygen, both

of which promote yeast growth, and minimise ester synthesis. The application of pressure

during fermentation also reduces both yeast growth and ester synthesis. Likely reasons for

this effect would appear to be that intracellular carbon dioxide accumulates causing a

decrease in cellular pH control and a disruption of enzyme function. The ionic composition

of wort may influence ester synthesis. Zinc, which as previously discussed is routinely

added to wort to ensure adequate yeast growth, may also encourage the formation of the

50

acetate esters of higher alcohols. The effect is probably a consequence of zinc stimulating

the production of the higher alcohol from the corresponding oxo-acid, thereby increasingthe supply of precursors for subsequent ester synthesis.

The major metabolic pathways that control ester synthesis in yeast are outlined in Figure 34.

From this figure and from the reaction seen below, one can see how ethyl acetate is the most

common ester produced by yeast. This is due to the fact that the most common alcohol in

yeast is ethanol, which is the alcoholic precursor of ethyl acetate. Esters of higher alcohols

and ethyl esters of long-chain fatty acids are also common.

CH,CH,OH + CHjCOSCoA

ethanol acetyl CoA

CH3CH2COOCH3 + CoASH

ethyl acetate

Carbonyls

Some 200 carbonyl compounds are reported to contribute to the flavour of beer and other

alcoholic beverages. Those influencing beer flavour, produced as a result of yeast metabolism

during fermentation, are various aldehydes and vicinal diketones, notably diacetyl. Also

carbonyl compounds exert a significant influence on the flavour stability of beer. Excessive

Pyruvate a-oxobutyrale

pyrophosphate)

a-acetohydroxy-butyrate

u-acetolactate

diacetyl

a-acetohydroxybutyrate

2,3-pentanedione

, Enzymatic | *\conversion y

Passive

diffusionNon-enzymaticdecomposition

Figure 33. Formation of diacetyl and 2,3-pentanedione as by-products of pathways

leading to the formation of the amino acids valine and isoleucinc.

51

Aceloin

^^, Enzymatic^^ conversion

Taken up or

excreted by cell

Butanediol

Figure 34. Reduction of diacetyl to acetoin

and 2,3-butanediol.

concentrations of carbonyl

compounds are known to cause

stale flavour in beer. The effects

of aldehydes on flavour stability

are reported as grassy notes

(propanol, 2-methyl butanol,

pentanol) and a papery taste

(fra/K-2-nonenal, furfural).

Quantitatively, acetaldehyde is

the most important aldehyde.

This is produced via the

decarboxylation of pyruvate

and is an intermediate in the

formation of ethanol. It may be

present in beer at concentrations

above its flavour threshold,

(approx. 10 mg/L), at which it imparts an undesirable "grassy" or "green apple" character.

Acetaldehyde accumulates during the period of active growth. Levels usually decline in the

stationary phase of growth late in fermentation. As with higher alcohols and esters, the

extent of acetaldehyde accumulation is determined by the yeast strain and the fermentation

conditions. Although the yeast strain is of primary importance, elevated wort oxygen

concentration, pitching rate and temperature all favour acetaldehyde accumulation. In

addition, the premature separation of yeast from fermented wort does not allow the re-

utilisation of excreted acetaldehyde associated with the latter states of fermentation.

Other important flavour-active carbonyls, whose presence in beer is determined in

the fermentation stage, are the vicinal diketones, diacetyl (2,3-butanedione) and 2,3-

pentanedione. Both compounds impart a "butterscotch" flavour and aroma to beer.

Quantitatively, diacetyl is the most important since its flavour threshold is approx. 0.1 mg/L

and is ten-fold lower than that of 2,3-pentanedione. The organoleptic properties of vicinal

diketones contribute to the

overall palate and aroma of 106

some ales but in most lagers they

impart an undesirable character.

A critical aspect of the

management of lager fermentations

and subsequent processing is to

ensure that the mature beer

contains concentrations of vicinal

diketones lower than their

flavour threshold.

1.05

1.04

1.03

1.02

1.01

Suspended

• yeast count

0.8

0.6

0.4<

0.2

60

50

40

30

20

10

Diacetyl and 2,3-pentanediones

arise in beer as by-products of the

pathways leading to the formation

of valine and isoleucine (Figure

0 20 40 60 80 100 120 140 160 160 200

Time (hours)

Figure 35. Pattern of diacetyl formation and

breakdown in relation to yeast growth

and wort gravity.

52

35). The a-acetohydroxy acids, which are intermediates in these biosyntheses, are in part

excreted into the fermenting wort. Here they undergo spontaneous oxidative

decarboxylation, giving rise to vicinal dikctones. Further metabolism is dependent on yeast

dehydrogenases. Diacctyl is reduced to acetoin and ultimately 2,3-butanediol, (Figure 36)

and 2,3- pentanedione to its corresponding diol. The flavour threshold concentrations of

these diols are relatively high and, therefore, the final reductive stages of vicinal dikctonc

metabolism are critical in order to obtain a beer with acceptable organoleptic properties.

The pattern of diacetyl formation and subsequent breakdown in relation to yeast growth and

gravity during a lager fermentation is shown in Figure 37. The diacetyl concentration peak

occurs towards the end of the period of active growth. The reduction of diacetyl takes place

in the latter stages of fermentation when active growth has ceased. In terms of practical

fermentation management the need to achieve a desired diacetyl specification may be the

factor which determines when the beer may be moved to the conditioning phase, filtered or

centrifuged (depending on the processing procedures). Thus, diacetyl metabolism is an

important determinant of overall vessel residence time, which clearly affects the efficiency

of plant utilisation.

The concentration of diacetyl present in fermenting wort is a function of the rate formation

of diacetyl precursor (a-acetolactate), oxidative decarboxylation of the precursor to form

diacetyl and reduction of diacetyl to acetoin. These reactions are influenced by the yeast

strain, both in terms of the biochemistry and technological behaviour and how these are

affected by wort composition, the type of fermenting vessel employed and the fermentation

conditions. Fermentation conditions that favour yeast growth rate, and consequently an

increased requirement for amino acid biosynthesis from pyruvate, would be expected to

lead to elevated levels of a-acetolactate. These conditions include high temperatures and

pitching rates and an increased level of wort oxygen, but may be modulated by wort

composition. Consequently, when the assimilable amino-nitrogen level is high, there will

be a reduced requirement for amino acid synthesis and potentially a lower level of

a-acetolactate. In addition, the presence of valine and isoleucine specifically inhibits the

formation of a-acetohydroxy acids.

Elevated levels of a-acctolactate in fermented wort do not inevitably lead to high diacetyl

concentrations in beer. However, this is undesirable since diacetyl formation may occur

during subsequent processing when no yeast is present to catalyse a-acetolactate reduction.

The non-enzymic oxidative decarboxylation of a-acetolactate is the rate-determining step

in the diacetyl cycle. The presence of oxygen is not essential since metal ions such as Cu2+,

Fe3+ and Al3+ may serve as alternative electron donors. The rate of formation of diacetyl

from a-acetolactate is also influenced by pH. Within the range encountered in fermenting

wort, a low pH promotes diacetyl formation but also high wort pH's at pitching (>5.3) will

promote yeast growth and elevated levels of a-acetolaclate and potentially, therefore,

diacetyl formation.

The reduction of vicinal diketones in the later stages of fermentation and during maturation

requires the presence of adequate yeast in suspension in the fermented wort. Thus, where

the yeast is particularly flocculent (this phenomenon will be discussed later), premature

separation will be reflected by low rates of diacetyl reduction and potentially elevated

levels in finished beer. Diacetyl removal is also affected by the physiological condition of

53

the yeast. When the pitching yeast is in poor condition, such that the primary fermentation

performance is suboptimal, the yeast present during the latter stages will be stressed and

the period of diacetyl reduction will be prolonged.

A number of strategies can be adopted to ensure that beer diacetyl specifications arc

achieved. Diacetyl removal can be attained post-fermentation in the conditioning stages of

brewing (traditional lagering). This is a slow process, expensive in terms of time and

conditioning capacity. Alternatively, it is desirable to ensure that minimum diacetyl

concentrations are achieved before the beer is removed from the fermenter. It is necessary

to select fermentation conditions (i.e. pitching rate, wort DO and attemperation regimes)

which provide an optimum profile. In practice, the aim is to promote the maximum a-

acetolactate levels as early as possible, such that the resultant diacetyl may be rapidly

reduced due to the presence of a high suspended yeast count. This reductive phase may be

stimulated by increasing the fermentation temperature approximately two-thirds through

the fermentation cycle.

There are a number of novel methods that are currently being developed to control beer

diacetyl levels. One (which is being used on a production basis in Finland) involves the use

of immobilised yeast technology and will be discussed later. Also, research has been

conducted on the genetic modification of brewer's yeast strains in order to reduce their

diacetyl formation potential. Four strategies have been investigated. The gene coding for a-

acetohydroxy acid synthetase (ILV2) may be deleted and thereby reduce the supply of

diacetyl precursor. Alternatively, the gene for a-acetohydroxy acid isomerase (ILV5), which

catalyses the reductive step in the synthesis of valine and isoleucine, could be amplified.

It is suggested that this would also reduce the pool size of diacetyl precursor by

promoting the synthesis of valine and isoleucine. A lager brewing strain with increased

levels of the ILV5 gene has been constructed which in laboratory-scale fermentations,

produced 70-80% less diacetyl than the wild type (the original strain). Other

fermentation properties have been found to be unaltered including the flavour of the

final beer.

The third strategy involves the enzyme oc-acetolactate decarboxylase which catalyses

the direct formation of acetoin from cx-acetolactate. Several bacterial species possess

this enzyme activity but it is not present naturally in brewing yeast strains. This cx-

acetolactate decarboxylase gene has been isolated from Acetobacter spp. (the bacteria

employed for vinegar manufacture) and inserted into brewing yeast. Diacetyl formation

with this cloned yeast is reduced. However, for reasons already discussed, these novel

strains have not been used in commercial brewing. No doubt when the benefits of the

new technology become more widely appreciated, adverse public reaction will

disappear.

The fourth strategy involves the addition of the enzyme oc-acetolactate decarboxylase, to

the cold wort prior to fermentation. This enzyme transforms the acetolactate directly into

acetoin, thus by-passing the diacetyl stage. The enzyme is available commercially under the

name of Maturex™ and in 1991 was approved for food grade application. Maturex™

is produced by Novo Nordisk A/S from Bacillus subtilis carrying the gene coding for

cc-acetolactate decarboxylase from Bacillus brevis.

54

Sulphur Compounds

Sulphur compounds make a significant contribution to the flavour of beer. Although small

amounts of sulphur compounds can be acceptable or even desirable in beer, in excess they

give rise to unpleasant off-flavours, and special measures such as purging with CO2 or

prolonged maturation times are necessary to remove them. Many of the sulphur compounds

present in beer are not directly associated with fermentation but are derived from the raw

materials employed. However, the concentrations of hydrogen sulphide (rotten egg aroma)

and sulphur dioxide (burnt match aroma) are dependent on yeast activity. Failure to

manage fermentation properly can result in unacceptably high levels of these compounds

occurring in the finished beer.

Sulphate

H2S

Organic

acids \J

Amino acids

I

Acetyl-CoA

J

Acetaldehyde

IEthanol

-► Keto (oxo)

acids V.

r

Vicinal

Diketones

Fatty acyl-CoA

Fatty

acids

Fusel

alcohols

Esters

Lipids

Figure 36. Inter-relationship between yeast metabolism and production

of flavour compounds.

The concentration of hydrogen sulphide and sulphur dioxide formed during fermentation

are primarily determined by the yeast strain used, although the wort composition and the

fermentation conditions are major factors, particularly where levels are abnormally high.

Both compounds arise as by-products of the synthesis of the sulphur-containing aminoacids cysteine and methionine from sulphate (Figure 30). These syntheses are influenced

by wort composition in that the yeast will preferentially assimilate sulphur-containing

amino acids. It is only when wort is depleted in such amino acids does the biosyntheticroute come into operation.

The peak of hydrogen sulphide and sulphur dioxide production occurs in the second or third

day of fermentation. Presumably, at this time the sulphur-containing amino acids in wort

55

ATP-Sulphuryiase

TATP

ADP

Adenosine - 5' phosphosulphate (APS)

ATP

I3/ phosphoadenosine - 5' phosphosulphate (PAPS)

NADPH

I| SULPHITE (SOJ +ADP |

Sulphite reductase

I

NADP

NADPH

NADP

SULPHIDE (H,S)

Cysteine synthase

(pantothenale requiring)Serine

contributing to beer flavour,

also has a number of other

functions in beer (and other

alcoholic beverages). It can

act as an antimicrobial agent,

an antioxidant and retard the

development of beer staling

character. Regarding its

antimicrobial activity, this

only occurs at concentrations

in excess of 50 mg/L which

is well above the permitted limit

in beer for most countries

except cast conditioned beer

Figure 38 (right). Structure

of cysteine, cystine,

methionine and glutathione.

Cysteine

Cystine

Methionine

Glutathione

Figure 37 (left). Pathway for the

synthesis of sulphur-containing

amino acids.

will have been utilised. Yeast growth

during fermentation is roughly

synchronous (cell division occurs at

the same time) and hydrogen sulphide

evolution seems to occur in a number

of peaks which correspond to the

phase of the yeast cell cycle just prior

to the onset of budding.

The formation of excessive levels of

hydrogen sulphide and sulphur

dioxide during fermentation is,

therefore, associated with conditions

that restrict yeast growth. In this

regard the provision of adequate

oxygen at the time of pitching is a

critical factor. Since both hydrogen

sulphide and sulphur dioxide arc

volatile, it follows that a vigorous

fermentation will promote its removal

via carbon dioxide stripping. The type

offermenting vessel is also influential.

Sulphur dioxide, as well as

HSCH^CHfNhyCOOH

NH,

INH,

HOGCCHCH2-S-S-CH2CHCOOH

CH2SCH.CH2CH(NH,)COOH

H2NCHCH2CH2CONHCHCONHCH2COOH

COOH CH2SH

56

and wine. Sulphur dioxide's retarding action on beer staling is two-fold. In the presence of

oxygen it is converted to sulphate and also the bisulphite will rcversibly bind to carbonyls,

some of which (as previously described), will give rise to the papery or cardboard

characteristics of stale beer. These sulphite complexes are flavour neutral. For many years

it has been traditional to add sodium or potassium metabisulphite to beer during maturation

in order to improve flavour stability. However, because of bisulphite's allergenic properties

this use is decreasing. However, research at the Carlsberg Technical Centre is developing

genetically manipulated brewing strains that hyper-produce sulphur dioxide. Preliminary

results with these strains would indicate that beer produced with them has enhanced flavour

stability. Dimethylsulphide (DMS) is one of the major flavour congeners found in

continental European lager beers. It has the aroma characteristics of cooked corn

(maize) or garlic. In beer it originates from two sources, from the hydrolysis of malt

S-methylmethionine (SMM) during mashing and from the reduction of dimethyl-

sulphoxide (DMSO) by the yeast. It is thought that usually the majority of the DMS is

produced by yeast and 80% of the DMS comes from DMSO. The DMS evaporation ratio

can vary between 0 and 65% throughout the formation of this compound during

fermentation. When the influence of wort DMSO concentration on the production of

DMS during fermentation was studied, it was observed that there is a proportional

relationship between the concentrations of these compounds at the end of fermentation and

at every stage of fermentation. The variety of malt has a direct influence on the DMSO

quantity and, therefore, an indirect influence on the level of DMS in beer. When the

concentration of DMSO in wort at pitching is high, then the concentration of DMS in the

beer will also be high.

To conclude this section, Figure 38 summarises the major metabolic interrelationships in

yeast affecting the formation of beer flavour compounds.

FLOCCULATION

As previously discussed, the flocculation property, or conversely, lack of flocculation, of aparticular yeast

culture is one of Non-flocculont Chain Former Flocculentthe major factors

when considering

important

characteristics

during brewing

and other ethanol

fermentations.

Unfortunately, a

certain degree of

confusion has

arisen by the use

of the term

flocculation in

the scientific

literature to Figure39. Flocculation inSaccharomycescerevisae.

57

2.0 -i

1.8-

1.6-

1.4-

1.2-

1.0-

Non-flocculent culture

Flocculent culture

describe different phenomena in

yeast cell behaviour. Specifically,

flocculation, as it applies to brewer's

yeast is "the phenomenon wherein

yeast cells adhere in clumps and

either sediment from the medium

in which they are suspended or

rise to the medium's surface".

