Food Biochemistry and Food Processing (Simpson/Food Biochemistry and Food Processing) || Biochemistry of Beer Fermentation

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33Biochemistry of Beer Fermentation

Ronnie Willaert

IntroductionThe Beer Brewing ProcessCarbohydrate Metabolism—Ethanol Production

Wort Carbohydrates Uptake and MetabolismMaltose and Maltotriose MetabolismGlycogen and Trehalose MetabolismWort Fermentation

Metabolism of Bioflavoring by-ProductsBiosynthesis of Higher AlcoholsBiosynthesis of EstersBiosynthesis of Organic AcidsBiosynthesis of Vicinal Diketones

Secondary FermentationVicinal DiketonesHydrogen SulfideAcetaldehydeDevelopment of Flavor Fullness

Beer Fermentation Using Immobilized Cell TechnologyCarrier MaterialsApplications of ICT in the Brewing Industry

Flavor Maturation of Green BeerProduction of Alcohol-Free or Low-Alcohol BeerProduction of Acidified Wort Using Immobilized

Lactic Acid BacteriaContinuous Main Fermentation

AcknowledgmentsReferences

Abstract: The carbohydrate metabolism and flavor formation dur-ing yeast primary and secondary fermentation (maturation) is re-viewed. Carbohydrate metabolism and ethanol production duringthe primary fermentation is discussed firstly. Next, the metabolismof the bioflavoring by-product formation, that is, higher alcohols,esters, organic acids, and vicinal diketones, is elaborated. The nextstep of the fermentation process is the maturation process wherethe vicinal diketones, acetaldehyde, and hydrogen sulfide concen-tration needs to be reduced to acceptable levels. The chapter isconcluded with a discussion about the use of immobilized cell tech-

nology to intensify the fermentation process and its impact on flavorproduction.

INTRODUCTIONThe production of alcoholic beverages is as old as history.Wine may have an archeological record going back more than7500 years, with the early suspected wine residues dating fromearly to mid-fifth millennium bc (McGovern et al. 1996). Clearevidence of intentional winemaking first appears in the repre-sentations of wine presses that date back to the reign of Udimuin Egypt, some 5000 years ago. The direct fermentation offruit juices, such as that of grape, had doubtlessly taken placefor many thousands of years before early thinking man devel-oped beer brewing and, probably coincidentally, bread baking(Hardwick 1995). The oldest historical evidence of formal brew-ing dates back to about 6000 bc in ancient Babylonia is a pieceof pottery found there, which shows workers either stirring orskimming a brewing vat.

Nowadays, alcoholic beverage production represents a sig-nificant contribution to the economies of many countries. Themost important beverages today are beer, wine, distilled spir-its, cider, sake, and liqueurs (Lea and Piggott 1995). In Bel-gium (“the beer paradise”), beer is the most important alcoholicbeverage, although the beer consumption declined in the last40 years: from 11,096,717 hL in 1965 to 9,703,000 hL in 2004(NN 2005). In this time frame, wine consumption doubled from1,059,964 to 2,215,579 hL. Another trend is the spectacular in-crease in waters and soft drinks consumption (from 5,215,056 to26,395,000 hL).

In this chapter, the biochemistry and fermentation of beer isreviewed. First, the carbohydrate metabolism in brewer’s yeastis discussed. The maltose metabolism is of major importance inbeer brewing, since this sugar is in a high concentration present inwort. For the production of a high-quality beer, a well-controlled

Food Biochemistry and Food Processing, Second Edition. Edited by Benjamin K. Simpson, Leo M.L. Nollet, Fidel Toldra, Soottawat Benjakul, Gopinadhan Paliyath and Y.H. Hui.C© 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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628 Part 5: Fruits, Vegetables, and Cereals

fermentation needs to be performed. During this fermentation,major flavor-active compounds are produced (and some of themare again metabolized) by the yeast cells. The metabolism ofthe most important fermentation by-products during main andsecondary fermentation is discussed in detail. The latest trendin beer fermentation technology is the process intensificationusing immobilized cell technology (ICT). This new technologyis explained and some illustrative applications—on small andlarge scale—are discussed.

THE BEER BREWING PROCESSThe principal raw materials used to brew beer are water, maltedbarley, hops and yeast. The brewing process involves extractingand breaking down the carbohydrate from the malted barley tomake a sugar solution (called “wort”), which also contains es-sential nutrients for yeast growth, and using this as a source ofnutrients for “anaerobic” yeast growth. During yeast fermenta-tion, simple sugars are consumed, releasing heat and producingethanol and other flavoring metabolic by-products. The major

biological changes, which occur in the brewing process, are cat-alyzed by naturally produced enzymes from barley (during malt-ing) and yeast. The rest of the brewing process largely involvesheat exchange, separation, and clarification, which only pro-duces minor changes in chemical composition when comparedto the enzyme catalyzed reactions. Barley is able to produce allthe enzymes that are needed to degrade starch, β-glucan, pen-tosans, lipids, and proteins, which are the major compounds ofinterest to the brewer. An overview of the brewing process isshown in Figure 33.1, where also the input and output flows areindicated. Table 33.1 gives a more detailed explanation of eachstep in the process.

CARBOHYDRATEMETABOLISM—ETHANOL PRODUCTIONWort Carbohydrates Uptake and Metabolism

Carbohydrates in wort make up 90–92% of wort solids. Wortfrom barley malt contains the fermentable sugars sucrose, fruc-tose, glucose, maltose, and maltotriose together with some

Malt

Wort cooling and aeration

Primary fermentation

Beer filtration

Maturation and conditioning

Beer stabilization

Beer packaging

Brewing water

Unmalted cereals

Hops/hopproducts

Syrups

Yeast

Spent grains

Hot trub

Spent hops

Yeast

Cold trub

Yeast

Milling

Mashing

Wort separation

Wort boiling

Wort clarification

Figure 33.1. Schematic overview of the brewing process (input flows are indicated on the left side and output flows on the right side).

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33 Biochemistry of Beer Fermentation 629

Table 33.1. Overview of the Brewing Processing Steps: From Barley to Beer

Process Action Objectives Time Temperature (◦C)

MaltingSteepingGerminationKilning

Moistening and aeration ofbarley

Barley germinationKilning of the green malt

Preparation for the germinationprocess

Enzyme production, chemicalstructure modification

Ending of germination andmodification, production offlavoring and coloringsubstances

48 h3–5 d24–48 h

12–222222–110

Milling Grain crushing withoutdisintegrating the husks

Enzyme release and increase ofsurface area

1–2 h 22

Mashing + wortseparation

Addition of warm/hot water Stimulation of enzyme action,extraction and dissolution ofcompounds, wort filtration,to obtain the desiredfermentable extract as quickas possible

1–2 h 30–72

Wort boiling Boiling of wort and hops Extraction and isomerization ofhop components, hot breakformation, wort sterilization,enzyme inactivation,formation of reducing,aromatic and coloringcompounds, removal ofundesired volatile aromacompounds, wortacidification, evaporationof water

0.5–1.5 h >98

Wort clarification Sedimentation orcentrifugation

Removal of spent hops,clarification (whirlpool,centrifuge, settling tank)

<1 h 100–80

Wort cooling andaeration

Use of heat exchanger,injection of air bubbles

Preparing the wort for yeastgrowth

<1 h 12–18

Fermentation Adding yeast, controlling thespecific gravity, removal ofyeast

Production of green beer, toobtain yeast for subsequentfermentations, carbondioxide recovery

2—7 d 12–22 (ale)4–15 (lager)

Maturation andconditioning

Beer storage in oxygen freetank, beer cooling, addingprocessing aids

Beer maturation, adjustment ofthe taste, adjustment of CO2

content, sedimentation ofyeast and cold trub, beerstabilization

7–21 h −1–0

Beer clarification Centrifugation, filtration Removal of yeast and cold trub 1–2 h −1–0Biological

stabilizationPasteurization of sterile

filtrationKilling or removing of

microorganisms1–2 h 62–72 (past.)

−1–0 (filtr.)Packaging Filling of bottles, cans, casks,

and kegs; pasteurization ofsmall volumes in packings

Production of packaged beeraccording to specifications

0.5–1.5 h −1–roomtemperature

dextrin material (Table 33.2). The fermentable sugars typicallymake up 70–80% of the total carbohydrate (MacWilliam 1968).The three major fermentable sugars are glucose, α-glucosidesmaltose, and maltotriose. Maltose is by far the most abundantof these sugars, typically accounting for 50–70% of the total

fermentable sugars in an all-malt wort. Sucrose and fructose arepresent in a low concentration. The unfermentable dextrins playlittle part in brewing. Wort fermentability may be reduced orincreased by using solid (e.g., corn grits, flakes, or rice) or liquidadjuncts (e.g., sugar syrups).

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Table 33.2. Carbohydrate Composition of Worts

Origin Type of WortDanish Lager

11◦PCanadian

Lager 13◦PBritish Pale

Ale 10◦PCanadian CornAdjunct Wort

All-Malt Wort18◦P

Fructose (g/L) (%)a 2.12.7

1.51.6

3.34.8

1.31.3

3.0

Glucose (g/L) (%)a 9.111.6

10.310.9

10.014.5

14.715.7

13.0

Sucrose (g/L) (%)a 2.32.9

4.24.5

5.37.7

1.81.9

Maltose (g/L) (%)a 52.466.6

60.464.2

38.956.5

62.867.0

80.0

Maltotriose (g/L) (%)a 12.816.3

17.718.8

11.416.5

13.214.1

24.0

Total ferm. sugars (g/L) 78.7 94.1 68.9 93.8 121.0Maltotetraose (g/L) 2.6 7.2 2.0Higher sugars (g/L) 21.3 26.8 25.2Total dextrins (g/L) 23.9 34.0 25.2Total sugars (g/L) 102.6 128.1 94.1 117.5

Source: Patel and Ingledew (1973), Huuskonen et al. (2010), and Hough et al. (1982).aPercent of the total fermentable sugars.

Brewing strains consume the wort sugars in a specific se-quence: glucose is consumed first, followed by fructose, maltose,and finally maltotriose. The uptake and consumption of maltoseand maltotriose is repressed or inactivated at elevated glucoseconcentrations. Only when 60% of the wort glucose has beentaken up by the yeast, the uptake and consumption of maltosewill start. Maltotriose uptake is inhibited by high glucose andmaltose concentrations. When high amounts of carbohydrateadjuncts (e.g., glucose) or high-gravity wort are employed, theglucose repression is even more pronounced, resulting in fer-mentation delays (Stewart and Russell 1993).

The efficiency of brewer’s yeast strains to effect alcoholic fer-mentation is dependent upon their ability to utilize the sugarspresent in wort. This ability very largely determines the fer-mentation rate as well as the final quality of the beer produced.In order to optimize the fermentation efficiency of the primaryfermentation, a detailed knowledge of the sugar consumptionkinetics, which is linked to the yeast growth kinetics, is required(Willaert 2001).

Maltose and Maltotriose Metabolism

The yeast Saccharomyces cerevisiae transports the monosaccha-rides across the cell membrane by the hexose transporters. Thereare 20 genes encoding hexose transporters (Dickinson 1999,Rintala et al. 2008): 18 genes encoding transporters (HXT1 toHXT17, GAL2) and two genes encoding (SNF3, RGT2). Thedisaccharide maltose and the trisaccharide maltotriose are trans-ported by specific transporters into the cytoplasm, where thesemolecules are hydrolyzed by the same α-glucosidase yieldingtwo or three molecules of glucose, respectively (Panchal andStewart 1979, Zheng et al. 1994a).

