forty years of brewing research

8/19/2019 Forty Years of Brewing Research

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VOL. 115, NO. 1, 2009 3

The Horace Brown Medal Lecture:

Forty Years of Brewing Research

Graham G. Stewart1,2

ABSTRACT

J. Inst. Brew. 115(1), 3–29, 2009

Horace Brown spent fifty years conducting brewing research inBurton-on-Trent, Dublin and London. His contributions wereremarkable and his focus was to solve practical brewing prob-lems by employing and developing fundamental scientific prin-ciples. He studied all aspects of the brewing process includingraw materials, wort preparation, fermentation, yeast and beerstability. As a number of previous presenters of the HoraceBrown Lecture have discussed Brown’s achievements in detail,the focus of this paper is a review of the brewing research that

has been conducted by the author and his colleagues during thepast forty years. Similar to Horace Brown, fundamental researchhas been employed to solve brewing problems. Research studiesthat are discussed in this review paper include reasons for pre-mature flocculation of ale strains resulting in wort underattenua-tion including mechanisms of co-flocculation and pure strainflocculation, storage procedures for yeast cultures prior to prop-agation, studies on the genetic manipulation of brewer’s yeaststrains with an emphasis on the FLO1 gene, spheroplast fusionand the respiratory deficient (petite) mutation, the uptake andmetabolism of wort sugars and amino acids, the influence ofwort density on fermentation characteristics and beer flavour andstability, and finally, the contribution that high gravity brewinghas on brewing capacity, fermentation efficiency and beerquality and stability.

Key words: Co-flocculation, flavour, flocculation, genetic ma-nipulation, high gravity brewing, petite mutation, propagation,stability, wort clarity and composition, yeast management.

INTRODUCTION

The Horace Brown Medal commemorates HoraceTabberer Brown (1848-1925), one of the “founding fath-ers of the Institute of Brewing”. Although largely self-taught, Horace Brown was a true polymath who left hismark on virtually all areas of science as applied to brew-ing, in a career which lasted over 50 years. His researchwork considered barley germination, beer microbiology,water composition, oxygen and fermentation, beer haze

formation, wort composition and beer analysis. His con-tributions were remarkable and his focus was to solvepractical brewing problems by employing and developingfundamental scientific principles. A number of previousrecipients of the Horace Brown Medal have reviewedBrown’s achievements in detail14,73,105. However, a briefsummary here of Brown’s research is appropriate.

In 1916, the London Section of the Institute of Brew-ing presented Horace Brown with a portrait of himself(Fig. 1) “as an expression of the affection, esteem, andhomage of the Members of the Institute”. In reply, Brown

presented a lecture15 entitled: “Reminiscences of FiftyYears’ Experience of the Application of Scientific Meth-ods to Brewing Practice”. Brown began his presentationwith the following statement: “The origin of this paper isthe result of a retrospect and a process of mental stock-

1 International Centre for Brewing and Distilling, Heriot-Watt

University, Riccarton, Edinburgh, EH14 4AS, Scotland.2 Present address: G.G. Stewart Associates, Brook House, Caerphilly

Business Park, Caerphilly, Wales, CF83 3GS.

Corresponding author. E-Mail: [email protected]

This lecture was presented at the IBD Africa Section Convention,

March 2, 2009.

Publication no. G-2009-0225-581

© 2009 The Institute of Brewing & Distilling Fig. 1. Horace Tabberer Brown.


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4 JOURNAL OF THE INSTITUTE OF BREWING

taking, supported by the fact that I have been closelyconnected for half-a-century with one of the most interest-ing industries in the world”. Brown discussed his researchactivities in Burton-on-Trent, Dublin and London and thathis focus was to solve practical brewing problems byemploying and developing fundamental scientific princi-ples. His studies on all aspects of the brewing process,including raw materials, wort preparation, fermentationand beer stability, were reviewed.

At the end of this wide ranging lecture (which waspublished as a 91-page paper in the Journal of the Instituteof Brewing)15 Brown expressed personal sentiments thatare, in part, relevant today. Brown stated that he wasdeeply disappointed that more had not been achieved toexploit science in brewing. He blamed two factors – thelack of education of brewers and the short-term vision ofbrewery directors. He concluded “Those who are reallythe responsible heads of manufacturing businesses seldomhave any desire to know anything of the inner meaning oftheir processes or the scientific principles which underlinethem”. One has to wonder if Brown overstated the prob-lem that prevailed at the time. A great deal of fundamentalresearch in brewing and related industries occurred in the

second half of the nineteenth century. It will be discussedin this paper that a great deal of relevant research has beenconducted since 1916. However, one cannot help wonder-ing if the industry has come “full circle” and its interest inlearning more about basic scientific principles is waning!

Most of the period of Brown’s research coincided withthe industrial revolution. Consequently, brewing (as inmany other industries) was being converted from a cot-tage craft into a major industry and with this conversioncame the need for scientific understanding. The brewingindustry became the centre of much scientific researchand it is interesting to note that, at this time, four Fellowsof the Royal Society (including Horace Brown) were em-ployed by breweries in the United Kingdom118. This same

phenomenon was also occurring in other countries. Forinstance in Denmark, J.C. Jacobsen, who founded theCarlsberg Brewing Company in 1847 (naming it after hisson – Carl), stated that, “The person who has the mostthorough knowledge in chemistry, and supporting sci-ences, together with the necessary technical proficiencyand insight, will be Europe’s foremost brewer in the com-ing generation”. This led to the establishing of the Carls-berg Foundation in 1876, which became, and still is, thehome of a number of distinguished scientists59.

This paper is largely (but not exclusively) a discussionof personal experiences with brewing research, mainlywith yeast, during the past forty years, working, first ofall, for twenty-five years in the R&D Department of the

Labatt Brewing Company Limited and subsequently inthe International Centre for Brewing and Distilling atHeriot-Watt University. However, interest in yeast re-search began as a PhD student at Bath University when,as with many other research workers, yeast proved to be asuitable and inexpensive micro-organism for biochemical(and later molecular biological) research. Indeed, it waspurchased in the mid-1960s from the local baker’s yeastfactory for 4 shillings (approximately £0.20)/pound. Thefocus of this paper is to discuss (similar to Horace Brown)how basic research has been employed to answer practical

brewing questions. Although most of the research dis-cussed in this paper has been conducted at Labatt andHeriot-Watt University, relevant research by other groupshas been included. All of the research discussed was ini-tially published elsewhere, mainly in peer-reviewed jour-nals.

BREWER’S YEAST STRAINS

For the past 50 years and longer, the brewing industryhas been going through a period of seeking scientific rea-sons for empirical knowledge, and fermentation and yeastresearch have been no exception to this generalisation.Efforts have been concentrated on “what the cell does andhow it does it”. The focus has been to discover the reasonsbehind the empirical facts of yeast growth, fermentationand flocculation. Early research concentrated upon bio-chemical and physiological aspects. During the past 25years, this has been expanded to include consideration ofgenetic and molecular biological aspects together with agreater understanding of the influence of the genotype ona culture’s physiology and function. In addition, 30 yearsago131 it was believed that brewer’s yeast strains would be

genetically in vitro constructed and particularly employedfor specific brewing purposes. However, for reasons thatwill be discussed later, although specific strains have beendeveloped by genetic manipulation, none of them havebeen used for brewing132.

Over the years, considerable efforts have been devotedto a study of the biochemistry and genetics of brewer’syeast (and other industrial yeast strains)130. The objectivesof these studies have been two fold: (1) to learn moreabout the biochemical and genetic make-up of brewingyeast strains; and (2) to improve the overall performanceof such strains, with particular emphasis being placed onbroader substrate utilization capabilities, increased etha-nol production, improved stress tolerance to environmen-

tal conditions such as high osmotic pressure, ethanol,temperature, salt and physical shear and to understand themechanism(s) of flocculation.

Understanding the substrate specificity of brewing andother industrial yeast strains is a major objective of manyzymologists. Progress in achieving these objectives hasbeen impeded for several reasons, which include the factthat such strains employed in the production of beer anddistilled products, are not readily amenable to geneticmanipulation by classical techniques. There are manymethods that can be employed in genetic research anddevelopment of industrial yeast strains. These methods,which will be discussed in greater detail later in this docu-ment130, include hybridisation, mutation and selection,

spheroplast (or protoplast) fusion and transformation, andassociated with it, DNA transformation and associatedgene manipulation techniques. Industrial yeast strains areoften polyploid or even aneuploid and, as a consequence,do not possess a mating type, have a low degree of sporu-lation and spore viability, rendering genetic analysis ofsuch strains difficult but not impossible154. However, mod-ifications to the incubation temperature and presporula-tion carbon source have significantly increased sporogeniccapabilities of industrial strains, thereby facilitating recov-ery of viable spores and thus providing an opportunity to


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manipulate such strains by classical (hybridisation) tech-niques5,6.

PROPAGATIONAND SEDIMENTATION

CHARACTERISTICS OF LAGERAND ALE STRAINS

The contribution of the Carlsberg Foundation to brew-ing research has already been discussed. One of their not-able scientists was Emil Christian Hansen (Fig. 2). Hesuccessfully isolated four strains of bottom fermentingyeast from the Carlsberg lager yeast culture. He studiedthem from the standpoint of brewery performance, andonly one of these strains proved to be suitable for beerfermentation. The strain, described as “Carlsberg Yeast No.1” was introduced into the Carlsberg Brewery for useon a production scale on 13 May 1883 and pure strainbrewing of lager can be said to have commenced from thisdate59. Due to the origin of “Carlsberg Yeast No.1” it wasnamed “Saccharomyces carlsbergensis” Hansen 1883.This grouping of lager yeasts is now known as Sac-

charomyces pastorianus91.Hansen soon found that it was too tedious and incon-

venient for the laboratory to regularly furnish the Carls-berg Brewery with pure cultures and it would be easier toobtain a specific apparatus for this purpose. With the as-sistance of the coppersmith, W.E. Jansen, Hansen startedto construct such an apparatus. By the beginning of 1886,the apparatus was working effectively in the CarlsbergBrewery. Jansen began to sell the apparatus and Heinekenwas one of the first breweries to purchase this equipment.

