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THIRD EDITION

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Brewing Microbiology Third edition

Edited bụ

Fergus G. Priest and lain Campbell

International Centre for Brewing and Distilling

Heriot-Watt University Edinburgh, UK

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Brewing microbiology / [edited by] Fergus G. Priest and Jain Campbell—3rd ed. <small>p.cm.</small> Includes bibliographical references and index. <small>ISBN 978-1-4613-4858-0 ISBN 978-1-4419-9250-5 (eBook)</small>

<small>© 2003 Springer Science+Business Media New York</small> Originally published by Kluwer Academic/Plenum Publishers New York in 2003 Softcover reprint of the hardcover 3rd edition 2003 <small>10987654321AC.LP. record for this book is available from the Library of Congress.All rights reservedNo part of this book may be reproduced, stored in a retrieval system, or transmitted in any</small> form or by any means, electronic, mechanical, photocopying, microfilming, recording, or <small>otherwise, without written permission from the Publisher, with the exception ofany material supplied specifically for the purpose of being entered and executed ona computer system, for the exclusive use by the purchaser of the work</small>

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2.2 The cell cycle

2.3 The growth and fermentation cycle

24 Cell composition, nutrition, and general metabolism 2.5 Energy and intermediary metabolism

2.6 Yeast biochemistry and beer production

3.2 Genetic features of Saccharomyces cerevisiae 3.3. The need for new brewing yeasts

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Molecular biological approaches to yeast differentiation The nature of the brewing yeast genome

The microbiota of barley The microbiota of malt

Effects of microorganisms on malting

Effects of the microbiota on beer and distilled spirit

Gram-negative brewery bacteria Hennie J.J. Van Vuuren and Fergus G. Priest

Miscellaneous non-fermentative bacteria Detection, enumeration, and isolation

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7 Wild yeasts in brewing and distilling

Properties for identification of yeasts Detection of wild yeasts

Identification of wild yeasts Effects of wild yeasts in the brewery Elimination of wild yeasts

8 Rapid detection and identification of microbial spoilage Inge Russell and Robert Stewart

Hybridization using DNA probes Polymerase chain reaction

Random amplified polymorphic DNA PCR Nucleic acid-based identification Techniques for examining proteins

Methods that examine aspects of cell composition Techniques for studying morphology and behavior

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10 Microbiology and sanitation in U.S. microbrewies Michael J. Lewis

101 Introduction 10.2 The raw materials 10.3. The process and product 10.4 Beer contact surfaces 10.5 Concluding remarks

11 Cleaning and disinfection in the brewing industry Manjit Singh and Jacqueline Fisher

111 Introduction 112 Definitions

11.3. Standards required in a brewery 114 Cleaning methods available

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Iain Campbell* International Centre for Brewing and Distilling, Heriot-Watt University, Edinburgh EH14 4AS, UK

Jacqueline Fisher* Diversey Lever, Weston Favell Centre, Northampton, NN3 8PD, UK

Brian Flannigan BioForce Associates, 3 Merchiston Avenue, Edinburgh EH10 4NT, UK

John R.M. Hammond BRF International, Lyttel Hall, Coopers Hill Road, Nutfield, Surrey, RH1 4HY, UK

Michael J. Lewis University of California Academic Director of Brewing Programs, University Extension, Davis, California 95616, USA

Fergus G. Priest International Centre for Brewing and Distilling, Heriot-Watt University, Edinburgh EH14 4AS, UK

Inge Russel* 467 Commissioners Road E., London, Ontario, Canada N6C 276

Manjit Singh* Diversey Lever, Weston Favell Centre, Northampton, NN3 8PD, UK

J. Colin Slaughter International Centre for Brewing and Distilling, Heriot-Watt University, Edinburgh EH14 4AS, UK

Robert Stewart Labatt Breweries of Canada, Research and Technical Services Department, 150 Simcoe Street, London, Ontario, Canada N6A 4M3 Hennie J.J. van Vuuren Food, Nutrition and Health, Faculty of Agricultural Sciences, The University of British Columbia, Vancouver, B.C. Canada V6T 1Z4

<small>*Retired or changed address recently</small>

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Much has happened in the brewing industry since the last edition of this book was published in 1996. In particular, there has been substantial con-solidation of larger brewing companies as major multinational concerns, and at the other end of the spectrum the microbrewing scene in various parts of the world has become established as a sustainable enterprise. For those involved in the scientific and technical aspects of fermented bever-age production the changes have been no less daunting. The complete genome sequence of Saccharomyces cerevisiae has been determined and studies are underway in numerous laboratories throughout the world to unravel the expression of the genome (transcriptomics and proteomics) and understand exactly “how a yeast works.” This will undoubtedly con-tribute to our understanding of yeast fermentation and flavor generation in a revolutionary way because it will enable the simultaneous monitor-ing of all genes in the organism durmonitor-ing the fermentation. In Chapters 2 and 3 of this volume Colin Slaughter and John Hammond bring the reader up-to-date in this rapidly moving area and cover the remarkable achievements of modern biochemistry and molecular biology. Iain Campbell has also revised the systematics of culture and wild yeasts in Chapter 7.

The other major technical change since the last edition of this book is the introduction of molecular characterization and detection of microor-ganisms based largely, but not exclusively, on the polymerase chain reac-tion (PCR) for amplificareac-tion of specific DNA fragments. Although few of these methods are yet used routinely in the brewery laboratory, the speed, accuracy, and scope that they offer are immensely attractive, and it is likely that as they become more automated and less expensive they will be incorporated into quality-assurance procedures. In addition to describ-ing these excitdescrib-ing developments, we have also included new chapters covering the role of microbiology in the brewing process and, in

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particu-Jar in the microbrewery, as well as a chapter devoted to the rationale behind microbiological analyses used in the brewery. We are grateful to Brian Flannigan for revising his definitive account of the microorganisms associated with barley and malt and finally, we thank Annie Hill for assis-tance with preparation of diagrams.

Brewing microbiology has a long and prominent history. We hope that the third edition of this book will persuade the reader that there is still much to learn of this ancient art.

Fergus G Priest, Iain Campbell (ICBD, Edinburgh)

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Microbiological aspects of brewing

Iain Campbell

1.1 INTRODUCTION

Not only are there microbiological aspects of brewing, one could argue that the science of microbiology developed from brewing. Louis Pasteur’s assistance with a problem of beer spoilage was an important stage in the development of modern microbiology.

Brewing is essentially a combination of engineering with botany, bio-chemistry, bio-chemistry, and microbiology. Archaeological evidence indicates that beer has been produced since before 4000 BC (Moll, 1994), but the microbiological nature of the process has been understood only for the past 150 years. The application of microbiology to optimize the process in terms of efficiency and quality is of even more recent date. There are two principal aspects to brewing microbiology: (a) the quality of the culture yeast itself, and (b) the control of various possible microbial contaminants. The specialized chapters later in this book provide a full explanation of the various microbiological aspects of beer production, but this brief intro-ductory chapter is intended to provide a microbiological overview of the production of alcoholic beverages, and beer in particular. For information on other, nonmicrobiological aspects of brewing the reader is referred to general texts, e.g. by Moll (1994), Hardwick (1995), or Lewis and Young (1995), but a general outline of the process is provided as Fig. 1.1.