Maltose utilization in yeast is conferred by any one of fiveMAL loci: MAL1 to MAL4 and MAL6 (Bisson et al. 1993,Dickinson 1999). Each locus consists of three genes (MALx1,where x stands for one of the five loci): gene 1 encodes amaltose transporter (permease), gene 2 encodes a maltase (α-glucosidase), and gene 3 encodes a transcriptional activator ofthe other two genes. Thus, for example, the maltose transportergene at the MAL1 locus is designated MAL61. The three genesof a MAL locus are all required to allow fermentation. Alterna-tively, some authors use gene designations such as for the MAL1locus: MAL1T (transporter = permease), MAL1R (regulator),and MAL1S (maltase). The genetic and biochemical analysis ofmaltose fermentation by yeast cells revealed a series of five un-linked telomere-associated multigene MAL loci: MAL1 (chro-mosome VII), MAL2 (chromosome III), MAL4 (chromosomeII), and MAL6 (chromosome VIII). The MAL loci exhibit a veryhigh degree of homology and are telomere linked, suggestingthat they evolved by translocation from telomeric regions ofdifferent chromosomes (Michels et al. 1992). Since a fully func-tional or partial allele of the MAL1 locus is found in all strainsof S. cerevisiae, this locus is proposed as the progenitor of theother MAL loci (Chow et al. 1983), as all S. cerevisiae strains,and even its closest related yeast species S. paradoxus, containMAL1 sequences near the right telomere of chromosome VII.The genes in the MAL loci show a high degree of sequenceand functional similarity, but there can be extensive variability,and several different alleles that determine distinct phenotypes(i.e., MAL-inducible and MAL-constitutive strains) have beendescribed (Novak et al. 2004). Gene dosage studies performedwith laboratory strains of yeast have shown that the transport ofmaltose in the cell may be the rate-limiting step in the utilizationof this sugar (Goldenthal et al. 1987). Constitutive expression

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of the maltose transporter gene (MALT) with high-copy-numberplasmids in a lager yeast strain has been found to acceleratethe fermentation of maltose during high-gravity (24◦P) brewing(Kodama et al. 1995). The constitutive expression of MALS andMALR had no effect on maltose fermentability.

The control over MAL gene expression is exerted at threelevels. The presence of maltose induces, whereas glucose re-presses, the transcription of MALS and MALT genes (Federoffet al. 1983a, 1983b, Needleman et al. 1984). The constitutivelyexpressed regulatory protein (MALR) binds near the MALS andMALT promotors and mediates the induction of MALS andMALT transcription (Cohen et al. 1984, Chang et al. 1988,Ni and Needleman 1990). Experiments with MALR-disruptedstrains led to the conclusion that MalRp is involved in glucoserepression (Goldenthal and Vanoni 1990, Yao et al. 1994). Rela-tively little attention has been paid to posttranscriptional control,that is, the control of translational efficiency, or mRNA turnover,as mechanisms complementing glucose repression (Soler et al.1987). The addition of glucose to induced cells has been re-ported to cause a 70% increase in the liability of an mRNA pop-ulation containing a fragment of MALS (Federoff et al. 1983a).The third level of control is posttranslational modification. Inthe presence of glucose, maltose permease is either reversiblyconverted to a conformational variant with decreased affinity(Siro and Lovgren 1979, Peinado and Loureiro-Dias 1986) orirreversibly proteolytically degraded depending on the physio-logical conditions (Lucero et al. 1993, Riballo et al. 1995). Thelatter phenomenon is called catabolite inactivation. Glucose re-pression is accomplished by the Mig1p repressor protein, whichis encoded by the MIG1 gene (Nehlin and Ronne 1990). It hasbeen shown that Mig1p represses the transcription of all threeMAL genes by binding upstream of them (Hu et al. 1995). TheMIG1 gene has been disrupted in a haploid laboratory strainand in an industrial polyploid strain of S. cerevisiae (Klein et al.1996). In the MIG1-disrupted haploid strain, glucose repressionwas partly alleviated; that is, maltose metabolism was initiatedat higher glucose concentrations than in the corresponding wild-type strain. In contrast, the polyploid �mig1 strain exhibited aneven more stringent glucose control of maltose metabolism thanthe corresponding wild-type strain, which could be explainedby a more rigid catabolite inactivation of maltose permease,affecting the uptake of maltose.

All α-glucoside transport systems so far characterized in yeastare H+-transporters that use the electrochemical proton gradientto actively transport these sugars into the cell (Crumplen et al.1996, Stambuk and de Araujo 2001). It has been shown thatmaltose uptake is the rate-limiting step of fermentation (Kodamaet al. 1995, Wang et al. 2002, Rautio and Londesborough 2003).At least three different maltose transporters have been identi-fied in S. cerevisiae, and while the MALx1 transporters (andprobably the two MPH2 and MPH3 alleles) encode high affinity(Km = 2–4 mmol l−1) maltose permeases, the AGT1 permease (agene present in partially functional mal1g loci) transports mal-tose with lower (Km ≈ 20 mmol l−1) affinity (Han et al. 1995,Stambuk and de Araujo 2001, Day et al. 2002a, Alves et al.2007, 2008). AGT1 is found in many S. cerevisiae laboratorystrains and maps to a naturally occurring, partially functional

allele of the MAL1 locus (Han et al. 1995). Agt1p is a highlyhydrophobic, postulated integral membrane protein. It is 57%identical to Mal61p (the maltose permease encoded at MAL6)and is also a member of the 12 transmembrane domain super-family of sugar transporters (Nelissen et al. 1995). Like Mal61p,Agt1p is a high-affinity, maltose/proton symporter, but Mal61p iscapable of transporting only maltose and turanose, while Agt1ptransports these two α-glucosides as well as several others in-cluding isomaltose, α-methylglucoside, maltotriose, palatinose,trehalose and melezitose. AGT1 expression is maltose inducibleand induction is mediated by the Mal-activator.

Brewing strains of yeast are polyploid, aneuploid, or, in thecase of lager strains, alloploid. Recently, Jespersen et al. (1999)examined 30 brewing strains of yeast (5 ale strains and 25 lagerstrains) with the aim of examining the alleles of maltose andmaltotriose transporter genes contained by them. All the strainsof brewer’s yeast examined, except two, were found to containMAL11 and MAL31 sequences, and only one of these strainslacked MAL41. MAL21 was not present in the 5 ale strainsand 12 of the lager strains. MAL61 was not found in any ofthe yeast chromosomes other than those known to carry MALloci. Sequences corresponding to the AGT1 gene (transport ofmaltose and maltotriose) were detected in all but one of theyeast strains.

Although maltose is easily fermented by the majority of yeaststrains after glucose exhaustion, maltotriose is not only the leastpreferred sugar for uptake by these Saccharomyces cells, butmany yeasts may not use this α-glucosidase at all (Zheng et al.1994a, Yoon et al. 2003). Incomplete maltotriose uptake dur-ing brewing fermentations results in yeast fermentable extractin beer, material loss, greater potential for microbiological sta-bility and sometimes atypical beer flavor profiles (Stewart andRussell 1993). Maltotriose uptake from wort is always slowerwith ale strains than with lager strains under similar fermenta-tion conditions. However, the initial transport rates are similarto those of maltose in a number of ale and lager strains. El-evated osmotic pressure inhibits the transport and uptake ofglucose, maltose and maltotriose with maltose and maltotriosebeing more sensitive to osmotic pressure than glucose in bothlager and ale strains. Ethanol (5% w/v) stimulated the transportof maltose and maltotriose, due in all probability to an ethanol-induced change in the plasma membrane configuration, but hadno effect on glucose transport. Higher ethanol concentrationsinhibited the transport of all three sugars.

Maltotriose uptake shows complex kinetics indicating thepresence of high- and low-affinity transport activities, and stud-ies on sugar utilization revealed that maltose and maltotrioseare apparently transported by different permeases (Zheng et al.1994b, Zastrow et al. 2001). Two genes are recognized as per-mease genes for transporting maltotriose in yeasts: AGT1 trans-porter from S. cerevisiae and MTY1 (also called MTT1) perme-ase from S. pastorianus. They are characterized as low-affinity(Km ≈ 20 mmol l−1) maltotriose transporters (Stambuk and deAraujo 2001, Dietvorst et al. 2005, Salema-Oom et al. 2005,Alves et al. 2007, 2008).

Recent reports regarding the observed patterns of maltose andmaltotriose utilization by yeast cells show contradicting results.

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Data indicated that all known α-glucoside transporters present inS. cerevisiae, including the maltose permeases MAL31, MAL61,MPH2, and MHP3, allowed growth of the yeast cells on bothmaltose and maltotriose (Day et al. 2002a, 2002b). Their kineticanalysis of maltose and maltotriose uptake indicated that allthese transporters, including AGT1 permease, could transportboth sugars with practically the same affinities and capacity.Recently, to better understand maltotriose utilization by yeaststrains, an analysis of maltotriose and maltotriose utilization by52 laboratory and industrial Saccharomyces yeast strains wasperformed (Duval et al. 2009). Microarray comparative genomehybridization (aCGH) was used to correlate the observed phe-notypes with copy number variations in genes known to be in-volved in maltose and maltotriose utilization by yeasts. Theresults showed that S. pastorianus strains utilized maltotriosemore efficiently than S. cerevisiae strains and highlighted theimportance of the AGT1 gene for efficient maltotriose utiliza-tion by S. cerevisiae yeasts. The fermentation performance ofa lager (S. pastorianus) strain was improved when its AGT1gene was replaced with the AGT1 gene of an ale (S. cerevisiae)strain, since the ATG1 gene of the lager strains studied con-tained a premature stop codon and did not encode functionaltransporters (Vidgren et al. 2005, 2009). The transformantswith repaired AGT1 had higher maltose transport activity, es-pecially after growth on glucose, which represses endogeneousα-glucoside transporter genes. The sequences of two AGT1-encoded α-glucosidase transporters with different efficiencies ofmaltotriose transport in two Saccharomyces strains were com-pared (Smit et al. 2008). The amino acids Thr505 and Ser557,which are respectively located in the transmembrane (TM) seg-ment TM11 and on the intracellular segment after TM12 of theAGT1-encoded α-glucosidase transporters, are critical for effi-cient transport of maltotriose in S. cerevisiae. It was also shownthat maltotriose utilization could be improved by attaching amaltase encoded by MAL32 to the yeast cell surface (Dietvorstet al. 2007).

Glycogen and Trehalose Metabolism

Glycogen and trehalose are the main storage carbohydrates inyeast cells (Panek 1991). The synthesis of both compoundscommences with the formation of uridine diphosphate (UDP)-glucose catalyzed by UDP-glucose pyrophosphorylase.

Glycogen is a reserve carbohydrate that is metabolized duringperiods of starvation. It is a branched polysaccharide composedof linear α-(1,4)-glycosyl chains with α-(1,6)-linkages (similarto starch but with a higher degree of branching). It is synthesizedstarting from glucose via glucose-6-phosphate and glucose-1-phosphate. Glycogen is synthesized from glucose, via glucose-6-phosphate and glucose-1-phosphate (Francois and Parrou 2001).UDP serves as a carrier of glucose units and is formed by atwo-step reaction, catalyzed by phosphoglucomutase and UDP-glucose phosphorylase. Glycogen synthesis is initiated by glyco-genin that produces a short α-(1,4)-glucosyl chain, which iselongated by glycogen synthase. The α-(1,6)-glucosidic bondsare formed by the branching enzyme Glc3. The dissimilationof glycogen occurs through the action of glycogen phosphory-

lase, which releases glucose-1-phosphate from the nonreducingends of the glycogen chains, and the debranching enzyme Gdb1,which transfers a maltosyl unit to the end of an adjacent linearα-(1,4) chain and releases glucose by cleaving the remainingα-(1,6)-linkage.

