An Introduction to
BREWING SCIENCE
& TECHNOLOGY Series III BREWER'S YEAST "The IBD Blue Book on Yeast"
THETHE INSTITUTE OF BREWING DISTILLING INSTITUTE OFAND BREWING
An Introduction to
BREWING SCIENCE & TECHNOLOGY Series III BREWER'S YEAST G.G. Stewart1 and I. Russell2
"The IBD Blue Book on Yeast" ('Heriot-Watt University, 2Labatt Brewing Company Limited)
G.G. Stewart and I. Russell
THE INSTITUTE OF BREWING AND DISTILLING 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 Haston Filsell and Janice Riddell for careful typing and editing of the manuscript.
ISBN No. 0900489 13 8 Copyright © 1998 The Institute of Brewing Allof rights of reproduction reserved in all countries of all texts illustrations. All rights reproduction andare distribution (hard copyinorrespect electronic) are and reserved in all No part may reproduced utilized any form without permission from the Institute of Brewing. countries in be respect of allortexts andinillustrations. Nowritten part may be reproduced or utilized in any form without written the Institute of Street, Brewing andW1Y Distilling. Published by: permission The Institute from of Brewing. 33 Clarges London 8EE, England. Published by: The Institute of Brewing and Distilling, 33 Clarges Street. London W1Y 8EE, England. Note - A number of small corrections to the 1998 copy were inserted in 2009 along with an updated reading list.
BREWER'S YEAST G.G. Stewart1 and I. Russell2
"The IBD Blue Book on Yeast" ( Heriot-Watt University, 2Labatt Brewing Company Limited) G.G. Stewart and I. Russell Contents Introduction Fundamentals
3 3
Characteristics of Brewing Yeasts
5
Yeast Morphology
7
Yeast Cell Growth and Division Genetic Characterisation of Yeast Genetic Tests for Typing Yeast Strains Brewer's Yeast Performance Uptake and Metabolism of Wort Nutrients Wort Sugars and Carbohydrates Control of Yeast Metabolism
13 14 23 28 29 29 35
Pasteur effect
35
Crabtree effect (glucose repression, catabolite repression)
Amino Acids, Peptides and Proteins
Oxygen
Vitamins Ions
Inorganic ions Hydrogen ions Potassium ions Sodium ions Divalent metal cations Magnesium ions Manganese ions Calcium ions Zinc ions Copper and iron ions Yeast Excretion Products
35
].".'.'."".'.'.
35
37
40 41
41 43 43 44
44 44 45 45 45
Organic and Fatty Acids Higher Alcohols
45 45 47 47
Esters
49
Carbonyls Sulphur Compounds Flocculation
51 55 57
Yeast Management Pure Yeast Cultures Preservation of Stock Yeast Culture Yeast Pitching and Cell Viability Yeast Collection Yeast Storage
62 62 64 64 65 66
Yeast Storage Conditions - Influence on Intracellular Glycogen
and Trehalose Levels Yeast Washing Contamination of Cultures with Bacteria Contamination of Cultures with Wild Yeast Yeast Cell Viability and Vitality Use of Specific Dyes for Assessing Cell Viability and Vitality
Capacitance The Power of Reproduction as a Viability Indicator Viability and Vitality Methods Based on Cell Metabolic State Adenosine triphosphate (ATP) NADH fluorosensor Specific oxygen uptake rate (BRF yeast vitality test) Acidification power Intracellular pH (ICP) method Measurement of yeast vitality by stress response Magnesium release test (MRT) Electrokinetics High Gravity Brewing Continuous Fermentation Immobilised Yeast Technology Production of Alcohol-free and Low Alcohol Beers Immobilised Lager Yeast to Reduce Maturation Times Primary Fermentation with Immobilised Yeast Distiller's Yeast Malt and Grain Whisky Ethyl Carbamate Supplementary Readings
Internet Web Sites Index Illustrations (Figures) Tables
66 69 71 72 74 74 74 74 75 75 75 77 77 77 77 77 78
78 81 84 86 87 89
93 94 97 99 100
102 106 108
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 unicellular fungi 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 EmbdenMeyerhof-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 pyruvate
» pyruvate decarboxylase
CO, + CHjCHO carbon dioxide + acetaldchydc
> alcohol dehydrogenase
CH3CH2OH cthanol
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'
Melibiase
' Fructose
Invertase
Figure 2. Utilisation of the sugar raffinosc and melibiosc by lager ISaccharomyces uvarum (carlsbergensis)] and ale
Melibiose Galactose ■
■ Glucose
(Saccharomyces cerevisiae) yeast.
Melibiase
(Note: Saccharomyces cerevisiae does
not possess the enzyme melibiase.) Saccharomyces cerevisiae Raff/nose 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
2009 Note : Saccharomyces pastorianus is the current correct taxonomic name for Saccharomyces carlsbergensis i.e., lager yeast.
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.
The distinguishing feature of a growing population of yeast cells is the presence of die 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
Figure 5. Electron micrograph of a yeast cell with multiple bud scars.
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
yeast strains (ale strains,
Fibrillar layer
very rarely lager strains),
new rounds of cell division occur before cell
manno-
manno-
") Manno
Glucan separation so clumps of protein protein > cells are produced - a J Glucan Glucan process known as chain formation. The site of cell mannoprotein Cell separation is marked on proteln wall the mother cell by a Glucan Glucan structure referred to as Glucan the bud scar and on the Glucan Glucan Glucan daughter cell by the birth scar. These scars cannot plasma membrane be seen under the light microscope but can be cytoplasm seen using fluorescence microscopy after staining Figure 6. Structure of the yeast cell wall. with fluorescent stains such as calcafluor or primulin. Bud scars also show up as very distinct structures
protein
layer
} Glucan layer
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
thethe complete sequence of the chromosomes has proteins synthesised in the cell. Now, Recently 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
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
applied to each of the genes in genes. New and existing molecular genetic methods are willbeing be applied are beingAapplied an attempt to define the function of their encoded product. similar, but complementary took place research programme, will take place in the United States. The Genome Project is therefore producing a a flow of information on yeast, much of which will provides set to produce 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
Inner membrane Matrix
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 of an aerobically grown yeast cell as spherical or rod-shaped
structures
Figure 7. Structure of the mitochrondrion. (A) A diagram showing the overall
surrounded by a double membrane. They 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 freezeetching 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 ••
1x10*
1x10'-
Figure 8. Batch growth curve for brewing yeast culture in shake flasks at 20"C (A) log phase,
1x10'I
(B) accelerating, (C) exponential phase, (D) decelerating phase,
and (E) stationary phase 0
5
10
IS
20
25
30
35
40
1X105
(adapted from Priest and
Campbell, Brewing Microbiology, 1996).