This definition excludes other

forms of aggregation, particularly

that of "clumpy-growth" and

"chain formation", which have

been discussed previously (Figure

39). This non-segregation of

daughter and mother cells during

growth has sometimes erroneously

been referred to as flocculation.

The term "non-flocculation"

therefore applies to the lack of cell

aggregation and, consequently, a much

slower separation of (dispersed)

yeast cells from the liquid medium. Flocculation usually occurs in the absence of cell

division, but not always, during late logarithmic and stationery growth phase and only

under rather circumscribed environmental conditions involving specific yeast cell surface

components (proteins and carbohydrate components) and an interaction of calcium ions.

Although yeast separation often occurs by sedimentation, it may also be by flotation

because of cell aggregates entrapping bubbles of CO2 as in the case of "top-cropping" ale

brewing yeast strains.

0.B-

0.6-

0.4-

0.2-

10 20 30 40 SO 60 70

Percentage attenuation

80 90 100

Figure 40. Static fermentation flocculation.

-• Protein sites

t* Mannan sites

Figure 41. Lectin theory of flocculation. Protein lectins on the yeast cell surface interact

with cither mannose containing and/or glucose containing carbohydrate determinants on

the cell walls of adjacent cells only in the presence of calcium.

58

Figure 42. Electron photomicrograph of Sacchawmyces cerevisiae flocculent

and non-flocculent strains shadow-cast with tungsten oxide.

Flocculent Yeast Non-flocculent Yeast

Adhering Culture Non-Adhering Cultures

Figure 43. Electron photomicrographs of adhering and non-adhering cultures of

Candida albicans (photograph courtesy ofJ. Douglas).

59

The requirement of yeast flocculation is a much discussed and disputed topic and there

is a dire need for some degree of standardisation of such tests. Due to the plethora of

flocculation tests and the fact that nearly every laboratory involved in this area of study

appears to have their own "pet" method, it is very difficult to interpret results from one

laboratory to another. The methods being employed to measure yeast flocculence can be

roughly divided into three groupings:

• Sedimentation methods (for example, Helm Sedimentation Test)

In this test, the yeast culture is removed from the growth medium and the cells washed

a number of times with deionised water containing 80 mg/mL of calcium ion, usually

as calcium chloride at pH 4.0 and depending on the scale of the test, the suspension

placed in a test tube (10 mL scale) or measuring cylinder (100 mL scale).

• Direct observation of floe formation in the growth medium

In this method, a small inoculum of the yeast strain is seeded into 20 mL screw capped

glass bottles containing 15 mL of medium. After three days incubation at 25°C, the

flocculation characteristics of the culture are determined by the nature of the floes

subsequent to the sediment being brought back into suspension by shaking of the bottle.

The method allows for routine flocculence determinations of a large number of cultures

and has been employed extensively in genetic studies on flocculation.

To express the flocculation results from the above flocculation tests, a subjective

graduation of flocculation is often used, for example: 5 - extremely flocculent; 4 - very

flocculent; 3 - moderately flocculent; 2 - weakly flocculent; 1 -rough; and 0 - non-flocculent.

An alternative measurement of flocculation has been to examine microscopically the

floes and determine the percentage of cells in floes compared to unflocculatcd cells.

• Static fermentation methods

In this method, the concentration of yeast in suspension is determined during the course

of the fermentation (Figure 40). The first two methods for measuring yeast flocculence

can be viewed as artificial in vitro tests for flocculence due to the fact that they are

conducted under artificial conditions in relation to the brewing process. This latter

method for assaying yeast flocculence is a more in vivo style test because it is carried out

under conditions more closely akin to the static fermentation conditions encountered in

a brewery.

Individual strains of brewer's yeast differ considerably in flocculating power. At one

extreme there are highly non-flocculent, often referred to as powdery, strains. At the other

extreme there are flocculent strains. The latter tend to separate early from suspension in

fermenting wort, giving an under-attenuated, sweeter and less fully fermented beer. Beers

of this nature, because of the presence of fermentable sugars, are liable to biological

instability. By contrast, poorly flocculcnt (non-flocculent or powdery) yeasts produce a dry,

fully fermented, more biologically stable beer in which clarification is slow, leading to

filtration difficulties and the possible acquisition of yeasty off-flavours. The disadvantages

presented by the two types of yeast strain are especially relevant to more traditional

fermentation systems where the fermentation process is dependent upon the sedimentation

characteristics of the yeast. Contemporary brewing technology has largely reversed this

situation where yeast sedimentation characteristics are now fitted into the fermenter design.

60

The efficiency, economy and speed of batch fermentations have been improved by the use

of cylindro-conical fermentation vessels and centrifuges [which are often (but not always)

employed in tandem). There is no doubt that differences in the flocculation characteristics

of various yeast cultures are primarily a manifestation of the culture's cell wall structure.

Several mechanisms for flocculation have been proposed. One hypothesis is that anionic

groups of cell wall components are linked by Ca2+ ions. In all likelihood, these anionic

groups are proteins. Another hypothesis implicates mannoproteins specific to flocculent

cultures acting in a Icctin-like manner to cross-link cells; here Ca2+ ions act as ligands to

promote flocculence by conformational changes (Figure 41). Most people working in the

field agree that the latter hypothesis is the most credible. In addition to flocculation there

is the phenomenon of co-flocculation. Co-flocculation is defined as the phenomenon

where two strains are non-flocculent alone but flocculent when mixed together. To date,

co-fiocculation has only been observed with ale strains, and there are no reports of

co-flocculation between two lager strains of yeast. There is a third flocculation reaction

which has been described, where the yeast strain has the ability to aggregate and

co-sediment with contaminating bacteria in the culture. Again this phenomenon appears to

be confined to ale yeast strains, and co-sedimentation of lager yeast with bacteria has notbeen observed.

As described above, flocculation requires the presence of surface protein and mannan

receptors. If these are not available or are masked, blocked, inhibited or denatured,

flocculation cannot occur. Onset of flocculation is an aspect of the subject where there

is great commercial interest but about which relatively little is known. As previously

discussed, the ideal brewing strain remains in suspension as fermenting single cells untilthe end of fermentation when the sugars in the wort are depleted, and only then does it

rapidly flocculate out of suspension. What signals the onset of activation or relief from

inhibition? This is still an unanswered question that is currently being studied by a numberof research laboratories.

Electron microscopy of flocculent and non-flocculent cultures shadowed with tungsten

oxide has revealed that flocculcnt cultures possess a "hairy" outer surface (Figure 42).

It is noteworthy that surface appendages have been implicated in many instances of microbial

flocculation, aggregation, and adhesion. For example, it is believed that adhesion of cells

of the pathogenic yeast Candida albicans to mucosal surfaces involves Icctin-like

interactions between the protein portion of mannoprotein located in fibrils on the cellsurface and glycoside receptors on epithelial cells (Figure 43).

Yeast flocculation is genetically controlled and research on this aspect of the phenomenon

dates from the early 1950's. However, because of the polyploid/aneuploid nature ofbrewing yeast strains, most, but not all, of the research on flocculation genetics has been

conducted on haploid/diploid genetically defined laboratory strains. Numerous genes have

been reported to directly influence the flocculent phenotype in Sacchawmvces spp. Four

dominant flocculation genes have been identified FLO1 (alleles are FLO2, FL04, FLOS),

FLOS, FL09 and FLOW, as well as a semi-dominant gene,/7o5, and two recessive genes,

flo6 and f\o7. In addition, mutations in several genes, including the regulatory genes TUP1

and SSN6, have been found to cause flocculation or 'flaky' growth in non-flocculent strains.In total, at least 33 genes have been reported to be involved in flocculation or cell aggregation.

Although, the role of many of these genes is far from understood FLO! has been successfully

61

cloned into brewing strains. Also, as the chromosomal location of FLO1 is known

(Chromosome I) and with knowledge of the yeast genome sequence, the amino acid

sequence of this gene has been deduced. A study of the genetics of yeast flocculation

affords an opportunity to study the genetics of structural (cell wall), rather than enzymatic,

proteins. This research also presents the possibility of being able to control and manipulate

one of the most impbrtant characteristics of a brewer's yeast strain.

YEAST MANAGEMENT

It has been previously discussed in this document (but is such an important fact that it is

worthy of repetition), that in brewing the cropped yeast is re-pitched into subsequent

brews. The quality of the cropped yeast will significantly affect the overall performance of

a subsequent fermentation into which this yeast is pitched, which in turn will influence the

resulting beer quality and stability.

It is normal procedure in many breweries to propagate fresh yeast (particularly lager yeast)

every 8-10 generations (fermentation cycles), or earlier if contaminated (the yeast could

also be acid washed), or if a fermentation problem is identified. Fermentation problems

include sluggish fermentations, usually slower rates of wort maltose and maltotriose

uptake, higher levels of sulphur dioxide and hydrogen sulphide, prolonged diacetyl

reduction times and increased flocculation and sedimentation rates.

Pure Yeast Cultures

The systematic use of clean, pure

and highly viable cells ensures

that bacteria, wild yeasts or yeast

mutations (such as respiratory

deficiency) do not lead to

inconsistent fermentations and

off-flavour development.

The practice of using a pure

yeast culture for brewing was

started by Emil C. Hansen in the

Carlsberg laboratory over 100

years ago. Employing dilution

techniques, he was able to

isolate single cells of brewing

yeast, test them individually and

select the specific yeast strains

that gave the desired brewing

properties. The first pure yeast

culture was introduced into a

Carlsberg brewery on a production

scale in 1883, and the benefits of

using a pure culture quickly

became clear. Soon, 23 countries

Exhaust

Spray ball

Condensate

or

coolant

Pitching

yeast to

fermenter

Temperature

probe

Yeast

inoculation

port

Sterile wort

Figure 44. Typical propagation vessel.

62

had installed Hanscn's pure culture plant, for example, in North America, Pabst, Schlitz,

Anhcuser Busch and 50 smaller breweries were using pure lager cultures by 1892.

Hansen's first propagation plant consisted of a steam-sterilised wort receiver and propagation

vessel equipped with a supply of sterile air and an impeller. The basic principles of propagation

devised in 1890 have changed little. Propagation can be batch or semi-continuous and

usually consists of three stainless steel vessels of increasing size, equipped with attemperation

control, sight glasses and non-contaminating venting systems (Figure 44). Each vessel is

equipped with a CIP system and often has in place heat sterilising and cooling systems for

both the equipment and the wort. Ideally the yeast propagation system should be located in

a separate room from the fermenting area with positive air pressure, humidity control, an

air sterilising system, disinfectant mats in doorways and limited access by brewing staff.

During yeast propagation, the aim is to obtain maximum yield of yeast but also to keep

the flavour of the beer similar to a normal fermentation so that it can be blended into the

production stream. As a result, the propagation is often carried out at only slightly higher

temperatures and with intermittent aeration to stimulate yeast growth. The propagation of

the master culture to the plant fermentation scale is a progression of fermentations of

increasing size (typically 5-20 X) until sufficient yeast is grown to pitch a half or full size

commercial brew.

Wort sterility is normally ensured by boiling for 30 minutes or it can be pasteurised using

a plate heat exchanger, passed into a sterile vessel and then cooled. Wort gravities typically

range from 10°P (1040 OG) to 16°P (1064 OG) but typically should be at the lower end of

the range. Depending on the yeast strain, zinc or a commercial yeast food can be added.

Aeration (oxygenation) is important for yeast growth, and ale wort is aerated using oxygen

or sterile air, and anti-foam may be added depending on the yeast. Agitation is not normally

necessary as the aeration process and CO2 evolved during active fermentation are sufficientto maintain the yeast in suspension.

A typical brewery yeast propagation schedule would be as follows, but details will vary

greatly with the size of the brewery and the particular propagation equipment available:

• Loop of culture from slope or petri dish;

• Transfer to 250 mL wort (1040, 10°P) or yeast extract-peptone broth in 500 mL flask,

place on shaker for 2 days at 20°C (lager) or 278C (ale);

• Transfer to 50 L vessel containing 25-35 L wort (1040-1048, 10-l2°P), 3 days, slowshaking;

• Transfer to 20-30 hL yeast vessel, 15-20 hL, wort (1040-1048, 10-12°P), aerate/oxygenate

(25 L/min.), 2O-22°C, 2-3 days;

• Transfer to larger culture vessel 100-150 hL, 75-100 hL wort, 20-22°C, 2-3 days;

• Transfer to fermenting vessel, 300 hL. Ferment using normal procedures;

• Crop yeast and blend "green" beer at low rate (20-30%);

• Hygiene during the whole procedure is critical!

63

Preservation of Stock Yeast Culture

The long term preservation of a brewing yeast culture requires that not only is optimal

survival important, but it is imperative that no change in the character of the yeast occurs.

Many yeast strains are difficult to maintain in a stable state and long term preservation by

lyophilization, (freeze drying), which has proven useful for mycelial fungi, has been found to

give poor results with many brewing yeast strains. Storage studies have been conducted with

a number of ale and lager brewing strains. The following storage conditions were investigated:

• Low temperature (-70°C refrigeration or liquid nitrogen);

• Lyophilization (freeze drying);

• Storage in distilled water;

• Storage under oil;

• Repeated direct transfers on culture media (subculture once a week for two years);

• Long term storage at 21 °C on solid nutrient medium - subcultures every six months;

• Long term storage at 4°C on solid nutrient medium - subcultures every six months.

After a two year storage period, wort fermentation tests including wort fermentation

rate and wort sugar uptake efficiency, flocculation tests, sporulation ability, formation of

respiratory deficient colonies and ease of revival were conducted, and the results compared

to the characteristics of the unstored control culture. Low temperature storage appears

to be the storage method of choice if cost and availability of the appropriate equipment

is not a significant factor. Cultures stored at -70°C had the lowest death rate and were

the easiest to revitalise. Also, the degree of flocculation, wort fermentation ability,

sporulation ability and proportion of respiratory deficient mutants present were all

unaffected by this storage method. Storage at 4°C on nutrient agar slopes, subcultured

every 6 months, was the next method of preference to low temperature storage.

Lyophilization and other storage methods revealed yeast instability which varied from

strain to strain. Today many breweries store their strains (or contract store) at -70°C.

Routine subculturing of cultures on solid media every six months is a less desirable but

very cost effective storage method. Lyophilisation of brewer's yeast cultures should

be avoided!

Yeast Pitching and Cell Viability

Microscopic examination of brewery pitching yeast is a rapid way to ensure that there is not

a major contaminant or viability problem with the pitching yeast culture. When a sample

of pitching yeast in either water, wort or beer is examined under the microscope, it

can be difficult if not impossible to distinguish a small number of bacteria from the trub or

other extraneous non-living material. Trub material, however, is irregular in size and outline,

and dissolves readily in dilute alkali.

A trained microbiologist becomes familiar with the typical appearance of the yeast

cytoplasm and shape of the yeast cells, whether the cells are normally chain formers, or in

clumps, etc., and thus one can sometimes identify the presence of wild yeasts due to cells

with an unusual shape or differences in budding or flocculating behaviours.

64

The use of viability stains such as methylene blue gives a good indication of the health of the

cells. Although there are a number of good stains and techniques available, in experiencedhands, methylene blue will quickly identify a problem if there is a known history of thetypical viability of the yeast strain prior to pitching.

Yeast pitching is governed by a number of factors such as wort gravity, wort constituents,

temperature, degree of wort aeration/oxygenation and previous history of the yeast. Ideally,

one wants a minimum lag in order to obtain a rapid start to fermentation, which then results

in a fast pH drop, and ultimately assists in the suppression of bacterial growth. Pitchingrates employed vary from 5-20 million cells/mL (depending on the original gravity of the

wort) but 10-12 million cclls/mL is considered an optimum level by many and results in

a lager yeast reproducing three to five times. Increasing the pitching rate results in fewer

doublings, since yeast cells under given conditions multiply only to a certain level ofcells/unit volume, regardless of the original pitching rate.

The pitching rate can be determined by a number of methods such as dry weight,

turbidimeter sensors, haemocytometer, and electronic cell counting. Recently, use has been

made of commercially available in-line biomass sensors which utilise the passive dielectricalproperties of microbial cells and can discriminate between viable and non-viable cells

and trub. The amount of yeast grown is limited by a number of factors including oxygensupply, nutrient exhaustion and accumulation of inhibitory metabolic products.