When yeast cells are pitched in aerated wort, an immediateglycogen mobilization is observed (Quain 1988, Boulton 2000).Glycogen accumulates during the exponential growth phase, af-ter oxygen has been consumed. When the yeast growth is ceasedtoward the end of the primary fermentation, are maximum glyco-gen levels obtained. In the stationary phase, the glycogen levelsdecline slowly. Glycogen provides energy for the synthesis ofsterols and unsaturated fatty acids during the aerobic phase of thebeer fermentation, and energy for the cellular maintanance func-tions during the stationary phase in the storage phase betweencropping and pitching (Quain and Tubb 1982, Boulton et al.1991). The glycogen content is directly related to subsequentfermentation performance. Therefore, yeast storage occurs bestat a low temperature, without agitation and under an atmosphereof nitrogen or carbon dioxide to minimize glycogen breakdown(Murray et al. 1984).

The regulation of the glycogen content is complex and occursin part by the cAMP/PKA pathway (Smith et al. 1998). Glycogenaccumulation is repressed by a high PKA-activity (Francois andParrou 2001). The glycogen content increases and the PKA-activity is reduced, when an essential nutrient is progressivelyconsumed from the growth medium.

Trehalose is a disaccharide (α-d-glucopyranosyl-1,1- α-d-glucopyranoside) which contains two molecules of d-glucose(Boulton and Quain 2006). Trehalose biosynthesis is catalyzedby the trehalose synthase complex, which forms trehalose-6-phosphate from UDP-glucose and glucose-6-phosphate, andnext dephosphorylates it to trehalose. Trehalose is degradaded bythe neutral (Nth1) or the acid (Ath1) trehalase (Panek and Panek1990, Francois and Parrou 2001). Like glycogen, trehalose alsoaccumulates in yeast under conditions of nutrient limitation.It has been observed that trehalose rapidly accumulates in re-sponse to environmental stress, such as dehydration, heatingand osmotic stress (during high gravity brewing) (Majara et al.1996, Hounsa et al. 1998). Under stress conditions, the higherlevels of trehalose protect the cells by binding to membranes andproteins (Francois and Parrou 2001). Trehalose accumulation inresponse to stress is, however, a transient phenomenon (Parrouet al. 1997).

In the beginning of the primary fermentation, high glucoselevels activate the camp/PKA pathway (Zahringer et al. 2000).This results in posttranslational activation of neutral treha-lase and in induction of its NTH1 gene, and in repression ofthe trehalose synthase genes, which results in reduced levelsof trehalose.

Wort Fermentation

Before the fermentation process starts, wort is aerated. This isa necessary step since oxygen is required for the synthesis ofsterols and unsaturated fatty acids, which are incorporated inthe yeast cell membrane (Rogers and Stewart 1973). It has been

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shown that ergosterol and unsaturated fatty acids increase bothin concentration as long as oxygen is present in the wort (e.g.,Haukeli and Lie 1979). A maximum concentration is obtained in5–6 hours after pitching, but the formation rate is dependent uponthe pitching rate and the temperature. Unsaturated fatty acids canalso be taken up from the wort, but all malt wort does not containsufficient unsaturated lipids to support a normal growth rate ofyeast. Adding lipids to the wort, especially unsaturated fattyacids might be an interesting alternative (Moonjai et al. 2000a,2000b).

The oxygen required for lipid biosynthesis can also be in-troduced by oxygenation of the separated yeast cells. The useof the preoxygenation technique resulted in more controllableand consistent fermentations, and as a consequence, in a morebalanced beer flavor profile (Jakobsen 1982, Ohno and Taka-hashi 1986a, 1986b, Boulton et al. 1991, Devuyst et al. 1991,Masschelein et al. 1995, Depraetere et al. 2003, Depraetere2007). Recently, the impact of yeast preoxygenation on yeastmetabolism has been assessed (Verbelen et al. 2009). Therefore,expression analysis was performed of genes that are of impor-tance in oxygen-dependent pathways, oxidative stress responseand general stress response during 8 hours of preoxygenation.The gene expressions of both the important transcription fac-tors Hap1 and Rox1, involved in oxygen sensing, were mainlyincreased in the first 3 hours, while YAP1 expression, which isinvolved in the oxidative stress response, increased drasticallyonly in the first 45 minutes. The results also show that stress-responsive genes (HSP12, SSA3, PAU5, SOD1, SOD2, CTA1,and CTT1) were induced during the process, together with theaccumulation of trehalose. The accumulation of ergosterol andunsaturated fatty acids was accompanied by the expression ofERG1, ERG11, and OLE1. Genes involved in respiration (QCR9,COX15, CYC1, and CYC7) also increased during preoxygena-tion. Yeast viability did not decrease during the process, andthe fermentation performance of the yeast reached a maximumafter 5 hours of preoxygenation. These results suggest that yeastcells acquire a stress response along the preoxygenation period,which makes them more resistant against the stressful conditionsof the preoxygenation process and the subsequent fermentation.

Different devices are used to aerate the cold wort: ceramic orsintered metal candles, aeration plants employing venturi pipes,two component jets, static mixers, or centrifugal mixers (Kunze1999). The principle of these devices is that very small air (oxy-gen) bubbles are produced and quickly dissolve during turbulentmixing.

As a result of this aeration step, carbohydrates are degradedaerobically during the first few hours of the “fermentation”process. The aerobic carbohydrate catabolism takes typically12 hours for a lager fermentation.

During the first hours of the fermentation process, oxidativedegradation of carbohydrates occurs through the glycolysis andKrebs (TCA) cycle. The energy efficiency of glucose oxidationis derived from the large number of NADH2

+ produced for eachmole of glucose oxidized to CO2. The actual wort fermentationgives alcohol and carbon dioxide via the Embden-Meyerhof-Parnas (glycolytic) pathway. The reductive pathway from pyru-vate to ethanol is important since it regenerates NAD+. Energy

is obtained solely from ATP-producing steps of the Embden-Meyerhof-Parnas pathway. During fermentation, the activityof the TCA cycle is greatly reduced, although it still servesas a source of intermediates for biosynthesis (Lievense andLim 1982).

Lagunas (1979) observed that during aerobic growth of S.cerevisiae, respiration accounts for less than 10% of glucosecatabolism, the remainder being fermented. Increasing sugarconcentrations resulting in a decreased oxidative metabolism isknown as the Crabtree effect. This was traditionally explained asan inhibition of the oxidative system by high concentrations ofglucose. Nowadays, it is generally accepted that the formationof ethanol at aerobic conditions is a consequence of a bottleneckin the oxidation of pyruvate, for example, in the respiratorysystem (Petrik et al. 1983, Rieger et al. 1983, Kappeli et al.1985, Fraleigh et al. 1989, Alexander and Jeffries 1990).

A reduction of ethanol production can be achieved bymetabolic engineering of the carbon flux in yeast resulting in anincreased formation of other fermentation product. A shift of thecarbon flux towards glycerol at the expense of ethanol formationin yeast was achieved by simply increasing the level of glycerol-3-phosphate dehydrogenase (Michnick et al. 1997, Nevoigt andStahl 1997, Remize et al. 1999, Dequin 2001). The GDP1 gene,which encodes glycerol-3-phosphate dehydrogenase, has beenoverexpressed in an industrial lager brewing yeast to reducethe ethanol content in beer (Nevoigt et al. 2002). The amountof glycerol produced by the GDP1-overexpressing yeast in fer-mentation experiments—simulating brewing conditions—wasincreased 5.6 times and ethanol was decreased by 18% com-pared to the wild-type strain. Overexpression did not affect theconsumption of wort sugars and only minor changes in theconcentration of higher alcohols, esters and fatty acids couldbe observed. However, the concentrations of several other by-products, particularly acetoin, diacetyl, and acetaldehyde, wereconsiderably increased.

Mutants of S. cerevisiae strains deficient in tricarboxylic acidcycle genes have been reported as suitable strains for the produc-tion of nonalcoholic beer (Navratil et al. 2002). Strains deficientin fumarase and α-ketoglutarate dehydrogenase made nonal-coholic beers with an alcohol content lower than 0.5% (v/v)(Selecky et al. 2008). The low ethanol content was compensatedby the considerable increase of organic acids (citrate, succinate,fumarate, and malate). Some of the mutants released high levelsof lactic acid, which protects beers against contamination andmasks an unacceptable worty off-flavor.

METABOLISM OF BIOFLAVORINGBY-PRODUCTSYeast is an important contributor to flavor development in fer-mented beverages. The compounds, which are produced dur-ing fermentation, are many and varied, depending on both theraw materials and the microorganisms used. The interrelationbetween yeast metabolism and the production of bioflavoringby-products is illustrated in Figure 33.2. The important flavorcompounds produced by yeast can be classified into five cat-egories: alcohols, esters, organic acids, carbonyl compounds

P1: SFK/UKS P2: SFK



Sulphite

Sulphate

Sulphite

Glucose

Glucose

Maltose Maltotriose

Sucrose homocysteine

H2S H2S

Amino acids Fructose-6-PFructose

Pyruvate Keto acids

Acetaldehyde

Vicinal diketones

Amino acids

Fatty acyl CoA

Acetyl CoA Organic acids

Fatty acids Esters

Ethanol

Higheralcohols

Lipids

Figure 33.2. Interrelation between yeast metabolism and theproduction of bioflavoring by-products.

(aldehydes and vicinal diketones), and sulfur-containing com-pounds (Hammond 1993). In addition, some speciality beers cancontain important concentrations of volatile phenol compounds(Vanbeneden et al. 2007).

Biosynthesis of Higher Alcohols

During beer fermentation, higher alcohols (also called “fuselalcohols”) are produced by yeast cells as by-products and repre-sent the major fraction of the volatile compounds. More than35 higher alcohols in beer have been described. Table 33.3gives the most important compounds, which can be classified

into aliphatic (n-propanol, isobutanol, 2-methylbutanol (or ac-tive amyl alcohol), and 3-methylbutanol (or isoamyl alcohol)),and aromatic (2-phenylethanol, tyrosol, tryptophol) higher alco-hols. Aliphatic higher alcohols contribute to the “alcoholic” or“solvent” aroma of beer, and produce a warm mouthfeel. Thearomatic alcohol 2-phenylethanol has a sweet rose-like aromaand has a positive contribution to the beer aroma. It is believedthat this compound masks the dimethyl sulfide (DMS) percep-tion (Hegarty et al. 1995). The aroma of tyrosol and tryptopholare undesirable, but they are only present above their thresholdsin some top-fermented beers.

Higher alcohols are synthesized by yeast during fermenta-tion via the catabolic (Ehrlich) and anabolic pathway (aminoacid metabolism) (Ehrlich 1904, Chen 1978, Oshita et al. 1995,Hazelwood et al. 2008). In the catabolic pathway, the yeastuses the amino acids of the wort to produce the correspond-ing α-keto acid via a transamination reaction. Isoamyl alcohol,isobutanol, and phenylethanol are produced via this route fromleucine, valine, and phenylalanine, respectively. An outsider inthis pathway is propanol, which is derived from threonine viaan oxidative deamination. The excess oxoacids are subsequentlydecarboxylated into aldehydes and further reduced (alcohol de-hydrogenase) to higher alcohols. This last reduction step alsoregenerates NAD+.

Dickinson and coworkers looked at the genes and enzymes,which are used by S. cerevisiae in the catabolism of leucineto isoamyl alcohol (Dickinson et al. 1997), valine to isobu-tanol (Dickinson et al. 1998), and isoleucine to active amylalcohol (Dickinson et al. 2000). In all cases, the general se-quence of biochemical reactions is similar, but the details forthe formation of the individual alcohols are surprisingly differ-ent. The branched-chain amino acids are first deaminated to thecorresponding α-ketoacids (α-ketoisocapric acid from leucine,α-ketoisovaleric acid from valine, and α-keto-β-methylvaleric

Table 33.3. Major Higher Alcohols in Beer (Partly Adapted From NN 2000.)