In most countries, barley malt is an obligatory ingredient of beer and represents a distinct environment with its own varied microbial flora (Chapter 4). The great majority of these microorganisms are unable to grow in beer under normal circumstances. Most fungi and bacteria are suppressed by one or more of the following effects: (a) the antimicrobial properties of hops, (b) falling pH during fermentation, from 5.0-5.2 in

<small>Brewing Microbiology, 3rd edn. Edited by F. G. Priest and I. Campbell.</small>

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Modification of barley starch (preparation for hydrolysis in the mash tun) Hydrolysis of protein => free amino nitrogen

Structural alteration to more friable consistency MILLING, MASHING

Milling to suitable particle size range

Enzymic hydrolysis and extraction of sugars, amino acids, other yeast nutrients and enzymes with hot water => sweet wort

Production of flavor compounds, by-products of yeast metabolism

Purging of unwanted volatiles (e.g. H;S) by evolution of CO,

Fig. 1.1 Outline of the brewing process.

wort to 3.8-4.0 in beer, (c) developing CO; and anaerobic conditions, and (d) increasing ethanol content. Lacking most of these protective effects, wort is highly susceptible to spoilage and must be pitched with culture yeast immediately after collection, or better still, during collection. Even before fermentation gets properly under way, a small number of contam-inant bacteria in wort would be swamped by the enormous excess of pitching yeast. Beer, with its intrinsic antimicrobial properties and a low level of residual fermentable sugar, is relatively stable but liable to spoilage by the few specialized types of bacteria and yeasts capable of growing anaerobically on the complex polysaccharides or other organic compounds still present after fermentation (Hammond, Brennan & Price, 1999). So, although malt carries only small numbers of the lactic and acetic acid bacteria and enterobacteria shown in Table 1.1, these are a likely source of contamination of wort or beer when a combination of other fac-tors favors the growth of these organisms. A wider range of contaminants is possible in unhopped or low-alcohol beers, each of which lacks one of the protective effects of normal beer. Table 1.1 also shows aerobic con-taminants, i.e. oxidative yeasts and acetic bacteria, as contaminants of beer. However, these organisms can flourish only as a result of a packag-ing fault allowpackag-ing access of atmospheric oxygen to the head space of the container.

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Table 1.1 Occurrence of microbial contaminants of the brewing industry

Beer, by definition, is prepared from cereals. The carbohydrate of cereals is starch, which brewing yeasts are unable to utilize, so the grains must first be malted to modify the starch for hydrolysis to fermentable sugars during mashing. Because the malting process is essentially a controlled version of natural germination, any cereal can be malted, but barley became the principal cereal for beer production because of its husk, which

limits the potential for fungal spoilage and provides a natural filter for

clarification of the extracted wort.

Not all barley is suitable for malting; only certain varieties are accept-able. In addition to the essential botanical characteristics such as lack of dormancy and efficient modification, an important property is the nitro-gen content. Too low a level of amino-N in the wort will restrict yeast growth, but normally the problem is too high a level of nitrogen in the barley, more than is necessary for yeast growth. The resulting excess nitro-gen, particularly amino-N, in the final beer encourages microbial spoilage of the final beer, especially by lactic acid bacteria (Chapter 5).

Because barley is harvested only in autumn but malting takes place throughout the year, barley must be dried from its 20-25% moisture con-tent at harvest to about 11% for storage prior to malting. That latter figure is a compromise between the requirement to prevent microbial, especially fungal, spoilage and its becoming too dry, thus affecting the viability. Drying and storage of malting barley must be carefully controlled to avoid risk of either dormancy or death of the grain. Additionally, within the constraints of preventing damage to the grain, the least energy-requiring drying regime is preferred (Palmer, 1989).

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In malting, the starchy endosperm of the grain is modified by the development of hydrolytic enzymes and the partial breakdown of its structure. Simultaneously, proteolytic enzymes develop to hydrolyze pro-tein to simple amino acids and peptides that yeast can utilize. The malt-ing process occurs in three stages—steepmalt-ing, germination and kilning—all of which have microbiological implications (Chapter 4). Steeping in water at 15-18°C over 48 h, traditionally in two or more stages with ‘air rests’ between, stimulates the growth of the embryo plant as would occur if it had been planted in soil. However, these moist, warm, aerated conditions of steeping and germination also encourage growth of the microbial flora that is inevitably present on the surface of the grain (Chapter 4). When sufficiently germinated and modified, the green malt must be dried for storage, but with the incidental advantage of develop-ing the characteristic ‘malty’ flavor. Because the critical factor in prevent-ing microbial spoilage is water activity, not the percentage moisture content, it is necessary to reduce the moisture content from the 46-50% reached during germination to about 5% for storage. Again this is a com-promise, this time between the requirement for microbiological stability and the energy cost of drying. Other aspects of kilning—the development of color and flavor but preservation of amylolytic and proteolytic enzymes for the hydrolytic activities of the mashing process—are mainly biochemical and chemical effects, but there is an incidental reduction in the numbers of the microbial flora.

1.3 BREWING: MASHING AND HOP BOILING

Next, the mashing process in the brewery extracts yeast nutrients from the grain. In the brewhouse the grist of malt, with or without other cereal adjuncts, is milled, mashed, and filtered to remove spent grains from the sweet wort. This is then boiled with hops, clarified again, and cooled to fermentation temperature. The main microbiological considerations are the start and finish of brewhouse operations.

Malt mills are designed to grind the contents of the grains finely and to improve extraction of sugars and other yeast nutrients, but yet to cause as. little damage as possible to the husks. Essentially this is an engineering problem, but there are several microbiological implications of milling. Because the microbial flora of the grain includes some potential beer-spoilage organisms, dust from milling should be prevented from reaching the fermentation area. In some breweries the malt is moistened, by either water or brief exposure to low-pressure steam, because the softened husks are less liable to damage in the mill and will give more efficient operation of the lauter tun. This also reduces the contamination, explo-sion, and health risks associated with fine dry dust, but microbial growth on any accumulation of moist grain or powder in the mill is a spoilage

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hazard. Even though the Bacillus and Clostridium spp. likely to grow in such conditions are not themselves beer-spoilage bacteria, some meta-bolic products, e.g. butyric acid, would persist to the final beer.

Over the history of brewing, many different herbs have been used as flavoring for beer, but hops are now standard. Perhaps they became the preferred flavoring because it was observed that beers brewed with hops were the most resistant to spoilage. The different hop varieties in common use vary in their content of bitter acids, resins, and oils, but all are mod-erately antimicrobial (Simpson, 1993). The exact nature of the antimicro-bial agent is not yet known but alpha acids tend to be inhibitory to many Gram-positive bacteria in particular. The flavor is extracted by boiling for 60-90 min, with the incidental advantages of sterilizing and concentrating the wort, isomerizing hop alpha- and beta-acids, and purging the wort of harsh grainy flavors. Also, if glucose, sucrose, or maltose crystals or syrup are included in the recipe, this is the appropriate stage for addition. These adjuncts are not necessarily sterile: a typical quality specification could

include a maximum microbial count, e.g. 2 X 10°/g, low enough to be

sterilized after addition in the last 15-30 min of hop boiling.

The majority of breweries now use processed hops, either pellets pre-pared from ground cone material or liquid CO, hop extract. If an already isomerized extract is used, the extract itself does not need to be boiled, but the sweet wort must still be boiled for the other reasons listed previously. Like sugar adjuncts, the isomerized extract is added late in the boiling process. Hop boiling generates a precipitate of protein/tannin complexes and insoluble calcium salts and phosphates that must be removed before fermentation, because the particulate material would adversely affect fer-mentation, and therefore flavor production by the yeast. After traditional hop boiling using cone hops, the wort may be clarified by filtration through the settled bed of spent hops. Pellets or extract do not provide a suitable filter medium, so the wort is clarified either by centrifugation, or increasingly commonly now, by a ‘whirlpool’ separator where hop debris and hot break collect at the center of the vortex, while clear wort is drawn from the side of the vessel. Provided it is kept clean, the whirlpool vessel operating at close to 100°C and with no moving parts (other than the wort itself) poses no microbiological problems. Then the hot wort has to be cooled to about 20°C before pitching with yeast inoculum. Additionally the cooled wort must be aerated, normally to 6-8 ppm dissolved oxygen (DO). The amounts of unsaturated fatty acids and sterols naturally present in wort are too low to support the amount of yeast growth required for an efficient fermentation, hence the requirement for initial aeration of the wort to allow the yeast to synthesize these compounds (see Chapter 2). These steps have important microbiological requirements: cleanliness of the heat exchanger, its structural integrity to prevent potentially contami-nated cooling water leaking into the wort stream, sterility of the pipework, and the fermentation vessel receiving the wort, sterility of the aeration air,

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and microbiological quality of the pitching yeast. Each of these factors has to be considered in the event of a ‘troubleshooting’ investigation of a con-taminated fermentation.