Time (hours)
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 Growth in brewer's yeast is associated almost entirely with the growth of the
Late
bud which reaches the size of the mature cell by the time it separates from the
/
nuclear A
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
division J
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 Figure 9. Cell cycle of Sacchammyces only occupies part of the cell cycle. The cerevisiae. 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 development of brewer's yeast strains. Classical approaches to strain improvement include mutation and selection, screening and selection, and cross-breeding (hybridisation). Mutation is any change 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
meiosis and sporulation
Diploid Phase (2n) p/a diploid
Haploid Phase (n)
Figure 10. 2 mating type a and
4 spored ascus
Saccharomyces cerevisiae.
2 mating type a
s
v,
Haploid/diploid life cycle of
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-OffFlavour) 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
Laboratory Haploid -Killer" Strain
"Non-Killer' Yeast
brewing Lager
Strain Rare Mating (Forced Mating)
S\ Hybrid
(Heterokaryon)
Segregation under Influence of Kargone
Figure 13. Rare mating protocol to produce brewing strains with zymocidal
(Kar = Karyogamy defective)
"killer" activity.
Brewing Lager
Laboratory Haploid
Strain
-Killer Strain
True Hybrid
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. A question often asked is whether the toxin is still active in the finished beer. The toxin is extremely heat-sensitive, and a brewery pasteurisation cycle of 8 PU's has been shown to
completely inactivate it.
The introduction of a "killer" strain into a brewery where several yeasts are employed for the production of different beers can present logistical problems. An error on an operator's part in kegging lines and yeast tank lines could have serious consequences, since accidental mixing would 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 commercially available 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, but they are usually recessive mutations, due to functional loss of a single gene. Since brewer's yeast 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 both complementary genes will the recessive character be expressed. If the mutation weakens the yeast, the mutated strain will be unable to compete and soon be outgrown by the non-mutated yeast 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 yeast management techniques are considered.
Only three characteristics are routinely encountered resulting from yeast mutation that arc harmful 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. The mitochondria are then unable to synthesise certain proteins. This type of mutation is also called 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). The respiratory 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 a result these yeasts are unable to metabolise non-fermentable carbon sources such as lactatc,
glycerol or ethanol. Many phenotypic effects (actual expressed properties, such as the yeast's ability to perform a particular chemical reaction) occur due to this mutation and these include alterations in sugar uptake, metabolic by-product formation, and tolerance to stress factors such as ethanol and temperature. Flocculation, cell wall and plasma membrane 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).
Saccharomyces
Saccharomyces
diastaticus
uvarum
(carlsbergensis)
Whole cells spheroplasting enzymes
Spheroplasts
IDEX fusing agent (polyethylene glycol)
Fusing spheroplasts ( DEX • FLO
Fused spheroplasts
/
t
cell wall regeneration in
complete growth medium Regenerated fused cell
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 of high 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).
o
Donor DNA
Plasmid DNA
Cut DNA Pieces
Cut Plasmid Pieces
(J
spheroplasting enzymes
anneal and ligate
o
Recombinant 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
•
a-Acetolactate decarboxylase activity from the bacteria Enterobacter aerogenes and
and barley; 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
• 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.
hybridisation
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:
map.
• DNA fingerprinting by hybridisation with a
Figure 17. Restriction patterns of (A) yeast
DNA, and (B) DNA
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 be prepared 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 of Figure 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 given to 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 bacterium Providencia 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
Step 1 -
Step 2 -
Collection of organisms on a filter matrix
Step 3 -
Cell lysis and DNA strand separation
Binding of DNA to filler matrix
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 4 -
Step 5 -
Addition of labeled DNA Probes
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 technique in 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 of bands 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 singlestranded) 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 place
overnight. 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 and
detection 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. Karyotyping is an electrophoretic technique that separates
Figure 19. Chromosomal fingerprints of three brewing lager yeast strains.
whole chromosomes based on their different sizes. As discussed 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 of common 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
Target DNA
51.
3'
31-
5'
9
B Cycle 1
a
Denature! ion
annealed (C) at 55°C and
5,
then primer extension (D) proceeds at 72CC. The cycle
3'
04-C
3'
(A-D) is then repeated (E) until 25-40 cycles have been completed. (F) time-
55"C
51-
60
30
3'
1—1
/
l~~\
1
/
Primer /
1
/
72
51 Primer Annealing
Oonalurolion
94
\
/
\
Extension /
1
1
\
' Primer'
/ /
«
Anneatng
F
1 min
1 min
2 min
1 min
■4— Cycle 1 —► 5'
3'-
D
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.
„
Primer Extension 72*C
3'-
•5'
5'-
-3'
E Cycle 2 5'
-5' •3'
Number of cycles
B
► 25~«> 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. —222
249
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 crosscontamination 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 nutrients from the growtli/fermentation medium (wort), able to tolerate the prevailing environmental conditions (for example, ethanol
tolerance) and impart the desired flavour to the beer, but the microorganisms themselves must be effectively removed from the wort by flocculation, centrifugation and/or filtration
after they have fulfilled 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, ami no 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 together with 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
sequence (or priority) (Figure 22), although some degree of overlap does occur. The majority
Glucose
Fructose Maltose
Maltotriose
of brewing strains leave the malto-tetraose and other dextrins unfermented, but Saccharomyces
Dextrins
diastaticus is able to
utilise dextrin material. The initial step in the utilisation of any sugar
24
48
72
96
120
by yeast is usually either its passage intact across the cell membrane or its hydrolysis outside the cell membrane followed
144
Fermentation time (hours)
Figure 22. Order of uptake of sugars by yeast from wort.