CompoundFlavor Threshold

(mg/L) Aroma or Taste(b)

Concentration Range(mg/L) BottomFermentation

Concentration Range(mg/L) Top Fermentation

n-Propanol 600(c), 800(b) Alcohol 7–19 (12)(a),(f), 6–30(j) 20–45(i)

Isobutanol 100(c), 80–100(g),200(b)

Alcohol 4–20 (12) (f), 6–32(j) 10–24(i)

2-Methylbutanol 50(c), 50–60(g), 70(b) Alcohol 9–25 (15) (a), 12–16(j) 80–140(i)

3-Methylbutanol 50(c), 50–60(g), 65(b) Fusely, pungent 25–75 (46) (a), 30–60(j) 80–140(i)

2-Phenylethanol 5(a), 40(c), 45–50(g),75(d), 125(b)

Roses, sweetish 11–51 (28) (f), 4–22(g),16–42(h), 4–10(j)

35–50(g), 8–25(a), 18–45(i)

Tyrosol 10(a), 10–20(e), 20(c),100(d), (g), 200(b)

Bitter chemical 6–9(a), 6–15(a) 8–12((g), 7–22(g)

Tryptophol 10(a), 10–20(e), 200(d) Almonds, solvent 0.5–14(a) 2–12(g)

Sources: (a) Szlavko (1973), (b) Meilgaard (1975a), (c) Engan (1972), (d) Rosculet (1971), (e) Charalambous et al. (1972), (f) Values in48 European lagers (Dufour unpublished date), (g) Reed and Nogodawithana (1991), (h) Iverson (1994), (i) Derdelinckx (unpublished data),(j) immobilised cells (Willaert and Nedovic 2006).aMean value.

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acid from leucine). There are significant differences in the wayeach α-ketoacid is subsequently decarboxylated. The catabolismof phenylalanine to 2-phenylethanol and of tryptophan were alsostudied (Dickinson et al. 2003). Phenylalanine and tryptophanare first deaminated to 3-phenylpyruvate and 3-indolepyruvate,respectively, and then decarboxylated. These studies revealedthat all amino acid catabolic pathways studied to date use a sub-tle different spectrum of decarboxylases from the five-memberedfamily that comprises Pdc1p, Pdc5p, Pdc6p, Ydl080cp, andYdr380wp. Using strains containing all possible combinations ofmutations affecting the seven AAD genes (putative aryl alcoholdehydrogenases), five ADH and SFA1 (other alcohol dehydroge-nase genes), showed that the final step of amino acid catabolismcan be accomplished by any one of the ethanol dehydrogenases(Ahd1p, Ahd2p, Ahd3p, Ahd4p, Ahd5p) or Sfa1p (formalde-hyde dehydrogenase).

In the anabolic pathway, the higher alcohols are synthesizedfrom α-keto acids during the synthesis of amino acids from thecarbohydrate source. The pathway choice depends on the indi-vidual higher alcohol and on the level of available amino acidsavailable. The importance of the anabolic pathway decreasesas the number of carbon atoms in the alcohol increases (Chen1978) and increases in the later stage of fermentation as wortamino acids are depleted (MacDonald et al. 1984). Yeast strain,fermentation conditions, and wort composition all have signif-icant effects on the combination and levels of higher alcoholsthat are formed (MacDonald et al. 1984).

Conditions that promote yeast cell growth such as high lev-els of nutrients (amino acids, oxygen, lipids, zinc, . . .) and in-creased temperature and agitation stimulate the production ofhigher alcohols (Engan 1969, Engan and Aubert 1977, Landaudet al. 2001, Boswell et al. 2002). The synthesis of aromatic al-cohols is especially sensitive to temperature changes. On theother hand, conditions that restrict yeast growth, such as lowertemperature and higher pressure, reduce the extent of higheralcohol production. Higher pressures can reduce the extent ofcell growth and, therefore, the production of higher alcohols(Landaud et al. 2001). The yeast strain, fermentation conditions,and wort composition have all significant effects on the patternand concentrations of synthesized higher alcohols. Supplemen-

tation of wort with valine, isoleucine, and leucine induces theformation of isobutanol, amyl alcohol, and isoamyl alcohol, re-spectively (Ayrapaa 1971, Kodama et al. 2001). The overexpres-sion of the branched chain amino acid transferases genes BAT1and BAT2 result in an increased production of isoamyl alcoholand isobutanol (Lilly et al. 2006).

Biosynthesis of Esters

Esters are very important flavor compounds in beer. They havean effect on the fruity/flowery aromas. Table 33.4 shows themost important esters with their threshold values, which areconsiderably lower than those for higher alcohols. The majoresters can be subdivided into acetate esters and C6–C10 medium-chain fatty acid ethyl esters. They are desirable components ofbeer when present in appropriate quantities and proportions butcan become unpleasant when in excess.

Esters are produced by yeast both during the growth phase(60%) and also during the stationary phase (40%) (NN 2000).They are formed by the intracellular reaction between a fattyacyl-coenzyme A and an alcohol:

R′OH + RCO − ScoA → RCOOR′ + CoASH (1)

This reaction is catalyzed by alcohol acyltransferases(AATases; or ester synthethases) of the yeast. Since acetyl CoAis also a central molecule in the synthesis of lipids and sterols,ester synthesis is linked to the fatty acid metabolism (see alsoFig. 33.2). The majority of acetyl-CoA is formed by oxidativedecarboxylation of pyruvate, while most of the other acyl-CoAsare derived from the acylation of free CoA, catalyzed by acyl-CoA synthase.

Several enzymes are involved in the synthesis, the best char-acterized are AATase I and II that are encoded by ATF1 andATF2. Alcohol acetyltransferase (AAT) has been localized inthe plasma membrane (Malcorps and Dufour 1987) and foundto be strongly inhibited by unsaturated fatty acids, ergosterol,heavy metal ions, and sulfydryl reagents (Minetoki et al. 1993).Subcellular fractionation studies conducted during the batchfermentation cycle demonstrated the existence of both cy-tosolic and membrane-bound AAT (Ramos-Jeunehomme et al.1989, Ramos-Jeunehomme et al. 1991). In terms of controlling

Table 33.4. Major Esters in Beer (Adapted From Dufour and Malcorps 1994.)

Compound Flavor Threshold (mg/L) AromaConcentration Range (mg/L) in

48 Lagers

Ethyl actetate 20–30, 30(a) Fruity, solvent-like 8–32 (18.4)(a), 5–40(b)

Isoamyl acetate 0.6–1.2, 1.2(a) Banana, peardrop 0.3–3.8 (1.72), <0.01–2.8(b)

Ethyl caproate (ethylhexanoate)

0.17–0.21, O.21(a) Apple-like with noteof aniseed

0.05–0.3 (0.14), 0.01–0.54(b)

Ethyl caprylate(ethyl octanoate)

0.3–0.9, 0.9(a) Apple-like 0.04–0.53 (0.17), 0.01–1.2(b)

2-Phenylethylacetate

3.8(a) Roses, honey, apple,sweetish

0.10–0.73 (0.54)

Source: (a) Meilgaard (1975b) and (b) immobilised cells (Willaert and Nedovic 2006).aMean value.

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ester formation on a metabolic basis, it has further been shownthat ester synthesizing activity of AAT is dependent on its po-sitioning within the yeast cell. An interesting feature of thisdistribution pattern is that specific rates of acetate ester for-mation varied directly with the level of cytosolic AAT activity(Masschelein 1997).

The ATF1 gene, which encodes AAT, has been cloned from S.cerevisiae and brewery lager yeast (S. cerevisiae uvarum) (Fujiiet al. 1994). An hydrophobicity analysis suggested that AATdoes not have a membrane-spanning region that is significantlyhydrophobic, which contradicts the membrane-bound assump-tion. A Southern analysis of the yeast genomes in which theATF1 gene was used as a probe revealed that S. cerevisiae hasone ATF1 gene, while brewery lager yeast has one ATF1 geneand another homologous gene (Lg-ATF1). The AAT activitieshave been compared in vivo and in vitro under different fermen-tation conditions (Malcorps et al. 1991). This study suggestedthat ester synthesis is modulated by a repression–induction ofenzyme synthesis or processing the regulation of which is pre-sumably linked to lipid metabolism. Other enzymes are Eht1(ethanolhexanoyl transferase) and Eeb1, which are responsiblefor the formation of ethyl esters (Verstrepen et al. 2003, Saerenset al. 2006).

The ester production can be altered by changing the synthesisrate of certain fusel alcohols. Hirata et al. (1992) increased theisoamyl acetate levels by introducing extra copies of the LEU4gene in the S. cerevisiae genome. A comparable S. cerevisiaeuvarum mutant has been isolated (Lee et al. 1995). The mutantshave an altered regulation pattern of amino acid metabolism andproduce more isoamyl acetate and phenylethyl acetate.

Isoamyl acetate is synthesized from isoamyl alcohol andacetyl coenzyme A by AAT and is hydrolyzed by esterases atthe same time in S. cerevisiae. To study the effect of balancingboth enzyme activities, yeast strains with different numbers ofcopies of ATF1 gene and isoamyl acetate-hydrolyzing esterasegene (IAH1) have been constructed and used in small-scale sakebrewing (Fukuda et al. 1998). Fermentation profiles as well ascomponents of the resulting sake were largely alike. However,the amount of isoamyl acetate in the sake increased with in-creasing ratio of AAT/Iah1p esterase activity. Therefore, it wasconcluded that the balance of these two enzyme activities isimportant for isoamyl acetate accumulation in sake mash.

The synthesis of acetate esters by S. cerevisiae during fer-mentation is ascribed to at least three acetyltransferase activi-ties, namely, AAT, ethanol acetyltransferase, and isoamyl AAT(Lilly et al. 2000). To investigate the effect of increased AATactivity on the sensory quality of Chenin blanc wines and distil-lates from Colombar base wines, the ATF1 gene of S. cerevisiaewas overexpressed. Northern blot analysis indicated constitu-tive expression of ATF1 at high levels in these transformants.The levels of ethyl acetate, isoamyl acetate and 2-phenylethylacetate increased 3- to 10-fold, 3.8- to 12-fold, and 2- to 10-fold, respectively, depending on the fermentation temperature,cultivar, and yeast used. The concentrations of ethyl caprate,ethyl caprylate, and hexyl acetate only showed minor changes,whereas the acetic acid concentration decreased by more thanhalf. This study established the concept that the overexpression

of acetyltransferase genes such as ATF1 could profoundly affectthe flavor profiles of wines and distillates deficient in aroma.

In order to investigate and compare the roles of the known S.cerevisiae AATs, Atf1p, Atf2p, and Lg-Atf1p, in volatile esterproduction, the respective genes were either deleted or overex-pressed in a laboratory strain and a commercial brewing strain(Verstrepen et al. 2003). Analysis of the fermentation productsconfirmed that the expression levels of ATF1 and ATF2 greatlyaffect the production of ethyl acetate and isoamyl acetate. GC-MS analysis revealed that Atf1p and Atf2p are also responsiblefor the formation of a broad range of less volatile esters, suchas propyl acetate, isobutyl acetate, pentyl acetate, hexyl acetate,heptyl acetate, octyl acetate, and phenyl ethyl acetate. With re-spect to the esters analyzed in this study, Atf2p seemed to playonly a minor role compared to Atf1p. The atf1�atf2� doubledeletion strain did not form any isoamyl acetate, showing thattogether, Atf1p and Atf2p are responsible for the total cellu-lar isoamyl AAT activity. However, the double deletion strainstill produced considerable amounts of certain other esters, suchas ethyl acetate (50% of the wild-type strain), propyl acetate(50%), and isobutyl acetate (40%), which provides evidence forthe existence of additional, as-yet-unknown ester synthases inthe yeast proteome. Interestingly, overexpression of different al-leles of ATF1 and ATF2 led to different ester production rates,indicating that differences in the aroma profiles of yeast strainsmay be partially due to mutations in their ATF genes.