Yeast Collection

Yeast collection techniques vary depending on whether one is dealing with a traditional ale

top fermentation system, a traditional lager bottom fermentation system, a non-flocculcnt

culture where the yeast is cropped with a centrifuge, or a cylindro-conical fermentation

system. With the traditional ale top fermentation system, although there are many variations

on this system, a single, dual or multi-strain yeast system can be employed and the timing

of the skimming can be critical to maintain the flocculation characteristics of the strains.

Traditionally, the first skim or "dirt skim", with the trub present, is discarded, as is the final

skim in most cases. The middle skim is normally kept for repitching. With the traditional

lager bottom fermentation system, the yeast is deposited on the bottom of the vessel atthe end of fermentation. Yeast cropping is non-selective and the yeast contains entrained

trub. With the cylindro-conical fermentation system (now widely adopted for both ale

and lager fermentations), the angle at the bottom of the tank allows for effective yeastplug removal.

The use of centrifuges for the removal of yeast and the collection of pitching yeast is now

commonplace. There are a number ofadvantages such as shorter process time, cost reduction,

increased productivity and reduced shrinkage. Care must be taken to ensure that hightemperatures (i.e. >20°C) are not generated during centrifugation and that the design

ensures low dissolved oxygen pickup and a high throughput. This is usually accomplished

by use of a self-desludging and low heat induction unit. Timing control of the desludge

cycle is important and allows for a more frequent cycle for yeast from the pitching tank andresultant lower solids and a longer frequency for yeast being sent to waste with the highsolids and resulting reduced product shrink.

65

Yeast storage

Ideally the yeast is stored in a room that is easily sanitised, contains a plentiful supply of

sterile water and a separate filtered air supply with positive pressure to prevent the entry of

contaminants and a temperature of 0°C. Alternatively, insulated tanks in a dehumidified

room are employed. When open vessels were commonly used, greater care had to be taken

to ensure that sources of contamination were eliminated. Reduction of moisture levels

to retard mould growth and elimination of difficult to clean surfaces and unnecessary

equipment and tools from the room should be the rule.

Yeast is most commonly stored under six inches of beer, or under water or 2% potassium

dihydrogen phosphate solution. When high gravity brewing is used, it is important to

remember that the ethanol levels are significantly increased [could be as high as 8.5% (v/v)

prior to dilution] and this can affect the viability of the stored yeast. The yeast slurry should

be diluted (usually with sterile water) to an alcohol concentration less than 6% (v/v).

As more sophisticated systems have become available, storage tanks with external cooling

(0-4°C) and equipped with low shear stirring devices have become popular. The need for

low shear stirring systems has been shown to be important. With high velocity agitation in

a yeast storage tank, the yeast cell surface can become disrupted and unfilterable mannan

hazes in the final beer can result.

Reduction of available oxygen (for reasons to be discussed below) is important during

storage, and minimal exposure of yeast surfaces to air is desirable. Low dead cell counts

and minimal storage times are sought with the yeast being cropped "just-in-time" if

possible, for repitching. In this context, when cylindro-conical fermenters are employed,

the yeast collected in the cone of one vessel is sometimes pitched directly into another

fermenter, without use of a yeast storage system.

Yeast storage conditions - influence on

intracellular glycogen and trehalose levels

As discussed above, one of the factors that will affect fermentation rate is the condition

under which the yeast culture is stored between fermentations. Of particular importance in

this regard is the influence of these storage conditions on the intracellular glycogen level of

the cell. Glycogen is the major reserve carbohydrate stored within the yeast cell and is

similar in formation and structure to plant amylopectin (Figure 45). It serves as a store of

biochemical energy during the lag phase of fermentation when the energy demand is

intense for the synthesis of such compounds as sterols and fatty acids (i.e. the lipids). Thus an

intracellular source of glucose is required to fuel lipid synthesis at the same time that oxygen

is available to the cell. As described already in this document, brewery fermentations are

somewhat unique in this regard because oxygen is supplied in limited amounts and on a one

time basis, usually with the incoming wort. The uptake of oxygen by the yeast cell is very rapid

and at the same time there is a delay in the passive diffusion of wort glucose into the cell. There

is no appreciable wort glucose uptake during the first 6 hours, or even later, after pitching

whilst the wort dissolved oxygen is almost completely depleted in this same time period.

In order to synthesise lipid, the yeast immediately mobilises its reserve of glycogen in

order to fulfil the requirement of the cell for glucose. The high levels ofATP resulting from

respiration, activate the phosphorylase system which is necessary for the hydrolysis of

66

A Glycogen (C6H10O,)n

Trehalose(C12H2,On)

HOH,C

O

Control (no storage)

27% yeast glyccgen

(116 Hra) Anaerobic storage

15% yeast gtyecgen

(116 Hra) Aerobic storage9% yeaat glycogen

24 48 72 96 120 144 168

Fermentation time (hours)

Figure 46. The effect of yeast glycogen at pitching

on a lager fermentation.

Figure 45. Chemical structure of

(A) glycogen - a high molecular

weight polymer with

branched-chain structure

composed of D-glucopyranose

residues, and (B) trehalose - an

a-D-glucopyranosyl-

a-D-glucopyranoside.

glycogen to glucose. The

phosphorylasc activity during wort

fermentation peaks coincidcntally

with glycogen hydrolysis which

is within the first 10 hours after

pitching. Dissimulation of glycogen

and the synthesis of lipid are both

rapid. The hydrolysis of glycogen

from approximately 27% to 5%

and the corresponding production

of lipid from 5% to 11.5% of the

cell dry weight occurs within the

first 6 hours after pitching.

Towards the later stages of

fermentation, the yeast restores its

reserve of glycogen. The actual

maximum of glycogen content is a

function of yeast strain, fermentation

temperature, wort gravity and a

plethora of other factors. However,

the concentration of glycogen stored

and the degree of depletion at the

end of fermentation will, to a great

extent, determine the ability of the

yeast culture to survive extended

storage periods and still ferment at

an acceptable rate when pitched into

wort (Figure 46). As previously

described, storage conditions for

most brewing yeast handling systems

are far from ideal for growth or

even maintenance, since limited

assimilable carbon and soluble

nitrogen are present, together with

a relatively high concentration of

ethanol. Under these conditions

the yeast must survive for an

67

10

-O- Aerobic storage at 4°C

—•- Aerobic storage at 15°C

24 48 72 96

Storage time (hours)

120 144

Figure 47. The effect of yeast storage temperature

on intracellular glycogen concentration.

30-l indeterminate period of time and

to do so requires a basal level of

metabolic energy. To a great

, ^ extent, glycogen must provide the

cell with these requirements. In

order to study the change in

glycogen content during storage,

its concentration has been

monitored as a function of time

and storage temperature. Storage

temperatures (Figure 47) have a

direct influence on the rate of

glycogen dissimulation, as might

be expected considering the

effect that temperature has upon

metabolic rates in general. Of

particular interest is the fact that

within 48 hours, the yeast stored aerobically at 15°C has only 15% of its original glycogen

concentration remaining.

In summary, conditions under which yeast is stored and collected and the time of storage

can result in detrimental changes to the yeast which will result in sluggish fermentation

rates and modifications to the flavour and stability of the final beer. Good yeast handling

practices should include collection and storage procedures which avoid inclusion of oxygen

in the slurry, cooling of the yeast slurry to 2-5°C as soon as possible after collection, and

perhaps most importantly, recognition prior to pitching of yeast that contains low

intracellular glycogen in order that appropriate corrections in the pitching rate can be made.

Trehalose is one of the major carbohydrates in yeast. It is a non-reducing disaccharide

consisting of two glucose units linked together by an a-1, l-glycosidic bond (Figure 45B).

Trehalose plays a protective role in osmorcgulation, in protecting cells during conditions

of nutrient depletion and starvation, and in improving cell resistance to high and low

temperatures. This protective role may be due to the stabilising effect of trehalose on cell

membranes. The effects of ethanol shock on the intracellular trehalose content of an ale

Table 5. Effect Of Ethanol Shock On Intracellular Trehalose Content Of An Ale And

Lager Yeast Strain [adaptedfrom Odumeru et al, 1993, J. Ind. Microbiol., 11(2), 113].

Yeast & Strain Number

Saccharomyces uvarum (carlsbergensis)

Saccharomyces uvarum (carlsbergensis)

Saccharomyces uvarum (carlsbergensis)

Saccharomyces cerevisiae

Saccharomyces cerevisiae

Saccharomyces cerevisiae

Treatment for 60 min.

control (21°C)

heat shock. (37°C)

10% (v/v) ethanol (21°C)

control (21°C)

heat shock (37°C)

10% (v/v) ethanol (21°C)

Trehalose

(Hg/mg dry wt)

8.2

22.5

11.8

6.5

13.7

8.0

68

Glucose Trehaloso |

Phosphatase

Trehalose

phosphate

Glucose-6-phosphate

Trehalose

phosphate

synthet

Urldlne

diphosphateglucose

Urldlne

diphosphate

glucose

pyrophosphorase

Fructose-6-phosphate

Figure 48. Pathways to glycogen and trehalose in yeast.

and lager yeast strain is shown in Table 5. Exposure of yeast cells to 10% (v/v) ethanol for

60 minutes resulted in a significant increase in the trehalose content of the cells. Theseresults indicate that ethanol shock induces accumulation of trehalose in yeast cells. Figure48 illustrates the pathways to glycogen and trehalose.

Yeast Washing

Some breweries incorporate a yeast wash into their process as a routine part of the

operation, especially if there are concerns over eliminating bacteria responsible for

the production of apparent total N-nitroso compounds (ATNC) which have been implicated

as possible carcinogenic agents. Other breweries only wash when there is evidence of

bacterial infection. There has been considerable controversy over the practice of yeastwashing and its effect on subsequent fermentations. Studies carried out at the Brewing

Research Foundation International suggest that the problems often ascribed to yeastwashing (for example, reduced cell viability and vitality, reduced rate of fermentation,

changes in flocculation, fining problems, smaller yeast crops and modifications in the

balance of flavour components) are only apparent if yeast washing is carried outincorrectly.

There are three commonly employed procedures for washing yeast: sterile water, acid washand acid/ammonium persulphate wash:

69

• Sterile Water Wash: Cold sterile water is mixed with the yeast slurry, the yeast is allowed

to settle and the supernatant water is discarded. Bacteria and broken cells are removed

through this process. This can be repeated a number of times.

• Acid Wash: There arc a number of acids that can be used. Most common are phosphoric,

citric, tartaric and sulphuric acid. The cooled (2-5°C) yeast slurry is acidified with dilute

acid to pH 2.0/2.2, and it is important that agitation is continuous through the acid

addition stage. The yeast is usually allowed to stand for a maximum period of two hours.

• Acid/Ammonium Persulphate: An acidified ammonium persulphate treatment has also

been found to be effective and can yield material cost savings. It is recommended that

0.75% (w/v) ammonium persulphate is added to a diluted yeast slurry (2 parts water:

1 part yeast).

Acid washing can influence yeast performance, including:

• Reduced yeast viability;

• Reduced yeast vitality;

• Reduced rate and/or degree of fermentation; and

• Changes in yeast quality parameters such as flocculation, fining, size of yeast crop and

excretion of cell components.

Acid washing of yeast can be summarised into the do's and do not's.

The Do's of acid washing are:

• Use food grade acid;

• Chill the acid and the yeast slurry before use to less than 5°C;

• Wash the yeast as a beer slurry or as a slurry in water;

• Ensure constant stirring whilst the acid is added to the yeast and preferably throughout

the wash;

• Ensure that the temperature of the yeast slurry does not exceed 5°C during washing;

• Verify the pH of the yeast slurry; and

• Pitch the yeast immediately after washing.

The Do Not's of acid washing arc:

• Do not wash for more than two hours;

• Do not store washed yeast;

• Do not wash unhealthy yeast; and

• Avoid washing yeast from high gravity fermentations prior to dilution.

There are a number of options to acid washing brewer's yeast:

• Never acid wash yeast;

• Low yeast generation (cycle) specification;

• Discard yeast when there is evidence of contamination (bacteria or wild yeast);

• Acid wash every cycle, this procedure can have adverse effects on yeast; or

• Acid wash when bacteria infection levels warrant the procedure.

70

SHf

It is important to remember that acid washing can effect yeast quality and performance.

Since yeast are more acid tolerant than most bacteria, the procedure does not kill the

culture yeast, and it will not eliminate contaminating wild yeast.

Contamination of Cultures with Bacteria

Detailed consideration of microbial contaminants of brewing yeast cultures are beyond

the scope of this publication, but a brief review of the most important aspects is

probably appropriate. Bacteria are common spoilage agents of beer. The most troublesome

Gram-positive bacteria

are the lactic acid : *"Jf 4» ,.*"bacteria belonging to zS - C^

the genera Lactobacillus

and Pediococcus. At

least ten species of

lactobacillus can cause

beer spoilage. When

viewed under a light

microscope, lactobacilli

are very pleomorphic

in appearance and can

range in shape from

long slender rods to

short coccobacilli.

Brewing lactobacilli are

heterofermentative

(producing lactic acid

as well as other acids

and /or alcohols and

some strains produce

diacetyl) and

homofermentative

(producing only lactic

acid). They are acid

tolerant and have

complex nutritional

requirements. Some

species such as

Lactobacillus brevis

and Lactobacillus

plantarum can grow

quickly during

fermenting, ageing or

storage, whereas others such as Lactobacillus lindneri grow relatively slowly. Lactobacillus

spoilage is most problematic during conditioning of beer and after packaging where

spoilage gives rise to a "silky turbidity" and off-flavours.

/■i.

,»1

,*l

.Vfv,'

X

*

x\

10um

<■

Figure 49. Photomicrographs of typical bacteria found as

brewing contaminants. Top - Pediococci and bottom - Lactobacilli.

71

Pediococci are homofermentative cocci that occur in pairs and tetrads. Six species of

Pediococci have been identified, but the species predominantly found in beer is

Pediococcus damnosus. Pediococcus infection in the beer is characterised by lactic acid

and diacetyl formation. Infection may also cause ropiness in beer due to the production of

polysaccharidc capsules.

Many Gram-positive bacteria are inhibited by hop bittering compounds, particularly the

iso-a-acids, but Gram-negative bacteria are usually, but not always, unaffected. Some

members of the Micrococcaceae can survive in beer, grow and cause spoilage as can

some aerobic spore forming bacteria belonging to the genus Bacillus. Generally, these two

genera are inhibited by hop components and prefer an aerobic environment and, therefore,

are not a serious threat.

Important Gram-negative beer spoilage bacteria include acetic acid bacteria (Acetobacter,

Gluconobacter) certain members of the family Enterobacteriaceae (Escherichia,

Aerobacter, Klebsiella, Citrobacter, Obesumbacterium) as well as Zymomonas, Pectinatus

and Megasphaera. Acetic acid bacteria can convert ethanol to acetic acid, producing a

vinegar flavour in the beer and tend to produce a ropy slime. This type of spoilage is most

often observed in draught beer. The bacteria are airborne and prefer an aerobic environment

but can survive under microaerophilic conditions and infect the kegs as a result of air

entering or beer standing too long on tap in a partly filled keg. The Enterobacteriaceae are

aerobes and facultative aerobes and do not tolerate high ethanol levels. They are usually

found early in the fermentation and can produce celery-like, cooked cabbage, cooked

vegetable and rotten-egg aromas, especially if pitching of the wort is delayed. Figure 49

illustrates some of the bacterial contaminants encountered in brewing fermentation.

Contamination of Cultures with Wild Yeast

A wild yeast is any yeast, other than the culture yeast, that was unintentionally pitched.

With breweries producing different types ofbeer, each with its own yeast or mixture ofyeasts,

it is important that cross contamination does not occur. Wild yeast can originate from a wide

variety of different sources, from beer, brewing yeast, empty bottles etc. Figure 50 is a

photomicrograph of wild yeast. In addition to various Sacchawmyces species, species of the

genera Brettanomyces, Candida, Debaromyces, Hansenula, Kloeckera, Pichia, Rhodotorula,

Torulaspora and Zygosccharomyces have been isolated. The potential of wild yeasts to

cause adverse effects varies with the specific contaminant. If the contaminating wild yeast

is another culture yeast, the primary concern is with rate of fermentation, final attenuation,

flocculation and taste implications. If the contaminating yeast is a non-brewing strain and

can compete with the culture for wort constituents, inevitably problems will arise as these

yeasts can produce a variety of off-flavours and aromas often similar to those produced by

contaminating bacteria. Some wild yeasts can utilise wort dextrins (Saccharomyces

diastaticus has also been discussed), resulting in an over-attenuated beer that lacks body.