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 usually accounts for 50-60% of the fermentable sugar in wort. Maltose fermentation in Saccharomyces yeasts requires at least one of five unlinked (each independent) MAL loci each
Maltose
Maltotriose
p^mease Maltose
Maltotriose
a-glucosidase
a-glucosidase
GLUCOSE
consisting of three genes encoding the structural gene
for a-glucosidase (maltase) (MAL S), maltose permease (MAL T) and an activator R) whose product co-ordinately regulates the expression of the aglucosidase and permease genes. The expression of MAL S and MAL T is
permease transporter
glucose
t
(MAL
glucoamylase
I
Starch/Dextrin
transporter permease transporter permease glu
\
I
fructose + fructrose invertase
I
Sucrose
Figure 23. Uptake of sugars by the yeast cell. 30
Maltose
cell
i /
membrane %
Maltotriose
I
"Carrier Protein
(maltose permease)
I
"Carrier Protein \ (maltotriose permease) /
t
t
Maltose
Figure 24 (left). Uptake and metabolism of maltose and maltotriose by the yeast cell.
Maltotriose
a-Glucosidase
Glucose
| glucose!
t Ale brewing 3train O—O Oorepressed variants
£ADP glucose 6-phosphate
fructose 6-phosphate 6-p
I
ATP ADP
12
fructose 1,6-diphosphate
24
38
48
60
72
84
86
106
55-
B
5-
glyceraldehyde
[ dihydroxyacetone phosphate
3-phosphate
L
JNADHj,
J
4-
1 3.5i '■
1": 1
ADP
05-
atp
3-phosphoglycerate
0
12
2-1
36
48
60
72
84
9B
108
120
Time (hours)
1
Figure 25 (above). (A) Degree plato reduction, and (B) ethanol production
2-phosphoglycerate
by an ale brewing strain and its depressed variants.
phosphoenol pyruvate
Ipyruvate]
45
2 1.5-
1,3-diphosphoglycerate
J{
120
Time (hours)
ADP
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, Alpha- ketoglutarate
Succinyl CoA Biosynthesis excretion
GTp
Biosynthesis
QDp
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
NADH
aldehydes
gtycerol
8thanol| and fusel alcohols
oxaloacetate
• malate
dehydrogenases fumarate
■ succinate
diacetyl
2,3-butanedbl
dimethyl sulfoxide
NAD+
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), a cofactor 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 the tricarboxylic 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 acids will spill over into the fermented beer. The additional substrates that are generated from the
Kreb's cycle may be used to supply additional substrates for biosynthesis (Figure 27). In respiring cells, molecular oxygen is used as the final H+ acceptor and glucose is completely oxidised. By the end of oxidative phosphorylation, one glucose molecule yields 2 ATP from the glycolytic pathway, 24 2 ATP from the Kreb's cycle, and 24 12 ATP from oxidative phosphorylation. Thus, respiration of 1 glucose molecule yields 28 38 molecules of ATP overall. In respiring yeast, NAD+ is regenerated using oxidative phosphorylation and the Kieb's cycle.
Under anaerobic conditions, the Kreb's cycle may operate partially, but the extent of operation has yet to be determined. When yeast are in the fermentative state, NAD+ is regenerated using a range of hydrogen acceptors (Figure 28). For example, yeast are 33
glycogen man nan
glucan trehalose
hexose monophosphate
glucose-6-phosphate
pathway
triose phosphates
phosphoenol pyruvate
nucleotides inoaci
Krebs'
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: ethanol
acetaldehydc
pyruvate
CH3CH2OH
■*- CH3CHO -
CHjCOCOOH
co2
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. for reasons that are unclear. 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 growth involves the uptake of nitrogen, mainly inmainly the form amino acids, ammonium Activeyeast yeast growth involves the uptake of nitrogen, inofthe form of amino acids, ions small peptides for the synthesis of proteins and other nitrogenousofcell compounds. Later in for and the synthesis of proteins and other nitrogenous compounds the cell. Later in the the fermentation, yeastmultiplication multiplication stops, uptake slows or ceases. In wort, fermentation as as yeast stops,nitrogen nitrogen uptake slows or ceases. Inthe 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
B - Intermediate Absorption
Glutamic acid
Valine Methionine Leucine Isolcucine Histidine
Aspartic acid Asparaginc Glutamine Serinc
C - Slow Absorption
D - Little or No Absorption
Glycine
Proline
Phenylalanine
Tyrosine Tryptophan Alaninc
Threonine Lysine
Ammonia
Arginine
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
Class 2
Class 3
Glutamic acid Asparaginc
Isolcucine
Lysine Histidine Arginine Leucine
Glutamic acid Glutamine Threonine Serinc
Valine Phenylalanine Glycine Alanine Tyrosine
Methionine Proline
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 acids must 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
clear that there are only three points in the brewing process where oxygen is beneficial. At pitching, during propagation of ainnew and for barley germination malting. evident that thisyeast is the only point theculture, brewing process where oxygen during 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 35 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 Production /adapted jmm Lentini el «/., 1994, Proc. Com hist. Brew. (Asia Pacific Sect.), Sydney, 23, 89-95}. High Trub Wort
Low Trub
Low Trub
Wort
Wort
Wort
6180
5510
880
510
8
4
8
4
18.3
26.5
24.2
34.6
High Trub
Linoleic Acid (Hg/gDW yeast) Wort oxygen
concentration (mg/L) Total esters (mg/L)
A typical lipid composition of brewing yeast would consist of 70-90% 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 aerobic and 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 dynamicstate 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
Level in Wort /100 ml
Some Metabolic Functions
Biotin
0.56 u.g
Thiamin(Bl)
60 ug
Carboxylation reactions, protein, nucleic acid, carbohydrate, fatty acid Decarboxylation of pyruvate
Calcium pantothenate Nicotinic acid
45-65 ug 1000-1200 Ug
Riboflavin
20-50 ug
Inositol
9.3 mg (free) and 18.9 mg (total)
Pyridoxine,
85 Hg
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
pyridoxal, & pyridoxaminc
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 sodiumphosphate 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 sodiumchloride 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, carriermediated translocation across the plasma membrane. This secondary phase involves energydependent 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 transmembrane 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 Mgorthophosphate 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+. Although Mn2+ can be substituted for Mg2+ as an enzyme cofactor in vitro, this is unlikely to
be 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 and decreases 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 is strongly influenced by the intracellular levels of Mg2+ ions. Calcium ions
Calcium stimulates yeast growth but it is not a growth requirement. It is involved in the membrane structure and function. Yeast cells maintain cytosolic Ca2+ at very low levels. 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 energydependent uptake of Ca2+ into the vacuole may be involved in regulating Ca2+ metabolism in 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 regulation and have been implicated in the transition from log to exponential phase in batch cultures of yeast. Also, culture media requirements for Ca2+ in yeast growth and division have been highlighted recently by findings that indicate Mn2+ can effectively 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 in Saccharomyces cerevisiae prevents budding and arrests cells in the G, cycle of the cell cycle. Zn2+ requirements for the growth of yeast cannot be met by other metal ions. 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 yeast alcoholic fermentation, namely, alcohol dehydrogenase is a zinc-metalloenzyme. The brewing significance of this lies in the phenomenon of "stuck" fermentations which may be ameliorated following appropriate supplementation with zinc salts (Figure 32).