Recently, it has been discovered that the Atf1 enzyme is lo-calized inside lipid vesicles in the cytoplasm of the yeast cell(Verstrepen 2003). Lipid vesicles are small organelles in whichcertain neutral lipids are metabolized or stored. This indicatesthat fruity esters are possibly by-products of these processes.

Ester formation is highly dependent on the yeast strain used(Nykanen and Nykanen 1977, Peddie 1990, Verstrepen et al.2003b) and on certain fermentation parameters such as tem-perature (Engan and Aubert 1977, Gee and Ramirez 1994,Sablayrolles and Ball 1995), specific growth rate (Gee andRamirez 1994), pitching rate (Maule 1967, D’Amore et al. 1991,Gee and Ramirez 1994), and top pressure (NN 2000, Verstrepenet al. 2003b). Additionally, the concentrations of assimilable ni-trogen compounds (Hammond 1993, Calderbank and Hammond1994, Sablayrolles and Ball 1995), carbon sources (Pfisterer andStewart 1975, White and Portno 1979, Younis and Stewart 1998,2000), dissolved oxygen (Anderson and Kirsop 1975a, 1975b,Avhenainen and Makinen 1989, Sablayrolles and Ball 1995),and fatty acids (Thurston et al. 1981, 1982) can influence theester production rate.

Acetate ester formation in brewer’s yeast is controlled mainlyby the expression level of the AATase-encoding genes (Ver-strepen et al. 2003b). Additionally, changes in the availabilityof the two substrates for ester production, higher alcohols andacyl-CoA, also influences ester synthesis rates. Any factor thatinfluences the expression of the ester synthase genes and/orthe concentrations of substrates will affect ester production ac-cordingly. Perhaps the most convenient and selective way toreduce ester production is applying tank overpressure, if neces-sary in combination with (slightly) lower fermentation temper-atures, low wort free amino nitrogen (FAN) and glucose levels

P1: SFK/UKS P2: SFK



and elevated wort aeration or wort lipid concentration (Ver-strepen et al. 2003b). Enhancing ester production is slightly morecomplicated.

If it is possible, overpressure or wort aeration can be reduced.Otherwise, worts rich in glucose and nitrogen combined withhigher fermentation temperatures and lower pitching rates orapplication of the drauflassen technique may prove helpful.

Biosynthesis of Organic Acids

Over hundred different organic acids have been reported inbeer (Meilgaard 1975b). Important organic acids detected inbeer include pyruvate, acetate, lactate, succinate, pyroglutamate,malate, citrate, α-ketoglutarate, and α-hydroxyglutarate; and themedium-chain length fatty acids, caproic (C6), caprylic (C8),and capric (C10) acid (Coote and Kirsop 1974, Meilgaard 1975b,Klopper et al. 1986). They influence flavor directly when presentabove their taste threshold and by their influence on beer pH.These components have their origin in raw materials (malt, hops)and are produced during the beer fermentation. Organic acids,which are excreted by yeast cells, are synthesized via aminoacid biosynthesis pathways and carbohydrate metabolism. Es-pecially, they are overflow products of the incomplete Krebscycle during beer fermentation. Excretion of organic acids isinfluenced by yeast strain and fermentation vigor. Sluggish fer-mentations lead to lower levels of excretion. Pyruvate excretionfollows the yeast growth: maximal concentration is reached justbefore the maximal yeast growth and is next taken up by theyeast and converted to acetate. Acetate is synthesized quicklyduring early fermentation and is later partially reused by theyeast during yeast growth. At the end of the fermentation, ac-etate is accumulated. The reduction of pyruvate results in theproduction of d-lactate of l-lactate (most yeast strains producepreferentially d-lactate). The highest amount of lactate is pro-duced during the most active fermentation period.

The change in organic acid productivity by disruption of thegene encoding fumarase (FUM1) has been investigated and ithas been suggested that malate and succinate are produced viathe oxidative pathway of the TCA cycle under static and sakebrewing conditions (Magarifuchi et al. 1995). Using a NAD+-dependent isocitrate dehydrogenase gene (IDH1, IDH2) disrup-tant, approximately half of the succinate in sake mash was foundto be synthesized via the oxidative pathway of the TCA cycle insake yeast (Asano et al. 1999).

Sake yeast strains possessing various organic acid productivi-ties were isolated by gene disruption (Arikawa et al. 1999). Sakefermented using the aconitase gene (ACO1) disruptant containeda twofold higher concentration of malate and a twofold lowerconcentration of succinate than that made using the wild-typestrain. The fumarate reductase gene (OSM1) disruptant producedsake containing a 1.5-fold higher concentration of succinate,whereas the α-ketoglutarate dehydrogenase gene (KGD1) andfumarase gene (FUM1) disruptants gave lower succinate con-centrations. In S. cerevisiae, there are two isoenzymes of fu-marate reductase (FRDS1 and FDRS2), encoded by the FRDSand OSM1 genes, respectively (Arikawa et al. 1998). Recentresults suggest that these isoenzymes are required for the reox-

idation of intracellular NADH under anaerobic conditions, butnot under aerobic conditions (Enomoto et al. 2002).

Succinate dehydrogenase is an enzyme of the TCA cycleand thus essential for respiration. In S. cerevisiae, this enzymeis composed of four nonidentical subunits, that is, the flavo-protein, the iron–sulfur protein, the cytochrome b560, and theubiquinone reduction protein encoded by the SDH1, SDH2,SDH3, and SDH4 genes, respectively (Lombardo et al. 1990,Chapman et al. 1992, Bullis and Lamire 1994, Daignan-Fournieret al. 1994). Sdh1p and Sdh2p comprise the catalytic domain in-volved in succinate oxidation. These proteins are anchored tothe inner mitochondrial membrane by Sdh3p and Sdh4p, whichare necessary for electron transfer and ubiquinone reduction,and constitute the succinate:ubiquinone oxidoreductase (com-plex II) of the electron transport chain. Single or double disrup-tants of the SDH1, SDH1b (which is a homologue of the SDH1gene), SDH2, SDH3, and SDH4 genes have been constructed andshown that the succinate dehydrogenase activity was retained inthe SDH2 disruptant and that double disruption of SDH1 andSDH2 or SDH1b genes is necessary to cause deficiency of suc-cinate dehydrogenase activity in sake yeast (Kubo et al. 2000).The role of each subunit in succinate dehydrogenase activity andthe effect of succinate dehydrogenase on succinate productionusing strains that were deficient in succinate dehydrogenase,have also been determined. The results suggested that succinatedehydrogenase activity contributes to succinate production un-der shaking conditions, but not under static and sake brewingconditions.

The medium-chain fatty acids account for 85–90% of thefatty acids in beer and impart an undesirable goaty, sweaty, andyeasty flavor (Chen 1980). These fatty acids are produced denovo by yeast during anaerobic fermentation and are not theresult of β-oxydation of wort or yeast long-chain fatty acids. Asa result, any change in fermentation conditions that promote theextent of yeast growth also favor increased levels of medium-chain fatty acids in beer. Higher temperature, increased wortoxygenation, and possibly elevated pitching rates are all effectivein this respect (Boulton and Quain 2006). The presence of thesemedium-chain fatty acids in beer is also related to yeast autolysis(Masschelein 1981). Yeast autolytic off-flavors are stimulatedat high temperatures, high yeast concentrations, and prolongedcontact times at the end of primary fermentation and duringsecondary fermentation.

Biosynthesis of Vicinal Diketones

Vicinal diketones are ketones with two adjacent carbonyl groups.During fermentations, these flavor-active compounds are pro-duced as by-products of the synthesis pathway of isoleucine,leucine, and valine (ILV pathway) (see Fig. 33.3) and thusalso linked to amino acid metabolism (Nakatani et al. 1984)and the synthesis of higher alcohols. They impart a “buttery”,“butterscotch” aroma to alcoholic drinks. Two of these com-pounds are important in beer, that is, diacetyl (2,3-butanedione)and 2,3-pentanedione. Diacetyl is quantitatively more impor-tant than 2,3-pentanedione. It has a taste threshold in lagerbeer from 17 µg/L (Saison et al. 2009) to 150 µg/L (Meilgaard

P1: SFK/UKS P2: SFK



Threonine

Threonine

α-Ketobutyrate

α-Acetohydroxybutyrate

Isoleucine

Pyruvate

α-Acetolactate α-Acetolactate

LeucineValine

α-Acetohydroxy- butyrate

Diacetyl

Acetoin

2,3-Butanediol

2,3-Pentane-dione

2,3-Pentanediol

Acetylethyl- carbinol

CO2CO2

Carbohydratemetabolism

Bacterialacetolactatedecarboxylase

CO2ILV5

Figure 33.3. The synthesis and reduction of vicinal diketones in Saccharomyces cerevisiae.

1975b), approximately 10 times lower than that of pentanedione(Wainwright 1973).

The excreted α-acetohydroxy acids are overflow products ofthe ILV pathway that are nonenzymatically degraded to thecorresponding vicinal diketones (Inoue et al. 1968). Tetraploidgene dosage series for various ILV genes have been constructedand the obtained yeast strains were used to study the influenceof the copy number of ILV genes on the production of vicinaldiketones (Debourg et al. 1990, Debourg 2002). It was shownthat the ILV5 activity is the rate-limiting step in the ILV path-way and responsible for the overflow (Fig. 33.3). The nonen-zymatic oxidative decarboxylation step is the rate-limiting stepand proceeds faster at a higher temperature and a lower pH (In-oue and Yamamoto 1970, Haukeli and Lie 1978). The producedamount of α-acetolactate is very dependent on the used yeaststrain. The production increases with increasing yeast growth.For a classical fermentation, 0.6 ppm α-acetolactate is formed(Delvaux 1998). At high aeration, this value can be increasedto 0.9 ppm and in cylindro-conical fermentations tanks evento 1.2–1.5 ppm.

It has been shown that valine inhibits the synthesis ofα-acetolactate through feedback inhibition (Magee and deRobinson-Szulmajster 1968). This inhibition is directed to theprotein Ilv6p, which is the regulatory subunit of acetohydroxyacid synthase (the catalytic subunit encoded by ILV2)(Pang andDuggleby 1999, 2001). Because the uptake of valine is delayedin a normal beer fermentation, the suppressive effect of va-line accounts for the postponed onset of total diacetyl (sumof actual diacetyl and α-acetolactate), and this effect persistslonger in worts with high levels of FAN content. In contrast,low FAN levels give two diacetyl peaks as a result of the re-quirement for valine biosynthesis. Therefore, a minimum FANlevel above the critical value of 50 ppm (Nakatani et al. 1984)

or 140 ppm (Pugh et al. 1997) should be maintained dur-ing the fermentation to ensure the presence of valine in thefermenting wort.

Yeast cells posses the necessary enzymes (reductases) to re-duce diacetyl to acetoin and further to 2,3-butanediol, and 2,3-pentanedione to 2,3-pentanediol (Bamforth and Kanauchi 2004).These reduced compounds have much higher taste thresholdsand have no impact on the beer flavor (Van Den Berg et al.1983). The reduction reactions are yeast strain dependent. Thereduction occurs at the end of the main fermentation and duringthe maturation. Sufficient yeast cells in suspension are necessaryto obtain an efficient reduction. Yeast strains that flocculate earlyduring the main fermentation needs a long maturation time toreduce the vicinal diketones. Diacetyl can be complexed usingSO2. These complexes cannot be reduced, but diacetyl can againbe liberated at a later stage by aldehydes. This situation is es-pecially applicable to yeast strains, which produce a lot of SO2.Worts, which are produced using a high content of adjuncts, canbe low in free amino acid content. These worts can give rise toa high diacetyl peak at the end of the fermentation.