As a result of Hansen and Jansen’s work, the practiceof employing a pure strain in lager production was soonadopted by breweries all over the world, particularly inthe United States. Ale-producing regions however, met

this “radical innovation” with severe opposition! Themethod was merely regarded as a means of reducinginfection by wild yeasts and bacteria. In 1959 it was re-ported60 that of 39 ale yeast cultures in use commerciallyin Britain, 12 contained a single strain, 16 had two major

strains and the rest contained three or more yeast strains.These ale strains are taxonomically classified as part ofthe species Saccharomyces cerevisiae91.

Co-flocculation

In the 1960s and 1970s ale consumption was popularin Canada. In 1970, it was 60% of the beer produced inOntario. However, similar to the situation in Britain, theale yeast cultures employed were largely uncharacterised.

The Labatt ale culture possessed top cropping propertiesand exhibited intermittent premature flocculation charac-teristics resulting in underfermented wort with residualsugars (mainly maltotriose – details later). This problemwas exacerbated during high gravity brewing trials. It wasof interest to enumerate the number of strains in this aleculture and characterise them. One of the most suitablemethods available in the late 1960s for this purpose wasthe Giant Colony Morphology Method106. This methodinvolved inoculating the yeast culture onto solid mediaand examining the colonial morphology that developedafter incubating under standard conditions for at leastthree weeks at 18°C. It has been found that gelatin, as thesolidifying matrix, tends to enhance the distinctive fea-

tures of the colonial morphology to a greater extent thanagar. It is usual to find that malt-wort, corn steep liquor,fruit and vegetable infusions induce greater colony differ-entiation than complete media based on nutrients fromanimal sources or defined synthetic media. This methodhas, however, one major shortcoming (as well as theprolonged incubation period necessary at below ambienttemperature). It gives no information on the value of abrewing strain for brewing purposes, to quote Cook 26 whostated in his address to the European Brewery ConventionCongress in 1969: “It is important to realize that this pro-cedure (i.e., the giant colony procedure) is rather like tak-ing photographs of those in the hall. The photographswould enable us to identify individuals elsewhere but

would tell us nothing of their performance as maltsters,brewers and scientists”. However, although the ale brew-ing strains exhibit characteristic colonial morphologieswhen grown on wort gelatin media; lager strains exhibitsimilar, uncharacteristic, and somewhat uniform morphol-ogy, strain to strain.

Analysis of the Labatt ale culture’s strain compositionshowed that two morphologically different colony types(Fig. 3) were present. On isolation, both colony typesproved to be stable respiratory sufficient separate strainsof the species Saccharomyces cerevisiae and they were

Fig. 2. Emil Christian Hansen.Fig. 3. Giant colony morphologies of Labatt ale yeast strains –LAB A/69 (left colony) and LAB B/69 (right colony).


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coded LAB A/69 and LAB B/69. Both strains, whencultured alone in wort, were non-flocculent during allphases of growth. However, when cultured together inwort in a 1:1 ratio, the culture was flocculent in the laterstages of a wort fermentation and sedimented out of sus-pension (Fig. 4). Also, when stationary phase cells of thetwo strains were mixed together in a 1:1 ratio in thepresence of calcium ions at pH 4.5 (a variation of theHelm’s sedimentation test57) flocs immediately began toappear and a very flocculent culture resulted that sedi-

mented out of suspension (Fig. 5). This type of behaviour,where two yeast strains are non-flocculent alone butflocculent when mixed together, has been termed co-flocculation137,128. Co-flocculation has also been termedmutual aggregation and mutual flocculation42,170.

To date, co-flocculation has only been observed withale strains, and there are no reports of co-flocculationbetween non-flocculent lager yeast strains. Another typeof co-flocculation reaction which has been described iswhere an ale yeast strain has the ability to aggregate andco-sediment with contaminating bacteria such as Hafnia protea, Lactobacillus brevis, Pediococcus sp. and Lacto-bacillus sp170,176.

It was decided that the two strain composition of the

Labatt production ale strain was undesirable, particularlybecause of its tendency for pre-mature flocculation andwort under-attenuation, resulting in failure to meet thebeer’s alcohol specification (this is prior to the introduc-tion of high gravity brewing on a production basis). Pro-duction trials with LAB A/69 strain were conducted inboth lower (sales) (12°Plato) and higher gravity (16°Plato)wort. This strain proved to be capable of successfullyfermenting both wort gravities but, because of its non-flocculent property, required centrifugation to harvest theculture for both clarification and re-use. This strain has

been employed for ale production by Labatt with highgravity worts for the past twenty-five years.

Pure strain flocculation

As well as co-flocculation between two or more brew-er’s yeast strains, the flocculation characteristics of indi-vidual strains are also important. As previously discussed,the flocculation property, or conversely, lack of floccula-tion, of a particular culture (strain) is one of the majorfactors when considering important characteristics during

brewing and other ethanol fermentations. Unfortunately, acertain degree of confusion has arisen by the use of theterm flocculation in the scientific literature to describedifferent phenomena in yeast behaviour. Specifically, floc-culation, as it applies to brewer’s yeast, is “ the phenome-non wherein yeast cells adhere in clumps and either sedi-ment from the medium in which they are suspended or riseto the medium’s surface”149. This definition excludesother forms of aggregation particularly that of clumpygrowth or chain formation148 (Fig. 6). This non-segrega-tion of daughter and mother cells during growth hassometimes erroneously been referred to as flocculation.Chain formation only occurs with some ale strains andhas not been observed in lager strains to the same extent.

The term “non-flocculation” applies to the lack of cellaggregation and, consequently, a much slower separationof (dispersed) yeast cells from a static liquid medium.Flocculation usually occurs in the absence of cell division(but not always) during late logarithmic and stationarygrowth phases and only under rather circumscribed envi-ronmental conditions, involving specific yeast cell surfacecomponents (proteins and carbohydrates) and the interac-tion of calcium ions (details later). Although yeast separa-tion often occurs by sedimentation (bottom cropping), asalready described in the discussion of co-flocculation, it

Fig. 4. Co-flocculation – cylinder wort fermentation test. Fig. 5. Co-flocculation – Helm’s sedimentation test.


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may also be by flotation because of cell aggregates en-trapping CO2 bubbles. These are top-cropping ale brewingstrains.

Calcium adsorptionThe importance of calcium ions during yeast floccula-

tion cannot be over emphasised147. With many flocculentstrains, the calcium can be removed from the yeast cellwall as a result of washing with deionised water and theculture will become reversibly non-flocculent156. If cal-cium is then added to this de-flocculated culture the cellsbecome flocculent again (Fig. 7). Some flocculent strainsare not de-flocculated by washing with water, the cellsneed to be treated with a solution of a chelating agentsuch as EDTA followed by washing with water to remove

the EDTA. This treatment will de-flocculate these culturesand the flocculation phenotype will be restored upon addi-tion of calcium ions (Fig. 8).

It has been proposed72 that flocculent yeast strains areable to adsorb considerably more Ca++ ion than non-floc-culent strains. It has also been suggested that cell wallsisolated from flocculent cultures bind more Ca++ ions than

walls isolated from non-flocculent cultures. Employingradiolabelled Ca45, studies have been conducted to com-pare the calcium binding ability of a number of ale andlager flocculent and non-flocculent brewer’s yeast cul-tures150,151. When the final calcium uptake of each culturewas analysed (Table I), it was clear that no direct corre-lation existed between the total calcium adsorbed andflocculation. There is strain-to-strain variation in calciumbinding, and furthermore this variation does not correlatewith flocculation and non-flocculation, when comparingone strain to another. However, with the knowledge that

Fig. 6. Flocculation in Saccharomyces cerevisiae.

Fig. 7. Water wash deflocculation.

Fig. 8. EDTA wash deflocculation.


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many flocculent yeast cultures can be de-flocculated bywashing with deionised water (Fig. 7), if one could cor-relate the amount of calcium washed off a yeast culturewith the visible loss of flocculation, an improved perspec-tive of calcium binding behaviour in yeast and its relation-ship to flocculation might be obtained.

To test this hypothesis, aliquots of flocculent and non-flocculent yeast suspensions were taken after 120 min

incubation with the Ca45 solution and centrifuged. Theyeast pellet was washed four times with 2 mL of deion-ised water pH 4.0 for 15 sec on a Vortex mixer and theactivity of each centrifuged supernatant determined with ascintillation counter. Using standard curves relating cal-cium concentration to radioactivity, the amount of cal-cium removed with each washing was determined. Thefirst wash did not de-flocculate the flocculent yeast cul-tures, but did remove loosely adhering calcium aroundand in the interstitial spaces between the yeast cells. Thissource of calcium should be relatively the same percent-age of total calcium bound for each yeast culture (Table I)and is in all probability not related to flocculation, sincethe visible observation of flocculation did not disappear

during this first wash.The subsequent washings gradually removed any ob-

servable flocculation. The sum of the calcium removed inwashings 2 to 4 were expressed as a percentage of thetotal calcium removed during washing. When the resultswere expressed in these terms (Table II) for flocculent andnon-flocculent cultures, the flocculent cultures bound 28to 40% more calcium after four water washings than non-flocculent cultures. As would be expected, there is strain-to-strain variation in calcium adsorption. This variation isin all likelihood a reflection of diversities in cell wall

structure from strain to strain. In addition, this strain-to-strain variation in calcium adsorption per se does not cor-relate with the flocculation phenotype, when comparingone strain to another. The only meaningful measure of

calcium behaviour that correlated with flocculation wasthe ease with which calcium washed off the cell and thiscoincided with the visible loss of flocculation.