45
Figure 30. The effect of
180-1
Strain A
zinc levels in wort on
Strain B
primary fermentation time (adapted from Skands et
160-
ai, Proc. EBC Cong., Maastricht, 1997, p. 413). 140-
Copper and iron ions
120-
100
Both copper and iron are essential nutrients for yeast which act as cofactors in several enzymes including the redox pigments of
02 0.3 Zinc addition mg/L
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 most of 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 wort
during 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 devolving from pyruvate are rate-determining.
Medium-chain-length fatty acids (C6-CUI) arise via the activity of fatty acid synthetase as intermediates in the formation of longcr-chain-length fatty acids, which are incorporated into the various classes of yeast lipids. In addition, a proportion are derived from the assimilation and further metabolism of wort lipids. The release of medium- and longchain-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 since acids 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 catabolism
or 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, then
reduced 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-methyI1-butanol from isoleucine.
47
aldehydes
sugar
carbohydrate metabolism
ethanol and fusel alcohols
oxoacids ammo
acids Figure 31. Production of higher alcohols (adapted from 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
saturated fats
and phospholipids
sterols
and phospholipids
!■ I
squalene
c
unsaturated acyl CoAs
/
mevalonic acid
/
^\
/di and tri- -4—pyruvate
(oxoacidsV-*--( carboxylic V acids
t
C acyl CoAs r.e\i
,
membrane
alcohols
_
\
M
i
T
ESTERS
amino acids
J
V.
nitrogen metabolism 1
i
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 of ethyl 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 longerchain-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 of oxygen 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 increasing the 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
+
ethanol
CHjCOSCoA
CH3CH2COOCH3
acetyl CoA
+
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-acetohydroxybutyrate
a-acetohydroxybutyrate
u-acetolactate
diacetyl
2,3-pentanedione
, Enzymatic
conversion
|
*\
Passive y diffusion
Non-enzymatic decomposition
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
concentrations of carbonyl compounds are known to cause stale flavour in beer. The effects
of aldehydes on flavour stability are reported as (propanol,
grassy notes
2-methyl
butanol,
pentanol) and a papery taste
(fra/K-2-nonenal, furfural). ^^, Enzymatic
^^ conversion
Quantitatively, acetaldehyde is
the most important aldehyde. This is produced via the
Taken up or excreted by cell
decarboxylation of pyruvate and is an intermediate in the formation of ethanol. It may be present in beer at concentrations above its flavour threshold,
Aceloin Butanediol
Figure 34. Reduction of diacetyl to acetoin and 2,3-butanediol.
(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 reutilisation 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 2,3pentanedione. 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
1.05
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. Diacetyl and 2,3-pentanediones arise in beer as by-products of the pathways leading to the formation of valine and isoleucine (Figure
60
Suspended 1.04
•
0.8
yeast count
50 40
0.6 30
1.03 0.4< 1.02 0.2
1.01
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
20 10
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 aacetolactate 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 aacetohydroxy 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 cxacetolactate 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
Amino acids
I
Organic acids
\J
Acetaldehyde
Ethanol r
Fusel alcohols
Fatty acyl-CoA Fatty acids
Vicinal Diketones
I
Acetyl-CoA
J
-► Keto (oxo) acids V.
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 amino acids 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 biosynthetic route 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
Figure 37 (left). Pathway for the
T
ATP-Sulphuryiase
synthesis of sulphur-containing
ATP
amino acids.
ADP
will have been utilised. Yeast growth during fermentation is roughly
Adenosine - 5' phosphosulphate (APS)
I I I
ATP
synchronous (cell division occurs at the same time) and hydrogen sulphide
3/ phosphoadenosine - 5' phosphosulphate (PAPS) NADPH
evolution seems to occur in a number of peaks which correspond to the
NADP
phase of the yeast cell cycle just prior to the onset of budding.
NADPH
The formation of excessive levels of hydrogen sulphide and sulphur
| SULPHITE (SOJ +ADP | Sulphite reductase
NADP
SULPHIDE (H,S) Cysteine synthase (pantothenale requiring)
contributing to beer flavour, also has a number of other
Serine
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 of fermenting vessel is also influential. Sulphur dioxide, as well as
Cysteine
HSCH^CHfNhyCOOH
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
Cystine
NH,
I
NH,
HOGCCHCH2-S-S-CH2CHCOOH
Methionine
CH2SCH.CH2CH(NH,)COOH
Glutathione H2NCHCH2CH2CONHCHCONHCH2COOH
Figure 38 (right). Structure of cysteine, cystine,
COOH
methionine and glutathione. 56
CH2SH
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 dimethylsulphoxide (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 a particular yeast
culture is one of
the 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
Non-flocculont
Chain Former
Flocculent
Figure39. Flocculation inSaccharomycescerevisae. 57
describe different phenomena in yeast cell behaviour. Specifically, flocculation, as it applies to brewer's
2.0 -i Non-flocculent culture
1.8-
Flocculent culture
yeast is "the phenomenon wherein yeast cells adhere in clumps and either sediment from the medium
1.61.4-
in which they are suspended or
1.2-
rise to the medium's surface". 1.0-
This definition excludes other forms of aggregation, particularly
0.B-
that of "clumpy-growth" and "chain
0.6-
formation",
which have
been discussed previously (Figure
0.4-
39). This non-segregation of
0.2-
10
20
30
40
SO
60
70
80
90
Percentage attenuation
Figure 40. Static fermentation flocculation.