These yeasts are found as both contaminants of fermentation and at post-fermentation

stages of the brewing process. In addition, as previously discussed, wild yeasts often produce

a phenolic off-flavour due to the presence of the POF gene. However, under controlled

conditions, such as in the production of a German wheat beer or "weissbier", this phenolic

clove-like aroma, produced when yeast decarboxylates wort ferulic acid to 4-vinyl

guaiacol (Figure 51), can be a desirable attribute of the beer.

72

Figure 50. Photomicrograph

of (A) wild yeast, and

(B) brewing yeast culture

contaminated with wild yeast.

CH = CHCOOH CO,

(Yeast with

POF gene

4-VG producer)

— OCH,

OH

Ferulic Acid(wort constituent)

CH = CH,

■OCH,

4-Vinyl Guaiacol(a source of phenolic

off-flavour in beer)

Figure 51. Decarboxylation of ferulic acid to 4-vinyl guaiacol by yeast.

73

YEAST CELL VIABILITY AND VITALITY

Yeast viability is defined as the percentage of live cells in a sample, and yeast vitality is a

measure of yeast activity or fermentation performance. Yeast vitality has been described as

a function of the total cell viability and the physiological state of the viable cell population.

Many criteria are used to assess yeast cell viability and vitality. Consequently, the perceived

viability of a yeast sample may vary depending on the criteria selected. It is often

beneficial to monitor a combination of parameters to gain a more complete understanding

of a yeast's physiological state. A number of methods of studying yeast cell viability and

vitality are summarised below.

Use of Specific Dyes for Assessing Cell Viability and Vitality

Methylene blue is the dye most commonly used for yeast cell viability staining. Viable cells

are able to reduce this stain making it colorless, whereas non-viable cells are unable to

reduce the stain rendering them a deep blue-purple shade. A viable yeast cell count may be

completed using a hemocytometer and a light microscope in less than ten minutes. When

buffered and supplemented adequately, methylene blue dye has no effect on yeast cell

viability. Methylene blue staining is considered to be an accurate method only when yeast

cell viability is greater than 90%. Other brightfield stains which have been used to monitor

yeast cell viability include Aniline Blue and Crystal Violet.

There are also many fluorescent stains designed to assess yeast cell viability and

vitality. When fluorescent stains are used in conjunction with confocal microscopy

or flow cytometry valuable information may be obtained on yeast cell growth and

metabolic state.

Capacitance

The principle of this method is that the application of a radio frequency to a viable

cell results in a charge buildup within the membrane, and a capacitance is generated.

Non-viable cells are unable to generate this capacitance. A linear correlation has been

demonstrated between capacitance and viable yeast biomass.

The Power of Reproduction as a Viability Indicator

Standard plate count measures the ability of yeast cells to proliferate and form colonies on

nutrient agar. It generally takes three days for visible colonies to form and viability is

assessed by counting the number of colony forming units (CFU). Care must be taken when

using this method on very flocculent yeast.

Yeast viability by slide culture is also based on the ability of yeast cells to proliferate. A

drop of yeast culture is placed on a film of nutrient agar and after approximately 18 hours

of incubation the formation of microcolonies is observed under the microscope. Cells which

have given rise to microcolonies are considered viable whereas single cells that have not

formed microcolonies are considered non-viable. It is relatively less time consuming than

standard plate counts but still much slower than the staining techniques. An advantage of

the slide culture method is that it is accurate at relatively low yeast cell viabilities.

74

Viability and Vitality Methods Based on Cell Metabolic State

Adenosine triphosphate (ATP)

ATP (adenosine 5' triphosphate) is a good indicator of cell viability since it is present in all

living cells and is degraded when cells die. ATP allows for the detection of viable cells in

a short amount of time (10-15 minutes) when compared with traditional plating techniques.

Since the quantity of ATP per cell does not vary significantly for a given strain (but varies

between strains), it can be inferred that the amount of ATP present in a biomass sample is

proportional to the number of viable cells present of that cell type. Another advantage of

using ATP as a viability indicator is that the amount of ATP present in a cell is roughly

independent of the growth rate. Therefore a correlation between ATP concentration and the

amount of viable cell mass can be made.

The "firefly assay" is used to determine the quantity of ATP present in a biomass sample.

This invasive method involves extracting the ATP from the cells and reacting it with firefly

luciferin in a two-step reaction which is catalyzed by the enzyme firefly lucifcrasc. Light

is one of the products of this reaction and a stoichiometric relationship exists between the

amount of light produced and the quantity of ATP in the biomass sample. Extractants used

to release intracellular ATP include boiling in buffers such as tris-EDTA, cationic detergents,

acids, and organic compounds such as acetone and ethanol. The reactions taking place arc

summarised below:

Lucifcrin + Lucifcrasc + ATP + Mg2+—*-(Luciferin-Luciferase-AMP) + Pyrophosphate

(Luciferin-Luciferase-AMP) + O2 * Oxyluciferin + Luciferase + CO2 + AMP + Light

ATP concentrations as low as 1012 g in 100 ml volume may be detected using the firefly

method (Figure 52).

NADHfluorosensor

NADH has successfully been used as a non-invasive, on-line method of monitoring yeast

cell metabolism. Viable cells contain nicotinamide adenine dinucleotide (NAD) coenzyme

whereas non-viable cells or spores normally lose their NAD. The oxidised form, NAD+ is used

by dehydrogenases to accept electrons from their substrates. For example, in the

enzymatic conversion of malate to oxaloacetate in the presence of oxygen,

malate dehydrogenase (MDE) first binds to NAD+ to form a complex of MDE-NAD+. This

complex then combines with nialate to form a ternary complex MDE-NAD^malate. From

here, NADH, H* ion, and oxaloacctatc are released:

malate + NAD+ * * oxaloacetate + NADH + H+

(oxidised) (reduced)

The reduced form, NADH, fluorcsces while the oxidised form, NAD\ does not. NADH

is strongly fluorescent with an emission maximum at 460 nm wavelength. The total NAD

is the sum of NADH and NAD*. The reducing state is defined as the ratio of the reduced

form to the total amount of NAD:

R = [NADH] / ([NAD+] + [NADHD

Cell metabolic state determines the reducing state which will remain constant unless there

is a shift in metabolism. Thus, the influence of substrates such as oxygen on the reducing

state may be predicted. When oxygen is in excess, the reducing state approaches zero because

75

non-microbial call

microbial coll

Microbial &

non-microbial cells

B

Selective release of ATP from

non-mlcroblal cells.

Microbial cells remain intact

Addition of

luerferin-lucifcraso

Microbinl A

non-mlcroblal cells

Selective release of ATP from

non-microbial cells.

Microbial cells remain Intact

Hydrolysis of non-microbial

ATP with ATPase.

Microbinl cells remain Intact

ATPaae inactivated. Selective

ralaaaa of mlcroblsl ATP

Addition of

tucifertn-lucllerase

Microbial celts

Solocllvo release

nf mlnrnhlal ATP

Addition of

luclferin-llieKara

Luminometer Readout

(Total non-microbial RLU)

Rparlmit

(Total microbial RLUt

Luminomiitar Roadniit

(Total microbial RLU)

Figure 52. Measurement of ATP-driven bioluminescence.

(A) Total non-microbial bioluminescence from a mixture of microbial and

non-microbial cells.

(B) Total microbial bioluminescence from a mixture of microbial and non-microbial cells.

(C) Total bioluminescence from microbial cells only. RLU, relative light units.

76

NADH is easily oxidised to form NAD+and H2O, and when there is a lack of oxygenavailable to the cells, R approaches one. The concentration of [NADH] as well as the intensity

of the fluorescent signal are influenced by the number of viable cells, the reducing state ofthe cells and environmental effects. Measuring NADH has an advantage over monitoringdissolved oxygen or pH because it directly measures, in real time, events occurring withinthe cell rather than changes outside the cell environment.

Specific oxygen uptake rate (BRF yeast vitality test)

Researchers at the Brewing Research Foundation International (BRFI) developed a method

to determine the vitality of pitching yeast by measuring its specific oxygen uptake rate.Various groups have shown a correlation between oxygen uptake rate of yeast and

fermentation performance if yeast viability is less than 90%. The method involves the pitchingof yeast into aerated media and the measurement of the oxygen uptake rate for one hour.

A reduced oxygen uptake rate parallels other yeast changes such as the reduction in yeast

lipids, glycogen, acidification power test value, and yeast viability. Under these conditions,oxygen uptake rate correlates well with yeast fermentation performance. However, other

researchers have found that oxygen uptake rate did not correlate well with fermentation

performance when yeast had been previously acid-washed. Even though acid-washed yeast

showed decreased specific oxygen uptake rates, they actually showed better fermentationperformance than non-acid-washed yeast.

Acidification power

The acidification power test developed by Opekarova and Sigler measures the drop inextracellular pH of a suspension of yeast cells after the addition of glucose. This method is

useful for detecting large differences in yeast metabolic activity, but requires extensiveyeast washing and multiple sample points.

Intracellular pH (ICP) method

The ICP method uses a pH-sensitive fluorescent reagent to measure the intracellular pH ofindividual cells and cell mass. It was found that the intracellular pH of more active yeastcells does not decrease, even if the extracellular pH is low, whereas the intracellular pH ofless active cells actually decreases under low extracellular pH conditions. This test may be

capable of detecting more subtle changes in yeast cell vitality than acidification power test.

Measurement ofyeast vitality by stress response

As stated earlier, vitality may be considered a measure of yeast activity or fermentation

performance. It has also been defined as the ability of cells to endure or overcome stress.Therefore one could relate vitality to the response of yeast cells to stresses such as ethanol,heat shock, and high salt concentrations. Methylene blue, fluorescent dyes, and standard platecounts may be used to assess the ability of cells to remain viable after being subjected to agiven stress.

Magnesium release test (MRT)

The magnesium release test is based on the observation that low molecular weight speciessuch as magnesium, potassium, and phosphate ions are released by yeast immediatelyfollowing inoculation into glucose containing medium. Trials performed on Saccltaromycescerevisiae showed that cells which released greater quantities of magnesium immediately

77

after inoculation into high gravity (16°P) wort had higher vitality and fermentationperformance than yeast which released lower amounts of magnesium. Subsequent

fermentations performed using the more vital yeast had shorter lag phases, higher cellcounts, higher end ethanol concentrations, and lower diacetyl levels. The magnesiumrelease test takes less than 15 minutes to perform and it uses a commercially available

magnesium test kit (Sigma) which allows the quantitative colourimetric measurement ofmagnesium in wort before and immediately after yeast inoculation.

Electrokinetics

The measurement of zeta potential (electrostatic charge) is very sensitive, in fact, it is up to

2.5X103 more sensitive than impedance measurements. When electrophoretic mobility is

applied to yeast cells it can distinguish between living and dead cells. This gives a direct

measure of viability and, by looking at the size of the charge or the zeta potential on the

cell, it allows one to make an accurate assessment of viability. It can give a precise, easyand rapid direct measurement of the number of dead and live yeast calls and consequently

the viability of the sample.

HIGH GRAVITY BREWING

High gravity brewing is a procedure which employs wort at higher than normal concentrationand, consequently, requires dilution with water (usually de-oxygenated), at a later stage inprocessing. By reducing the amount of water employed in the brewhouse, increased

production demands can be met without expanding existing brewing, fermenting and storage

facilities. Reconstitution with water can occur either entirely or in part, at almost any

stage in the process, including: kettle (copper), strikeout, pre-wort cooler, during or after

fermentation, during maturation and pre- and post- beer filter. Generally, the lower the

hopping levels and the higher

the adjunct level, the more suited

the beer will be to higher gravity

without significant flavour

changes.

High gravity brewing has

been progressively introduced

into breweries around the world

for the past twenty-five years.

However, internationally it

cannot be said that its use is

universal, because some

companies have chosen (or

are compelled), for product

and legal/taxation reasons, not

to adopt this process. Slowly

the legal and taxation issues

are being addressed to permit

the production of high gravity

worts without undue financial

120-1

50 100 160

Fermentation time (hours)

200 250

Figure 53. Fermentation of 16°P and 25°P wort by

production lager strain A in shake flasks at 21°C,

inoculum 0.35% (w/v).

78

penalties. Nevertheless, the impact on flavour of brewing and fermenting certain producttypes at high gravity remains a concern and challenge to some breweries.

There are a number of advantages and disadvantages to this process. The advantages canbe summarised as follows:

• Increased brewing capacity, more efficient use of existing plant facilities;

• Reduced energy (heating, refrigeration, etc.), labour cleaning and effluent costs;

• Improved beer physical and flavour stability;

• More alcohol per unit of fermentable extract because of reduced yeast growth and moreof the wort sugars being converted to alcohol;

• High gravity worts may contain higher adjunct rates;

• Beer produced from high gravity worts are often rated smoother in taste; and

• High gravity brewing offers greater flexibility in product type. From one "mother" liquida number of products can be brewed as a result of dilution and/or use of malt and hopextracts and syrups.

The disadvantages can be summarised as follows:

• Due to the more concentrated mash (increased rate of carbohydrate to water), there is a

decreased brewhouse material efficiency and reduced hop utilisation. Although it isbeyond the scope of this document to discuss this aspect of high gravity brewing indetail, recent studies have found that this problem can be alleviated by the use ofmodern mash filters in place of lauter tuns and/or kettle syrups.

• Decreased foam stability (head retention). Hydrophobic polypeptides have been shownto form the backbone of foam and, therefore, their presence in beer is essential. It has

been shown that both high and low gravity wort loses hydrophobic polypeptides

throughout the brewing process, with the high gravity process suffering a more rapid

120 n

p

80 120 160

Fermentation time (hours)

200 240

Figure 54. Percent viability of brewer's yeast strains during fermentation of 27°P wort inshake flasks at 21°C, inoculum 0.35% (w/v).

79

loss. It would appear that high gravity mashing does not extract high molecular

weight polypeptidcs, which includes the hydrophobic polypeptides, as efficiently as low

gravity mashing. Also during fermentation, there is a disproportionate loss of hydrophobic

polypcptides from high gravity wort when compared to low gravity wort.

• There can be a difficulty in achieving flavour match to comparable lower gravity beers.

The effects of high gravity wort on ester formation during fermentation have already

been discussed. However, flavour problems with high gravity worts have been

exaggerated and adjustments to the process can be made (for example, yeast pitching

rate, fermentation temperature profile, DO at pitching and the spectrum of wort sugars,

particularly the ratio of glucose to maltose).

• High gravity worts can influence yeast performance with effects apparent upon

fermentation and flocculation. The increased osmotic pressure, elevated alcohol

concentration and modified nutrient balance, all have a profound influence on yeast

performance during the fermentation of high gravity worts. Stress tolerance during the

fermentation of the worts by brewer's yeast is strain dependent. Figure 53 illustrates the

effect of 16°P (1064) and 258P (1100) (a distinctly experimental wort gravity) on the

viability (determined with mcthylene blue stain) of a production lager strain during

fermentation. This strain fermented the 16°P wort efficiently, with nearly 100% viable

cells in the culture at the end of fermentation. This culture could be repitched into fresh

wort with confidence. However, in 25°P wort, this strain exhibited sluggish fermentation,

and poor cell viabilities such that the culture could not be re-pitched.

In order to study yeast strain variability and diversity in high gravity wort, four lager strains

[the original lager strain A studied plus an additional three (B, C, D)] were pitched into

27°P (1108) wort (Figure 54). The fermentation performance and cell viability of these four

strains was diverse. Strain B maintained a high viability throughout the fermentation,

whereas both strains C and D had a viability of approximately 75% at the end of the

fermentation, and again strain A exhibited poor viability (<20%) as a result of fermenting

27°P wort. Another major negative effect of high gravity worts on yeast performance

concerns the number of generations (yeast cycles) that can be fermented by a single yeast

culture. Significant strain to strain variation has been observed and, although there are

exceptions, it would appear that ale strains are more susceptible than lager strains to repeated

re-pitching in high gravity wort (i.e. >16°P).