Yeast flocculation and cell surface fimbriae

The yeast cell wall is a complex structure consisting ofmannan, glucan, protein, chitin, lipid and a number ofother compounds (Fig. 9)66. Flocculation requires thepresence of cell surface proteins and mannan receptors80.If these are not available, masked, blocked, inhibited ordenatured, flocculation cannot occur. Onset of floccula-tion is an aspect of the subject where there is significantcommercial interest, but about which little is understood.The ideal brewing strain is one which in a typical fermen-

tation, without the use of a centrifuge, remains in suspen-sion as fermenting single cells until close to the end offermentation, when the wort sugars and most amino acidshave been utilised (details later) and the vicinal diketones(diacetyl, etc.) are reduced. Only then will the culture rap-idly flocculate and settle out of suspension to be harvestedand re-pitched into a subsequent fermentation126,172. Whatsignals the onset of flocculation? This is still an unan-swered question162.

Electron microscopy of flocculent and non-flocculentbrewer’s yeast cultures shadowed with tungsten oxide hasrevealed that flocculent cultures possess a “hairy” outersurface (called fimbriae), whereas, non-flocculent culturesdo not contain cell surface fimbriae (Fig. 10)40. This

observation has been re-confirmed by recent studies125,163.There are a number of treatments that will result in de-

flocculation of flocculent cultures that contain fimbriaeprior to treatment. Treatments include protease and shearin a blender, both of which result in irreversible loss offlocculation and these cultures no longer contain cell sur-face fimbriae. It is interesting to note that when these de-flocculated fimbriae-less cultures were recultured in wort,they became flocculent again when they entered late loga-rithmic growth phase and they also once again containedcell surface fimbriae.

Table I. Total calcium adsorbed by flocculent and non-flocculent yeastcultures.

Yeast culture

Flocculation

characteristics

Calcium adsorbed (mg/mg

of dry weight yeast)

Ale Non-flocculent 258Ale Non-flocculent 200Ale Non-flocculent 144Ale Flocculent 203Ale Flocculent 244Ale Flocculent 207

Lager Non-flocculent 214Lager Non-flocculent 256Lager Flocculent 272Lager Flocculent 189Lager Flocculent 191

Table II. Calcium removed from flocculent and non-flocculent culturesduring de-flocculation washings.

Yeast cultureFlocculation

characteristic

Total calcium washed off

yeast (mg/100 mg of dryweight yeast)

Ale Non-flocculent 18Ale Non-flocculent 19Ale Flocculent 30

Ale Flocculent 42Lager Non-flocculent 12Lager Non-flocculent 14Lager Flocculent 20Lager Flocculent 22

Fig. 9. Schematic diagram of the architecture of the yeast cellwall. Components such as mannoprotein are found distributedthroughout the entire wall and therefore the layered arrangementshows zones of enrichment.


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Genetic control of yeast flocculation

Genetic studies on yeast flocculation began over 50years ago. The first publication on this subject was byPomper and Burkholder97 who reported crossing a haploidculture that possessed a “dispersed” character (non-floc-culent) with a haploid culture that possessed a “non-dis-perse” character (flocculent). The “disperse” characterwas reported to be dominant over the non-disperse charac-ter. In the early 1950’s, Gilliland and Thorne independ-ently carried out extensive studies on the genetics of yeast

flocculation. Gilliland49 studied two non-brewing strainsof S. cerevisiae which differed only in their flocculationproperties. It was proposed that a single gene was respon-sible for the flocculence phenotype. Thorne166 confirmedGilliland’s studies and demonstrated that flocculence wasan inherited characteristic and that flocculence was domi-nant over non-flocculence.

As has already been discussed, and will be described ingreater detail later, genetic work involving brewer’s yeaststrains is fraught with difficulties because of their frequenttriploid, polyploid, or aneuploid nature147. Consequently,studies have focussed on haploid and diploid flocculentand non-flocculent strains. The flocculation characteristicstudied was reversible and induced by calcium ions. The

flocculent haploid strain (coded 169) was of oppositemating type to the non-flocculent haploid strain (coded168). These two strains were mated using micromanip-ulation techniques and the resulting diploid hybrid(169/168) was found to be flocculent, confirming previousfindings that the flocculent character was dominant andstable159.

Tetrad analysis of spores isolated from asci of the169/168 hybrid revealed that the dominant flocculence ofstrain 169 was controlled by a single gene locus (i.e. 2:2segregation). This gene has been coded FLO1. The next

question was the location of the gene on one of the 16chromosomes of Saccharomyces. A detailed discussion ofthe chromosome mapping procedures employed in thisstudy is beyond the scope of this paper. Suffice to say, ithas been shown that FLO1 is located on Chromosome I,33 cM from the centromere on the right hand side of thechromosome109,159.

The mapping of the FLO1 gene employed traditionalgene mapping techniques (mating, sporulation, microma-nipulation, tetrad analysis, etc.). Today novel genetic tech-

niques have been developed, the principle of which is thesequencing of the Saccharomyces genome76. This has ex-panded our knowledge of the genetic control of yeastflocculation. Flocculation genes identified to date includeFLO1, FLO2, flo3, FLO4, FLO5, flo6, FLO7, FLO8,FLO9, FLO10, FLO11 and MUC1165. Of all the floccula-tion genes identified, FLO1 is the most extensively stud-ied and perhaps the most important and capable of con-ferring flocculation when transformed into non-flocculentS. cerevisiae strains165.

STORAGE AND PRESERVATIONOF STOCK YEAST CULTURES

The development of yeast propagation techniques byHansen and Jansen has already been discussed. Of equalimportance to the actual scale-up facilities and proceduresis the maintenance of the stock yeast cultures betweenpropagations. This became even more important with theadvent of the use of ale yeast single strains, along withlager strains. The long term preservation of a brewingyeast strain requires that not only is optimal survival im-portant, but it is imperative that no change in the characterof the yeast strain occurs. Many yeast strains are difficultto maintain in a stable state and long term preservation by

Fig. 10. Electron photomicrographs of Saccharomyces cerevisiae flocculent and non-flocculent shadow – cast withtungsten oxide.


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lyophilisation (freeze drying), which has proven useful formycelial fungi and bacteria64, has been found to give poorresults with brewing yeast strains65. Storage studies havebeen conducted with a number of ale and lager brewingstrains108. The following storage conditions were inves-tigated:

• Low temperature [70°C refrigeration or liquid nitro-gen (196°C)];

• Lyophilisation (freeze drying);

•

Storage in distilled water;• Storage under oil;• Repeated direct transfer on culture media (subcultured

once a week for two years);• Long term storage at 21°C on solid nutrient medium –

subcultured every six months;• Long term storage at 4°C on solid nutrient medium –

subcultured every six months.After a two year storage period, wort fermentation tests

including fermentation rate and wort sugar uptake effi-ciency, flocculation characteristics, sporulation ability,formation of respiratory deficient colonies and ease ofsurvival were conducted and the results compared to thecharacteristics of the stored control culture. Low tempera-

ture storage appears to be the storage method of choice, ifcost and availability of the appropriate equipment is not asignificant factor. Cultures stored either at 70°C or inliquid nitrogen168 had the lowest death rate and were theeasiest to revitalise. Also, the degree of flocculation, wortfermentation properties, sporulation ability and proportionof respiratory deficient mutants present were all unaf-fected by this storage method. Storage at 4°C on nutrientagar slopes, subcultured every 6 months, was the nextmethod of preference to low temperature storage. Lyo-philisation and other storage methods revealed yeast insta-bility, which varied from strain to strain. Currently, manybreweries store their strains (or contract store them) at70°C. Routine subculturing of cultures on solid media

every six months is less desirable, but a very cost effectivestorage method. Lyophilisation of brewer’s yeast culturesshould be avoided!

GENETIC MANIPULATIONOF BREWER’S YEAST STRAINS

The behaviour, performance and quality of a yeaststrain is influenced by two sets of determining factors, col-lectively called nature-nurture effects. The nurture effectsare all the environmental factors (i.e. the phenotypes), towhich the yeast is subjected from pitching onwards. Onthe other hand, the nature influence is the genetic make-

up (i.e. the genotype) of a particular yeast strain.It has already been stated in this paper that the expec-

tation that genetically manipulated yeast strains would beemployed in brewing has not been achieved. In 1986 westated132: “The use of manipulated yeast strains in brewingwill become commonplace within the next decade withyeast strains specifically bred for such characteristics asextra-cellular amylases, β-glucanases, proteinases, β-glu-cosidase production, pentose and lactose utilisation, car-bon catabolite repression and production of a plethora ofheterologous proteins. There is no doubt that prior to the

introduction of such strains at the production level, theenvironmental and legal impact of such a move will haveto be assessed”. Over 20 years later, genetically manipu-lated brewer’s yeast strains are still not employed com-mercially, due in large part because of opposition frompublic opinion. Whether this will change, only time willtell! Nevertheless, genetic techniques have been used tostudy the genetic composition and function of suchstrains.