100
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.
-•
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. (Fig. 39). 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 con formational 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 not been 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 until the 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 number
of 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 cell surface 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 of brewing 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 thethe flocculent phenotype in Saccharomyces spp. A spp. number of been reported reported to todirectly directlyinfluence influence flocculent phenotype in Sacchawmvces Four dominant flocculation been identified including FLO4,FL04, FLO8, FLOS), FLO5, dominant flocculationgenes geneshave have been identified FLO1 FLO1, (allelesFLO2, are FLO2, FLO9, FLO10 and FLOW, FLO11, as gene, flo3, andand twotwo recessive genes, FLOS, FL09 and aswell wellasasa asemi-dominant semi-dominant gene,/7o5, 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 Spray ball
The systematic use of clean, pure
Exhaust
and highly viable cells ensures that bacteria, wild yeasts or yeast mutations (such as respiratory deficiency) do not lead to
inconsistent fermentations and
beer off-flavour development. 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
Condensate or
coolant
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 strain culture was introduced into a Carlsberg brewery on a production scale in 1883, and the benefits of using a single pure strain culture quickly became clear. Soon, 23 countries
Temperature
probe Yeast inoculation port
Pitching
Sterile wort
yeast to fermenter
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 the 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 sufficient to 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, slow
•
Transfer to 20-30 hL yeast vessel, 15-20 hL, wort (1040-1048, 10-12°P), aerate/oxygenate
shaking;
(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%);into the regular beer stream.
•
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);at minus 198 degrees C);
•
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 experienced hands, methylene blue will quickly identify a problem if there is a known history of the typical 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. Pitching
rates 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 of cells/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 dielectrical properties 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 oxygen supply, 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 at the 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 yeast plug removal.
The use of centrifuges for the removal of yeast and the collection of pitching yeast is now commonplace. There are a number of advantages such as shorter process time, cost reduction, increased productivity and reduced shrinkage. Care must be taken to ensure that high temperatures (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 and resultant lower solids and a longer frequency for yeast being sent to waste with the high
solids and reducedproduct productshrink. shrink. Centrifugation can induce hydrodynamic shear solids and resulting resultinginreduced
on centrifuged yeast cells resulting in haze, foam and off-flavour problems in the resulting beer. 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 of ATP resulting from respiration, activate the phosphorylase system which is necessary for the hydrolysis of 66
A
Glycogen (C6H10O,)n
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-glucopyranosyla-D-glucopyranoside.
glycogen to glucose. The phosphorylasc activity during wort fermentation peaks coincidcntally with glycogen hydrolysis which is within the first 10 hours after
Trehalose(C12H2,On) HOH,C O
Control (no storage)
27% yeast glyccgen
(116 Hra) Anaerobic storage 15% yeast gtyecgen
(116 Hra) Aerobic storage 9% 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.
67
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
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
10
-O- Aerobic storage at 4°C
—•- Aerobic storage at 15°C
24
48
72
96
120
144
Storage time (hours)
Figure 47. The effect of yeast storage temperature on intracellular glycogen concentration.
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]. Treatment for 60 min.
Yeast & Strain Number
Trehalose (Hg/mg dry wt)
Saccharomyces uvarum (carlsbergensis)
Saccharomyces uvarum (carlsbergensis) Saccharomyces uvarum (carlsbergensis) Saccharomyces cerevisiae Saccharomyces cerevisiae
Saccharomyces cerevisiae
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)
68
8.2 22.5 11.8 6.5 13.7
8.0
Trehaloso |
Glucose
Phosphatase
Trehalose phosphate Trehalose phosphate synthet
Urldlne diphosphate
Glucose-6-phosphate
glucose
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. These results indicate that ethanol shock induces accumulation of trehalose in yeast cells. Figure
48 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 yeast washing and its effect on subsequent fermentations. Studies carried out at the Brewing Research Foundation International suggest that the problems often ascribed to yeast washing (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 out incorrectly.
There are three commonly employed procedures for washing yeast: sterile water, acid wash and 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
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
bacteria belonging to the genera Lactobacillus and Pediococcus. At
:
*"Jf zS
4»
-
,.*"
C^
SHf
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 hetero fermentative
(producing lactic acid as well as other acids and /or alcohols and
some strains produce diacetyl) and
/ ■i.
homofermentative (producing only lactic
,»1
acid). They are acid tolerant and have complex nutritional requirements. Some species such as Lactobacillus brevis and
Lactobacillus
plantarum can grow quickly during
X
x\
<■
,*l
.Vfv,'
*
10um
Figure 49. Photomicrographs of typical bacteria found as brewing contaminants. Top - Pediococci and bottom - Lactobacilli.
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.
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 of beer, each with its own yeast or mixture of yeasts, 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,
CH = CH,
(Yeast with POF gene 4-VG producer)
— OCH,
■OCH,
OH Ferulic Acid
4-Vinyl Guaiacol
(wort constituent)
(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). NADH fluorosensor
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+ *
*
(oxidised)
oxaloacetate + NADH + H+ (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
B
Microbial &
Microbinl A
non-microbial cells
non-mlcroblal cells
Selective release of ATP from non-mlcroblal cells. Microbial cells remain intact
Selective release of ATP from non-microbial cells. Microbial cells remain Intact
Microbial celts
Hydrolysis of non-microbial ATP with ATPase. Microbinl cells remain Intact
ATPaae inactivated. Selective
Addition of luerferin-lucifcraso
nf mlnrnhlal ATP
Addition of tucifertn-lucllerase
Addition of
Luminometer Readout
(Total non-microbial RLU)
Solocllvo release
ralaaaa of mlcroblsl ATP
Rparlmit
(Total microbial RLUt
luclferin-l lie Kara
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 oxygen available 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 of the cells and environmental effects. Measuring NADH has an advantage over monitoring dissolved oxygen or pH because it directly measures, in real time, events occurring within the 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 pitching of 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 fermentation performance than non-acid-washed yeast. Acidification power
The acidification power test developed by Opekarova and Sigler measures the drop in extracellular 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 extensive
yeast washing and multiple sample points. Intracellular pH (ICP) method
The ICP method uses a pH-sensitive fluorescent reagent to measure the intracellular pH of individual cells and cell mass. It was found that the intracellular pH of more active yeast cells does not decrease, even if the extracellular pH is low, whereas the intracellular pH of less 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.