Dilution of high gravity wort before or after fermentation requires that the water employed

be given special treatment. The specifics of the treatment procedure will vary depending on

the dilution point. Dilution in the fermenter improves fermentation vessel capacity as less

headspace is required. Water used for this purpose should be of the following quality (i.e.

carbon and diatomaceous earth filtered, pH adjusted and microbiologically sterile and

temperature adjusted). The requirement for expensive oxygen deaeration equipment is

circumvented because oxygen will be removed by the yeast. However, the longer the beer

is maintained undiluted, the greater is the capacity efficiency. Consequently most breweries

add the water to the concentrated beer immediately prior to the final polishing filter. The

water for dilution at this point in the process requires special treatment, in order to ensure

the quality and stability of the finished beer. Such treatment is to secure biological purity

and chemical consistency and encompasses filtration, pH adjustment and occasionally

80

ozonization, UV treatment or pasteurisation. In addition, most importantly, the dissolved

oxygen content of the water must be reduced to approximately 50-100 ug/L. This can be

achieved by vacuum deaeration using either a hot or cold process. The hot system flashes

water at 77°C, and the cold system flashes water at a temperature of 3-24°C through the

vacuum deaerator. Also water deaeration can be achieved by purging with an inert gas such

as carbon dioxide.

♦ Beer + Surplus Yeast

Yeast reservoir

Yeast recycle

flow controller

CONTINUOUS FERMENTATION

Brewers only have limited

control over yeast and

fermentation. The major

means of control lies in altering

the composition of wort through

choice of grist materials and

mashing conditions in the

brewhouse and in yeast pitching

rates and temperature

adjustments in the fermentation

cellars. There was great

expectation in the 19S0's and

I 960's that significant

improvements in process

control would be gained by

switching from batch to

continuous fermentation. Another

important motive for engaging

in continuous processing was

economy, particularly in the

quantity and the overall cost

of required plants.

Continuous fermentation for the

production of beer was first

attempted prior to 1900. Indeed,

by 1906 at least five separate

systems had been proposed

including simple stirred tanks,

multiple arrangements of such

vessels and towers packed

with supporting materials upon

which a culture of yeast was

maintained (the genesis of cell immobilisation applied in brewing, more of this later).

The reasons why these systems failed to gain a foothold in commercial operations atthat time, are obscure, but it is likely that the inability to adequately guard against

contamination and the resistance of traditional brewers to change were majorfactors.

CO,-

Temperature

control panels

Oxygenatedwort

Wort flow controller

Figure 55. Multi-stage tower fermenter (adaptedfrom

Portno, EBC Monograph V, EBC Fermentation and

Storage Symposium, Zoeterwoude, 1978, p. 149).

81

Stirror

I drive

CO, Outlet

Wort in

Pump

SterilizerYeast out

Figure 56. Stirred tank continuous fermentation system

[adaptedfrom Bishop, JIB, 1970, 76(9), p. 173].

A re-awakening of interest was stimulated in the late 1950's when multivessel systems were

in operation in Canada and in New Zealand. This was shortly followed in the U.K. by a

novel tower system which exploited the ability of flocculent strains to sediment, thus

enabling a high concentration of cells to be held within the system (Figure 55). This opened

up the spectre of much more rapid fermentation than had hitherto been possible. In the

decade between 1960 and 1970 substantial interest arose in the brewing industry in

the field of continuous fermentation. Enhanced knowledge of brewing science, together

with advances in engineering and electronic control equipment, offered real hope that

continuous fermentation could be developed into a viable process. It was anticipated that

the following advantages would result from the use of continuous fermentation in brewing:

• Reduced capital cost as a result of higher reactor productivity;

* Less beer tied up in process as a result of much faster throughput;

* Reduced labour costs due to less down time and, therefore, less cleaning and automatic

control of steady state; and

• Lower product cost resulting from the production of more cthanol and less yeast,

reduced beer losses, improved hop utilisation and reduced detergent usage.

The major economic gains were, therefore, with respect to capital investment, labour costs

and value of the in-process product (i.e. inventory costs). Since that time, this view has

changed substantially. With the exception of a brewer in New Zealand (and experiments

with immobilised cell technology which will be discussed later), no major company is

dependent upon continuous fermentation for commercial production of beer. An increase

in its use in the U.K. for ale products in the late 1960's proved transitory. Continuous

fermentation never found acceptance for lager production. Two separate designs (based on

different fermentation principles) were installed in breweries in the U.K. and in 1970, a

stirred tank system with a maximum output of 32,000 hL per week was installed in four

breweries. Key features were two stirred fermenters in series and a sedimentation vessel for

harvesting yeast (Figure 56). Yeast was not recycled and the residence time was 15 hours.

Similar outputs to those achieved with cylindro-conical vessels (which at this time were

being extensively introduced into many breweries), with a 5 day turn around time, would

have required the stirred fermenter system to be 5'/2 times greater in capacity.

82

A system employing flocculent yeast and tower fermcnters was being introduced

concurrently to the development, installation and commissioning of the stirred system. In

order for the tower system to be attractive to brewers, the following requirements neededto be satisfied:

• High yeast concentrations in order to permit high throughputs;

• The use of a wide choice of different yeast strains. A number of flocculent ale strains

were found to meet this requirement;

• The production of a consistent end product at different flow rates so that output could

be modified to meet fluctuations in consumer demand;

• A fermentable sugar gradient (i.e. wort sugars being metabolised in the usual priority -

glucose, maltose and maltotriose) through the tower, in order to prevent high levels ofesters, deflocculation and reduced fermentation capacity; and

• Control of the overall amount of yeast formed and of the growth rate in order to

produce acceptable levels of flavour compounds. This would be achieved by carefullycontrolling the wort oxygen level.

By the late I970's to early 1980's all of the continuous fermentation systems employed on

a production scale, with the exception of the New Zealand system, had not performed up

to expectation and therefore ceased to be in operation.

Why did the brewing industry fail to make a commercial success of continuous fermentationin the past? Essentially batch fermentation was simpler in concept. A vessel is cleaned,

sterilised and rinsed, and then filled with wort and pitched with the required quantity of

yeast. The primary fermentation cycle can be pre-programmed and little further attention is

required until maturation; typically 4 to 7 days later for ale, or 7 to 10 days for lager.

Operation by trained but not highly qualified staff is required. On the other hand, continuous

fermentation requires on-going laboratory monitoring and complex automatic control offlow rates, temperature gradients, yeast recycle rates (if immobilisation is not employed)

and oxygen levels. Cell morphology and fermenting wort gravity require regular checking.

Engineering support to correct possible faults in control systems, pumps, heat exchangers

must be available 24 hours a day, 7 days a week. The advent of on-line control and rapid

microbiological and analytical methodology could make what was an impossibility in theearly 1980's a reality in the new millennium.

There are a number of other factors that complicate the use of continuous fermentation in

brewing. Among them, the effect of continuous fermentation on the rest of the brewing

process must be addressed. How will the brewhouse be impacted? It simply may be

possible to have a series of wort reservoirs to feed the bank of continuous fermenters.

Although the residence time within continuous fermenters may be shorter than batch

fermenters, economic consideration should not be based on this factor alone. Of much more

relevance is the volumetric bioreactor productivity (i.e. volume of beer fermented per unit

fermenter volume per unit time). The impact of continuous fermentation on the flexibility

of the brewery needs to be addressed. Not all consumers drink the same beer, they drinkmore in summer than in winter, more on a hot dry weekend than on a cool wet one. The

ability to provide the required diversity of products in varying and unforeseeable amounts

83

is a prerequisite of a successful brewing operation. Through extensive market research,

years of trend monitoring and improvements in forecasting techniques, traditional

breweries have managed to maintain flexibility by employing banks of batch fermenters.

How then can a continuous process which is best suited for the production of a high volume

product at an unvarying rate meet this requirement? The advent of immobilised yeast cell

technology has allowed the development of novel continuous beer fermentation systems

which aim to satisfy today's brewery needs.

IMMOBILISED YEAST TECHNOLOGY

In traditional brewery fermentations, die yeast cell exists in two metabolic states: the

growth phase, in which the specific fermentation rate for each of the fermentable wort

sugars reaches a maximum, and the longer stationary phase, where growth is terminated,

fermentative power progressively declines, and maintenance activities take precedence.

For synthesis of its cellular materials, a growing yeast cell employs intermediates of

the catabolic glycolytic process as intermediates for anabolic synthetic reactions to

polysaccharides, proteins, lipids and nucleic acids. Hence, for the production of primary

and secondary products of yeast metabolism that define the alcoholic strength and flavour-

active quality of beer, a comprehensive understanding of growth regulated activities is useful.

This understanding becomes even more important when fermentation systems with high

volumetric productivities but with possibilities of growth limitations are considered for

matching products of traditional batch systems.

Immobilised cells have been defined as "those physically confined or localised to a certain

defined region of space with retention of their catalytic activity and viability". Beer

production with immobilised yeast has been the subject of research for a number of years

but has so far found limited application within the industry. When research into beer

production using immobilised yeast began, many questions needed to be answered including:

• The ideal specifications of the immobilisation matrix;

• The nature of interactions between yeast and the support surface;

• Interactions between yeast cells within the support;

• The mechanism of immobilisation;

• The influence of wort composition on beer quality;

• The genetic and phenotypic stability of immobilised yeast;

• The flexibility of immobilised yeast bioreactors;

• The influence of yeast immobilisation on the production of beer flavour components; and

• The viability of repeated and continuous usage of immobilised cells.

A further issue that needs to be addressed relates to whether beer produced by immobilised

yeast in continuous cultures can ever be the same as that made by free yeast cells under

batch conditions. In beer production, unlike in fuel ethanol processing where the attainment

of high yields of one major metabolic product is desired, the aim is to achieve a particular

balance of cell products and metabolic compounds. This raises the issue as to whether any

modifications to the beer production process could be realistically expected to produce exactly

the same balance of such compounds and hence a beer with an unaltered flavour profile.

84

The most widespread cell immobilisation technique is entrapment within a matrix. This

matrix, commonly a non-toxic polymer, is gelled around the cells to be immobilised.

Typical examples of the polymers used for entrapment of cells include: alginate,carrageenan, chitosan, agar, polyacrylamide, pectin, gelatin, epoxy resin and silica gel. Cell

entrapment by this technique is usually followed by cell growth in a nutrient medium to

fully colonise the matrix with cells. The polymeric mixture is either gelled immediately

into the desired form or gelled into sheets or blocks and subsequently cut into particles of

the desired dimension. The most common form is spherical beads ranging in size from 0.3

to 3.0 mm in diameter, although the smaller beads are generally preferred because of the

more favourable mass transfer characteristics for the entrapped cells.

This aforementioned technique characteristically allows a considerably higher biomass

loading than immobilisation in or on preformed supports. The essential concept of

this immobilisation method is that the matrix is porous enough to allow the diffusion of

substrates and products. The retention of cells maintained within the immobilisation matrix

should be as complete as possible, however, cellular outgrowth should not be restricted.

The mechanical strength of the gel matrix is important for minimising gel splitting or

stripping of yeast cells from the matrix due to the evolution of carbon dioxide by theimmobilised yeast during fermentation. Abrasion caused by particle-to-particle contact,

particularly in fluidised beds or stirred reactors, can cause problems in gels with weak

mechanical structures. Particle compression, seen commonly in packed bed reactors, may

also lead to immobilised cell aggregate breakdown and has been a further reason for theoptimisation of mechanical strength of the gel particle.

An alternative to entrapment is the immobilisation of cells in or on preformed non-porous

or porous supports. It is the most gentle fixation technique since, for the most part, no

changes in the cultivation conditions are necessary to produce the immobilised biocatalysts.

Typical examples of successfully used preformed supports for the immobilisation of

Saccharvmyces spp. include wood chips, diatomaccous earth, volcanic rock, stainless steel,

porous brick, porous sintered glass, porous silica, DEAE cellulose, PVC chips, glass fibres

and plant cell matrices. Cells immobilised on surfaces in direct contact with the liquid substrates

reduce mass transfer problems associated with more intrusive immobilisation techniques.

Direct comparison among different immobilising mechanisms is complicated because more

than one immobilising mechanism may occur in the same matrix. Indeed, certain porous

preformed supports may represent a combined form of cell immobilisation, involving

adsorption, cell growth, self-aggregation of cell population (flocculation), and, finally,entrapment of the aggregate within the porous network of the carrier.

The immobilisation of yeast cells for successful application in brewing implicates the

retention of whole catalytic cells within a bioreactor. In order to be a viable alternative to

traditional free cell fermentation and maturation systems, immobilised cells must have

considerably longer working lifetimes, characteristically measured in weeks or months.

Mass transfer limitations of substrate into, and products out of, the immobilised cells and

associated matrix are of critical interest. Criteria for the commercial feasibility of employingimmobilised cell systems are as follows:

• Low capital cost

- High productivity

- Mechanically simple

85

• Low operating cost

- Continuous operation

- Simple operation

- Low energy input

• Operational control and flexibility

- Controlled oxygenation

- Controlled yeast growth

- Rapid start-up and shut-down

- Control of contamination

• Quality control and flexibility

- Desired flavour profile

- Consistent product

- Wide choice of yeast

- Complete attenuation.

Commercial viability of immobilised yeast brewing systems depends on the optimisation

of the inter-related factors of cell physiology, mass transfer, immobilisation procedures, and

reactor design in order to ensure high specific rates of fermentation independent of yeast

growth. A consistently produced beer with the desired sensory and analytical profile is

further necessary for commercial success. Significant progress has been made in recent

years and a number of alternatives to conventional batch technology exist today. Among

these, specific immobilised cell systems for maturation and for special malt beverages are

now commercially available. Essentially there are three applications of immobilised cell

technology in brewing:

• Production of alcohol-free and low alcohol beers;

• Maturation or secondary fermentation; and

• Primary fermentation.

Production of Alcohol-free and Low Alcohol Beers

The production of alcohol-free and low alcohol beers is possible by three basic methods:

• Normal fermented beer is the starting liquid and the alcohol is removed employing

techniques such as reverse osmosis, dialysis or evaporation. However, it is impossible

to remove only alcohol, without removing other essential flavour components.

Consequently, in alcohol-free beer produced using these methods, the flavour is not

identical to normal beer.

• Fermentation is stopped (or arrested) early in the cycle. A normal wort is brought into

contact with yeast at low temperature (0-5°C) for up to 24 hours. In most cases, a high

yeast concentration (>100 million cells/mL) is employed. After a maximum of 24 hours

the yeast is removed usually with a centrifuge; highly flocculent strains and filtration

can also be used. There are a number of disadvantages to this method which include:

yeast is never homogeneously distributed in the fermentation vessel resulting in

inconsistent beer quality; yeast has to be removed quickly in order to prevent an

alcohol overshoot; during flocculation, the effectiveness of the yeast is reduced; and

beer is retained in the yeast slurry, resulting in liquid losses.

86

• An immobilised cell system has been developed in an attempt to overcome the

disadvantages of the methods discussed above. Controlled ethanol production hasbeen achieved using yeast immobilised on DEAE cellulose in packed beds. A majoradvantage of this type of carrier is that transport restrictions and diffusional limitationsare minimised since the yeast cells are bound to the positively charged surface. This is

an ideal situation provided that negatively charged wort components or particles do not

adversely affect the binding capacity of brewing yeasts to the carrier. Accordingly, wort

treatment and filtration are essential to secure efficient and controlled fermentations. An

industrial scale packed bed reactor has been successfully operating at the BavariaBrewery in the Netherlands for the production of alcohol-free beer.

The immobilised alcohol free beer process when compared to the classical arrested batchfermentation is reported to produce a better tasting low alcohol beer and to improve productconsistency. Bavaria BV of the Netherlands is using the Cultor packed bed immobilised

yeast bioreactor (capacity 150,000 hL/annum) for the production of alcohol free beer.

Several other companies, have also purchased this technology and are presently producingimmobilised cell alcohol free beer.

The Bavaria system employs DEAE cellulose as the immobilisation material andimmobilisation of the cells is achieved as a result of ionic bonding between carrier (positivecharge) and yeast cells (negative charge). Lactic acid (produced with Lactobacillus

amylovorus in a bioreactor of similar design to the one used for the production of alcohol-

free beer) is added to the wort before fermentation in order to adjust the pH to 4.0. This lowwort pH prevents the growth of contaminating bacteria while exerting a positive influenceon yeast activity.

The pre-treated wort is then pumped to the top of the reactor and allowed to percolate

through the fixed bed of carrier. The fermentation is normally run at 0-1 °C with a flowrate

of 20 hL/hr. Under these conditions, the yeast preferentially consumes the wort glucose.Due to glucose repression of the maltose and maltotriosc transport systems, these sugarsare therefore not readily metabolised by the yeast. Of the total amount of glucose, only 20%

is utilised and no more than 0.08% ethanol will be produced. The beer produced is low incarbonyl and sulphur compounds, and possesses good flavour quality and stability.