There are a number of methods that can be employedin the genetic research and development of brewer’s134 and related yeast strains55. Classical approaches to strainimprovement include mutation and selection7, screeningand cross-breeding (hybridisation)146,48. The use of hy-bridisation to map FLO1 on Chromosome I of the Sac-charomyces genome has already been described. Mutationis any change that alters the structure of the DNA mol-ecule, thus modifying the genetic material. The muta-genised strains often no longer exhibit many desirableproperties of the parent strain and exhibit slower growthrates and produce undesirable taste and aroma compoundsduring fermentation145. Mutagenesis is seldom employedwith brewing strains due to their polyploid/aneuploid na-

ture.Spontaneous yeast mutations are a common occurrence

throughout the growth and fermentation cycle, but theyare usually recessive, due to functional loss of a singlegene56. Because of the aneuploid or polyploid nature ofmost strains, the dominant gene will function adequatelyin the strain, as it will be phenotypically normal. Only ifthe mutation takes place in all complementary genes willthe recessive character be expressed. However, if the mu-tation weakens the yeast, the mutated strain will be unableto compete and will soon be outgrown by the non-mutatedyeast population. The characteristics that are routinely en-countered resulting from mutations that are harmful to awort fermentation are:

•

The tendency of yeast strains to mutate from flocculentto non-flocculent133;

• The loss of ability to ferment maltotriose111;• The presence of respiratory deficient mutants85.

The respiratory deficient (RD) or “petite” mutation isthe most frequently identified mutant found in brewingyeast strains. This mutant arises spontaneously when asegment of the DNA in the mitochondria becomes defec-tive to form a flawed mitochondrial genome. The mito-chondria are then unable to synthesise certain proteins.This type of mutation is also called the “petite” mutationbecause colonies of such a mutant are usually muchsmaller than respiratory sufficient (RS) colonies (alsocalled “grande”) (Fig. 11). The RD mutation usually

occurs at frequencies of between 0.5 and 5% of the popu-lation, but in some strains, levels as high as 50% havebeen reported121. RD mutants can also occur as a result ofdeficiencies in nuclear DNA, but these are much rarer.

Deficiencies in mitochondrial function result in dimin-ished ability to function aerobically and as a result theseyeasts are unable to metabolise non-fermentable carbonsources such as lactate, glycerol or ethanol (Fig. 12).Many phenotypic effects occur as a result of this mutationand include alteration in sugar uptake (particularly malt-ose and maltotriose), by-product formation and subse-


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VOL. 115, NO. 1, 2009 11

quent metabolism (for example, diacetyl), and intoleranceto stress factors such as ethanol, osmotic pressure andtemperature. Also, further to the discussion of “storageand preservation of stock yeast cultures”, RD mutants aredifficult to store and liquid nitrogen and 70°C refrigera-tion have both been found to be the most effective storagemethods108. Flocculation, cell wall and plasma membranestructure, and cellular morphology are affected by this RDmutation.

Beer produced with a yeast culture that is RD is likelyto have flavour defects and fermentation problems. Forexample, beer produced from these mutants contained ele-vated levels of diacetyl and higher alcohols122. Wort fer-mentation rates were slower, higher dead cell counts wereobserved and biomass production and flocculation abilitywere reduced45,51.

The advent of the “new biotechnology” has stimulatedthe development of novel methods of genetic manipu-lation141 including spheroplast (protoplast) fusion andrecombinant DNA. Spheroplast fusion is a technique thatcan be employed in the genetic manipulation of brewer’syeast strains110. The method does not depend on ploidyand mating type and consequently has great applicabilityto such strains because of their polyploid nature andabsence of mating type characteristics. Details of the

procedure can be found elsewhere155. Examples ofsuccessful fusions with commercial brewing and relatedstrains are:

• The construction of a brewing yeast with amylolyticactivity by fusion of S. cerevisiae and S. diastaticus113;

• A polyploid strain capable of high ethanol productionby fusion of a flocculent strain with saké yeasts90 and

• Construction of strains with improved osmotoleranceby fusion of S. diastaticus and S. rouxii (an osmotol-erant yeast strain)112,123.Although spheroplast fusion is an extremely efficient

technique, it relies mainly on trial and error and is notspecific enough to modify strains in a predictable manner.The fusion product is nearly always very different from

both the original fusion partners, because the genome ofboth strains becomes integrated. Spheroplast fusion hasbeen found to be a viable technique when flavour of thefinal product is not critical. For example, fusion productswere constructed that could survive high osmotic pres-sure, elevated fermentation temperatures (ca>40°C) andshowed increased ethanol tolerance152. Such strains arebeing used successfully in the industrial alcohol industry,but produce beer with unsatisfactory beer flavour/tasteprofiles.

Recombinant DNA techniques can also be used tomake thousands of copies of the same DNA molecule toamplify DNA, thus generating sufficient DNA for variouskinds of experiments or analysis. Although the author and

his colleagues have not employed these techniques to im-prove brewer’s yeast strains, other groups55 have success-

Fig. 12. Growth of respiratory sufficient (RS) and respiratory deficit (RD) cultures on fermentable (glucose) and non-fermentable (lactate) carbon sources.

Fig. 11. Respiratory sufficient and respiratory deficient mutants– triphenyl tetrazolium chloride overlay.


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fully conducted such experiments to “improve” brewer’syeast strains. Examples include:

• Glucoamylase activity from the fungus Aspergillus niger ;

• Glucanase activity from the bacterium Bacillus subti-lis, the fungus Trichoderma reesii and from barley;

• α-Acetolactate decarboxylase activity from the bacte-ria Enterobacter aerogenes and Acetobacter spp. fordiacetyl control;

•

Extracellular protease for chill-proofing beer; and• Modification of the yeast’s flocculation properties.

The future prospects for the use of recombinant DNAwith brewer’s and distiller’s yeast in the industry are un-clear. It is surprising that recombinant brewer’s yeasts arenot commercially in use today, in both brewing anddistilling, but public opinion does not support their use.Since the early days of yeast genetic manipulation therehas been considerable activity, which has been succinctlydescribed by Boulton and Quain10. Permission wasgranted over a decade ago from the appropriate authoritiesin the United Kingdom for the use of a baker’s yeaststrain that is genetically manipulated for more rapid malt-ose use, leading to enhanced baking properties, and for a

brewing strain cloned with DNA from S. diastaticus, thatsecretes glucoamylase to utilise wort dextrins and producelow calorie beer48.

The sequencing of the S. cerevisiae genome began in1989 and was completed with the publication of thesequence in 199650. Although a major achievement, theDNA sequence of S. cerevisiae is relatively small, with agenome size of 13.5 Mb compared to the genome of manwith a genome size of the order of 3500 Mb88.

Recently, the Australian Wine Research Institute, incollaboration with the Australian Genome Research Fa-cility, has sequenced the DNA of a wine yeast strain9. It isinteresting to note that this study took approximately sixmonths to complete. The first yeast strain to be sequenced

involved 70 laboratories, took 10 years and cost millionsof dollars!

The sequencing of the yeast genome, in conjunctionwith gene expression analysis, has enabled the identifica-tion of genes that have altered gene expression patterns inresponse to stressful environmental conditions18. Alltech,in collaboration with Heriot-Watt University, has studiedthe inhibitory effects of the stress factors most commonlyencountered during alcoholic fermentation of corn mash-es53. The key stress related yeast genes with different re-sistance levels to environmental stress have been assessed.A “stress model” has been developed to assess yeast stressresistance and evaluate the suitability of a specific strainfor use in industrial ethanol fermentations52. This “stress

model” could potentially be used for screening candidateyeast strains for relative stress resistance in the fuelethanol industry, and other fermentation industries, whereyeast encounters similar stresses.

UPTAKE AND METABOLISMOF WORT SUGARS, AMINO ACIDS

AND PEPTIDESCompared to other media employed for the production

of fermentation alcohol, both industrial and potable, wort

is by far the most complex. As a consequence of this,when yeast is pitched into wort it is introduced into anintricate environment because it consists of simple sugars,dextrins, amino acids, peptides, proteins, vitamins, ionssuch as zinc, magnesium, manganese, calcium, sodiumand potassium, nucleic acids and other constituents toonumerous to mention. One of the major advances in brew-ing science, during the past 40 years, has been the eluci-dation of the mechanisms by which the yeast cell utilises,in an orderly manner, the plethora of wort nutrients.

Reference has already been made to the uptake of wort

sugars, in the context of premature flocculation of co-flocculent ale cultures, resulting in residual maltotriose inthe fermented wort. Quantitative determination of wortsugars has evolved during the past 40 years. The initialexperiments employed paper and thin layer chromatogra-phy95, followed by gas chromatography (GC) whichinvolved pre-derivatisation to make the sugar volatilewhen injected into the GC94. Current methods for wortsugar determination employ high performance liquidchromatography (HPLC)89. Similar to wort sugars, thedetermination of the 19 amino acids in wort has alsodeveloped over time. The pioneering studies on wortamino acid composition and uptake by yeast were con-ducted by Jones and Pierce62 (Fig. 13) who employed

rudimentary liquid chromatography, compared to today’sHPLC techniques, however, they still established the fun-damentals of the order of amino acid uptake.