Flow cytometry methods are now available to measure intracellular pH.
Measurement of yeast 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 plate
counts may be used to assess the ability of cells to remain viable after being subjected to a given stress.
Magnesium release test (MRT)
The magnesium release test is based on the observation that low molecular weight species such as magnesium, potassium, and phosphate ions are released by yeast immediately following inoculation into glucose containing medium. Trials performed on Saccltaromyces cerevisiae showed that cells which released greater quantities of magnesium immediately 77
after inoculation into high gravity (16°P) wort had higher vitality and fermentation
performance than yeast which released lower amounts of magnesium. Subsequent
fermentations performed using the more vital yeast had shorter lag phases, higher cell counts, higher end ethanol concentrations, and lower diacetyl levels. The magnesium
release test takes less than 15 minutes to perform and it uses a commercially available
magnesium test kit (Sigma) which allows the quantitative colourimetric measurement of magnesium 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, easy and 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 concentration and, consequently, requires dilution with water (usually de-oxygenated), at a later stage in processing. 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
120-1
changes.
High gravity brewing has been progressively introduced into breweries around the world for the past twenty-five years. thirty However, internationally it However, internationally cannot be said that its use is
adoption was slow in some some cases universal, because for product, legal and taxation companies have chosen (or reasons. are compelled), for product and legal/taxation reasons, not
The legal and issues to adopt thistaxation process. Slowly associated with this procedure the legal and taxation issues are to permit arebeing beingaddressed addressed to permit the production of high gravity the production of high gravity worts without undue financial worts without undue financial
50
100
160
200
250
Fermentation time (hours)
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 product types at high gravity remains a concern and challenge to some breweries.
There are a number of advantages and disadvantages to this process. The advantages can be 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 more
of 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" liquid
a number of products can be brewed as a result of dilution and/or use of malt and hop
extracts 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 is beyond the scope of this document to discuss this aspect of high gravity brewing in detail, recent studies have found that this problem can be alleviated by the use of modern mash filters in place of lauter tuns and/or kettle syrups.
•
Decreased foam stability (head retention). Hydrophobic polypeptides have been shown to 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
200
240
Fermentation time (hours)
Figure 54. Percent viability of brewer's yeast strains during fermentation of 27°P wort in shake 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 polypeptides high gravity wortwort whenwhen compared to low gravity Foam stability problems polypcptidesfrom from high gravity compared to low wort. gravity wort. •
can be overcome by the use of hop extracts and wheat in the grist.
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 . The requirement for expensive oxygen deaeration equipment is temperature adjusted). 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.
CONTINUOUS FERMENTATION Brewers only have limited control over yeast and
♦ Beer + Surplus Yeast
Yeast reservoir Yeast recycle
flow controller
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
Temperature control panels
Oxygenated
CO,-
wort
Wort flow controller
Figure 55. Multi-stage tower fermenter (adaptedfrom Portno, EBC Monograph V, EBC Fermentation and Storage Symposium, Zoeterwoude, 1978, p. 149).
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 at that time, are obscure, but it is likely that the inability to adequately guard against contamination and the resistance of traditional brewers to change were major factors.
81
Stirror
I drive
CO, Outlet Wort in
Pump
Sterilizer
Yeast 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 needed to 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;
sugar gradient (i.e. (i.e. wortwort sugars being being metabolised in the usual priority glucose, • AAfermentable fermentable sugar gradient sugars metabolised in the usual- priority maltose and maltotriose see Fig. 22) through tower, in order to prevent high levels of esters, glucose, maltose and -maltotriose) throughthe the tower, in order to prevent high levels of deflocculation and reduced fermentation capacity; andcapacity; and esters, deflocculation and reduced fermentation •
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 carefully controlling the wort oxygen level.
By to early 1980's, all ofall theofcontinuous fermentation systems systems employedemployed on Bythe thelate late1970's I970's to early 1980's the continuous fermentation on aa production hadwith not performed up to expectations therefore ceased to be operation, up productionscale scale, the exception of the New and Zealand system, had notinperformed with the exception of the New Zealand which is still in use today. to expectation and therefore ceasedsystem, to be in operation.
Why did the brewing industry fail to make a commercial success of continuous fermentation in 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 of
flow 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 the
early 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 drink more 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 the immobilised 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 the optimisation 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 employing immobilised 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 has been achieved using yeast immobilised on DEAE cellulose in packed beds. A major advantage of this type of carrier is that transport restrictions and diffusional limitations
are 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 Bavaria Brewery in the Netherlands for the production of alcohol-free beer.
The immobilised alcohol free beer process when compared to the classical arrested batch fermentation is reported to produce a better tasting low alcohol beer and to improve product
consistency. 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 producing
immobilised cell alcohol free beer.
The Bavaria system employs DEAE cellulose as the immobilisation material and immobilisation of the cells is achieved as a result of ionic bonding between carrier (positive
charge) 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 low wort pH prevents the growth of contaminating bacteria while exerting a positive influence
on 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 sugars are 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 in carbonyl 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 the appropriate operating conditions. It is recommended that the entire reactor including carrier particles 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
Heat Treatment
Maturation Immobilized it Fermentors
From Primnry
Fermentation
Yeast
Cooling
Figure 57. Cultor's two hour continuous maturation system (adapted from 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 following advantages 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
Bulk
concentration
(gradient ol substrate)
Stagnant/1 " layer
—■
—'
'
' Concentration at the
' surfaofl ot tho bead
Active layer
Bead core
External mass transfer
Figure 58. Mass transfer diagram of an entrapment carrier. 89
insufficient free amino nitrogen (FAN) consumption
resulting
in
Stainless Steel —►V
Head Plate
\
T—*
—
unbalanced levels of ester and
T—► "Green" Beer
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
-
Inoculated Beads
1 :
► :
:
4—Thermal Jacket
<— Bioreactor Draft Tube
Sparging
Plant Wort
caused by mass transfer limitations. The low oxygen availability in early immobilised cell processes
Figure 59. Labatt gas lift draft tube bioreactor
provoked a decoupling between
[Mensour el ai, JIB, 1997, 103(6), p. 363-370].