1.4 FERMENTATION

For microbiologists, this is the most interesting and important part of beer production, but the fermentation itself requires little attention in this overview. The metabolism of brewing yeasts is fully explained in Chapter 2, and Chapters 5-7 include an assessment of the precautions against contam-ination of the fermentation by bacteria and wild yeasts. The fermentation stage is subject to microbial contamination from a number of sources, some already identified in the preceding section. Apart from the wort itself the principal hazards are air, pitching yeast and the fermentation vessel and associated pipework and control equipment.

1.4.1 Yeast

Throughout most of the long history of brewing, fermentation was carried out in open vessels, which are still used in many small traditional brew-eries. No doubt the fermentations of the early brewers were naturally inoculated by yeasts present in the air or on brewery equipment, as is still the case today with Belgian lambic beer (van Oevelen et al., 1977). A good fermentation was recognized, among other features, by vigorous evolu-tion of CO, that carried a proporevolu-tion of the yeast to the surface as a thick frothy ‘head,’ hence the term ‘top yeast.’ The yeast head of a successful fermentation was collected by removing a side panel of the vessel or by using some specific procedure such as the Burton Union system, which is still used by Marstons Brewery in Burton-on-Trent. The stored yeast is used to inoculate the next fermentation, and so on. Long before the requirements could have been expressed in the microbiological terms of Table 1.2, brewers recognized the value of preserving a good brewing yeast with these properties.

The origin of the most widely used yeast of the modern brewing indus-try has not been recorded, but certainly it originated in a Bavarian monastery-brewery at least 300 years ago. It is likely that the original strain was a chance contamination or hybridization of a traditional brew-ing yeast with wild yeast, perhaps a local wine yeast (Martini & Kurtzman, 1985). The ‘bottom yeast’ of the new type of fermentation did not form a true head, only a foam that contained too little yeast to pitch a subsequent fermentation. The yeast for the next fermentation, then, had to be recovered from the bottom of the vessel after settling at the end of fermentation. Perhaps at the same time it was discovered that the new yeast was ideal for a secondary fermentation at low temperature, which

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Table 1.2 Essential properties of brewing yeast

Consistent production of desirable flavor and aroma metabolites Rapid fermentation

Efficient fermentation (maximum yield of ethanol, minimum production of yeast biomass)

Tolerance to the inhibitory effects of wort and beer (osmotic stress of initial

sugar, toxic effect of final alcohol and CO,)

Suitable flocculation and sedimentation properties at the end of fermentation (and for ‘top fermentations,’ head formation)

High final viability for pitching the next fermentation

High genetic stability over successive fermentations

improved the flavor and CO, content. That type of beer became estab-lished in Bavaria, but the fame of the beer spread. In 1842 the yeast was stolen by Czech brewers to begin brewing it in Plsen (or Pilsen), which, perhaps unjustly, became the name for that type of beer in many coun-tries, although the name Lager is normally used in Britain. Several decades later the yeast culture was stolen from the Czechs by one of the Jorgensen family of the Danish Carlsberg company—according to legend, hidden in his top hat where it survived the journey by stagecoach back to Denmark. There in the 1880s, the pioneer yeast taxonomist and technolo-gist E. C. Hansen first isolated pure yeast cultures and showed the differ-ence between the ‘top yeast’ of the traditional beers of Belgium, Britain, and Germany (which he named Saccharomyces cerevisiae) and the ‘bottom yeast’ of the Bavarian and Czech beers (S. carlsbergensis, although that species is no longer valid; see Chapter 7). Pure cultures of S. carlsbergensis were then exported worldwide, initiating the worldwide production of the pilsener type of beer.

Enclosed fermentation vessels have become common in the brewing industry since about 1970. Although initially simply a covered version of the original rectangular open vessel, modern equipment is now a cylindro-conical fermentation vessel (CCFV). The vigorous circulation during fermentation caused by a central upward movement with bubbles of CO, and downward movement by the cooling jackets on the wall increases yeast activity, and it also allows more effective control over tem-perature. These vessels are particularly suited to bottom yeast. Not only is it impossible to skim off the head of top yeast in a CCFV, but the vigor-ous circulation creates even more head than in shallow rectangular ves-sels. Half the volume of a CCFV would be filled with yeast head, which is very inefficient use of the expensive vessel. However, for beers that must use top yeast, head formation can be suppressed by antifoam, although a non-head-forming mutant with exactly the same flavor characteristics as the original yeast is preferable. It is unlikely that lager yeast could be used as a substitute, because it would cause an obvious difference in flavor.

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A specific strain of culture yeast, or possibly a consistent mixture of several strains, is used for a particular beer. In the latter case, a check on the relative proportions of the various strains in pitching yeast is advis-able. In the past, serial subculturing was carried out indefinitely, but mod-ern breweries tend to maintain active pure cultures by replacing the yeast after a predetermined number of ‘generations’: not actual cell generations but that number of successive fermentations. The yeast may still be in per-fect condition, but replacement of yeast cultures according to a fixed pro-gram in the yeast propagation plant simplifies planning of brewery operations. However, in the case of ‘high-gravity fermentations,’ the high concentration of ethanol (at least 8%) and other metabolites, as well as general osmotic stress, may cause such degeneration that the yeast is unsuitable for reuse, and a freshly propagated culture is required for each fermentation.

There are two aspects of pitching yeast quality: the yeast must be (a) in an active state and (b) free from beer-spoilage bacteria and wild yeasts. Various methods are available for assessment of yeast viability and the rather more exacting concept of vitality (see Chapters 2 and 12), but the simplest method, which is sufficiently accurate for most purposes, uses methylene blue stain to detect dead cells microscopically. Because that test can be carried out in about 15 min, there is no excuse for neglecting to confirm a high-percentage viability, preferably 99-100% but certainly at least 95%, before deciding to use the yeast. Yeast-pitching rate is usually in the range of 1-2 x 107 cells/ml (higher for high-gravity worts), but on a production scale, initial measurement of the yeast by weight is often more convenient. It is advisable to confirm this count immediately after pitching, either microscopically or by one of the in-line automated meth-ods now available (Carvell, 1997).

Although the viability and/or vitality of yeast can, and should, be measured before each fermentation, examination for contaminants is a more lengthy process and it is impracticable to test yeast by standard microbiological culture methods prior to reuse. ‘Instant’ bioluminescence, immunology or polymerase chain reaction methods are sufficiently rapid to screen yeast for contaminants and have the result available before the yeast is pitched into the next fermentation (see Chapters 8 and 12) but are seldom used at present. Fortunately, it is highly unlikely that a trouble-some level of contamination by either bacteria or wild yeast would appear suddenly; the numbers of contaminants would normally increase steadily over a number of successive fermentations. Therefore, routine microbiological testing of the pitching yeast will give an indication of a developing problem. As an absolute minimum level of testing available to even the simplest of breweries, flavor assessment of beers during fermen-tation will warn the brewer of the presence of troublesome levels of con-taminants and suggest that the yeast should not be reused.