Low alcohol beer production may be stopped for several weeks by simply circulating wort

through the reactor at low temperature (2-4°C) in order to prevent excessive yeast growth.The production of low-alcohol beer can be resumed by restarting the wort feed at theappropriate operating conditions. It is recommended that the entire reactor including carrierparticles be cleaned and re-sterilised twice a year.

Immobilised Lager Yeast

to Reduce Maturation Times

As previously discussed, the removal of diacetyl and 2,3- pentanedione and their

precursors cc-acetolactate and a- acetohydroxybutyrale is one of the major features of

flavour maturation. This stage is the most time-consuming in traditional lager beer production.The Finnish company Cultor, who worked in association with the Sinebrychoff and Bavaria

87

Yeast

Removal by

Centrifugat:on

From Primnry

Fermentation

Heat

Treatment

Maturation

Immobilized

it Fermentors

Yeast Cooling

Figure 57. Cultor's two hour continuous maturation system (adaptedfrom Pajunen, EBC

Symposium: Immobilised Yeast Applications in the Brewing Industry, Espoo, Finland,

1995, p. 26).

breweries from Finland and the Netherlands respectively, and with the German engineering

firm Tuchenhagen have developed a process utilising immobilised cells for the

accelerated maturation of beer.

Their proprietary carrier Spczyme® (DEAE cellulose particles) is at the heart of the above

technologies. The immobilisation of the yeast cells on the carrier was accomplished

through surface adsorption in a downflow packed bed continuous biorcactor through

which a yeast slurry was recirculated. The main advantage of this technology is its high

volumetric productivity with corresponding residence times of only a few hours. The

maturation process involving purely physical processes is viewed by this group as a more

acceptable alternative from the consumer's point of view as opposed to technologies using

free or immobilised a-acetolactate decarboxylase enzymes or the genetic engineering

approach with low diacetyl producing yeast strains.

The system developed by Cultor and their associates is industrially available and has been

operational at an industrial scale (1 million hL per year) in Finland since 1993

(Sinebrychoff Ab, Kereva Brewery). Figure 57 provides a schematic of the maturation

system developed by Cultor. In order to achieve rapid reduction of diacetyl in the "green

beer", the freely suspended yeast cells are centrifuged and the resulting beer is subjected to

a heat treatment process (65-90°C for a holding time of 7-20 minutes). The non-enzymatic

conversion of the diacetyl precursor, a-acetolactate, to acetoin is quickened in this step. The

beer is then introduced into a packed bed column containing yeast cells immobilised on

DEAE cellulose particles. In this final stage, the yeast cells complete the conversion of the

remaining diacetyl into acetoin while other flavour maturation also occurs.

The road for accelerated maturation of "green beer" has been well paved by Cultor. High

levels of diacetyl can be effectively reduced by adopting a strategy similar to that of Cultor.

Companhia Cervejaria Brahma from Brazil purchased a maturation system from Cultor in

1994. The initial trials with the accelerated maturation unit showed very promising results.

However, problems also occurred, especially during the start-up of the immobilised cell

reactors (first 12 hours of operation) when off-flavours in the product were noticed

(resinous flavour). The rapid maturation product differed from the traditionally aged

product in pH and foam collapse rate. The taste panel also found a difference in the

product from the immobilised cell system compared to the regularly aged beer.

The preliminary preference from the taste panel, however, was for the immobilised cell

treated beer. Brahma stopped their testing in 1994 because of company restructuring and

reappraisal of priorities.

The Belgian company Alfa Laval, in association with Schott Engineering from Germany,

have also developed a rapid maturation process similar to that of Cultor, employing their

own porous glass bead carrier called Siran®. Schott has underlined the followingadvantages of using porous glass as the carrier material:

• High surface area and, therefore, high biomass loading capacity;

• Good mass transfer properties;

• Robustness of the material meaning easy regeneration;

• Chemical inertness;

• Possibility of steam sterilisation; and

• Good flow properties.

The Alfa Laval/Schott process is now being employed commercially at the Hartwall

Brewing Company in Finland for the rapid maturation of a high quality beer. Both the

above rapid maturation processes have allowed the respective breweries to reduce their beer

maturation times from weeks to hours.

Primary Fermentation with Immobilised Yeast

The use of continuous fermentation employing free cells for beer production has already

been discussed in this document. The application of immobilised cell technology in

brewing for primary fermentation has been studied since the early 1980's. Most of the early

attempts to produce beer with immobilised cells in a continuous reactor were plagued by

Bulkconcentration

(gradient ol substrate)

' Concentration at the

Stagnant/1 " —■ —' ' ' surfaofl ot tho beadlayer

Active layer

Bead core

External

mass transfer

Figure 58. Mass transfer diagram of an entrapment carrier.

89

Stainless Steel —►VHead Plate \

Inoculated

Beads

Sparging

Gas

T—* —

- 1 :

► : :

T—► "Green" Beer

4—Thermal Jacket

<— Bioreactor

Draft Tube

Plant Wort

Figure 59. Labatt gas lift draft tube bioreactor

[Mensour el ai, JIB, 1997, 103(6), p. 363-370].

insufficient free amino nitrogen

(FAN) consumption resulting in

unbalanced levels of ester and

higher alcohols. The resulting

flavour profile was not always

intrinsically unacceptable, but was

generally outside the flavour range

of the breweries investigating this

new technology. The main reason

for this unbalanced metabolic

behaviour was the altered growth

pattern of immobilised cells

caused by mass transfer limitations.

The low oxygen availability in

early immobilised cell processes

provoked a decoupling between

biomass and ethanol production,

which would be desirable in fuel

ethanol production (increased yield) but not in a brewing fermentation (impaired flavour).

In an attempt to circumvent this problem, Kirin Breweries designed a process where a free

cell chemostat (continuous fermenter) preceded the immobilised cell bioreactor. Important

yeast growth occurred in the first stage, with the resulting desirable FAN consumption. The

remaining attenuation occurred in an anaerobic packed bed reactor with alginate entrapped

yeast cells. The alginate matrix was subsequently replaced by porous ceramic beads. This

system produced a beer with

acceptable flavour. With a diacetyl sta9e' stago2

reduction step similar to the one

described by the Cultor process,

beer was produced within 3 to 5

days. However, the added complexity

of the immobilised chemostat and

the loss of productivity involved

suggest that this process could be

improved.

The consumption of FAN can be

improved by the fluidization of

alginate-entrapped immobilised

yeast cells. The objective is to

enhance mass transfer in immobilised

cell bioreactors to the point where

"normal" yeast growth and resulting

"normal" flavour profiles are

possible. Internal mass transfer

refers to the transfer of nutrients

within the carrier (Figure 58). As a

Wort Beor

Vessel

Wort Supply Immob

Pump Yeast Reactor

Figure 60. Schematic of Meura Delta two stage

multi-channel immobilised loop reactor for the

continuous production of beer (adaptedfrom Krikilion

et ai, Proc. EBC Cong., Brussels, 1995, p. 419).

90

result of the formation of substrate gradients within entrapment carriers, most of thebiomass is concentrated in an active layer located near the interface with the external medium

(the wort). The most common option to improve internal mass transfer in immobilised cellsystems is to reduce bead diameter. The choice of bead or particle size is a compromisebetween the smallest possible size and technological properties such as pressure drop in apacked bed reactor and separation from bulk medium in all reactors. For beer production,a particle diameter between 0.2 and 1.5 mm has been found to minimise mass transferlimitations.

External mass transfer refers to the transfer of nutrients from the bulk medium to the carrier

surface. The main issue when considering external mass transfer in immobilised cellsystems is the choice between a packed bed reactor or a fluidiscd or agitated reactor. Packed

bed reactors suffer from several engineering problems linked to their limited external masstransfer. Channelling can be reduced by the use of upward flow reactors or the use of

incompressible carriers in downward flow processes. Extensive growth may result in

plugging of the reactor leading to an excessive pressure drop. In addition extensive CO-,production, linked to active fermentation, is difficult to remove from a packed bed reactor"

Consequently, packed bed reactors are only used primarily for processes with limitedgrowth (maturation or alcohol-free beer).

Fluidised bioreactor configurations are preferred for primary beer fermentations and, forthis purpose, alternative carriers are usually considered. Siran glass beads can be attractive,

but cost is high (approx. $100 US/litre) and regeneration requires the destruction of allpreviously entrapped biomass with chemicals such as peroxide. When considering the cost

of currently available carriers, gel entrapment is one of the best options for industrial

primary fermentation of beer. With the appropriate polymer material, the ingredient cost for

inoculated gel beads could be as low as S0.50 US/litre. A gel-forming polymer that falls

into this category is kappa-carrageenan. This polysaccharide, extracted from seaweed, isalso known as "Irish Moss". In solution with potassium ions, ic-carrageenan forms athermoreversible gel.

Canadian researchers have developed a novel continuous beer fermentation system usingimmobilised yeast cells. A 50 L gas lift draft tube bioreactor (Figure 59) was designed andinstalled in a pilot plant for use with the carrageenan gel beads for the primary fermentation

of beer. An expanded head region provides an increased surface area so that completegas-fluid separation occurs. By allowing the gas phase to escape as head gas, optimal solid-

liquid mixing results in the annulus area or outer perimeter of the reactor. Gas lift systemsprovide good mixing with minimum shear on the solid matrix. As a result, they significantlyimprove mass transfer between the liquid medium (beer wort) and the catalyst(immobilised yeast cells). In brewing, the uptake of free amino nitrogen from the wort is

critical to the formation of flavour-active compounds. Poor contact between the yeast cellsand the liquid medium can result in insufficient consumption of free amino nitrogen andconsequently yield a product with unbalanced flavour and aroma.

A mixture of air and carbon dioxide were utilised as the sparging gas. The specific

proportion of air to CO2 determined the level of yeast growth within the bioreactor andthus, directly influenced the resulting flavour profile of the finished product. Air proportionsbetween 2% and 5% were judged by a taste panel to produce a beer with an acceptable

91

flavour profile, although not a perfect match to the traditionally produced control. This

system operated with a minimum wort residence time of 20 hours, corresponding to seven

to ten-fold time savings as compared to traditional free cell batch fermentation. Such an

immobilised cell system, used in conjunction with an accelerated maturation process, is

capable of producing a finished product with a turnover time of two days.

Meura Delta, a Belgian brewing equipment manufacturer, has also been involved in

research on immobilisation systems for the production of beer. Utilising silicon carbide

rods as a matrix, Meura Delta has developed a bioreactor with an external liquid recirculation

loop for the production of both alcohol free and regular beer (Figure 60). Yeast cells are

immobilised by adsorption on the internal surface of the silicon carbide rods. The fermenting

medium is then circulated through the internal channels of the carbide rods as well as the

external surface of the rods via a recirculation pump. "Green beer" is drawn from the top

of the reactor at a rate dictated by the fresh medium feed pump. The immobilisation matrix

is 900 mm in length, 25 mm in diameter and has a void volume of 60%. Meura Delta has

performed extensive research so that an appropriate method to fix their immobilisation

matrix in the reactor could be developed. Such a design allows them to achieved a fluid

flow pattern which facilitates cleaning and immobilisation procedures.

There are a number of questions that still remain to be addressed when contemplating the

industrial production of beer in one to two days. Although immobilised cell technology can

deliver such a fast fermentation time there are still problems. One of the main drawbacks

of rapid fermentation is the relatively high level of diacetyl and its precursor in the green

beer. As previously described, there is a rapid method for reduction of vicinal diketones but

this process represents added complexity, stability threats and cost. Use of genetically

modified yeast or the addition of a-acetolactate decarboxylase is one alternative but as

already discussed these approaches could have a negative impact on the consumer's

perception of such beer.

I

Performance

improvement required

by mainstream market

Expected trajectory of

performance Improvement

Current performance of potentially

disruptive technology

Time

Figure 61. How to assess a disruptive technology (adapted

from Bower and Christensen, Harvard Business Review,

1995, 73, p. 43).

The second important

challenge requiring

attention concerns the

operation of industrial

continuous immobilised

cell reactors. Many of

these challenges are

similar to those faced by

the free cell continuous

fermenters that were

developed in the late

1960's and early 1970's.

Brand proliferation by

many breweries has

rendered production

flexibility an important

production parameter.

Once an immobilised cell

92

reactor has been loaded with the inoculated carrier, it must be capable of operating forseveral months to be cost effective. How is it possible to produce different types of beer

with (he same reactor? Obviously, if these beers require different yeast strains, then a seriesof reactors each containing a specific yeast strain would be required. However, if the

differences depend on wort composition and fermentation parameters, then the possibilityexists that a well-defined procedure will enable a switch from one brand to another, by

modifying wort composition and operating parameters. Continuous fermentation is stillgenerally perceived to be an inflexible process. The challenge then remains to demonstrate

that immobilised cell fermentations can indeed be versatile.

The future of immobilised cell technology for primary fermentation in breweries is difficultto predict. However, it is a technology that cannot be ignored. It has already been introduced

into a small number of micro breweries and it is anticipated that this development will continue.ft has the potential of being a "disruptive technology" (Figure 61). A disruptive technology

is one that completely changes the manner in which a particular industry conducts its business.

Currently, the performance of immobilised technology in brewing still lies below that oftraditional batch fermentations because of its lack of acceptance and unproven long term

performance. However, its potential to revolutionise the industry is increasing rapidly!

DISTILLER'S YEAST

The origins of distilling processes are difficult to trace but are more recent than the

production of undistilled alcoholic beverages such as beer and wine. Although the early

Chinese and Greek cultures appear to have been aware of distilled beverages, the earliestdescriptions of distilling processes appear to have been more concerned with the production

of drugs by alchemists than with beverages. There are several references in the literature todistilling around Europe dating from the twelfth and thirteenth centuries. However, the firsttreatise on distilling was written in the fourteenth century by a French chemist, Arnold de

Villeneuve, and during the following two centuries, the use of distilling processes expanded

rapidly throughout Europe. Diagrams of batch distillation equipment with a characteristicpear-shaped flask and a

worm to increase the

cooling surface area date

from a German publication

in 1551. Scotch Whisky

production is generally

recognised as dating from

reports of the supply of

barley to a Friar Cor in

1494. Over the next 400

years, various factors

influenced the evolution

of Scotch Whisky into its

present international status.

Crucial has been the

design and operation of

stills and a number of

Mashing r> Fermentation

Wort

16%

Sugar

Wash

8%

Alcohol

Distillation

<

(

(

Rectifier

)

)

)

>

CoKey

Still

Spirit

04%

Alcohol

Figure 62. Grain whisky - continuous column

distillation.

93

complex configurations suitable for distilling, as distinct from evaporation, date from the

sixteenth century. All types used worm condensers; tubular-coiled or rectangular-coiled pipes

immersed in large tubs of water. The simplest metal to work for making these relativelycomplex shapes for stills and worms was copper. This was fortunate since, as was later

discovered, the use of copper is essential for the production of high-quality spirit,

particularly in pot-distilling.

The development of continuous distillation dates from the early nineteenth century and in

1830, the continuous two-column still, designed and patented by Aeneas Coffey was

accepted by the excise authorities. The continuous still consists of two sections, analyser

and rectifier, which were initially located one above the other but later placed side by side.

This continuous still design is still employed today for the distillation of grain alcohol usedin the production of blended Scotch Whisky (Figure 62). A further advantage of these stills

is that they have simplified the production of relatively pure spirit and has consequently

made them suitable for the production of gin and vodka.

A detailed description of distillation processes is beyond the scope of this publication.

Nevertheless, some background on Scotch Whisky is appropriate prior to a discussion of

the yeasts employed in the process. Scotch Whisky has been defined in United Kingdom

law since 1909. The current definition is that contained in the Scotch Whisky Act 1988 and

states: "Scotch Whisky" means whisky:

• Which has been produced in a distillery in Scotland froni water and malted barley (to

which only whole grains of other cereals may be added) all of which have been:

- processed at that distillery into a mash;

- converted to a fermentable substrate only by endogenous enzyme systems; and

- fermented only by the addition of yeast.

• Which has been distilled to an alcoholic strength by volume of less than 94.8% so that

the distillate has an aroma and taste derived from the raw materials used in, and the

method of, its production.

• Which has been matured in an excise warehouse in Scotland in oak casks of a capacity

not exceeding 700 litres, the maturation period being not less than three years.

• Which retains the colour, aroma and taste derived from the raw materials used in, and

the method of, its production and maturation.

• To which no substance other than water and spirit caramel has been added.

The Scotch Whisky Act 1988 prohibits the production in Scotland of whisky other than

Scotch. The Scotch Whisky Act 1988 and the associated European Union legislation both

specify a minimum alcoholic strength of 40% by volume, which applies to all Scotch

Whisky bottled and/or put up for sale within or exported from the community.