Wort sugar uptake

Wort contains the sugars sucrose, glucose, fructose,maltose and maltotriose together with dextrin material. Inthe normal situation, brewing yeasts are capable of uti-lising sucrose, glucose, fructose, maltose and maltotriose,in this approximate sequence (or priority), although somedegree of overlap does occur, leaving maltotetraose and

Fig. 13. John Pierce and Margaret Jones.


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the other dextrins unfermented36 (Fig. 14). Brewer’s yeastis also capable of utilising sugars such as galactose, butnot lactose, unless it is hydrolysed to its constituentmonosaccharides31,32. In addition, there is a closely relatedspecies (regarded as the same species by some) to S. cere-visiae, designated as S. diastaticus. This yeast speciesproduces an extracellular glucoamylase that is capable ofhydrolysing wort dextrins to glucose, a sugar that is easilymetabolised46,47.

Maltose and maltotriose are the major sugars in brew-

er’s wort (Table III), and as a consequence, a brewingyeast’s ability to use these two sugars is vital and dependsupon the correct genetic complement153. Brewer’s yeastcultures possess independent uptake mechanisms (maltoseand maltotriose permease) to transport these two sugarsacross the cell membrane into the cell119. Once inside thecell, both sugars are hydrolysed to glucose units by the α-glucosidase system (Fig. 15). The transport, hydrolysisand fermentation of maltose are particularly important inbeer production, in Scotch whisky production and inbaking, since maltose is the major component of brewing

wort, spirit mash in Scotland and wheat dough. Maltosefermentation by Saccharomyces sp. requires the presencein the genome of one of five unlinked MAL loci, eachconsisting of three genes encoding the structural gene forα-glucosidase (maltase) ( MALS ), maltose permease( MALT ) and an activator ( MALR), whose product co-ordinately regulates the expression of the α-glucosidase

and permease genes41. The expression of MALS and MALT is regulated by maltose induction and repressed byglucose25.

Indeed, a major limiting factor in the fermentation ofwort is the repressing influence of glucose (and possiblyfructose) upon maltose and maltotriose uptake. Onlywhen approximately 50% of the wort glucose has beentaken up by the yeast cells will the uptake of maltosecommence (Fig. 14). This is yeast strain and wort com-position dependent38,39. In other words, in most strains ofS. cerevisiae and related species, maltose utilization issubject to control by carbon catabolite repression115. In asimilar manner, the presence of glucose will repress theproduction of glucoamylase by S. diastaticus, thereby in-

hibiting the hydrolysis of wort dextrins and starch75. Re-pression of this nature has a negative effect on overallfermentation rate.

Studies have also been conducted where glucose isadded to fermenting wort when the yeast strain is metabo-lising maltose and has already taken up all of the availablewort glucose. The added glucose caused inhibition (re-pression) of the maltose uptake. Once this glucose hadbeen taken up by the yeast culture, the metabolism ofmaltose recommenced43,44,146,147.

In order to try to overcome this repression, the glucoseanalogue, 2-deoxy-glucose (2-DOG) has been success-fully employed for the selective isolation of spontaneousmutants of yeasts114,167 and other fungi4 (Fig. 16). These

mutants were derepressed for the production of carbohy-drate-hydrolysing enzymes employing this non-metabolis-able glucose analogue. Derepressed mutants of brewingand other industrial strains have been isolated that are ableto metabolise wort maltose and maltotriose in the pres-ence of glucose (Fig. 17). Fermentation and ethanol for-mation rates in 12°Plato wort were also increased in the 2-DOG mutants, when compared with the parental strain. Inaddition, 2-DOG starch mutants of S. diastaticus havebeen isolated that exhibited increased fermentation abilityin brewer’s wort, cassava and corn mash171.

Fig. 14. Order of uptake of sugars by yeast from wort.

Table III. Typical sugar spectrum of wort.

Percent composition

Glucose 10–15Fructose 1–2Sucrose 1–2Maltose 50–60Maltotriose 15–20

Dextrins 20–30

Fig. 15. Uptake of sugars by the yeast cell.

Fig. 16. Structure of D-glucose and 2-deoxy-D-glucose.


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In order to illustrate the phenomenon of derepressionand the effect of 2-DOG mutants on overall fermentationrate of a brewer’s wort, several strains of S. cerevisiae have been studied63,143. In all instances, the presence ofglucose derepressed the uptake of maltose. Numerous sta-ble 2-DOG mutants were found to be capable of utilisingmaltose in the presence of significant concentrations ofglucose82,83 (Fig. 17).

All of our studies with 2-DOG mutants discussed

above were conducted with ale and distilling yeast strains.We were unable to isolate 2-DOG mutants from the lageryeast strains that were screened. This is the major reasonwhy large scale trials were not conducted with 2-DOGmutants in a country (Canada), which by the late 1980s,was predominantly a lager producer. Recently, a major

Spanish brewing company, with university collaborators,has re-examined the use of 2-DOG mutants to ferment25°Plato wort, this time with a lager yeast strain96. Stable2-DOG mutants of their lager yeast strain were isolatedand their fermentation characteristics in 25°Plato wort,using 2 L EBC tubes, were assessed at 13°C. Improvedfermentation capacity, where wort glucose did not repressmaltose uptake, was achieved without changes in the beerflavour profile.

Uptake of wort maltose and maltotriose –differences and similarities between aleand lager yeast strains

A number of ale and lager yeast strains have beenemployed in order to explore the mechanisms of maltoseand maltotriose uptake from wort. A 16°Plato all maltwort was used in a 30 L static fermentation (Fig. 18).Under these conditions, lager strains utilised maltotriosemore efficiently than ale strains, whereas maltose utilisa-tion efficiency was not dependent on the type of brewingstrain employed158,177. Although strain to strain variationswere observed, this supports the proposition, already dis-cussed, that maltotriose and maltose possess independent,

but closely linked, uptake (permease) systems178. In addi-tion, this consistent difference between ale and lagerstrains supports the observation93 that ale strains appear tohave greater difficulties completely fermenting wort (par-ticularly high gravity wort) than lager strains.

Free amino nitrogen in wort and beer

The individual wort amino acids, ammonium ions andsmall peptides (di- and tripeptides) are known collectivelyas free amino nitrogen (FAN)103. FAN is believed to be agood index for potential yeast growth and fermentationefficiency164. Adequate levels of FAN in wort ensure effi-cient yeast cell growth and hence, a desirable fermenta-tion performance84. FAN is only a general measurementand a “blunt” instrument for setting wort and, ultimately,malt specifications. The objective of this study was to elu-cidate the role of different nitrogen wort components onyeast fermentation.

Static fermentations were conducted in 2 L cylindersusing 15°Plato wort containing 30% very high maltose

Fig. 18. Maltotriose (A) and maltose (B) uptake profiles from 16°Plato wort – 30 L static fermentation at 15°C.

Fig. 17. Degree of Plato reduction (A) on ethanol production (B)by an ale brewing strain and its 2-DOG derepressed variants.


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syrup (VHMS), supplemented with zinc sulphate (0.2mg/L). A lager and an ale yeast strain were employed forfermentation at 13°C and 20°C respectively. Sampleswere taken throughout the fermentations and yeast in sus-pension, specific gravity, total FAN (Fig. 19), ammonia,individual amino acids, di and tripeptide levels andproteinase activity determined69 (Fig. 20). A novel methodfor the determination of di- and tripeptides was em-ployed70. Following yeast removal and protein precipita-

tion, the samples were filtered through an ultra-filtrationmembrane (molecular weight exclusion of 500 Daltons)and hydrolysis followed by HPLC was employed to deter-mine the resulting amino acids.

The results confirmed Jones and Pierce’s findings62 thatamino acid uptake can be divided into four groups (TableIV) (Fig. 21) with amino acid uptake completed, with theexception of proline, within the first 48 h of fermentation.Peptide removal commenced during the first 19 h of

fermentation, increased between 19–24 h of fermentationand between 24–67 h of fermentation decreased gradually(Fig. 22). The important finding is that yeast fermentationactivity does not cease when wort FAN is depleted70. Dur-ing fermentation, oligopeptides are produced as a result oflarger peptide hydrolysis due to protease excretion/secre-tion (Fig. 23). Both lager and ale yeast strains can simul-taneously use amino acids and small peptides as sourcesof assimilable nitrogen. The implications of yeast protease

secretion on beer foam stability, particularly during highgravity brewing, will be discussed later.

Fig. 20. Total nitrogen fermentation absorption profile for atypical lager yeast strain with 15°Plato wort. □, Totaloligopeptides; , proline; , total ammonia; , total aminoacids.

Fig. 22. Total nitrogen absorption profile for a typical ale yeaststrain during fermentation of a 15°Plato wort. □, Totaloligopeptides; , proline; , total ammonia; , total aminoacids.

Fig. 21. Amino acid absorption pattern for a typical ale yeaststrain with a 15°Plato wort. □, Group A; , group B; , group C;, proline.

Fig. 23. Proteinase activity for ale and lager yeast strains duringfermentation of a 15°Plato wort. , Proteinase lager activity; □,proteinase ale activity.

Table IV. The order of wort amino acid uptake during fermentation.

Group A: fastabsorption

Group B:

intermediateabsorption

Group C: slowabsorption

Group D:

little or noabsorption

Glutamic acid Valine Glycine ProlineAspartic acid Methionine PhenylalanineAsparagine Leucine TyrosineGlutamine Isoleucine TryptophanSerine Histidine AlanineThreonine AmmoniaLysineArginine

Fig. 19. Fermentation profile of a typical lager yeast strain with15°Plato wort. □, FAN; , S.G.; , pH; , number of suspendedcells.