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The ‘top yeast’ of traditional ale fermentations is collected as the yeast head that develops during the most active phase of the fermentation: the middle of the logarithmic phase in terms of yeast cell growth. There are two advantages of this system: (a) non-head-forming wild yeast and bac-terial contaminants are mainly left behind in the fermenting beer, and (b) even though the yeast population is intentionally a mixed culture, the mixture collected at the same stage of successive fermentations maintains the same proportion of the several yeast strains. Many traditional brew-eries have used the same yeast over many years, perhaps in some cases over several centuries. In well-hopped strong ales, the combination of high hop rate, high original gravity and high final alcohol concentration greatly reduces the risk of successful competition of wild yeasts with the culture yeast. Additionally, many breweries routinely wash the yeast cul-ture with acid at regular intervals to eliminate bacterial contaminants, on the basis that most bacteria are less acid tolerant than S. cerevisiae (Simpson & Hammond, 1989).

In lager fermentations, or with ale fermentations in enclosed CCFVs where non-head-forming yeast strains have been selected to make best use of the volume of the fermentor, yeasts are harvested on settling out at the end of the fermentation. Usually a cooling jacket is fitted to the cone to maintain the settled yeast in good condition. It is accepted that the via-bility of the yeast will fall by several percent as a result of the long expo-sure to the ethanol and other metabolic by-products in the beer, but there may also be the incidental protective effect that bacteria and wild yeast contaminants are selectively killed. However, it is now recognized that yeast cells of different age settle at different rates (Smart, 1999), partly because of the larger number of bud scars on old cells. This results in lay-ers of varying age, viability, and vitality in the cone of the FV and yeast for propagation should best be taken from the youngest, upper layer.

Although when the first CCFVs were introduced they were of similar volume to the older vessels they replaced, it is now common practice to build larger CCFVs that are filled by several successive cycles of the brew-house. This creates a microbiological problem. To prevent contamination, the first batch of wort must be inoculated immediately. But should the inoculum be the normal amount for that volume of wort, or the total amount of yeast required for (say) the three successive brews required to fill the vessel? The similar question that applies to the amount of dis-solved oxygen is more serious, because more yeast can be added later if required, but it would create flavor problems to aerate late in the fermen-tation. In practice, it is part of the commissioning program to determine the best mode of operation. Certainly there is a maximum size of CCFV dictated by the capacity of the brewhouse, or ultimately the yeast will fer-ment the wort already in the vessel faster than the next batch of wort can be produced.

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1.4.2 Fermentation vessels and associated pipework

As stated previously (Section 1.3), cleaning rather than sterilization is suf-ficient for brewhouse vessels, but for fermentation vessels and their pipework and instrumentation, both cleaning and sterilization are essen-tial. These procedures and their rationale are explained in detail in Chapter 11, but acceptable microbiological standards depend not only on satisfactory sterilization. It is equally important to design and build ves-sels and associated equipment to reduce the risk of contamination in the first place.

A few examples indicating the basic principles of plant design for ster-ile or pure culture operation can be discussed here. Perfectly horizontal surfaces are a hazard, because when drained, puddles of dilute wort or beer rinsings provide sufficient nutrient for growth of contaminant microorganisms. Therefore pipework must have a continuous downward slope throughout its run, or if that is impossible, a drain point at the low-est point of the dip. Alternately, pipework can be kept full of sterilized water when out of service, but then it is important to design the layout of pipes, and valves in particular, for minimal mixing between the protective water and the incoming charge of wort or beer. A conical or dished bot-tom to a tank with, of course, the outlet at the lowest point, is the best design for cleaning. A tank or fermentation vessel: with a flat bottom should have a slight slope toward the drain point, and all corners must be rounded, rather than right angled, to facilitate complete cleaning. All inner surfaces of vessels should be smooth and unobstructed. Internal obstructions such as cooling coils make proper cleaning impossible, so attemperation should be by wall panels. Some types of valve are incom-patible with sterile or pure-culture operation. From a microbiological point of view, the essential features of a valve in brewery pipework are (a) there is no possibility of contact between beer or other process fluid and the potentially contaminated environment, and (b) the entire inner sur-face in contact with beer can be sterilized in a single operation. Other fea-tures, e.g. mixproof design, are also important but are not microbiological

14.3 Air

Air is a microbiological hazard in two ways. The first hazard concerns breweries with traditional open vessels. The atmosphere of the fermenta-tion cellar may contain spoilage microorganisms from three important sources: spray from an adjacent fermentor undergoing cleaning; airborne cereal contaminants drifting in from the mill room; or bacteria and yeasts, most likely of agricultural or horticultural origin, blown in from outside. At certain times of the year, particularly in autumn, plant surface microor-ganisms from farms or orchards reach large numbers in the air—up to

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2.5 x 105 yeastlike fungi/ mổ (Last & Price, 1969). Anecdotal evidence sug-gests an increased risk of airborne microbial contamination from the dis-turbance to plant life during harvest time, even for city-center breweries some distance from a farm.

Second, aeration of the wort, although biochemically essential (Chapter 2), can introduce microbial contamination. Adiabatic heating in the compressor is insufficient to sterilize the air, so it should pass through a sterilizing filter. Many breweries now use cylinders of compressed oxy-gen to provide the higher DO levels required for high-gravity fermenta-tions (using wort 1.5-2 times normal strength and therefore an equal increase in DO), but because pure oxygen is self-sterilizing, filtration is

1.5 POST-FERMENTATION TREATMENTS

Most of the postfermentation processes of the brewing industry are pri-marily engineering operations, but there are biochemical and microbio-logical aspects. The biochemistry of maturation is discussed in Chapter 2, but the microbiological implications of postfermentation processes deserve some attention here.

1.5.1 Clarification

With the exception of cask-conditioned beers, which are clarified as much as possible by addition of ‘fining’ agents (Section 1.5.2), it is normal prac-tice to filter beer to brilliant clarity. Now that asbestos is out of favor, breweries use either fibers of cellulose or particles of diatomite or pumice as filter medium. For all three of these media a precise range of particle size is required for optimum performance. Cellulose filters can be either (a) a sufficient number of single-pass sheets to provide the required sur-face area for the planned flow rate, or (b) layers of fibers on a stack of per-forated stainless-steel supports. Type (b) is equally suitable for particulate filter media, but with both (a) and (b) it is common practice to bleed a sus-pension of diatomite into the rough beer stream to build up an additional depth of filter during the run. This provides increased particle retention as the amount of trapped material increases.

It is theoretically possible that any of these three materials could be used to sterilize the beer, but they are not normally used in that way because sufficiently fine filters would cause unacceptably slow flow rates (Leeder, 1998). Many spaces between the fibres or particles are larger than the yeast cells, but microbial and inert haze-forming material are adsorbed in the depths of the filter by electrostatic attraction. Therefore excessive flow rate or pressure differential across the filter would force microorganisms through. The filters are operated in the cold, usually near

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0°C. The principal reason is to form chill haze that can then be filtered out of the beer, but there is also the incidental value of preventing or restrict-ing microbial growth in the beer or on the filter. If growth did occur, a newly formed bud could escape in the filtrate even though its parent yeast cell was firmly attached to the filter medium.

It is possible to use such filters to reduce the yeast count to less than 10 cells/1, well below the 10° yeast cells/ml required to form a visible haze. For large-volume outlets, such filtered beer without further treatment can be dispensed from cellar tanks of 1-5 hl with the assurance that over its short storage time (no longer than a week, but preferably only about 3 days) there is no risk of these small numbers increasing to dangerous lev-els of contamination. Such large tanks are rare, however, and normally the beer is subsequently pasteurized to achieve an acceptable shelf life. In that case, a faster flow rate through the filter is possible. Beer with up to 100 cells/1 of culture yeast can be protected by pasteurization, although obvi-ously the aim is to keep the yeast count as low as possible before pas-teurization.