Malt and Grain Whisky

Two different types of whisky, malt and grain, are produced each of which has quitedifferent characteristics. Malt whisky, which has a pronounced bouquet and taste, is made

exclusively from malted barley and yeast by the pot-still method, a process that does not

enable continuous production (Figure 63). Consequently, the whisky is made in separate

94

Mashing Fermentation

Wort

15%

Sugar

Wash

8%

Alcohol

Distillation

Figure 63. Malt whisky pot-still process - double

stage distillation.

batches, each of

which is similar

but not identical.

The flavour produced

is determined by a

variety of factors

and the most

important is the

location of the

distillery. For

example, whiskies

produced on Islay

(West of Scotland)

have a peaty flavour

as the malted barley

employed is kilned

using peat.

Grain whisky is made from a mixture of malted barley, maize (corn), wheat and yeast in

the proportion of approximately 16% barley malt and 84% maize and / or wheat, although

this varies from one distillery to another. As previously described, the grain spirit (unlikemalt whisky) is produced by a continuous process based on the Coffey still. Grain whisky

has less well defined characteristics than malt, thus making it eminently suitable for blending

purposes. Unlike malt, grain whisky varies little in taste from one distillery to another. The

main features of grain distilleries are their large capacity. Until comparatively recently,

most grain distilleries used only maize but currently the favourable price of wheat compared

to maize has resulted in many distillers using wheat even though the yield from wheat has

historically been less than maize.

The major differences between malt and grain distilling can be summarised as follows:

• In malt distilling, barley malt is employed whereas unmalted cereals such as maize and

wheat along with a small proportion of barley malt (to provide the hydrolytic enzymes)are employed in grain distilling.

• In malt distilling, the spent grains are removed from the wort prior to yeast pitching,

whereas in grain distilling the spent grains are not removed, they are present in the

fermenter along with the yeast and become a part of the still change. In both malt and

grain distilling the wort is not boiled prior to fermentation and therefore, is non-sterileand still contains active enzymes from the malt.

• Distillation in malt distilleries is a batch process whereas in grain distilling it is

continuous. As a consequence, grain distilling is far more efficient (approx. 3000 litrespirit/hr for grain compared to 800 litre spirit/hr for malt).

• The spent alcohol strength at maturation is 66-73% (v/v) for malt and 94% (v/v) for grain.

Following distillation, malt and grain whisky are stored separately in oak casks since oak

is the only wood that permits air to pass freely and yet has the ability to absorb certain

impurities and impart other constituents, thereby improving the quality of the spirit.

Traditionally, most whisky has been stored in second-hand casks made from American oak,

95

although the supply of the latter could decrease significantly if a relaxation in United States

distilling law to permit the re-use of bourbon casks (or barrels as they are called in North

America) is ever implemented. Currently, bourbon distillers are required by law to use

casks only once and these are subsequently sold to the Scotch Whisky industry and others.

Some whiskey has traditionally been matured in used sherry casks, with the spirit absorbing

some of the colour and sweetness of the sherry wine. It is estimated that approximately 4%

of malt fillings are regularly stored in sherry casks. The use of new wood still represents a

very small percentage of casks employed, but greater interest is currently being taken in

view of the longer term position of bourbon casks.

The yeasts employed in distilling are on the whole less clearly defined and characterised.

Prior to a discussion of their characteristics, the following is a summary of the differences

between Scotch Whisky production and brewing:

• As previously discussed, the production of Scotch Whisky is very closely regulated

through the Scotch Whisky Act 1988. The only country where the production of beer

is as closely regulated is in Germany with the Beer Purity Law which was originally

drafted in 1516.

• Malt and grain spirit are mashed, fermented, distilled and matured separately, whereas

in brewing malt and adjuncts are usually processed together to produce a single wort.

• In Scotch Whisky production, higher DP (diastatic power) malts are employed and

extract, attenuation and carbohydrate to alcohol efficiencies are critical.

• In the production of whisky, spent grains are not removed from the grain wort. Also,

unlike brewing, hops are not employed in whisky production.

• In the production of whisky, unlike brewing, the yeast is rarely, if ever, recycled.

Prior to this century, the yeast strains were "selected" empirically and specialised distilling

yeasts were not employed. It is possible that it would have been a mixed culture that was

recycled from one fermentation to the next. It may well have arisen from brewing and baking

yeasts and will certainly have been heavily contaminated with alcohol-tolerant bacteria

such as Lactobacillus spp. In Japan, studies on the physiology of Sake1 yeast,

Saccharvmyces sake, which is now considered to be a strain of Saccharomyces cerevisiae,

date back to the early twentieth century.

Many distilling companies still use a baker's or brewer's yeast for fermentation rather than

a specially developed yeast. The first attempts to produce a specialised distilling yeast date

back to early this century when yeasts specially adapted to produce industrial spirit were

selected and grown in pure cultures. However, the requirements of a distilling yeast are

somewhat subjective and dependent upon the process, although a high yield of ethanol

approaching theoretical maximum would be expected. However, other parameters that

should be considered include: rate of fermentation, substrate utilisation, ethanol tolerance

and economy of production.

In whisky production, as in most distilled beverage processes, the yeast is transferred into

the still along with the rest of the wash, and is destroyed during the distillation and removed

as part of the stillage. Presence of the yeast in the still has a significant effect on the nature

of the spirit, since many of the esters of longer chain fatty acids are present in the yeast cells.

In such processes, aerobically grown yeast cells are added to the wort at a concentration of

96

between lOMO7 cells/mL at the beginning of each fermentation. Many whisky distillersadd both specifically grown distilling yeast strains (the Quest M strain or similar strain) aswell as some brewing (usually ale) yeast. M strain is characterised by an ability to"super-attenuate wort". This means that the strain is capable of utilising lower molecularweight dextrins such as maltotctraose (G4, G5, G6, etc.). The exact mechanisms of usingthese dextrins is unclear.

As previously described, gene cloning techniques have led to rapid advances in the

molecular biology of the yeast Saccharomyces cerevisiae. Attention has turned to distillingyeasts. The Scotch Whisky Act 1988 would permit the use of genetically manipulated

strains but, similar to brewing, there is considerable scepticism regarding the commercialuse of such strains. One of the main objectives of genetic manipulation and straindevelopment of distilling yeasts is increased efficiencies of carbohydrate conversion tocthanol and extension of the range of utilisation lo abundant and inexpensive substrates.Cloning of several genes encoding enzyme systems for fermentation of simple sugars hasled to applications relevant to higher yields of conversion to ethanol. For example, analysisof the family of MAL genes (maltose uptake and hydrolysis system) has resulted inelucidation of the oc-glucosidase (maltase) and permease systems. Also, distillery yeastsoften lack the ability to utilise melibiose and transfer of the MELI gene from lager strainshas led to gain of this property. Higher ethanol yields can be obtained with substrates (forexample, beet molasses) containing significant levels of raffinose (a trisaccharide consistingmelibiose plus fructose).

Ethyl Carbamate

Ethyl carbamate (C2H5OCONH2), otherwise known as urethane, is a naturally occurringcompound present in many fermented foods and beverages including Scotch Whisky. Itis a chemical carcinogen, and this fact has led to concern in recent years regarding itspresence at trace levels in some alcoholic beverages. As a consequence of this, steps havebeen taken to impose acceptable limits on levels present in such products. From a distillationpoint of view, two outstanding properties of ethyl carbamate are its low volatility and itsrelatively high chemical stability. Although ethyl carbamate does not distill readily, transferto distillates may occur under suitable conditions.

Because of volatility considerations, any ethyl carbamate formed by the reaction betweenethanol and ureido compounds in the wash would not normally be expected to be presentin the final spirit. However, chemical analysis indicates that ethyl carbamate is virtuallyabsent from fermented wash. If it is going to form it can be detected during distillation. Itcan occur in fresh distillates and may continue to form in the spirit during maturation. Theobservations have indicated that a volatile

precursor is involved in its formation. If ethyl

carbamate is going to form, then its formation Ethy' Carbamate (C,H7NO2)

is accompanied by a corresponding decrease H o

in the distillate of a group of compounds \j_J'_orH rHoriginally described as "measurable cyanide". / ulh2lh,Subsequent analysis has shown that the first

measurable cyanide component to appear in Figure 64. Chemical structure of ethylfresh distillate is hydrogen cyanide. When carbamate (urethane).

97

radio-labelled hydrogen cyanide is added to distilled spirit, the label appears in ethylcarbamate. Thus it appears that the volatile precursor of ethyl carbamate is hydrogen

cyanide, formed in trace amounts during distillation.

Systematic attempts have been made to trace the origin of cyanide in materials and processes

used for whisky manufacture. Barley, wheat and maize have been examined along withwater, yeast and malted barley. The effects of cooking, malting, mashing and fermentation

have also been considered. No free cyanide is detectable in fermented wash, suggesting that

a heat-labile precursor of hydrogen cyanide is present at the end of fermentation. If microbialenzymes are used instead of malted barley to convert cooked maize or wheat, virtually nocyanide appears in the distillate after fermentation. Further research has confirmed that

malted barley contains a cyanide precursor which is located exclusively in the acrospire ofthe germinated kernel which can account for the hydrogen cyanide appearing in the distillate. This

cyanogen has been identified as a water-soluble cyanogenic glycoside known as

epiheterodendrin (EPH). EPH is thermostable, which enables it to survive kilning and mashing.

Suitable enzymes readily hydrolyse EPH. Although malt a-glucosidases are inactivated

during mashing, hydrolysis of EPH takes place during fermentation as a result of the actionof yeast enzymes. All yeasts examined are able to hydrolyse EPH to glucose andisobutyraldehyde cyanohydrin (IBAC). At -50°C, EPH breaks down to release hydrogen

cyanide. From a distillation point of view, hydrogen cyanide is characterised by two

properties. Firstly, its volatility, which imparts a high mobility to the compound during

distillation. Secondly, its high chemical reactivity which enables it to form, amongst other

products, ethyl carbamate (Figure 64).

Thus, the route is available for hydrogen cyanide to enter the spirit from at least one majorraw material. Over one hundred currently available and "historical" barley varieties have been

examined for their ability to produce hydrogen cyanide. About 10% lack this property, with

the remainder producing cyanide to differing degrees. Cyanide-producing capacity appears to

be a stable varietal characteristic, largely unaffected by crop year and region of cultivation.Thus, varieties such as Pipkin, Grit and Kaskadc are found to be consistently low-producers.

The following guidelines have been suggested for the reduction of ethyl carbamate levels

in distilled spirits:

• Select a barley variety for malting which is a low cyanide producer;

• If low cyanide varieties are unavailable, attempt to control malting conditions in order

to minimise acrospire growth, whilst maintaining the required levels of modification

and enzymic activity;

• In grain mashes, use high-enzyme malt and reduce the proportion of malt to a level

which is compatible with spirit recovery;

• Monitor all raw materials for other sources of cyanide;

• Choose appropriate still design and materials of construction to minimise copper

exposure on the reflux side and to minimise the amount of soluble copper entering the

final spirit; and

• Maintain appropriate distilling operating procedures. For example, foreshots and feints

will tend to contain relatively high levels of ethyl carbamate. As a consequence,

distillation rates and cut-points may be critical.

98

SUPPLEMENTARY READINGS

The following list is intended as a guide for those wishing to gain

more detailed knowledge than is given in this monograph.

Many additional excellent books can be found through the brewing organization websites

( MBAA, ASBC, AOB and IBD ).

Bamforth, C. (2009) Beer: Tap into the Art and Science of Brewing. 3rd edition. Oxford Univ. Press.

Bamforth, C.W. (ed.) (2008) Beer: A Quality Perspective.

Handbook of Alcoholic Beverages Series, Academic Press.

Berry, D.R. (1982) Biology of Yeast. Edward Arnold (Publishers) Ltd., London.

Briggs, D.E , Boulton, C.A., Brooks, P.A. and Stevens, R. (2004) Brewing Science Practice,

Woodhead Publishing Limited, Cambridge UK.

Boulton, C. and Quain, D. (2001). Brewing Yeast and Fermentation. Blackwell Science Limited, Oxford, UK.

McCabe, J.T. (ed.) (1999) The Practical Brewer: A Manual for the Brewing Industry.

3rd Edition, Master Brewers Association of the Americas, Madison, WI.

Gump, B.H. (Ed.) (1993) Beer and Wine Production; Analysis. Characterization, and

Technological Advances, American Chemical Society, Washington, DC.

Hardwick, W.A. (1995) Handbook of Brewing. Marcel Dekker, New York.

Inoue, T., (2008) Diacetyl in Fermented Foods and Beverages. American Society of Brewing Chemists, MN.

Jacques, K.A., Lyons T.P. and Kelsall, D. R. (eds.) 2003. The Alcohol Textbook. 4th Edition, Nottingham University press, Nottingham, UK.

Kunze, W. (1996) Technology Brewing and Malting, International Edition, (translated by T. Wainwright), VLB.

Lewis, M.J. and Young, T.W. (1995) Brewing. Chapman & Hall, London.

Moll, M. (1991) Beers and Coolers, (translated from the original French edition by T. Wainwright), Intercept Ltd. England.

Pollock, J.R.A. (1979) Brewing Science (3 volumes). Academic Press, London.

Priest, F.C and Campbell, I. (eds.) (2003) Brewing Microbiology, Kluwer Academic, New York.

Priest, F.G. and Stewart, G.G. (eds.) (2006) Handbook of Brewing, 2nd Edition, Taylor and Francis, NY.

Reed, G. and Nagodawithana, T.W. (1991) Yeast Technology, 2nd Edition, Van Nostrand Reinhold, New York.

Russell, I. (ed.) (2003) Whiskey: Technology, Production and Marketing, Academic Press, London.

Russell, I. and Stewart, G.G. (1995) Biotechnology (Vol. 9), H.J. Rehm and G. Reed, (eds.),

VCH, Weinheim, pp 419-462.

Stewart, G.G. (2009) The Horace Brown Medal Lecture. Forty Years of Brewing Research.

Journal of the Institute of Brewing. Vol. 115 issue 1, pp. 3-29 .

Walker, CM. (1998) Yeast Physiology and Biotechnology, John Wiley & Sons, Chichester, U.K.