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INFLUENCE OF WORT CLARITYON ESTER FORMATION

The level of wort solids (trub) carried over from cerealmashing and grape crushing has been the subject of

considerable debate in the production of beer, whisky andwine54,77,116.

Wort solids influence:• The removal of CO2 from solution during fermentation

by acting as nucleation sites. Removal of CO2 fromsolution can occur with no suspended particles but re-quires a great deal of energy68.

• Solids confer nutritive value to the wort. The rate offermentation is faster in the presence of insoluble ma-terial. Wort solids are generally associated with higherlevels of fatty acids71.

• In some instances, yeast cells are able to attach them-selves to wort solids and display enhanced growth be-cause of the concentration of nutrients at the particle

surface.In order to assess the influence of wort clarity on ester

formation (ethyl acetate and isoamyl acetate), 15°Platowort was produced in the ICBD 2 hL pilot plant (Fig. 24)and a number of wort types fermented in 1 L volumes in 2L cylinders at 27°C employing the S. cerevisiae “M”strain – a distilling strain obtained from Kerry Biosci-ences, Menstrie, Scotland. Carbon dioxide evolution ratesduring fermentation and ethyl acetate and isoamyl acetateconcentrations at the end of fermentation were deter-mined129. The following wort types were studied:

• Cloudy wort• Clear wort• Clear wort plus 0.2 g/L diatomaceous earth (DE)• Clear wort plus DE and 5.5 mg/L C16:1 fatty acid• Clear wort plus 0.2g/L bentonite.

The concentration of CO2 during fermentation of the15°Plato wort types was determined (Table V). Cloudywort, containing trub, and wort with DE acted as nuclea-tion sites and increased CO2 evolution out of the wort.Clear wort and wort plus bentonite did not function asnucleation sites and consequently CO2 remained in the

fermenting medium to a much greater extent. Why wasthere a difference between trub, DE and bentonite? Inorder to visualise each type of particle and obtain data ontheir surface characteristics, environmental scanning elec-tron microscopy (ESEM) was conducted (Fig. 25). In DE,there was a heterogeneous mix of particle shapes andsizes. The surface of most of the particles had an ex-tremely porous structure, as would be expected. The mi-crograph of the cloudy wort solids showed a mix ofdifferent structures, again porous in nature. Bentonite hada more homogeneous structure. In addition, it possessed a

Fig. 24. The ICBD 2 hL pilot brewery.

Table V. Concentration of carbon dioxide present during fermentation.

24 hours (g/L) 48 hours (g/L)

Cloudy wort 2 4Clear wort 5 8Clear wort plus diatomaceous earth 2 4Clear wort plus bentonite 5 8


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much different surface topology. It did not appear topossess the same porous nature as DE or wort solids.

Ester levels are also influenced by the particle concen-tration and type in wort. Ethyl acetate and isoamyl acetateconcentrations are high in cloudy wort and DE containingC16:1 fatty acid (Table VI). This reflects the fact thatunsaturated fatty acids, that would be absorbed onto worttrub, induce the synthesis of esters74. The influence ofmashing conditions under controlled circumstances hasbeen studied using the ICBD 2 hL pilot brewery157. Mash-ing conditions influence wort solid material levels. Thesesolids enhance the wort fatty acid composition, which re-sults in increased yeast growth and viability. This in turn,results in decreased ester and increased higher alcoholconcentrations in the fermented wort.

HIGH GRAVITY BREWING –ITS EFFECTS ON YEAST STRAIN

AND BEER STABILITY

During the past 30 years, process optimization and in-creased efficiencies have been priorities for many brewingcompanies worldwide138. Process intensification has be-come part of this endeavour and has focussed on reducedcapital expenditure142, increased fermentation rates and

final attenuation37, high quality yeast viability and vital-ity17, decreased maturation times92, more efficient stabili-sation and filtration140, enhanced beer quality and stabil-ity135 and high gravity brewing81.

Various studies with high gravity worts have alreadybeen discussed in this paper. High gravity brewing em-ploys wort at higher than normal concentration, and con-sequently requires dilution with water (usually de-oxy-genated) at a later stage in processing81. By this means, in-creased production demands can be met without expand-

ing brewing, fermenting and storage facilities.

The use of high gravity brewing,followed by appropriate dilution

Dilution of high gravity wort, before or after fermenta-tion, requires that the water employed be given specialtreatment. The specifications of the treatment procedurewill vary depending on the dilution point81. The longer thefermented wort is maintained undiluted, the greater thecapacity efficiency. Consequently, most breweries addwater to the concentrated beer immediately prior to thefinal polishing filter. Dilution at this point requires thatthe water is specially treated to secure biological purityand chemical consistency. Most importantly, the dissolved

oxygen (DO) content of the dilution water must be re-duced to approximately 50–100 μg/L (DO), in order toenhance beer flavour stability.

High gravity brewing began in the United States in theearly 1960s and spread throughout North America, Aus-tralia and South Africa. Taxation and regulation difficul-ties impeded its implementation in a number of Europeancountries. Regulation problems have now largely beenovercome and high gravity brewing can now be imple-mented worldwide without financial penalties. Neverthe-less, the impact on the flavour and foam stability of cer-

Table VI. Concentration of ethyl acetate and isoamyl acetate following160 h fermentation of different wort types.

Ethyl acetate(mg/L)

Isoamyl acetate(mg/L)

Cloudy wort 30 0.95Clear wort 16 0.55Clear wort plus diatomaceous

earth and C16:1 fatty acid 25 0.75

Fig. 25. Environmental scanning electron microscopy (ESEM) of different solid materials.


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tain product types at high gravity remains a concern andchallenge to some breweries.

There are a number of advantages and disadvantages tothis process. The advantages can be summarised as fol-lows81:

• Increased brewing capacity, more efficient use of exist-ing plant facilities – reduced capital expenditure;

• 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 consequently more of thewort sugars being converted to alcohol;

• High gravity worts may contain higher adjunct rates;• Although largely anecdotal, beers produced from high

gravity worts are often rated smoother in taste; and• High gravity brewing offers greater flexibility in prod-

uct type. From one “mother” liquid a number of prod-ucts can be brewed as a result of dilution and/or use ofmalt and hop extracts, syrups and stabilisers.Although these advantages are significant, as with

most important processes, high gravity brewing has anumber of disadvantages142:

•

Due to the more concentrated mash (increased ratio ofcarbohydrate to water), there is decreased brewhousematerial efficiency and reduced kettle hop utilisation.This problem can be alleviated by the use of modernmash filters58 (instead of a lauter tun), kettle syrups127 and/or hop extracts107;

• Decreased foam stability (head retention)11,30 – detailslater;

• There can be difficulty in achieving beer flavour matchcompared to comparable lower gravity beers1,169,175.The effects of high gravity wort on ester formation andthe influence of wort spectra (ratio of glucose to mal-tose) will be discussed later. However, flavour prob-lems with high gravity brewed beers have been exag-

gerated and adjustments to the process can make adifference. Particularly important is the yeast pitchingrate and the wort dissolved oxygen level at the begin-ning of fermentation86. As a “rule of thumb”, 1 mil-lion/mL viable yeast cells are pitched for every wort°Plato (i.e., a 16°Plato wort would be pitched with 16million cells/mL) and 1 ppm dissolved oxygen (DO)for every wort °Plato (i.e., a 16°Plato wort would re-quire 16 ppm DO) at the beginning of fermentation;and

• High gravity worts can influence yeast performancewith effects apparent upon fermentation and floccula-tion101. The increased osmotic pressure, elevated alco-hol concentration and modified nutrient balance, all

have a profound influence on yeast performance duringthe fermentation of high gravity worts – details later.Another negative effect of high gravity worts on yeastperformance concerns the number of generations(yeast cycles) that can be fermented by a single yeastculture. The number of cycles that can be employed isreduced with increasing wort gravity. The use of theLabatt lager yeast strain exhibited the following speci-fications:

12°Plato 16°Plato)136.

Influence of high gravity worts

on yeast viabilityWhen yeast is first pitched into high gravity wort,

passive diffusion of water out of the cell occurs and thisresults in a decrease in cell viability (determined bymethylene blue and methylene violet staining). Figures 26and 27 illustrate experiments with 12 and 20°Plato wortsfermented with a lager and an ale yeast strain. Cell via-bility decreased in both strains within the first 24 h offermentation99. However, the decrease in viability wasexacerbated with the 20°Plato compared to the 12°Platowort. However, with both yeast types (which are repre-sentative of a number of lager and ale strains studied 99 –data not shown) the viability recovered later in the fer-mentation. In addition, for reasons that are unclear, ale

strains maintained higher viability than lager strains102.

Fig. 26. Effect of wort gravity on the viability of a lager yeaststrain. Viability determined by the methylene blue stainingmethod.

Fig. 27. Effect of wort gravity on the viability of an ale yeaststrain. Viability determined by the methylene blue stainingmethod.


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Effects of stress on yeast intracellular storagecarbohydrates

Saccharomyces and related species, including brewer’syeast strains, contain two major intracellular storage car-bohydrates – trehalose and glycogen. Trehalose is a disac-charide containing glucose units (Fig. 28). It protects thecell against stress (for example, osmotic pressure, ethanol,high and low temperature and desiccation)87. It has beencorrelated with cell survival under adverse conditions and

is also an important stress indicator in brewing yeast cul-tures during high gravity wort fermentation (Figs. 29 and30). There was rapid synthesis of trehalose in 20°Platowort during the first 24 h of fermentation. As the culturesacclimatised to the stress conditions imposed by this wort,the intracellular trehalose levels decreased. It is interestingto note that lager strains maintained higher trehalose lev-els than ale strains99.