However, membrane filtration, which does sterilize, is becoming increasingly popular, especially in small and medium-sized breweries, but some larger breweries also use sterile filtration and aseptic filling (Tagaki, 1993). Pasteurization is unnecessary for sterile-filtered beer, so membrane filtration avoids the substantial energy cost and the potential flavor defects from heating the beer. Membrane filters are true mechani-cal filters: the pore size, usually 0.45 pm, retains bacteria, yeasts and inert particulate material on the surface. The advantage of a membrane filter is that the operating pressure is not restricted, but there is the disadvantage that any significant amount of particulate material in the beer will quickly ‘blind’ the filter. To some extent this can by minimized by cross-flow fil-tration, but even so, two filters are normally used in series. A rough pre-filter removes as much inert particulate material and microbial biomass as possible before the second, sterilizing, membrane filter, so an acceptable flow rate and long filter life can be achieved. The sterilizing filters can be composed of either inorganic material (ceramic or sintered metal) or organic (e.g. cellulose acetate or polycarbonate; Dunn et al., 1996).

1.5.2 Fining

Various collagens and other proteins of mammalian origin are effective clarification agents over a period of several weeks and are widely used as such in the wine industry. That is too slow for the brewing industry, so the traditional fining agent for the shorter timescale of clarifying traditional British cask-conditioned beer is isinglass. Isinglass is prepared from the swim bladders of various species of large tropical fish: large, for convenient-sized bladders and tropical, for greatest resistance to heat denaturation. The dried bladders are dissolved in food-grade organic acid: citric, malic

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and tartaric are all equally effective. It is essential that the mixing is in the cold, with minimal shear. The molecules of the fish collagen ichthyocoll are sensitive to both heat and mechanical damage, substantially reducing their fining activity that is dependent on large molecular weight, ideally at least 400 kDa. The viscous isinglass solution is stored at 24°C with SO, as an antibacterial preservative, but which as a reducing agent inciden-tally prevents any risk of introducing dissolved oxygen at such a sensitive late stage of the process.

Much of the yeast settles out at the end of fermentation but typically

10°-10° yeast cells/ml remain in suspension. These yeasts carry out the

secondary fermentation in the cask of the 1% residual fermentable sugar, or priming sugar added at racking (cask-filling) to restore the sugar to 1%. Casks are dispatched to the point of sale while the secondary fermenta-tion is still underway, so the brewer is completely dependent on the isin-glass and the bar staff to ensure acceptable clarity of the beer. Isinisin-glass is not a particularly powerful coagulant, and it is ineffective during the dis-turbance of transport and CO, evolution of fermentation in the cask. Only later is the yeast trapped by the netlike structure of ichthyocoll and settles to the lowest point of the cask, normally the side. Therefore isinglass is an ideal coagulant for use remote from the brewery: fast-acting in undis-turbed beer, within 24 h, and retaining its activity over several distur-bances during transport or in the bar cellar. Also, some breweries now use isinglass finings for preliminary clarification of beer for the filtration that precedes bottling, canning or kegging, to reduce the load on the filters.

1.5.3 Microbiological aspects of packaging

Cask-conditioned beer represents a very small percentage of British and North American beer production and is virtually nonexistent elsewhere. So, worldwide, there are two principal packaging arrangements in the brewing industry: (a) sterilization of beer prior to sterile filling into cleaned and sterilized containers (usually kegs), and (b) filling filtered beer into clean containers (usually glass bottles or metal cans) and subse-quently ensuring a satisfactory shelf life (at least 6 months, now often 1 year) by pasteurization. In both systems the heat treatment is equal in terms of pasteurization units (1 PU = 60°C/ min), but the ‘tunnel’ pas-teurizer for bottles and cans uses a lower temperature (typically 60-62°C) for a longer time. Individual breweries have their own preferred pasteur-ization treatment. In theory, 5 PU are sufficient to kill the small numbers of brewing yeast likely to pass througha cellulose or diatomite filter, but it would be difficult to provide such a low level accurately, and to elimi-nate the more heat-resistant bacterial or wild-yeast contaminants that could be present, up to 30 PU may be necessary. Because increased heat treatment could adversely affect flavor, particularly if atmospheric oxy-gen has accidentally gained access to the head space of the bottle or can

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during filling, choice of PU value must be a compromise between poten-tial risks of stale oxidized flavors and of microbial spoilage.

Bottles and cans are both popular packaging for ‘small-pack’ beer. Whether cans are constructed of aluminium or steel does not affect the microbiology of the canning process, but different systems are required for returnable glass bottles or nonreturnable ‘one-trip’ bottles of glass or plastic (usually polyethylene terephthalate, PET).

1.5.4 Glass bottles

Returnable bottles are obligatory in many countries for ecological/ environmental reasons. Because they may have been badly contaminated on their previous trip, rigorous cleaning and sterilization are required before reuse. The conveyor of the bottle-washing machine passes through the following sections: (a) caustic detergent/sterilant to remove labels and clean and sterilize the interior, (b) hot-water rinse to remove residual caustic, and (c) draining to dry. Bottles pass immediately to the filling area. Microbiological cleanliness and correct operation of the equipment are confirmed by routine checks on randomly selected bottles from the conveyor between cleaning and filling. Nonreturnable bottles do not require the rigorous cleaning of returned bottles; a jet of hot sterile water into the temporarily inverted bottle just before filling is sufficient to remove any material that may have fallen in during storage, but, again, occasional bottles should be sampled to confirm cleanliness. Returnable and one-trip bottles are not interchangeable because they are manufac-tured to different specifications but, other than washing, the packaging process is the same for both.

Although mechanically complex high-speed machinery is used, the basic process is that bright beer, filtered to a microbial count <100 cells/litre, is accurately measured into each bottle. To achieve acceptable beer stability, it is essential that the oxygen content of the head space is virtually zero. Replacing the air in each bottle by CO, or N, immediately before filling is common practice; also the beer is caused to foam, just enough to fill the head space of the bottle but not enough to overflow. A little hammer used to strike each bottle as it approaches the capping unit is one possibility, but a squirt of a small quantity of water (<1 ml) is com-monly used to induce controlled foaming. It is essential that the CO, or N, is sterile, and that the jet of water is not only sterile but also oxygen free. After capping—and it is important to confirm that caps too are in sterile condition—the bottles are conveyed through the pasteurizer, where the temperature is raised gradually to 60-62°C and held for the requisite number of PUs. They are then sprayed with cold water to cool to ambient

temperature to prevent overpasteurization by gradual cooling.

Subsequent labelling and packaging of the bottles have no microbiologi-cal implications.

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1.5.5 Cans

Most of the canning process is essentially the same as for nonreturnable glass bottles, i.e. from preliminary internal cleaning to cooling after pas-teurization. Subsequent differences are minor: adhesive labels are unnec-essary and less protective packaging will suffice. ‘Widgets’ to mimic draught beer are also a possible source of contamination of canned beer and obviously these units, and the gas they contain, must also be subject to routine microbiological testing.

1.5.6 Packaging of sterile-filtered or flash-pasteurized beer PET or other plastic bottles are insufficiently heat resistant to permit pas-teurization; therefore it is necessary to sterilize the beer before filling into sterile containers. Also, some breweries use sterile filtration to avoid the energy costs and flavor deterioration associated with pasteurization. In both cases there is a requirement not only for sterile beer but for sterile containers and filling equipment that protects against chance contamina-tion at that stage. The temperature of the manufacturing process ensures that new glass or PET bottles are sterile, but however well the containers are protected during transport and storage, some contamination is possi-ble before filling. Again, a jet or spray of warm sterile water is used imme-diately before the filling head. Reliable machinery is now available for aseptic packaging of foods and drinks, usually requiring the filling area to be protected with sterile gas (either CO, or N, must be used for beer), so in practice the filling problems are little different from beer proceeding to pasteurization.