Gas
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
Beor
Wort
Vessel
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 Supply Pump
Immob 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 the biomass 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 cell systems is to reduce bead diameter. The choice of bead or particle size is a compromise between the smallest possible size and technological properties such as pressure drop in a packed 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 transfer limitations.
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 cell systems 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 mass transfer. 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 limited
growth (maturation or alcohol-free beer).
Fluidised bioreactor configurations are preferred for primary beer fermentations and, for this 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 all previously 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, is also known as "Irish Moss". In solution with potassium ions, ic-carrageenan forms a
thermoreversible gel.
Canadian researchers have developed a novel continuous beer fermentation system using immobilised yeast cells. A 50 L gas lift draft tube bioreactor (Figure 59) was designed and installed 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 complete
gas-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 systems provide good mixing with minimum shear on the solid matrix. As a result, they significantly improve 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 cells and the liquid medium can result in insufficient consumption of free amino nitrogen and
consequently 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 and thus, directly influenced the resulting flavour profile of the finished product. Air proportions between 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. The second important challenge requiring
Performance
attention concerns the
improvement required
operation of industrial continuous immobilised cell reactors. Many of
by mainstream market
Expected trajectory of performance Improvement
I
Current performance of potentially disruptive technology
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
Time
breweries
has
rendered production
Figure 61. How to assess a disruptive technology (adapted from Bower and Christensen, Harvard Business Review, 1995, 73, p. 43). 92
flexibility an important production
parameter.
Once an immobilised cell
reactor has been loaded with the inoculated carrier, it must be capable of operating for several 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 series of reactors each containing a specific yeast strain would be required. However, if the differences depend on wort composition and fermentation parameters, then the possibility exists that a well-defined procedure will enable a switch from one brand to another, by modifying wort composition and operating parameters. Continuous fermentation is still generally 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 difficult to 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 of traditional 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 earliest descriptions 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 to distilling around Europe dating from the twelfth and thirteenth centuries. However, the first treatise 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 characteristic
pear-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
Distillation Mashing
)
)
<
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
Rectifier
r> Fermentation
(
)
(
Wort
Wash
16 - 2016% % sugar
8% % 8 -10 Alcohol alcohol
Sugar
>
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 relatively complex 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 used in 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 quite different 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
Distillation
Mashing
Fermentation
Wort
Wash
15%
8%
Sugar
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
Alcohol
Figure 63. Malt whisky pot-still process - double stage distillation.
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 (unlike
malt 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-sterile and 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 litre spirit/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.
and the fermentation temperature is 28°C- 32°C.
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 distillers add both specifically grown distilling yeast strains (the Quest M strain or similar strain) as well 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 molecular weight dextrins such as maltotctraose (G4, G5, G6, etc.). The exact mechanisms of using
these 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 distilling yeasts. The Scotch Whisky Act 1988 would permit the use of genetically manipulated strains but, similar to brewing, there is considerable scepticism regarding the commercial
use of such strains. One of the main objectives of genetic manipulation and strain development of distilling yeasts is increased efficiencies of carbohydrate conversion to cthanol and extension of the range of utilisation lo abundant and inexpensive substrates. Cloning of several genes encoding enzyme systems for fermentation of simple sugars has led to applications relevant to higher yields of conversion to ethanol. For example, analysis of the family of MAL genes (maltose uptake and hydrolysis system) has resulted in elucidation of the oc-glucosidase (maltase) and permease systems. Also, distillery yeasts often lack the ability to utilise melibiose and transfer of the MELI gene from lager strains has led to gain of this property. Higher ethanol yields can be obtained with substrates (for example, beet molasses) containing significant levels of raffinose (a trisaccharide consisting melibiose plus fructose).
Ethyl Carbamate Ethyl carbamate (C2H5OCONH2), otherwise known as urethane, is a naturally occurring
compound present in many fermented foods and beverages including Scotch Whisky. It
is a chemical carcinogen, and this fact has led to concern in recent years regarding its
presence at trace levels in some alcoholic beverages. As a consequence of this, steps have
been taken to impose acceptable limits on levels present in such products. From a distillation point of view, two outstanding properties of ethyl carbamate are its low volatility and its relatively high chemical stability. Although ethyl carbamate does not distill readily, transfer
to distillates may occur under suitable conditions.
Because of volatility considerations, any ethyl carbamate formed by the reaction between ethanol and ureido compounds in the wash would not normally be expected to be present in the final spirit. However, chemical analysis indicates that ethyl carbamate is virtually absent from fermented wash. If it is going to form it can be detected during distillation. It
can occur in fresh distillates and may continue to form in the spirit during maturation. The observations have indicated that a volatile precursor is involved in its formation. If ethyl
carbamate is going to form, then its formation is accompanied by a corresponding decrease
in the distillate of a group of compounds
originally described as "measurable cyanide".
Subsequent analysis has shown that the first
measurable cyanide component to appear in fresh distillate is hydrogen cyanide. When 97
Ethy' Carbamate (C,H7NO2) H o
\j_J'_orH rH
/
ulh2lh,
Figure 64. Chemical structure of ethyl carbamate (urethane).