99

A partial list of associations, universities and publications

American Society of Brewing Chemists

http://www.asbcnet.org/

Brauwelt International

http://www.brauweltinternational.com/

Brewer and Distiller International

http://www.ibd.org.uk/publications/brewer-and-distiller-international/

Brewers’ Guardian

http://www.brewersguardian.com/

Brewery Convention of Japan

http://www.brewers.or.jp/english/index.html/

Brewing Research International

http://www.brewingresearch.co.uk/

Brewing Science Monatsschrift fur Brauwissenschaft

http://www.brewingscience.de/

European Brewing Convention

http://www.europeanbreweryconvention.org/

Heriot-Watt University - International Center for Brewing and Distilling

www.bio.hw.ac.uk/icbd/icbd.htm/

Brewers Association

http://www.brewersassociation.org/

Institute of Brewing and Distilling

http://www.ibd.org.uk/

Journal of the Institute of Brewing

http://www.scientificsocieties.org/jib/

Master Brewers Association of the Americas

http://www.mbaa.com/

100

Technical Quarterly of the Master Brewers Association of the Americas

http://www.mbaa.com/TechQuarterly/

The American Malting Barley Association

http://www.ambainc.org/

The New Brewer

http://www.beertown.org/craftbrewing/newbrewer.html

The Saccharomyces genome database - excellent source of online information on yeast

http://www.yeastgenome.org/

The VLB Berlin Research and Teaching Institute for Brewing (VLB)

http://www.vlb-berlin.org/

UC Davis campus program - Department of Food Science and Technology

http://www-foodsci.ucdavis.edu/bamforth/

University of California-Davis – Extension

http://www.extension.ucdavis.edu/brewing/

University of Nottingham – Brewing Science

http://www.nottingham.ac.uk/brewingscience/

Weihenstephan - Brewing University

www.wzw.tum.de/wzw/english/weihenstephan.html

University of Ballarat, Australia - Brewing School

http://www.ballarat.edu.au

Yeast – A Newsletter for Persons Interested in Yeast (M-A Lachance editor)

http://publish.uwo.ca/~lachance/YeastNewsletter.html

101

INDEX

acetaldchyde, 5, 52, fig.38

acidification power: .sec YEAST METABOLISM

alcohol-free beer, 86: see also LOW-ALCOHOL

BEER

amino acids, 35ff, 49, Tables 1 & 2

containing sulphur, 42, fig.30

formation of isoleucine and valine, fig.35

inorganic sulphur, and, 42

see also: CYSTEINE

METHIONINE

SULPHATE

WORT NUTRIENTS

Bbacteria, 72-3, fig.49

acetic acid, and, 72

Bacillus, 72

Enterobacteriaceae, 72

Lactobacillus brevis, 72

Laclobacillus Iindueri, 72

LactobacUlus plantarum, 72

Micmcoccaceae, 72

bioluminscence. ATP-driven, 75, fig.52

see also: VIABILITY

brewing yeast,

ale and lager differences, 6, fig.2

characteristics of, 5ff.

oxygen and, 37

performance of, 28

Sacchawmyces cerevisiae, 5, 6

Saccharomyces uvarum (carlsbergensis), 6

species of, 5

systematics and taxonomy of, 5

see also: SACCHAROMYCES

CEREVISIAE

cCandida albicans,

cultures of, 59, fig.43

see also: FLOCCULATION

carbohydrate catabolism, 34, fig.29

see also: AMINO ACIDS

carbon dioxide, 5

carbonyls, 51 ff

cell cycle, 13

cell immobilisation, 84

nature of technique, 84

see also: IMMOBILISATION OF

YEAST

cell viability, 74ff

based on cell metabolic slate, 75

Adenosinc triphosphate (ATP), 75

ATP-driven bioluminescencc, fig. 52, 75

capacitance, 74

dyes, use of, 74

power of reproduction as indicator, 74

see also: CELL VIABILITY

METHODS

CELL VITALITY

cell viability methods, 74ff

acidification power, 77

effect of adding glucose, 77

Adenosine triphosplwte, 75

correlation with viable cell mass, 74

BRF yeast vitality test, 77

Electrokinetics, 78

measurement methods compared, 75

Intracellular pH (1CP) method, 77

compared with acidification power, 77

Magnesium release test, 77

trials on Sacchammyces cerevisiae, 74

measurement by stress response, 77

examples of stress, 77

NADH as fluorosensor, 75

complex MDE-NAD* - malate, 75

measurement of ATP-driven

bioluminescence, 75

specific oxygen uptake rate, 77

pitching yeast into aerated media, 77

cell vitality,

specific oxygen uptake rate (BRF vitality

test), 77

see also: CELL VIABILITY

characteristics, 5ff, fig.3

see also: YEAST

contamination of cultures,

with bacteria, 71

with wild yeast, 72, fig.50

continuous fermentation. 81 ff, figs.56-7

advantages of, 82

cell immobilisation, and, 84

commercial failure, why, 81

features of, 81

flocculent yeast, and, 81

cystine, structure of, 56

Ddecarboxylation of ferulic acid, 17, 73. fig.51

diacetyl,

formation of, 51. figs.35 & 37

102

reduction of. 51. ligs.36 & 37

wort gravity, and, 52

yeast growth, and, 52

see also: CARBONYLS

disruptive technology, 92, fig.61

distiller's yeast, 93

ethyl carbamate, and, 97

whisky and, 94

DNA genetic tests, 23-8, figs. 16, 18 & 20

chromosomal fingerprinting, 26, figs. 19 & 21

hybridisation, fig.l7(b), fig. 18

polymerase chain reaction, figs.21-2

yeast DNA, fig.l7(a)

DNA technology and brewer's yeast, 23

E

Embden-Meyerhof-Parnas pathway,

and Kreb's cycle, 32

and oxidative phosphorylation, 33

entrapment, 84, fig.58

see also: IMMOBILISATION OF YEAST

enzymes, 14

catalysts and inorganic ions, 41

melibiose, 6

ester formation, 49, fig.34

see also: YEAST EXCRETION

PRODUCTS

ester production,

see OXYGEN

ethanol, 33, 78, fig.25

formation of, 5

processing of, 84

reaction formulas, 5

see also: WORT

ethanol tolerance, 78

ethyl carbamate, 97. fig.64

see DISTILLERS YEAST

fermentation, continuous, 81, fig.56

flocculation,

continuous fermentation, and, 81

lectin, theory of, 58, flg.41

measurement of,

by direct observation, 60

sedimentation methods, 60

static fermentation. 60, fig.40

non-flocculcnt yeast, 58, fig.42

poorly flocculent yeast, 58

Sacchammyces cewvisiae, in, 57, 58,

figs.39 & 42

foam, hydrophobic polypeptides, in, 79

genetic characterisation, 14-22, figs. 10-16

2-deoxy-glucose, 16

formation of diplotd zygotes, 15

mating of haploids, 15

reduction division of diploids, 15

spheroplast fusion, 20

triphenyl tetrazolium overlay, 18, fig. I

zygotes. 15

see also: DNA GENETIC TESTS

SACCHAROMYCES

CEREVIS1AE

glucose, 5, 29, 78, fig.23

analogue, (2-DOG) in maltose fermentation, 32

biochemical conversion of, 5

glutathione,

structure of, 56

glycogen, 66-69, figs.45-8

glycolysis, 29, fig.26

see also: WORT

HHansen, Emil C, 62

and Carlsberg, 62

high gravity brewing, 78ff, figs.53-4

decreased foam stability, 79

effect on ester formation, 79

yeast strain variability, 80

see also: WORT

higher alcohols, 47, fig.33

see also: YEAST EXCRETION

hydrophobic polypeptides,

essentially present in beer, 79

Iimmobilisation of yeast, 84-8. figs.57-61

nature of experiments, 82

Sacchammyces spp, and, 85

inorganic cations,

and yeast cells, 41

inorganic ions,

cellular anionic units, and, 41

enzyme catalysis, and, 42

enzyme formation, and, 42

ethanol production, and, 41

see also: PHOSPHORUS

inorganic sulphur,

and synthesis of amino acids, 42

intracellular Trehalose levels, 68, Table 5

ethanol shock, 68, Table 5

see also: YEAST STORAGE

CONDITIONS

103

ions, 41-7

cysteine, structure of, 56, fig.31

glutathionc, structure of, 56, fig.31

methionine, structure of, 56, fig.31

zinc levels, 45, and, fig.32

see also: INORGANIC IONS

WORT NUTRIENTS

Japan, sak6 yeast in, 96

K

Karyotyping, 26

Kreb's cycle (tricarboxylic acid cycle).

31-2, fig.27

Embden-Meyerhof-Parnas pathway, and,

3I.fig.26

fermentative pathway as alternative, 32

occurrence of, 32

oxidative phosphorylation, and, 33

yeast metabolism, and, 33

see also: PYRUVIC ACID

YEAST METABOLISM

lactobacillus, 71, fig.49

lifecycle, 15, fig. 10

lipids, 39

low alcohol beer, 86-87

alcohol-free beer, compared with, 86

production methods compared, 86-7

immobilised cell system, 87

controlled cthanol production, 87

premature fermentation arrest, 87

disadvantages during flocculation, 87

Mmaltose,

conversion into pyruvate, 31

fermentation, 31

genes, 10

glucose analogue, 32

maltotriose. and. 30, fig.24

Saccharomxces, and, 30

see also: WORT

maltotriose,

see also: MALTOSE

metabolism.

oxidative decarboxylation, 51

vicinal diketoncs, 51

yeast dehydrogenases,

how dependent on, 51

see also: METABOLISM

methionine. structure of, 56

mitochondria, 11, fig.4

see also: MORPHOLOGY

morphology, 7ff, figs.3-6

see also: MITOCHONDRIA

WORT

N

NAD+, fermentation of, 33, fig.28

ooxidative phosphorylation, 33

and Kreb's cycle. 33

and Embden-Meyerhof-Parnas pathway, 31

oxygen, 37ff,

and brewer's yeast performance, 35

and ester production, 39, Table 3

pediococcus, 71, fig.49

phosphorus, 42

as inorganic orthophosphate (H2 PO4). 42

orthophosphate transport, 42

translocation of orthophosphate, 42

polymerase chain reaction,

see: DNA GENETIC TESTS

preservation of culture, 62ff

see also: YEAST MANAGEMENT

pynivate, 31

see also: MALTOSE

PYRUVIC ACID

pyruvic acid, 5

conversion of. 35

R

rare mating, 17

respiratory deficiency, 18

Saccharomyces carlsbergensis (uvarum), 68,

fig. 15. Table 5

see also: YEAST STRAIN

Saccharomyces cerevisiae, 35, 66, figs.9. 10

& 12, Table 5

transport of insoluble ions, 45

copper resistance and, 4

flocculation in, 57-60, figs.39 & 42

immobilisation of yeast and, 85

maltose fermentation in, 30, fig.24

zinc ions and, 45

Saccharomyces diastaticus, 21, 23, fig. 15

104

Sacchammyces rvuxii, 21

see also: IMMOBILISATION OF YEAST

IONS

MALTOSE

YEAST CELLS

sake yeast,

in Japan, 96

Sphcroplast fusion, 20, Tig.IS

sporulation, 15, fig. 11

sugars, 29, 30, figs.22-4

see also: WORT

sulphur compounds, 55ff, figs.3O, 31 & 38

dimethylsulphide (DMS), 57

hydrogen sulphide, 55

sulphur dioxide, 55

see also: YEAST METABOLISM

Trehalose, 66ff, fig.45,48 and Table 5

tricarboxylic acid cycle,

see: KREB'S CYCLE

vitamins, 40, Table 4

see also: WORT

w

whisky,

and ethyl carbamate, 97, fig.64

grain, 93,94ff, fig.62

malt, 91,94ff, fig.63

malt and grain,

defined, 94

distillation and brewing distinguished, 95

distillation of, 95

distinguished from each other. 95

ingredients of, 95

storage of, following distillation. 95

wild yeast,

contamination of yeast culture, 73, 72. fig.5O

wort,

diacctyl and, 51

fermentation of, 16°P and 25°P, 78,

fig.53

gravity. 78

sugars, 80

treatment of components, 86

viability of yeast strains, 74ff and fig.54

wort nutrients,

ions, 41 ff

calcium, 45

conversion, 47

copper and iron, 46

divalent metal cations, and, 44

ferrous, 46

insoluble (Fe3+), 46

soluble (Fe2-), 46

hydrogen, 43

magnesium, 44

manganese, 45

potassium, 43

sodium, 44

zinc, 45

metabolism of, 30

oxygen, 37ff

sugars and carbohydrates, 30

uptake of, 30

vitamins, 40ff

yeast metabolism, control of, 35ff

wort production,

brewing and distillation distinguished, 96

wort sterility, 63

yeast,

acid washing of, 70

do's and don't's, 70

cell growth and division of, I2ff

cell viability of, 74-78

cell vitality of, 74-78

cell wall, 9

culture, fig.8

definition of, 3

genetic characterisation of, 14

genetic tests, 23

immobilised cells, 84

application of, 86

alternatives to entrapment, 85

commercial criteria, 86

comparative results of, 84

definition of. 84

entrapment technique, 84

gel matrix, 84

research into, 84

inorganic ions, and, 41

see also: BREWING YEAST

DISTILLERS YEAST

MORPHOLOGY

YEAST CELLS

YEAST MANAGEMENT

life cycle, 15

yeast cells, fig.l

division, 13

growth, 13-15

105

inorganic ions and cations, 41

polysaccharides, 14

viability and vitality, 74ff

see also: CELL VIABILITY

CELL VIABILITY METHODS

yeast excretion products, 46ff

carbonyls, 51

esters, 49

higher alcohols, 47

organic and fatty acids, 47

sulphur compounds, 55

see also: YEAST

yeast management,

collection, 65

contamination of cultures,

with bacteria, 71, fig.49

with wild yeast, 72, fig.50

pitching and cell viability, 64

preservation of culture, 64

pure culture. 62ff

storage. 66

storage conditions, 66

see also: CELL VIABILITY

yeast metabolism,

changes detected by acidification power test,

77

control of, 35

Crabtree effect, 35

fermentative pathway, 34

Kreb's cycle, 33

Pasteur effect. 35

yeast pitching, 64

see also: YEAST MANAGEMENT

yeast propagation, 62, fig.44

yeast sporulation. 15, fig. 11

yeast storage, 66

conditions, 66ff

intracellular glycogen levels, 66, fig.45

see also: GLYCOGEN

yeast strains, 23

alcohol tolerant bacteria in, 96

Lactobacillus spp, 11

Sacchammyces sake (Japan), 96

extracellular pH, 43

intracellular pH, 43

nature of, 5

translocation of orthophosphate, 42

z

zinc, 46, fig.32

zymocidal ("killer") activity, fig. 13

mating protocol, 17

ILLUSTRATIONS (FIGURES)

Figure

1 Electron micrograph of a budding cell

2 Utilisation of the sugar raffinose and melibiose by lager and ale yeast

3 Giant colony morphology on wort gelatin plates of typical ale and lager yeast strains

4 Main features of a typical yeast cell

5 Electron micrograph of yeast cell with multiple bud scars

6 Structure of the yeast cell wall

7 Structure of the mitochondrion: (A) diagram, (B) electron micrograph

8 Batch growth curve for brewing yeast culture

9 Cell cycle of Sacchammyces cerevisiae

10 Haploid/diploid life cycle of Sacchammyces cerevisiae

11 Sporulating yeast cell

12 Sacchammyces brewing yeast, with and without "killer" activity

13 Rare mating protocol to produce brewing strains with zymocidal "killer" activity

14 Triphenyl tetrazolium overlay of yeast colonies

15 Spheroplast fusion of two yeast strains

16 Production of a recombinant DNA brewer's yeast

17 Restriction patterns involving yeast DNA

18 DNA-DNA hybridisation test

19 Chromosomal fingerprints of three brewing lager strains

Page

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18

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22

24

25

106

Figure page

20 Polymerasc chain reaction with target DNA 27

Fingerprint patterns using polymerase chain reaction 27

Order of uptake of sugars by yeast from wort 30

Uptake of sugars by the yeast cell 30

Uptake and metabolism of maltose and maltotriose by the yeast cell 31

Degree plato reduction and ethanol production 31

EMP/glycolytic/glycolysis pathway 31

Kreb's cycle 32

Regenerating NAD+ by fermenting yeast 33

Contribution of carbohydrate catabolism to intermediate components 34

Effect of zinc levels in wort on primary fermentation time 46

Production of higher alcohols 48

Metabolic inter-relationships leading to ester formation 49

By-products of pathways leading to formation of amino acids valine and isoleucinc 31

Reduction of diacetyl to acetoin and 2,3-butanediol 52

Diacetyl formation and breakdown in relation to yeast growth and wort gravity 52

Inter-relationship between yeast metabolism and production of flavour compounds 55

Pathway for synthesis of sulphur-containing amino acids 56

Structure of cysteine, mcthionine and glutathione 56

Flocculation in Sacchammyces cerevisiae 57

Static fermentation flocculation 58

Lectin theory of flocculation 58

Electron photomicrograph of Sacchammyces cerevisiae 59

Electron photomicrographs of Candida albicans 59

Typical propagation vessel 62

Chemical structure of (A) glycogen and (B) trehalose 67

The effect of yeast glycogen at pitching on a lager fermentation 67

The effect of yeast storage temperature on intracellular glycogen concentration 68

Pathways to glycogen and trchalose in yeast 69

Photomicrographs of typical bacteria found as brewing contaminants 71

Photomicrograph of (A) wild yeast and (B) brewing yeast culture contaminated

with wild yeast 73

Decarboxylation of fcrulic acid to 4-vinyl guaical by yeast 73

Measurement of ATP-driven bioluminescence 76

Fermentation of 16°P and 25°P wort by production lager strain A 78

Viability of brewer's yeast strains during fermentation of 27°P wort 79

Multi-stage lower fermenter 81

Stirred tank continuous fermentation system 82

Cultor's 2-hour continuous maturation system 88

Mass transfer diagram of an entrapment carrier 89

Labatt gas lift draft tube bioreactor 90

Schematic of Meura Delta two-stage multi-channel immobilised loop reactor 90

How to assess a disruptive technology 92

Grain whisky - continuous column distillation 93

Mall whisky pot-still process - double stage distillation 95

Chemical structure of ethyl carbamate (urethanc) 97

107

TABLESTable Page

1 Classification of amino acids by speed of absorption from wort by ale yeast 36

2 Classification of amino acids by nature of Keto-Acid Analogues in yeast metabolism 36

3 Effect of linoleic acid and oxygen on ester production 39

4 Vitamins in sweet wort and functions in yeast metabolism 41

5 Effect of ethanol shock on intracellular trehalose content of ale and lager yeast strain 68

108