There are several strategies, which can be chosen to reducethe vicinal diketones amount during fermentation:

1. Since the temperature has a positive effect on the reductionefficiency of the α-acetohydroxy acids, a warm rest periodat the end of the main fermentation and a warm maturationare applied in many breweries. In this case, temperatureshould be well controlled to avoid yeast autolysis.

2. Since the rapid removal of vicinal diketones requires yeastcells in an active metabolic condition, the addition of5–10% Krausen (containing active, growing yeast) is aprocedure, which gives enhanced transformation of vic-inal diketones (NN 2000). This procedure can lead to

P1: SFK/UKS P2: SFK



overproduction of hydrogen sulfide, depending upon theproportions of threonine and methionine carried forwardfrom primary fermentation.

3. Heating up the green beer to a high temperature (90◦C)and hold it there for a short period (ca. 7–10 min) to de-carboxylate all excreted α-acetohydroxy acids. To avoidcell autolysis, yeast cells are removed by centrifugationprior to heating up. The vicinal diketones can be further re-duced by immobilized yeast cells in a few hours (typicallyat 4◦C) (see further).

4. Adding the enzyme α-acetolactate decarboxylase (Godt-fredsen et al. 1984, Rostgaard-Jensen et al. 1987): Thisenzyme decarboxylates α-acetolactate directly into ace-toin (see Fig. 33.3). It is not present in S. cerevisiae, buthas been isolated from various bacteria such as Enter-obacter aerogenes, Aerobacter aerogenes, Streptococcoslactis, Lactobacillus casei, Acetobacter aceti, and Aceto-bacter pasteurianus. It has been shown that the additionof α-acetolactate decarboxylase from L. casei can reducethe maturation time to 22 hours (Godtfredsen et al. 1983,1984). An example of a commercial product is Maturex Lfrom Novo Nordisk (Denmark) (Jensen 1993). Maturex Lis a purified α-acetolactate decarboxylase produced by agenetically modified strain of Bacillus subtilis, which hasreceived the gene from Bacillus brevis. The recommendeddosage is 1 a 2 kg per 1000 hL wort, to be added to thecold wort at the beginning of fermentation.

5. Using genetic modified yeast strains:a. Introducing the bacterial α-acetolactate decarboxylase

gene into yeast chromosomes (Fujii et al. 1990, Suihkoet al. 1990, Blomqvist et al. 1991, Enari et al. 1992,Linko et al. 1993, Yamano et al. 1994, Tada et al. 1995,Onnela et al. 1996). Transformants possessed a veryhigh α-acetolactate decarboxylase activity that reducedthe diacetyl concentration considerably during beerfermentations.

b. Modifying the biosynthetic flux through the ILV path-way by partially deactivation of ILV2. Spontaneousmutants resistant to the herbicide sulfometuron methylhave been selected. These strains showed a partial in-activation of the α-acetolactate synthase activity andsome mutants produced 50% less diacetyl compared tothe parental strain (Gjermansen et al. 1988).

c. Increasing the flux of α-acetolactate acid isomerore-cuctase activity encoded by the ILV5 gene (Dillemanset al. 1987). Since α-acetolactate acid isomerore-cuctase activity is responsible for the rate-limitingstep, increasing its activity reduces the overflow of α-acetolactate. A multicopy transformant resulted in a70% decreased production of vicinal diketones (Villa-neuba et al. 1990), whereas an integrative transformantgave a 50% reduction (Goossens et al. 1993). A tan-dem integration of multiple ILV5 copies resulted alsoin elevated transciption in a polyploidy industrial yeaststrain (Mithieux and Weiss 1995). Vicinal diketonesproduction could be reduced by targeting the mito-chondrial Ilv5p to the cytosol (Omura 2008).

SECONDARY FERMENTATIONDuring the secondary fermentation or maturation of beer, severalobjectives should be realized:

� Sedimentation of yeast cells� Improvement of the colloidal stability by sedimentation of

the tannin–protein complexes� Beer saturation with carbon dioxide� Removal of unwanted aroma compounds� Excretion of flavor-active compounds from yeast to give

body and depth to the beer� Fermentation of the remaining extract� Improvement of the foam stability of the beer� Adjustment of the beer color (if necessary) by adding color-

ing substances (e.g., caramel)� Adjustment of the bitterness of beer (if necessary) by

adding hop products

In the presence of yeast, the principal changes that occur arethe elimination of undesirable flavor compounds, such as vicinaldiketones, hydrogen sulfide, and acetaldehyde, and the excretionof compounds enhancing the flavor fullness (body) of beer.

Vicinal Diketones

In traditional fermentation lagering processes, the eliminationof vicinal diketones required several weeks and determined thelength of the maturation process. Nowadays, the maturationphase is much shorter since strategies are used to acceleratethe vicinal diketones removal (see the preceding text). Diacetylis used as a marker molecule. The objective during lagering isto reduce the diacetyl concentration below its taste threshold(<0.10 mg/mL).

Hydrogen Sulfide

Sulfite and hydrogen sulfide are intermediates in the biosynthe-sis of the sulfur-containing amino acids methionine and cysteine(Van Haecht and Dufour 1995, Duan et al. 2004). Hydrogen sul-fide plays an important role during maturation. Inorganic sulfateis taken up by the yeast cells via a permease. Subsequently,it is reduced to sulfide via the intermediates adenylyl sulfate,phosphoadenylyl sulfate and sulfite (see Fig. 33.4). H2S andSO2, which are not incorporated in S-containing amino acids,are excreted by the yeast cell during the growth phase (Ryderand Masschelin 1983, Thomas and Surdin-Kerjan 1997). Theexcreted amount depends on the used yeast strain, the sulfatecontent of the wort and the growth conditions (Romano andSuzzi 1992). Methionine causes decreased production of sulfurcompounds by feedback inhibition, while threonine increasesthe production (Thomas and Surdin-Kerjan 1997). H2S and SO2

can also be formed from the catabolism of S-containing aminoacids (Dual et al. 2004). The production of H2S and SO2 de-pends on the yeast strain, sulfate content in the wort, and growthconditions. The production of H2S could be reduced by the ex-pression of cystathione synthase genes from S. cerevisiae in abrewing yeast strain (Tezuka et al. 1992).

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Hydrogen Sulphide

Sulphate Sulphate

Adenylyl sulphate

Phospoadenylyl sulphate

Sulphite Sulphite

Hydrogen sulphide

Homocysteine

Methionine

S-adenosylmethionine

Cystathionine

Cysteine

o-Acetylhomoserine

1

Gluthatione

S-adenosylhomocysteine

CH3-THF

CH2-THF

THF

Serine

Glycine CH3-X

X

2

3

4

5

6

7

8

910

11

12

13

14

Figure 33.4. The remethylation, transulfuration, and sulfur assimilation pathways. Genes and enzymes catalyzing individual reactions are asfollows: 1, sulfate permease; 2, ATP sulfurylase; 3, MET14: adenylylsulfate kinase (EC 2.7.1.25); 5, MET10: sulfite reductase (EC 1.8.1.2); 6,sulfite permease; 7, MET17: O-acetylhomoserine (thiol)-lyase (EC 2.5.1.49); 8, CYS4: cystathionine β-synthase (CBS; EC 4.2.1.22); 9,CYS3: cystathionine γ-lyase (EC 4.4.1.1), 10, MET6: methionine synthase (EC 2.1.1.14); 11, SAM1 and SAM2: S-adenosylmethioninesynthetase (EC 2.5.1.6); 12, SAH1: S-adenosylhomocysteine hydrolase (EC 3.3.1.1); 13, SHM1 and SHM2: serine hydroxymethyltransferase(SHMT; EC 2.1.2.1); 14, MET12 and MET13: methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20). “X” represents any methyl groupacceptor; THF, tetrahydrofolate; CH2-THF, 5,10-methylenetetrahydrofolate; CH3-THF, 5-methyltetrahydrofolate. (Partly adapted from Chanand Appling 2003.)

At the end of the primary fermentation and during the matu-ration, the excess H2S is reutilized by the yeast. A warm con-ditioning period at 10 a 12◦C may be used to remove excessivelevels of H2S.

Brewing yeasts produce H2S when they are deficient in thevitamin pantothenate (Walker 1998). This vitamin is a precursorof coenzyme A, which is required for metabolism of sulfate intomethionine. Therefore, panthothenate deficiency may result inan imbalance in sulfur amino acid biosynthesis, leading to excesssulfate uptake and excretion of H2S (Slaughter and Jordan 1986).

Sulfite is a versatile food additive used to preserve a largerange of beverages and foodstuffs. In beer, sulfite has a dualpurpose, acting both as an antioxidant and an agent for maskingof certain off-flavors. Some of the flavor stabilizing propertiesof sulfite is suggested to be due to complex formation of bisul-fate with varying carbonyl compounds, of which some wouldgive rise to off-flavors in bottled beer (Dufour 1991). Especially,

the unwanted carbonyl trans-2-nonenal has received particularattention, since it is responsible for the “cardboard” flavor ofsome types of stale beer. It has been suggested that it would bebetter to use a yeast strain with reduced sulfite excretion duringfermentation and to add sulfite at the point of bottling to ensuregood flavor stability (Francke Johannesen et al. 1999). There-fore, a brewer’s yeast disabled in the production of sulfite hasbeen constructed by inactivating both copies of the two allelesof the MET14 gene (which encodes for adenylylsulfate kinase).Fermentation experiments showed that there was no qualitativedifference between yeast-derived and artificially added sulfite,with respect to trans-2-nonenal content and flavor stability ofthe final beer.

The elimination of the gene encoding sulfite reductase(MET10) in brewing strains of Saccharomyces results in in-creased accumulation of SO2 in beer (Hansen and Kielbrandt1996a). The inactivation of MET2 resulted into elevated sulfite

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concentrations in beer (Hansen and Kielbrandt 1996b). Beersproduced with increased levels of sulfite showed an improvedflavor stability.

Acetaldehyde

Aldehydes—in particular, acetaldehyde (green apple-likeflavor)—have an impact on the flavor of green beer. The accu-mulation of acetaldehyde is dependent on the kinetic propertiesof enzymes responsible for its formation (pyruvate decarboxy-lase and acetaldehyde dehydrogenase) and dissimilation (alco-hol dehydrogenase) (Boulton and Quain 2006). Acetaldehydesynthesis is linked to yeast growth (Geiger and Piendl 1976).Its concentration is maximal at the end of the growth phase andis reduced at the end of the primary fermentation and duringmaturation by the yeast cells. As with diacetyl, levels may beenhanced if yeast metabolism is stimulated during transfer, es-pecially by oxygen ingress. Removal also requires the presenceof enough active yeast. Fermentations with early flocculatingyeast cells can result in too high acetaldehyde concentrations atthe end.

Development of Flavor Fullness

During maturation, the residual yeast will excrete compounds(i.e., amino acids, phosphates, peptides, nucleic acids, . . .) intothe beer. The amount and “quality” of these excreted materialsdepend on the yeast concentration, yeast strain, its metabolicstate and the temperature (NN 2000). Rapid excretion of materialis best achieved at a temperature of 5–7◦C during 10 days (Vande Meersche et al. 1977).