radio-labelled hydrogen cyanide is added to distilled spirit, the label appears in ethyl carbamate. 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 with water, 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 microbial enzymes are used instead of malted barley to convert cooked maize or wheat, virtually no cyanide appears in the distillate after fermentation. Further research has confirmed that malted barley contains a cyanide precursor which is located exclusively in the acrospire of the 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 action of yeast enzymes. All yeasts examined are able to hydrolyse EPH to glucose and
isobutyraldehyde 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 major
raw 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
•
Maintain appropriate distilling operating procedures. For example, foreshots and feints
final spirit; and
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. 4 th 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
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
METHIONINE
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
SULPHATE
WORT NUTRIENTS
B bacteria, 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
c
Candida 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
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
D
decarboxylation 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
genetic characterisation, 14-22, figs. 10-16
yeast growth, and, 52
2-deoxy-glucose, 16 formation of diplotd zygotes, 15
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
mating of haploids, 15 reduction division of diploids, 15 spheroplast fusion, 20
triphenyl tetrazolium overlay, 18, fig. I zygotes. 15
chromosomal fingerprinting, 26, figs. 19 & 21 hybridisation, fig.l7(b), fig. 18 polymerase chain reaction, figs.21-2
see also: DNA GENETIC TESTS SACCHAROMYCES CEREVIS1AE
yeast DNA, fig.l7(a)
glucose, 5, 29, 78, fig.23
DNA technology and brewer's yeast, 23
analogue, (2-DOG) in maltose fermentation, 32 biochemical conversion of, 5
E
glutathione, structure of, 56
Embden-Meyerhof-Parnas pathway, and Kreb's cycle, 32
glycogen, 66-69, figs.45-8 glycolysis, 29, fig.26 see also: WORT
and oxidative phosphorylation, 33 entrapment, 84, fig.58
see also: IMMOBILISATION OF YEAST
H
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
Hansen, 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
I immobilisation of yeast, 84-8. figs.57-61 nature of experiments, 82 Sacchammyces spp, and, 85 inorganic cations,
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
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
M maltose,
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
o oxidative 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
copper and iron, 46
divalent metal cations, and, 44 ferrous, 46
IONS MALTOSE
insoluble (Fe3+), 46 soluble (Fe2-), 46
YEAST CELLS sake yeast,
hydrogen, 43
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
magnesium, 44 manganese, 45
potassium, 43 sodium, 44 zinc, 45 metabolism of, 30 oxygen, 37ff
sugars and carbohydrates, 30 uptake of, 30 vitamins, 40ff
sulphur dioxide, 55
see also: YEAST METABOLISM
yeast metabolism, control of, 35ff wort production,
Trehalose, 66ff, fig.45,48 and Table 5 tricarboxylic acid cycle,
brewing and distillation distinguished, 96 wort sterility, 63
see: KREB'S CYCLE
yeast,
vitamins, 40, Table 4
acid washing of, 70
see also: WORT
do's and don't's, 70 cell growth and division of, I2ff
w
cell viability of, 74-78 cell vitality of, 74-78 cell wall, 9 culture, fig.8 definition of, 3
whisky,
and ethyl carbamate, 97, fig.64 grain, 93,94ff, fig.62 malt, 91,94ff, fig.63 malt and grain, defined, 94
genetic characterisation of, 14 genetic tests, 23 immobilised cells, 84
distillation and brewing distinguished, 95
distillation of, 95
application of, 86 alternatives to entrapment, 85 commercial criteria, 86 comparative results of, 84 definition of. 84
distinguished from each other. 95 ingredients of, 95 storage of, following distillation. 95 wild yeast,
contamination of yeast culture, 73, 72. fig.5O
entrapment technique, 84 gel matrix, 84
wort,
diacctyl and, 51
research into, 84
fermentation of, 16°P and 25°P, 78, fig.53
inorganic ions, and, 41 see also: BREWING YEAST DISTILLERS YEAST MORPHOLOGY YEAST CELLS
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
YEAST MANAGEMENT
life cycle, 15 yeast cells, fig.l division, 13 growth, 13-15
105
control of, 35
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
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 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
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) Page
Figure 1
Electron micrograph of a budding cell
3
2 3
Utilisation of the sugar raffinose and melibiose by lager and ale yeast Giant colony morphology on wort gelatin plates of typical ale and lager yeast strains
6
4
Main features of a typical yeast cell
8
5
Electron micrograph of yeast cell with multiple bud scars
9
6
Structure of the yeast cell wall
7
Structure of the mitochondrion: (A) diagram, (B) electron micrograph
11
8
Batch growth curve for brewing yeast culture
12
9
Cell cycle of Sacchammyces cerevisiae
13
10
Haploid/diploid life cycle of Sacchammyces cerevisiae
15
11
Sporulating yeast cell
15
12 13
Sacchammyces brewing yeast, with and without "killer" activity Rare mating protocol to produce brewing strains with zymocidal "killer" activity
17
14
Triphenyl tetrazolium overlay of yeast colonies
18
15
Spheroplast fusion of two yeast strains
20
16
Production of a recombinant DNA brewer's yeast
22
17
Restriction patterns involving yeast DNA
24
18
DNA-DNA hybridisation test
25
19
Chromosomal fingerprints of three brewing lager strains
7
9
106
17
Figure
20
page
Polymerasc chain reaction with target DNA Fingerprint patterns using polymerase chain reaction Order of uptake of sugars by yeast from wort
27 27 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 Effect of zinc levels in wort on primary fermentation time Metabolic inter-relationships leading to ester formation
34 46 48 49
By-products of pathways leading to formation of amino acids valine and isoleucinc Reduction of diacetyl to acetoin and 2,3-butanediol
31 52
Diacetyl formation and breakdown in relation to yeast growth and wort gravity Inter-relationship between yeast metabolism and production of flavour compounds Pathway for synthesis of sulphur-containing amino acids Structure of cysteine, mcthionine and glutathione Flocculation in Sacchammyces cerevisiae
52 55 56 56 57
Production of higher alcohols
Static fermentation flocculation
58
Lectin theory of flocculation
58
Electron photomicrograph of Sacchammyces cerevisiae Electron photomicrographs of Candida albicans Typical propagation vessel
59 59 62
Chemical structure of (A) glycogen and (B) trehalose The effect of yeast glycogen at pitching on a lager fermentation The effect of yeast storage temperature on intracellular glycogen concentration Pathways to glycogen and trchalose in yeast
67 67 68 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 Measurement of ATP-driven bioluminescence
73 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 How to assess a disruptive technology Grain whisky - continuous column distillation
90 92 93
Mall whisky pot-still process - double stage distillation Chemical structure of ethyl carbamate (urethanc)
95 97
107
TABLES Table
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 4
Effect of linoleic acid and oxygen on ester production 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
39
Institute of Brewing and Distilling, 33 Clarges Street, London W1Y 8EE