1.5.7 Keg beer

In the kegging plant, returned kegs are conveyed through the processes of (a) rinsing and draining, (b) cleaning by a suitable detergent, (c) steam sterilization, with the condensate acting as a final rinse, and (d) refilling, although some equipment operates on a three-station layout where (a) and (b) are combined. In most plants, kegs pass through the system on parallel linear conveyors, but some modern plants have a carousel layout, like bottling and canning. The choice of detergent requires some care. Caustic preparations must never be used for aluminium kegs (NaOH reacts with Al, generating H, gas; see Chapter 11) and would be inacti-vated by any significant amount of residual CO, in stainless steel kegs. In the limited time available for treatment of each individual keg, steam is the only practicable method of sterilization. Because the filling unit is physically attached to the keg, risk of microbial contamination at that stage is low provided the pipework and connections of the filling system are cleaned and sterilized at intervals during each shift.

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1.5.8 Beer dispense

Finally, as with any catering industry, serving beer in the bar must meet the statutory food standards of the public health authorities. It is also in the brewer’s interest to ensure that the public are sufficiently impressed by beer of good quality to order more. Without a hygienic cellar and reg-ular cleaning and sterilization of the beer-dispense pipework, microbial spoilage of the beer is inevitable. Although it would be good microbio-logical practice, it is unreasonable to demand daily cleaning. Certainly, though, at least once per week the beer in the lines at closing time should be drained off and the pipes filled with detergent/sterilant overnight. The most likely points for contamination are (a) the coupling between the keg and dispense pipe, (b) any joints along the pipe, and (c) the dispense noz-zle on the bar, but Harper (1981) also noted development of contaminant microorganisms at zones of roughness within old pipes that were exam-ined after replacement. Any suspicion of spoilage of the company’s beer by careless bar operation should be investigated microbiologically, most conveniently by swab samples of the points (a), (b) and (c) identified here.

1.6 CONCLUDING REMARKS

The brewing process is essentially a microbiological one. In the current technology-driven climate it is easy to forget that we rely on our yeasts to effect the transformation of sugars into alcohol in such a way as to pro-vide exquisitely balanced flavors in the final product be it beer, wine or whisky. We are now beginning to understand yeast at an increasingly complex genomic level and in Chapters 2 and 3 the physiology and genet-ics of yeast in the context of brewery fermentations are discussed fully. However, the fermentation must also be balanced in terms of unwanted, spoilage organisms, be they nonculture yeasts or bacteria. The origins of such organisms on barley and malt are covered in Chapter 4 and the ensu-ing chapters deal with their characteristics, their effects on the fermenta-tion and final product, and methods of detecfermenta-tion and finally eliminafermenta-tion. Careful attention to the microbiological basis of brewing will undoubt-edly result in a more consistent product and the associated benefits that such reliability provides.

Carvell, J.P. (1997) Ferment, 10, 261.

Dunn, A.E., Leeder, G.I, Molloy, F. and Wall, R. (1996) Ferment, 9, 155.

Hammond, J.R.M., Brennan, M. and Price, A. (1999) Journal of the Institute of Brewing, 105, 113.

Hardwick, W.A. (ed.), (1995) Handbook of Brewing, Dekker, New York.

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Harper, D.R. (1981) Brewers Guardian, 110 (7), 23.

Last, ET. and Price, D.T. (1969) In The Yeasts, Vol. 1, Biology of Yeasts (eds A.H. Rose and J.S. Harrison), Academic Press, London, p. 183.

Leeder, G.I. (1998) Ferment, 11, 108.

Lewis, M.J. and Young, T.W. (1995) Brewing, Chapman and Hall, London. Martini, AV. and Kurtzman, C.P. (1985) International Journal of Systematic

Bacteriology, 35, 508.

Moll, M. (1994) Beers and Coolers, Intercept, Andover.

Palmer, G.H. (1989) Cereal Science and Technology, Aberdeen University Press,

Simpson, WJ. (1993) Journal of the Institute of Brewing, 99, 405.

Simpson, W.J. and Hammond, J.R.M. (1989) Journal of the Institute of Brewing, 95, 347-354.

Smart, K.A. (1999) Brewers Guardian, 128, 19. Tagaki, S. (1993), Ferment, 6, 185.

Van Oevelen, D., Spaepen, M., Timmermans, P. and Verachtert, H. (1977) Journal of

the Institute of Brewing, 83, 356.

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Biochemistry and physiology of yeast growth

J. Colin Slaughter

2.1 INTRODUCTION

Yeast is a eukaryotic, single-celled organism that, at the biochemical level, is very similar to all other eukaryotic cells. Indeed, much yeast research of recent times has used the organism as a model for more complex eukary-otes because of its ease of culture, the high level of genetic understanding, including the complete genome structure (Goffeau et al., 1996), and the ease of manipulation rather than an intrinsic interest in the industrial fer-mentation properties of the organism. We thus have a very considerable amount of laboratory-derived information on the biochemistry and molecular biology of yeast, and the aim of this chapter is to discuss aspects that are important in the production of beer rather than attempt-ing an overall survey of the topic. The background material is widely available in general textbooks (Stryer; 1995; Lodish et al., 1995; Zubay, 1998), in books that address yeasts in a more specific manner (Strathern, Jones and Broach, 1982; Walker, 1998; Dickinson and Schweizer, 1999) and at a number of web sites for example, the Saccharomyces Genome Database at http:/ /genome-www.stanford.edu /Saccharomyces or the many facili-ties for Saccharomyces genomics and proteomics available at http:// scientistcentral.com.

2.2 THE CELL CYCLE

When yeast cells are introduced into a nutritious aqueous medium, such as wort, with a temperature range between about 5°C and 35°C, the cells begin to grow and continue to do so until one of the essential nutrients is

<small>Brewing Microbiology, 3rd edn. Edited by F. G. Priest and I. Campbell.</small>

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exhausted. Each cell repeats an obligatory series of events known as the cell cycle (Walker, 1998). In summary, cell mass and dimension increase (G, phase) and, once a critical size has been achieved, synthesis of DNA occurs over a discrete period along with commencement of bud formation (S phase). This is followed by mitosis (M phase) in which the chromo-somes separate into two sets, one of which, along with essential cytoplas-mic organelles, moves into the developing bud. A septum is then synthesized to divide the bud from the mother cell. The newly indepen-dent daughter cell separates from the mother cell at some later point. In Saccharomyces cerevisiae, S and M phases merge into each other so that there is no clear G, phase between the two events as in some other cell types. In some brewing strains cell separation may be defective so that short chains of cells form. As mentioned previously, a minimum cell size is necessary before the S phase starts, so normally the mother cell will bud again before the new daughter cell. Before starting on the cell cycle, cells sense the nutrient status of the medium. If this is inadequate, G, does not occur but cells move into stationary phase (G,) instead. These cells show a lower metabolic rate and greatly increased resistance to stress compared to growing cells.

Under certain conditions of stress, pseudomycelial growth occurs. This represents a major change in growth pattern and the cell cycle. The cells become elongated, budding is unipolar and the buds do not detach from the mother cell (Gancedo, 2001).