When the conditioning period is too long or when the temper-ature is too high, yeast cell autolysis will occur. Some enzymesare liberated (e.g., α-glucosidase), which will produce glucosefrom traces of residual maltose (NN 2000). At the bottom ofa fermentation tank, the amount of α-amino-nitrogen can riseto 40–10,000 mg/L, which account for an increase of 30 mg/Lfor the total beer volume. The increase in amino acid concen-tration in the beer has a positive effect on the flavorfullness ofthe beer. Undesirable medium-chain fatty acids can also be pro-duced in significant amounts if the maturation temperature istoo high (Masschelein 1981). Measurement of these compoundsindicates the level of autolysis and permits the determination ofthe most appropriate conditioning period and temperature.

BEER FERMENTATION USINGIMMOBILIZED CELL TECHNOLOGYThe advantages of continuous fermentation—such as greaterefficiency in utilization of carbohydrates and better use ofequipment—led also to the development of continuous beerfermentation processes. Since the beginning of the twentiethcentury, many different systems using suspended yeast cellshave been developed. The excitement for continuous beer fer-mentation led—especially during the 1950 and 1960s—to thedevelopment of various interesting systems. These systems canbe classified as (i) stirred versus unstirred tank reactors, (ii)

single-vessel systems versus a number of vessels connected inseries, (iii) vessels that allow yeast to overflow freely with thebeer (“open system”) versus vessels that have abnormally highyeast concentrations (“closed” or “semiclosed system”) (Well-hoener 1954, Coutts 1957, Bishop 1970, Hough et al. 1982,).However, these continuous beer fermentation processes werenot commercially successful due to many practical problems,such as the increased danger of contamination (not only duringfermentation but also during storage of wort in supplementaryholdings tanks that are required since the upstream and down-stream brewing processes are usually not continuous), changesin beer flavor (Thorne 1968) and a poor understanding of thebeer fermentation kinetics under continuous conditions. One ofthe well-known exceptions is the successful implementation of acontinuous beer production process in New Zealand by MortonCoutts (Dominion Breweries), which is still in use today (Coutts1957, Hough et al. 1982).

In the 1970, there was a revival in developing continuousbeer fermentation systems due to the progress in research onimmobilization bioprocesses using living cells. Immobilizationgives fermentation processes with high cell densities, resultingin a drastic increase in fermentation productivities compared tothe traditional time-consuming batch fermentation processes.

The last 30 years, ICT has been extensively examined andsome designs have reached already commercial exploitation.Immobilized cell systems are heterogeneous systems in whichconsiderable mass transfer limitations can occur, resulting in achanged cell yeast metabolism. Therefore, successful exploita-tion of ICT needs a thorough understanding of mass transfer andintrinsic yeast kinetic behavior of these systems.

Carrier Materials

Cell immobilization can be classified into four categories basedon the mechanism of cell localization and the nature of sup-port material: (i) attachment to the support surface, which canbe spontaneous or induced by linking agents; (ii) entrapmentwithin a porous matrix; (iii) containment behind or within a bar-rier; and (iv) self-aggregation, naturally or artificially induced(Karel et al. 1985, Willaert and Baron 1996). Various cell im-mobilization carrier materials have been tested and used forbeer production/bioflavoring. Selection criteria are summarizedin Table 33.5. Depending on the particular application, reac-tor type and operational conditions, some selection criteria willbe more appropriate. Examples of selected carrier materials forparticular applications are tabulated in Table 33.6.

Cell immobilization by self-aggregation is based on the for-mation of cell clumps or flocs. Actually, flocs are formed at theend of the primary fermentation when flocculent strains are used.Flocculent strains can also be used in continuous fermentationsystems (see “Coutts continuous beer fermentation system” de-scribed in the preceding text). Very high yeast concentrationsmay be achieved by the use of inclined tubes, still zones aroundoutlet pipes and by holding the yeast in a filter (Hough et al.1982). Growth of the sedimentary yeast may be controlled bythe amount of air injected, while carbon dioxide is used to causesome mixing. A flocculent strain has also been used in the tower

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Table 33.5. Selection Criteria for Yeast CellImmobilization Carrier Materials

� High cell mass loading capacity� Easy access to nutrient media� Simple and gentle immobilization procedure� Immobilization compounds approved for food applications� High surface area-to-volume ratio� Optimum mass transfer distance from flowing media to

centre of support� Mechanical stability (compression, abrasion)� Chemical stability� Highly flexible: rapid start-up after shut-down� Sterilizable and reusable� Suitable for conventional reactor systems� Low shear experienced by cells� Easy separation of cells and carrier from media� Readily up-scalable� Economically feasible (low capital and operating costs)� Desired flavor profile and consistent product� Complete attenuation� Controlled oxygenation� Control of contamination� Controlled yeast growth� Wide choice of yeast

Source: Nedovic et al. (2005) and Verbelen et al. (2010).

fermenter system, which comprises a vertical tube into the baseof which wort is pumped (Royston 1966). The sedimentary yeastforms a solid plug at the base of the vessel and through it thewort permeates. Fermentation proceeds as the wort rises, withthe rate of wort injection being so adjusted that at the top of thetower the wort is completely fermented.

Yeast flocculation is a reversible, asexual, and calcium-dependent process in which cells adhere to form flocs consistingof thousands of cells (Stratford 1989, Bony et al. 1997, Jinand Speers 1999). Many fungi contain a family of cell wall“adhesines” (which are glycoproteins) that confer unique ad-hesion properties (Teunissen and Steensma 1995, Guo et al.2000, Hoyer 2001, Sheppard et al. 2004). These molecules arerequired for the interactions of fungal cells with each other (floc-culation and filamentation) (Teunissen and Steensma 1995, Loand Dranginis 1998, Guo et al. 2000, Viyas et al. 2003), withinert surfaces such as agar and plastic (Gaur and Klotz 1997, Loand Dranginis 1998, Reynolds and Fink 2001, Li and Palecek2003) and with mammalian tissues/cells (Cormack et al. 1999,Staab et al. 1999, Fu et al. 2002, Li and Palecek 2003). Theyare also crucial for the formation of fungal biofilms (Baillie andDouglas 1999, Reynolds and Fink 2001, Green et al. 2004). Theadhesin proteins in S. cerevisiae are encoded by FLO genes,including FLO1, FLO5, FLO9, FLO10, and FLO11 (Verstrepenet al. 2004). These proteins are called flocculins (Caro et al.1997) because these proteins promote cell–cell adhesion to formmulticellular clumps that sediment out of solution.

The flocculation phenomenon is genetically controlled by33 genes (Teunissen and Steensma 1995). Recently, the fivemembers of the FLO-adhesine family were studied by selectively

overexpressing each FLO gene in the laboratory strain S288C(Van Mulders et al. 2009). As all FLO genes are transcriptionallysilent in the S288C-strain background, each FLO gene can beactivated one by one and each resultant phenotype can be investi-gated. The FLO1, FLO5, FLO9, and FLO10 genes share consid-erable sequence homology. The member proteins of the adhesinfamily have a modular configuration that consists of three do-mains (A, B, and C) and an N-terminal secretory sequence thatmust be removed as the protein moves through the secretorypathway to the plasma membrane (Hoyer et al. 1998). The N-terminal domain (A) is involved in sugar recognition (Kobayashiet al. 1998). The adhesins undergo several posttranslational mod-ifications, that is, N- and O-glycosylations. They move from theendoplasmic reticulum (ER) through the Golgi and pass throughthe plasma membrane and find their final destination in the cellwall, where they are anchored by a glycosyl phosphatidylinositol(GPI) (Teunissen et al. 1993a, 1993b, Bidard et al. 1994, Bonyet al. 1997, Hoyer et al. 1998). The GPI anchor is added tothe C-terminus in the ER, and mannose residues are added tothe many serine and threonine residues in domain B in the Golgi(Udenfriend and Kodukula 1995, Bony et al. 1997, Frieman et al.2002, De Groot et al. 2003). The FLO1 gene product (Flo1p)has been localized at the cell surface by immunofluorescent mi-croscopy (Bidard et al. 1995). The amount of Flo proteins inflocculent strains increased during batch yeast growth and theFlo1p availability at the cell surface determined the flocculationdegree of the yeast. Flo proteins are polarly incorporated into thecell wall at the bud tip and the mother–daughter neck junction(Bony et al. 1997). The transcriptional activity of the flocculationgenes is influenced by the nutritional status of the yeast cells aswell as other stress factors (Verstrepen et al. 2003a). This impliesthat during beer fermentation, flocculation is affected by numer-ous parameters such as nutrient conditions, dissolved oxygen,pH, fermentation temperature, and yeast handling and storageconditions.

Applications of ICT in the Brewing Industry

Beer production with immobilized yeast has been the subject ofresearch for approximately 40 years, but has so far found lim-ited application in the brewing industry, because of engineeringproblems, unrealised cost advantages, microbial contaminations,and an unbalanced beer flavor (Linko et al. 1998, Branyik et al.2005, Nedovic et al. 2005, Willaert and Nedovic 2006, Verbelenet al. 2010). The ultimate aim of this research is the productionof beer, of desired quality, within 1–3 days. Traditional beer fer-mentation systems use freely suspended yeast cells to fermentwort in an unstirred batch reactor. The primary fermentationtakes approximately 7 days with a subsequent secondary fer-mentation (maturation) of several weeks. A batch culture systememploying immobilization could benefit from an increased rateof fermentation. However, it appears that in terms of increasingproductivity, a continuous fermentation system with immobi-lization would be the best method (Verbelen et al. 2006). Animportant issue of the research area is to whether beer can beproduced by immobilized yeast in continuous culture with thesame characteristic as the traditional method.

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Table 33.6. Some Selected Applications of Cell Immobilization Systems Used for Beer Production

Carrier Material Reactor Type Reference

Flavor maturationCalcium alginate beads Fixed bed Shindo et al. (1994)DEAE-cellulose Fixed bed Pajunen and Gronqvist (1994)Polyvinyl alcohol beads Fixed bed Smogrovicova et al. (2001)Porous glass beads Fixed bed Linko et al. (1993), Aivasidis (1996)Alcohol-free beerDEAE-cellulose beads Fixed bed Collin et al. 1991, Lomni et al. 1990Porous glass beads Fixed bed Aivasidis et al. (1991)Silicon carbide rods Monolith reactor Van De Winkel et al. (1991)

Acidified wortDEAE-cellulose beads Fixed bed Pittner et al. (1993)

Main fermentationCalcium alginate beads Gas lift White and Portno (1979), Nedovic et al. (1997)Calcium pectate beads Gas lift Smogrovicova et al. (1997)κ-Carrageenan beads Gas lift Mensour et al. (1996), Decamps et al. (2004)Ceramic beads Fixed bed Inoue (1995)Corncobs Gas lift Branyik et al. (2006a)Gluten pellets Fixed bed Bardi et al. 1997Polyvinyl alcohol beads Gas lift Smogrovicova et al. (2001)Polyvinyl alcohol Lentikats R© Gas lift Smogrovicova et al. (2001), Bezbradica et al. (2007)Polyvinyl chloride granules Gas lift Moll et al. (1973)Porous glass beads Fixed bed Virkajarvi and Kronlof (1998)Porous chitosan beads Fluidized bed Unemoto et al. (1998), Maeba et al. (2000)Silicon carbide rods Monolith reactor Andries et al. (1996)Spent grains Gas lift Branyik et al. (2002, 2004)Stainless steel fibre cloths Gas lift Verbelen et al. (2006)Wood (aspen beech) chips Fixed bed Linko et al. (1997), Kronlof and Virkajarvi (1999),

Pajunen et al. (2001)

Started in 1971, one of the first ICT systems for the fer-mentation and maturation of beer was developed by TREPAL(the R&D Centre of the Konenbourg Brewery and the Euro-pean Brewery) with INSA (Institut National des Sciences Ap-pliquees, Toulouse, France) and INRA (Institus National desRecherche Agronomique, Dijon, France) (Moll et al. 1973,Moll and Dueurtre 1996, Moll 2006). A continuous pilot in-stallation for fermentation and maturation of beer with a flowrate of 5 L/h was developed and worked for 9 months withoutany microbial contamination. Around the same time, Narzissand Hellich (1971) developed an ICT process where yeast cellswere immobilized in kieselguhr (which is widely used in thebrewing industry as a filter aid) and a kieselguhr filter was em-ployed as bioreactor (called the “bio-brew bioreactor”). Theiryeast cell immobilization method was based on a method forthe immobilization of enzymes (Berdelle-Hilge 1966). Fromthe beginning of the nineteen seventies, various systems havebeen developed and some have been implemented on anindustrial scale.