Glycogen is an intracellular glucose polysaccharide

with a structure similar to starch consisting of α 1,4 link-ages with α 1,6 branch points (Fig. 31). It is the majorreserve energy storage material in yeast cells and manyother organisms (including humans). Glycogen accumu-lates in yeast under nutrient limiting conditions. It has arole in providing carbon and energy for the maintenanceof cellular activities104. During the first 6–8 h of wortfermentation there is rapid utilisation of intracellular gly-cogen (Fig. 32). This utilisation is directly proportional tothe synthesis of lipids [mainly unsaturated fatty acids(UFA) and sterols (ergosterol)]. These lipids are employedby the cells to produce de novo membrane material duringcell division. Once cell division begins to decrease, glyco-

Fig. 30. Effect of wort gravity on trehalose metabolism in an aleyeast strain.

Fig. 32. Intracellular concentrations of glycogen and lipids in alager yeast strain during fermentation of a 15°Plato wort.

Fig. 29. Effect of wort gravity on trehalose metabolism in alager yeast strain.

Fig. 28. Structure of trehalose.

Fig. 31. Structure of glycogen.


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gen accumulates. It is important that maximum levels ofintracellular glycogen are present in the yeast culturewhen it is harvested for storage, prior to being re-pitchedinto a subsequent wort fermentation. It is critical that gly-cogen levels in yeast are conserved during storage be-cause depleted glycogen levels will lead to incompletefermentation.

Another form of stress imposed upon brewer’s yeast isthe process of acid washing. It has been shown to be aneffective procedure to remove bacterial contaminants fromyeast slurries2. The physiological condition of the yeastand various environmental factors have been shown toaffect the resistance of brewer’s yeast to acid washingconditions124. One such environmental condition is highgravity brewing35. Acid washing adversely affected yeastviability from a 20°Plato wort fermentation, whereasyeast from a 12°Plato wort fermentation were not affectedto the same extent34. Strain variations were observed be-

tween lager yeast strains in their resistance to high gravityconditions and acid washing33. The resistance to acidwashing was also influenced by storage conditions, withyeast that was stored poorly having the lowest yeastviability. Yeast management procedures must be opti-mised when repitching yeast from high gravity fermen-tation to ensure that the yeast is in good physiological

condition and can maintain its resistance to acid washing.

Yeast morphological changes inducedby high gravity worts

The yeast vacuole is an oval, intracellular body, whichexists as a single organelle or with several distinctcompartments and occupies approximately one third ofthe volume of the cell. It acts as a reservoir for storagenutrients and specific enzymes100. Its volume changeswith growth phase and environmental conditions. Yeastcells modify their vacuolar volume by increasing in size

Fig. 33. Effect of wort gravity on vacuole size with an ale yeast strain.

Fig. 34. Changes in the vacuolar morphology during high gravity wort fermentations with an ale yeast strain.


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VOL. 115, NO. 1, 2009 21

during the fermentation of 20°Plato wort compared to thesame yeast strain fermenting 12°Plato wort (Figs. 33 and34)98. As well as changes in vacuolar size, high gravitywort modifies the cell surface morphology of a yeastculture (Fig. 35). Viewed with a scanning electron micro-scope, cells fermenting a 20°Plato wort had a very unevencell surface topology compared to cells fermenting a

12°Plato wort. Those fermenting the low gravity wortexhibited a much smoother surface structure98.

Effect of high gravity brewingon beer foam stability

Beers brewed at higher gravities, followed by dilution,have poorer foam stability compared to similar beersbrewed at lower gravities37. It is known that specific hy-drophobic polypeptides play an important role in foamformation and stability3. Also important are isoalphaacids, metal ions and melanoidins. The level of hydro-phobic polypeptides has been determined throughout thebrewing and fermentation of high gravity (20°Plato) andlow gravity (10°Plato) worts. During brewing, there is aproportionately greater loss of hydrophobic polypeptideswith the 20°Plato wort than with the 10°Plato counter-

part16 (Fig. 36). When the high gravity beer was diluted to4.5% alcohol by volume, equivalent to the low gravitybeer, it contained a level of hydrophobic polypeptides thatwas less than 50% of the level produced in a low gravitybeer (Fig. 37).

The head retention of the diluted high gravity brewedbeer was less than that of the low gravity brewed beer28.This difference was apparent when both beers werepoured into 100 mL measuring cylinders and the timetaken for foam collapse determined. Two minutes afterpouring, there was very little foam or cling with thediluted high gravity beer sample, but substantial foam andcling remained on the low gravity beer sample (Fig. 38).Four minutes after pouring, differences in the foam sta-

bility of the two beer types were even more exacerbated(Fig. 39).

Fermentation is a key stage where hydrophobic poly-peptides are lost (Fig. 37). At least three factors wouldaccount for this loss27. Firstly, losses occur due to fer-menter foaming. During wort fermentation, a high gradi-ent of hydrophobic polypeptides towards the surface wasshown to occur12. This enhances adhesion of foam-activecompounds to the side of the fermenter vessel duringtransfer to the conditioning vessel. Secondly, foam-posi-tive hydrophobic polypeptides are lost as a result of hot

Fig. 35. Effect of wort gravity on the cell surface morphology of an ale yeast strain.

Fig. 36. Changes in hydrophobic polypeptide levels from kettlefull to final beer. Final beers diluted to 4.5% abv.

Fig. 37. Changes in hydrophobic polypeptide levels during thefermentation of low and high gravity worts. Final beers dilutedto 4.5% abv.


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and cold break formation12. Due to increasing polyphenol

levels in high gravity worts, a disproportionately greateramount of hydrophobic polypeptides are lost in high grav-ity worts due to hot and cold break precipitation, com-pared to lower gravity wort. Finally, yeast “secretes” pro-teolytic enzymes into the fermenting wort and theseappear to have a negative effect on the foam stability offinished beer due to polypeptide degradation that occursduring fermentation and storage. Proteinase A increasedthroughout fermentation (Fig. 40)29. Higher amounts ofproteinase A were released during a 20°Plato wort fer-mentation compared to the 10°Plato wort fermentation.

During high gravity wort fermentations, increased stresson the yeast, in the form of elevated osmotic pressure andethanol concentration, appears to have stimulated the se-cretion of proteinase A into the wort during fermenta-tion13.

Influence of wort sugar spectrumand gravity on ester formation

It has already been briefly discussed that one of the

disadvantages of this process is that fermentation of highgravity worts induces the production of disproportionatelyhigh levels of esters (Table VII)37. Varying the wort sugarsource has been reported173 to modify the level of manymetabolites, including esters, although reasons for thesedifferences are unclear. Entry of the hexose sugars, glu-cose and fructose, into the yeast cell is facilitated by thesame protein system, although utilisation of glucoseoccurs more quickly than fructose, when the two sugarsare fermented separately, possibly due to the differingaffinities of the sugars for the transporter8. It has alreadybeen discussed that the disaccharide maltose in wort isinternalised by the cell only when 40–50% of the glucosehas been removed from the wort38 and occurs via an active

transport system, whereas the uptake of glucose and fruc-tose is by passive transport173.

Initially 4% glucose and maltose in a synthetic me-dium (yeast extract – peptone medium) were fermentedseparately with shaking at 21°C, in order to eliminate anypossible inhibition of sugar uptake and the production ofethyl acetate and isoamyl acetate was monitored173. Thefermentation performance of three ale and three lagerbrewing yeast strains employed in this study was similar.Tables VIII and IX show the viabilities (determined bymethylene blue staining) and vitalities (determined by the

Table VIII. Percentage viability of brewing yeast strains after 96 hfermentation of synthetic mediaa.

Glucose Maltose

Ale 1 96 98Ale 2 92 98Ale 3 94 98Lager 1 97 99Lager 2 96 98Lager 3 95 99

aPeptone – yeast extract – 4% sugar medium. Methylene blue andmethylene violet stains employed.

Fig. 38. Beer foam collapse characteristics 2 min after pouring.Beer on left produced with low gravity wort (10°Plato). Beer onright produced with high (20°Plato) gravity wort.

Fig. 39. Beer foam collapse characteristics 4 min after pouring.Beer on left produced with low gravity wort (10°Plato). Beer onright produced with high (20°Plato) gravity wort.

Table VII. Influence of wort gravity on beer ester levels.

12°Plato 20°Plato

Ethanol (v/v) 05.1 05.0Ethyl acetate (mg/L) 14.2 21.2Isoamyl acetate (mg/L) 00.5 00.7

Table IX. Vitality of brewing yeast strains after a 96 h fermentation ofsynthetic mediaa.

Glucose Maltose

Ale 1 0.8 1.3Ale 2 0.9 1.3Ale 3 1.1 1.4

Lager 1 0.7 0.9Lager 2 0.8 1.2Lager 3 0.9 1.0

aPeptone – yeast extract – 4% sugar medium. Acidification power test.

Fig. 40. The effect of low (10°Plato) and high (20°Plato) wortson proteinase A release by yeast during fermentation.


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VOL. 115, NO. 1, 2009 23

acidification power test) of the cells, respectively, follow-ing four days of fermentation. For all six strains studied,cells cultured in maltose consistently had higher viabili-ties and enhanced vitalities compared to their glucose cul-tured counterparts. Reasons for these differences are notimmediately apparent. It may be the result of slowerinitial uptake rates of maltose compared to glucose andconsequent reduced growth rates. In addition, the fact thatmaltose uptake occurs by active transport and glucose bypassive transport is no doubt relevant.