2.3 THE GROWTH AND FERMENTATION CYCLE In the brewery context, yeast cells are grown for two different purposes. First, in yeast propagation the aim is to produce large quantities of yeast from tiny amounts, i.e. from laboratory stock culture to pitching yeast. The cells are maintained in well-oxygenated nutrient medium through several batches of increasing volume to allow as many cell cycles as needed to attain the desired quantity of yeast. Second, in fermentation the mass conversion of wort to beer in a single operation is the important task. The inoculum is large with growth limited to one or two cell cycles. Biochemically there is little difference between the two procedures except that the growth phase is greatly extended in yeast culture and cells are unlikely to experience lack of oxygen. Fermentation in many ways pre-sents more challenges as there is more than one aim—an economical con-version of wort to beer, delivery of the desired beer qualities of pH, ethanol content, and profile of flavor-active compounds, and the genera-tion of a crop of yeast cells with sufficiently high viability and vitality to be used as pitching yeast in subsequent fermentations.

During growth, sugars, nitrogenous compounds, inorganic ions and vitamins are taken up and the medium is acidified to about pH 4, and

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ethanol and carbon dioxide are released in the approximate range of 35 g to 70 g/1 depending on wort strength and degree of attenuation achieved. Several organic compounds and inorganic sulfur compounds are also released at much lower concentrations (approximate range of 0.1 mg to 50 mg/1). Many of these compounds are important in flavor and are the most difficult to control in industrial practice. Reproducibility from one batch of beer to another is achieved through use of a constant amount of pitch-ing yeast of a high viability and vitality, a constant fermentation period at a controlled temperature, and a maturation period in the presence of the yeast. An important aspect of yeast biochemistry not related to flavor is the clumping together of cells to form flocs that may rise to the surface, associated with gas bubbles, or sink to the bottom. This phenomenon led to the nomenclature of ‘top’ or ‘bottom’ fermenting yeast and has been exploited in separation of yeast from beer at the end of fermentation in all brewing systems. It is vitally important that the yeast flocculates at the right time. If it occurs too early and fermentation will not be completed with dire consequences for beer properties; too late and separation of the yeast from the finished beer will be made more difficult.

2.4 CELL COMPOSITION, NUTRITION AND GENERAL METABOLISM

Like other living cells the yeast cell is composed mainly of water. Most of the nonaqueous material is polymeric. Carbohydrates, proteins, and nucleic acids constituted by the six elements C, H, O, N, P, and S form the bulk of the material with a large range of low-molecular-weight organic compounds and inorganic ions making up the remainder. Yeast cells can grow on relatively simple media. They need a supply of fermentable sugar, e.g., glucose, fructose, galactose, sucrose, maltose, or maltotriose; a nitrogen source, e.g., ammonium, urea or amino acids; phosphate; sulfate; chloride; oxygen, and a range of metal ions, e.g., K, Mg, Ca, Fe, Cu, Zn, and Mn. Most yeast strains need in addition one or more of a small number of vitamins, e.g. biotin, pyridoxine, pantothenic acid, thiamine, meso-inositol, p-aminobenzoic acid, and nicotinamide, although exact requirements and amounts are strain specific. Yeast can grow anaerobi-cally but for it to do so for more than one or two generations, the medium must contain long-chain unsaturated fatty acids and ergosterol.

2.5 ENERGY AND INTERMEDIARY METABOLISM This is the area of most longstanding knowledge. Considerable detail is available in standard textbooks and web sites and the area is dealt with here in outline only. The energy required to drive the biosynthesis, and to

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some extent the controlled degradation, of the polymeric cell components is gained from the breakdown of monosaccharides, particularly glucose, and a few simple oligosaccharides as mentioned previously. One mol of glucose is metabolized through the long-established route known as the Embden-Meyerhof, or glycolytic, pathway (Fig. 2.1) to yield two moles of pyruvic acid. Each mole of this compound is converted to one mole of ethanol and one mole of carbon dioxide by the sequential action of pyru-vate decarboxylase and alcohol dehydrogenase. The biological function of these last two reactions is to oxidize the NADH formed earlier in the path-way by 3-phosphoglyceraldehyde dehydrogenase, back to nicotinamide adenine dinucleotide (NAD*) to allow a continuous flux of carbon and synthesis of adenosine triphosphate (ATP). The pathway involves both utilization and formation of ATP and creates a net gain in ATP of two mol per mole of glucose metabolized. This represents capture of only about 4% of the energy available in the sugar. Except under low sugar concen-trations, e.g. less than about 5 g/l, and in the presence of oxygen, S. cere-visiae utilizes sugar by this inefficient process of alcoholic fermentation rather than by the efficient process of mitochondrial oxidative rylation employed more commonly by eukaryotes. In oxidative phospho-tylation, acetyl coenzyme A (acetyl CoA), rather than acetaldehyde, is formed from pyruvate followed by metabolism in the mitochondria via the tricarboxylic acid (TCA) cycle and the electron transfer chain to yield about 30 moles of ATP per mole of glucose. The sugar is completely

oxi-dized to water and carbon dioxide.

ATP is used by the cell as the thermodynamic driving force to maintain and adjust cell composition and increase cell mass. The major reactions are the biosynthesis of polysaccharides, proteins and nucleic acids during growth (Stryer, 1995; Lodish et al., 1995; Zubay, 1998). The monomeric car-bon components of the polymers are derived from intermediates of glycol-ysis and through operation of parts of the TCA cycle. For example, the storage polysaccharide glycogen is derived from glucose 6-phosphate as is the cell wall glucan, chitin and mannosy] units of the glycoproteins (Fig. 2.2); lipids are formed from acetyl CoA and glycerol with the nitrogenous com-ponent of phospholipids and sphingolipids coming from serine (Fig. 2.3); the amino acid precursors of nucleic acids and proteins are formed from 3-phosphoglycerate, pyruvate, phosphoenolpyruvate, erythrose 4-phosphate, oxaloacetate, and 2-oxoglutarate (Table 2.1). Use of oxaloac-etate and 2-oxoglutarate as metabolic intermediates requires a continuous synthesis of oxaloacetate by reaction of carbon dioxide with pyruvate or phosphoenol pyruvate (Fig. 2.4). This is often referred to as an anaplerotic sequence in the sense that it serves to ‘fill up’ the intermediates in a path-way that otherwise acts as a cycle with self-replenishing intermediates (the TCA cycle in oxidative respiration).

Nitrogen enters the cell through the active transport of ammonium,

intact L-amino acids, small peptides or urea. After absorption urea is

con-verted to ammonium (Fig. 2.5a), which is then assimilated in the same

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Fig. 2.2 Formation of polysaccharides from glucose 6-phosphate

way as external ammonium. S cerevisiae cannot utilize nitrate or proteins. Ammonium is converted to the amino acid glutamate in a single reaction catalyzed by the enzyme nicotinamide adenine dinucleotide phosphate (NADP) glutamate dehydrogenase (Fig. 2.5b). This amino acid can be converted to three other amino acids, glutamine, proline and arginine, by conversions of the carbon skeleton leaving the a-amino group intact. In other cases the a-amino group of glutamate can be donated to a 2-oxoacid in a transamination reaction to form a new amino acid and 2-oxoglutarate (Fig. 2.5b). In some cases these amino acids are the final products, e.g. tyrosine, alanine, valine, leucine, and isoleucine, but in others more amino acids are formed by conversions of the carbon skeleton (Table 2.1). For

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Stearoyl CoA ——Ì* Oleoyl CoA Squalene

<small>Gh 3-4</small>

S201 be Glyeerol 3-phdsphate Serine p8

Serine Sl ý

Triacylglycerols Phospholipids Sphingolipids Sterols

Fig. 2.3 Formation of lipids from acetyl CoA.

example, aspartate is formed by transamination of oxaloacetate and then goes on to be the precursor for asparagine, threonine and methionine and the carbon precursor for isoleucine. Thus, glutamate acts as a direct car-rier of nitrogen from ammonium into amino acids. In the case of several other amino acids—serine, glycine, cysteine, lysine, histidine, and trypto-phan—the metabolic route is more complex (Table 2.1). There is no direct 2-oxoacid equivalent of the amino acid in the biosynthetic route, which is an important feature for beer quality as will be seen later. The source of nitrogen is still glutamate, however, directly or indirectly.