ICT processes have been developed for (i) the flavor mat-uration of green beer, (ii) the production of alcohol-free orlow-alcohol beer, (iii) the production of acidified wort usingimmobilized lactic acid bacteria, and (iv) the continuous beer

fermentation (Branyik et al. 2005, Verbelen et al. 2006, Willaertand Nedovic 2006, Willaert 2007, Verbelen et al. 2010).

Flavor Maturation of Green Beer

The objective of flavor maturation is the removal of di-acetyl and 2,3-pentanedione, and their precursors α-acetolactateand α-acetohydroxybutyrate, which are produced during themain fermentation (see the preceding text). The conversionof α-acetohydroxy acids to the vicinal diketones is the rate-limiting step. This reaction step can be accelerated by heat-ing the beer—after yeast removal—to 80–90◦C during a cou-ple of minutes. The resulting vicinal diketones are subse-quently reduced by immobilized cells into their less-flavor-activecompounds.

The traditional maturation process is characterized by a near-zero temperature, low pH and low yeast concentration, resultingin a very long maturation period of 3–4 weeks. Different strate-gies have been developed to accelerate diacetyl removal (seeSection “Vicinal diketones”). One of the techniques is the useof an ICT process to reduce the maturation period to about2 hours.

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Two continuous maturation systems have been implementedindustrially so far: one at Sinebrychoff Brewery (Finland, capac-ity: 1 million hL per year) (Pajunen 1995) and another system,developed by Alfa Laval and Schott Engineering (Mensour et al.1997). They are both composed of a separator (to prevent grow-ing yeast cells in the next stages), an anaerobic heat treatmentunit (to accelerate the chemical conversion of α-acetolactateto diacetyl, but also the partial directly conversion to acetoin),and a packed bed reactor with yeast immobilized on DEAE-cellulose granules or porous glass beads (to reduce the remain-ing diacetyl), respectively (Yamauchi et al. 1994). Later on, theDEAE-cellulose carriers were replaced by cheaper wood chips(Virkajarvi 2002). The heat treatment has been replaced by anenzymatic transformation in a fixed bed reactor in which the α-acetolactate decarboxylase is immobilized in special multilayercapsules, followed by the reduction of diacetyl by yeast in asecond packed bed reactor (Nitzsche et al. 2001).

Production of Alcohol-Free or Low-Alcohol Beer

The classical technology to produce alcohol-free or low-alcoholbeer is based on the suppression of alcohol formation by ar-rested batch fermentation (Narziss et al. 1992). However, theresulting beers are characterized by an undesirable wort aroma,since the wort aldehydes have only been reduced to a limiteddegree (Collin et al. 1991, Debourg et al. 1994, van Iersel et al.1998). The reduction of these wort aldehydes can be quicklyachieved by a short contact time with immobilized yeast cells ata low temperature without undesirable cell growth and ethanolproduction. A disadvantage of this short contact process is theproduction of only a small amount of desirable esters.

Controlled ethanol production for low-alcohol and alcohol-free beers have been successfully achieved by partial fermen-tation using DEAE-cellulose as carrier material, which waspacked in a column reactor (Collin et al. 1991, Van Dieren1995). This technology has been successfully implemented byBavaria Brewery (The Netherlands) to produce malt beer on anindustrial scale (150,000 hL/year) (Pittner et al. 1993). Severalother companies—that is, Faxe (Denmark), Ottakringer (Aus-tria), and a Spanish brewery—have also implemented this tech-nology (Mensour et al. 1997). In Brewery Beck (Germany), afluidized-bed pilot scale reactor (8 hL/day) filled with porousglass beads was used for the continuous production of nonal-coholic beer (Aivasidis et al. 1991, Breitenbucher and Mistler1995, Aivasidis 1996). Yeast cells immobilized in silicon carbiderods and arranged in a multichannel loop reactor (Meura, Bel-gium) have been used to produce alcohol-free beer at pilot scaleby Grolsch Brewery (The Netherlands) and Guinness Brewery(Ireland) (Van De Winkel 1995).

Nuclear mutants of S. cerevisiae that are defective in thesynthesis of tricarboxylic acid cycle enzymes; that is, fu-marase (Kaclıkova et al. 1992) or 2-oxoglutarate dehydrogenase(Mockovciakova et al. 1993) have been immobilized in calciumpectate gel beads and used in a continuous process for the pro-duction of nonalcoholic beer (Navratil et al. 2000). These strainsproduced minimal amounts of ethanol and they were also ableto produce much lactic acid (up to 0.64 g/dm3).

Production of Acidified Wort Using Immobilized LacticAcid Bacteria

The objective of this technology is the acidification of the wortaccording to the “Reinheidsgebot”, before the start of the boil-ing process in the brewhouse. An increased productivity ofacidified wort has been obtained using immobilized Lactobacil-lus amylovorus on DEAE-cellulose beads (Pittner et al. 1993,Meersman 1994). The pH of wort was reduced below a valueof 4.0 after contact times of 7–12 minutes using a packed-bedreactor in downflow mode. The produced acidified wort wasstored in a holding tank and used during wort production toadjust the pH.

Continuous Main Fermentation

During the main fermentation of beer, not only ethanol is beingproduced but also a complex mixture of flavor-active secondarymetabolites, of which the higher (or fusel) alcohols and estersare the most important (Verbelen et al. 2010). In addition, di-acetyl and some sulfury compounds can cause off-flavors. Sincethis complex flavor profile is closely related to the amino acidmetabolism and consequently to the growth of the yeast cells,differences in the growth metabolic state between freely sus-pended and immobilized yeast cell systems are most probablyresponsible for the majority of alterations in the beer flavor. Forthat reason, it is important that the physiological and metabolicstate of the yeast in conventional batch systems is mimicked asmuch as possible during the continuous fermentation with im-mobilized yeast. In the continuous mode of operation, cells arenot exposed to significant alterations of the environment, influ-encing the metabolism of the cells and consequently the flavor.Hence, the microbial population of continuous systems lacks thedifferent growth phases of a batch culture. To imitate the batchprocess as much as possible, plug-flow reactors or a series ofreactors can be used. As can be assumed, both the continuousmode of operation and the immobilization of yeast cells caninfluence the beer flavor.

The Japanese brewery Kirin developed a multistage contin-uous fermentation process (Inoue 1995, Yamauchi et al. 1994,Yamauchi and Kasahira 1995). The first stage is a stirred tankreactor for yeast growth, followed by packed-bed fermenters,and the final step is a packed-bed maturation column. The firststage ensures adequate yeast cell growth with the desirable FANconsumption. Ca-alginate was initially selected as carrier ma-terial to immobilize the yeast cells. These alginate beads werelater replaced by ceramic beads (“Bioceramic R©”). This systemallowed to produce beer within 3–5 days.

The engineering company Meura (Belgium) developed a reac-tor configuration with a first stage with immobilized yeast cellswhere partially attenuation and yeast growth occurs, followedby a stirred tank reactor (with free yeast cells) for complete at-tenuation, ester formation, and flavor maturation (Andries et al.1996, Masschelein and Andries 1995). Silicon carbide rods areused in the first reactor as immobilization carrier material. Thestirred tank (second reactor) is continuously inoculated by freecells that escape from the first immobilized yeast cell reactor.

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Labatt Breweries (Interbrew, Canada) in collaboration withthe Department of Chemical and Biochemical Engineering atthe University of Western Ontario (Canada) developed a contin-uous system using κ-carrageenan immobilized yeast cells in anairlift reactor (Mensour et al. 1995, 1996, 1997). Pilot scale re-search showed that full attenuation was reached in 20–24 hourswith this system compared to 5–7 days for the traditional batchfermentation. The flavor profile of the beer produced using ICTwas similar to the batch fermented beer.

Hartwell Lahti and VTT Research Institute (Finland) devel-oped a primary fermentation system using ICT on a pilot scaleof 600 L/day (Kronlof and Virkajarvi 1999). Woodchips wereused as the carrier material that reduced the total investment costby one-third compared to more expensive carriers. The resultsshowed that fermentation and flavor formation were very simi-lar compared to a traditional batch process, although the processtime was reduced to 40 hours.

Andersen et al. (1999) developed a new ICT process in whichthe concentration of carbon dioxide is controlled in a fixed-bedreactor in such a way that the CO2 formed is kept dissolved andis removed from the beer without foaming problems. DEAE-cellulose was used as carrier material. High-gravity beer of ac-ceptable quality has been fermented in 20 hours at a capacityof 50 L/h.

A well-explored concept for main beer fermentation is the useof a gas-lift bioreactor system (see Table 33.6). In this system,mixing established by the circulation of liquid and solid phases,provided high liquid circulation rates, low shear environment,and good mass transfer properties (Siegel and Robinson 1992,Vunjak-Novakovic et al. 1992, Chisti and Moo-Young 1993,Baron et al. 1996). Additionally, they possess the following pos-itive characteristics: high loadings of solids, simple construction,low risk of contamination, easy adjustment and control of op-erational parameters and simple capacity enlargement (Vunjak-Novakovic et al. 1998). Various carrier materials for gas-liftbioreactors have been studied to perform the main fermentation(see Table 33.6).

The optimization of temperature, wort gravity, feed volume,and wort composition seems to be an important tool for thecontrol of the flavor-active compounds formation in immobi-lized beer fermentation systems (Verbelen et al. 2006, 2010,Willaert and Nedovic 2006, Branyik et al. 2008). Many re-searchers have concluded that the optimization of aeration duringcontinuous fermentation is essential for the quality of the finalbeer (Virkajarvi et al. 1999). Oxygen is needed for the forma-tion of unsaturated fatty acids and sterols that needed for growth(Depraetere et al. 2003). However, excess oxygen will lead tolow ester production but to excessive diacetyl, acetaldehyde, andfusel alcohol formation (Okabe et al. 1992, Wackerbauer et al.2003, Branyik et al. 2004). It is possible to adjust the flavor ofthe produced beer by ensuring the adequate amount of dissolvedoxygen by sparging with a mixture of air, nitrogen, or carbondioxide (Kronlof and Linko 1992, Branyik et al. 2004). How-ever, it remains difficult to predict the right amount of oxygenbecause the oxygen availability to the immobilized yeast cellsis dependent of external and internal mass transfer limitations(Willaert and Baron 1996, Willaert et al. 2004).

Also, the reactor design, the carrier, and yeast strain can havea dominant effect on flavor formation (Cop et al. 1989, Linko,et al. 1997, Smogrovicova and Domeny 1999, Tata et al. 1999,Virkajarvi and Pohjala 2000).

ACKNOWLEDGMENTSThis work was supported by the Belgian Federal Science Pol-icy Office and European Space Agency PRODEX program, theInstitute for the Promotion of Innovation by Science and Tech-nology in Flanders (IWT), and the Research Council of theVUB.

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Food Biochemistry and Food Processing (Simpson/Food Biochemistry and Food Processing) || Biochemistry of Beer Fermentation