Despite the apparent sturdiness of the maltose growncells, the production of ethyl acetate and isoamyl acetate

was lower than in the glucose grown cells (Table X). Thelower levels of ester production, with maltose as the sub-strate, could be due to a number of reasons. It is possiblethat fermentation with maltose inhibits the transport ofesters out of the cell, perhaps by modifying the plasmamembrane, thus giving the impression that fewer estersare produced. However, in the light of the increasedviability and vitality of the maltose grown cells, this is un-likely. Another possibility is that maltose metabolism pro-duces lower levels of acetyl-CoA, which has been sug-gested as resulting in fewer esters due to a lack of

intermediate metabolites. It has also been proposed thatester production is linked to lipid metabolism144. If this isthe case, or if for some reason maltose metabolismproduces fewer toxic fatty acids, it would be reasonable toassume that reduced toxic fatty acids would be produced

in wort containing elevated levels of maltose120.It is generally agreed that a reduction in ester levels

particularly ethyl acetate and isoamyl acetate from highgravity brewed beers, would be welcome. In order tostudy the influence of maltose and glucose levels in highgravity worts, two 20°Plato worts were prepared, one con-taining 30% maltose syrup (MS) and the other containing30% very high maltose (VHMS) syrup. The sugar compo-sition of the two brewing syrups are shown in Table XI. Inaddition, a 12°Plato wort containing 70% (w/v) maltosesyrup (MS) was prepared and used as a control. The sugarspectra of the three worts is shown in Fig. 41. The maltoseplus maltotriose concentration in the 20°Plato VHMSwort had increased compared to the 20°Plato MS wort

with a corresponding decrease in the concentration of glu-cose plus fructose.

The three worts were fermented in the ICBD 2 hL pilotbrewery with a lager yeast strain at 13°C, and the concen-trations of ethyl acetate and isoamyl acetate determinedthroughout the fermentation (Figs. 42 and 43). The pro-files were similar for both esters. The concentration ofboth esters in the 20°Plato (MS) fermented wort wastwice that observed in the 12°Plato (MS) fermented wort.However, the ester concentration in the 20°Plato (VHMS)was approximately 25% reduced compared to the

Table X. Ethyl acetate and isoamyl acetate produced by brewing yeaststrains during the fermentation of a synthetic mediaa.

Ethyl Acetate (mg/L) Isoamyl Acetate (mg/L)

Glucose Maltose Glucose Maltose

Ale 1 4.13 2.79 0.14 0.14Ale 2 2.97 2.59 0.06 0.04Ale 3 3.13 2.71 0.05 0.03Lager 1 6.00 5.22 0.22 0.21Lager 2 3.75 3.28 0.26 0.22Lager 3 4.13 3.51 0.23 0.17

a Peptone – yeast extract – 4% sugar medium

Fig. 43. Isoamyl acetate concentration (mg/L) in fermentingworts of differing gravities and sugar composition. , 12°Plato(30% MS); , 20°Plato (30% MS);, 20°Plato (30% VHMS).

Table XI. Percent sugar composition of brewing syrups.

Maltose syrup

(MS)

Very high maltose

syrup (VHMS)

Glucose 15 5Maltose 55 70Maltotriose 10 10Dextrins 20 15

Fig. 42. Ethyl acetate concentration (mg/L) in fermenting wortsof differing gravities and sugar composition. , 12°Plato (30%MS);, 20°Plato (30% MS);, 20°Plato (30% VHMS).

Fig. 41. Wort sugar profiles of 12°Plato and 20°Plato wortscontaining either 30% (w/v) maltose syrup or 30% (w/v) very

high maltose syrup.


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20°Plato (MS) wort174. This confirms the findings em-ploying synthetic media with single sugars, that maltose

fermentations produce less ethyl acetate and isoamyl ace-tate than glucose fermentations. In addition, similar to thesynthetic media fermentations, the wort with elevatedconcentrations of maltose produced yeast with higher via-bilities than the wort containing lower levels of maltose139 (Tables XII and XIII).

THE APPLICATION OF CONFOCALMICROSCOPY AND FLOWCYTOMETRY TO ASSESSYEAST PERFORMANCE.

Recent applications of fluorescent analytical methods

for assessing yeast performance and quality utilise novelanalytical instruments. Specifically, confocal imaging andflow cytometry are beginning to be invaluable techniquesin the brewing research laboratory but are still underused.Studies to date have focussed on the implementation offluorescence optical methods and laser scanning confocalmicroscopy for monitoring yeast performance during highgravity wort fermentation79 and to examine hydrodynamicstresses160 imposed by yeast centrifugation20.

Confocal microscopy

A number of physiological parameters and cell com-pounds in yeast cells (glycogen, neutral lipids, trehalose,bud scars, DNA and intracellular proteinases) have been

successfully visualised with the aid of highly specificfluorochromes. In particular, the expression and sub cellu-lar localisation of proteinase A during yeast fermentationhas been studied employing a green fluorescent proteinclone117. These studies confirm previous findings13, usingthe Kondo et al. assay67 method for proteinase A, whichdemonstrated that this enzyme is located primarily in theyeast vacuole, but under stress conditions significant ac-tivity appears in the cell cytoplasm. As previously de-scribed in this paper, the cell’s sorting mechanism thattargets proteinase A to the vacuole is partially failing un-

der stress conditions (for example, high gravity wort fer-mentations) and proteinase A remains in the cytoplasm oris excreted into the environment. Laser scanning confocalmicroscopy as a qualitative tool, in conjunction with flowcytometry61,177 – details later, is a powerful tool to gaininsight into the response of yeast cells to the changingenvironmental conditions occurring during fermentation.

Flow cytometry

Flow cytometry is a technology that simultaneouslymeasures then analyses multiple physical characteristicsof single particles, usually cells, as they flow in a fluidstream through a beam of light. It can assess yeast phy-sical and chemical characteristics based on cell size, rela-tive granularity and fluorescence. Flow cytometry meth-ods have been developed to measure cell viability,damaged cells, intracellular pH(pHi), mannan residues21 and intracellular glycogen and trehalose22.

An example of the application of flow cytometry inbrewing research is the recently conducted study of discstack centrifuge operating parameters and their impact onyeast physiology24. Modern centrifuges produce forces inexcess of 10,000 times the earth’s gravitational force,

achieving solid separation in seconds with reduced equip-ment volume. Centrifuges have a number of applicationsin a brewery and can be used for:

• Cropping of non-flocculent yeast cultures at the end ofprimary fermentation which may then be re-pitchedinto a subsequent fermentation;

• Reducing the yeast quantity from “green” beer beforethe start of secondary fermentation;

• Beer recovery from cropped yeast;• Separation of the hot break after wort boiling;• Removal of cold break and yeast at the end of matura-

tion.This study has concentrated on the effect that centrifu-

gation has on yeast which is intended to be re-pitched.

The passage of yeast through a centrifuge exposes cells tomechanical and hydrodynamic shear stresses161. We haveshown23 that these stresses can cause a decrease in cellviability and flocculation, cell wall damage, increased ex-tracellular proteinase A (PrA) levels, hazier beers andreduced foam stability. In these studies, a commercial aleyeast was subjected to differing operating conditions dur-ing centrifugation with 5–6 hL/h Westfalia Separator (Fig.44). Yeast cultures, following centrifugation, were ana-lysed using flow cytometry techniques. Cell viability andintracellular pH decreased due to the processing condi-tions encountered during yeast cropping with a centrifuge.A relationship has been established whereby yeast cellwall mannan, an unfilterable haze constituent, as a func-

tion of G-force and centrifugation cycles is released fromthe cell wall while concurrently, particle sizes between0.5–2.8 mm and beer haze increased. Furthermore, yeastintracellular glycogen and trehalose levels were depletedas a result of centrifugation. During these experiments,the centrifuge was operated under conditions similar tothose encountered during commercial production. It is im-portant for yeast management systems that utilize centri-fuges, that they are designed and operated properly, tominimize negative consequences on beer quality and sta-bility19. Recent investigations found that implementation

Table XII. Percentage viability of an ale and a lager brewing strain afterfermentation in 12°Plato and 20°Plato wortsa.

12°Plato 20°Plato

MSb VHMSc MS VHMS

Ale 95 98 93 96Lager 94 97 95 98

a Methylene blue and methylene violet stains employed.b Maltose (55) syrup.c Very high maltose (70) syrup.

Table XIII. Vitality of an ale and a lager brewing strain afterfermentation in 12°Plato and 20°Plato wortsa.

12°Plato 20°Plato

MSb VHMSc MS VHMS

Ale 0.9 1.1 0.6 0.8Lager 0.7 0.9 0.6 0.9

a Acidification power test.bMaltose (55) syrup.c Very high maltose (70) syrup.


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VOL. 115, NO. 1, 2009 25

of newer model centrifuges and redesigned pipe workimproved “green” beer quality.

Flow cytometry analytical methods have also beenused to assess yeast physiological status during high and

low gravity wort fermentation. Analyses such as cell via-bility, damaged cells, pHi and intracellular glycogen andtrehalose content, were conducted. The results demon-strate that high gravity fermentations, compared to lowgravity fermentations, initially contain increased numbersof damaged cells and lower glycogen and trehalose levels,confirming the presence of stressed cells.

EPILOGUE

Although Horace Brown conducted his fifty years ofresearch over one hundred years ago, the results of hisstudies are still apparent. The award that has been estab-lished in his name is “to recognise eminent services on the

scientific or technical side of the fermentation ind

forty years of brewing research

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