Although the route catalyzed by NADP-dependent glutamate dehy-drogenase is the main way in which yeast assimilates ammonium, the Ky, value of this enzyme for ammonium is quite high and at low concentra-tions of the nitrogen source an additional system is also utilized. This relies on the joint action of two enzymes: glutamine synthetase, which forms glutamine from glutamate by addition of ammonium and has a low Ky value for ammonium, followed by NADPH-dependent glutamate synthase (glutamate oxoglutarate amino transferase or GOGAT), which forms two molecules of glutamate from the glutamine and 2-oxoglutarate. The net metabolic effect is the same as the glutamate dehydrogenase action; ammonium is transferred to 2-oxoglutarate to form glutamate.

Yeast can efficiently synthesize all the required amino acids from car-bohydrate and ammonium but in practice a complex system of metabolic control ensures that amino acids in the medium are taken up and used in preference to production through the biosynthetic pathways. Small pep-tides (less than six amino acyl residues) are taken up and hydrolyzed to the constituent amino acids in the vacuole. Once inside the cell, the exter-nally derived amino acids are indistinguishable from biosynthesized

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Table 2.1 The biosynthetic relationships of the amino acids

2-Oxoacid Equivalent amino acid* Derived amino acids 3-Phosphohydroxypyruvate Phosphoserine Serine, Glycine, Cysteine,

(from 3-phosphoglycerate) Tryptophan, (from oxaloacetate via

aspartate and threonine)

Oxaloacetate Aspartate Asparagine, Methionine,

Threonine, Isoleucine

2-Oxoglutarate Glutamate Glutamine, Arginine, Proline

2-Oxoadipate a-Aminoadipate Lysine

(from 2-oxoglutarate)

2-Oxo-phenylpyruvate (from _ Phenylalanine Tyrosine (Oxygen phosphoenol pyruvate dependent)

<small>*Several amino acids are formed by direct transamination of a 2-oxoacid. In some cases thesecompounds are then further metabolized to yield additional amino acids. The main excep-tions to this generalization are serine, lysine, glutamate, tryptophan and histidine.</small> In the case of serine the transamination product is phosphoserine and serine is formed from <small>this compound by action of a phosphatase.</small>

<small>The a-amino group of lysine is introduced by transamination at an intermediate point in thepathway to yield a-aminoadipic acid. A second amino group is introduced from glutamatevia the intermediate, saccharopine, to yield lysine.</small>

<small>Glutamate is normally synthesized from its oxoacid equivalent, 2-oxoglutatrate, by theenzyme NADPH glutamate dehydrogenase. The transaminase functions in the biosyntheticreactions of other amino acids where the glutamate amino group is transferred to an oxoacidto yield the equivalent amino acid and 2-oxoglutarate.</small>

<small>Tryptophan is synthesized directly from indolglycerol phosphate (derived from </small> phospho-enol pyruvate and erythrose 4-phosphate) by substitution of an intact molecule of serine for <small>the glyceraldehyde 3-phosphate moiety.</small>

<small>Histidine is synthesized from AMP by a unique route. The a-amino group is introduced bytransamination of imidazolacetol phosphate and the product is converted to histidine bytwo further enzymic steps.</small>

compounds and are metabolized in the same way. When external amino acids that involve transamination as the final step in biosynthesis are in excess of metabolic demand, a common pattern of degradation is through donation of the amino group to a metabolically derived 2-oxoacid through transamination (Fig. 2.5c). The 2-oxoacid corresponding to the carbon skeleton of the external amino acid is then decarboxylated and the

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Fig. 2.4 TCA cycle and anaplerotic formation of oxaloacetate. The solid arrows show the synthetic routes to aspartate and glutamate from glycolytic

intermedi-ates with the heavy arrows indicating the two anaplerotic routes. The dotted

arrows show the reactions necessary to complete the TCA cycle. The cycle func-tions in fully differentiated mitochondria in the presence of oxygen to effect com-plete oxidation of glucose.

resultant aldehyde reduced to a higher alcohol that is lost from the cell (see section 2.6.5 on flavor). Deamination of amino acids to release ammonium seems unlikely to be of general importance under brewing conditions as molecular oxygen is usually required. However, specific

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Fig. 2.5(a) Sources of nitrogen and general relationships. Most of the ammonium within the cell is converted to amino acids via glutamate. The enzymology of

car-bamoyl phosphate synthesis is complex but the compound can be formed with

either ammonium or glutamine acting as a nitrogen donor. It is required for syn-thesis of arginine and the pyrimidine bases.

enzymes are known that catalyze anaerobic deamination of aspartate, phenylalanine, cysteine, serine, and threonine. Alanine can be deami-nated by action of a specific NAD-dependent dehydrogenase. The ammo-nium released can be reassimilated through action of NADP-dependent glutamate dehydrogenase. Glutamate can also be deaminated by action of a specific NAD-dependent dehydrogenase, but control of transcription prevents yeast from producing the NAD- and the NADP-dependent glu-tamate dehydrogenases at the same time. The concentration of the enzymes is adjusted to the availability of glutamate in the medium. Individual amino acids vary widely in their ability to act as sole sources of nitrogen, and yeast growth is usually at its best when the medium contains a range of amino acids. Special notice should be taken of the utilization of proline in the brewery context. Strictly speaking, this is an imino acid but is a nor-mal component of proteins. Proline is prevalent in barley proteins and is the most common low-molecular-weight nitrogenous compound in wort. However, it is very poorly used under brewery conditions and remains mainly in the beer. This is because its degradation by yeast is a mitochon-drial function that requires oxygen. The same restriction limits the

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<small>use-From glucose via glycolysisand TCA cycle reactions</small>

<small>From glucose via glycolysis, TCA cycle</small> reactions, pentose phosphate pathway

To glutamine, proline and arginine reactions and anaplerotic reactions

Fig. 2.5 (b) Assimilation of ammonium.

<small>Amino acid (R2) 2-Oxoacid (R1)</small>

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a’ Proline oxidase Glutamicsemi g aldehyde SPAN ¡2 rolne carboxylate

Fig. 2.6 Degradation of proline and arginine.

fulness of arginine as a general nitrogen source in a brewery fermentation (Fig. 2.6).

The main quantitative function of amino acids is to act as the monomeric units of proteins but several are also very actively involved in other vital areas of metabolism. Serine is the main source of the nitrogenous compo-nent of phospholipids and sphingolipids. Methionine, through the inter-mediate S-adenosyl methionine, is active in methylation reactions and in the formation of the polyamines. Glycine, aspartic acid and glutamine all contribute to the biosynthesis of nucleotides and hence nucleic acids. Glycine acts as a precursor for heme.

The ribose phosphate component of the nucleotides is derived from glu-cose 6-phosphate via action of the pentose phosphate pathway (Fig. 2.7). This pathway also supplies erythrose 4-phosphate, a precursor of the aro-matic amino acids, and NADPH, which is needed for biosynthesis of many compounds, including glutamate. Under the conditions of brewery fermentation, the main function of the pentose phosphate pathway is in providing intermediary compounds to support biosynthesis rather than contributing to glycolytic flux and ATP production.

Brewery yeasts have strain-dependent requirements for a few vita-mins such as nicotinamide, biotin, pyridoxine, thiamine, pantothenate,

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E +hoaphsie Fructose 6-phosphate

Se Giy yyde3 > Glycolysis

Fig. 2.7. The pentose phosphate pathway.

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