Appl Microbiol Biotechnol (2003) 62:140–150DOI 10.1007/s00253-003-1340-5

M I N I - R E V I E W

B. Vanderhaegen · H. Neven · S. Coghe ·K. J. Verstrepen · G. Derdelinckx · H. Verachtert

Bioflavoring and beer refermentation

Received: 27 January 2003 / Revised: 10 April 2003 / Accepted: 11 April 2003 / Published online: 21 May 2003� Springer-Verlag 2003

Abstract Various techniques are used to adjust theflavors of foods and beverages to new market demands.Although synthetic flavoring chemicals are still widelyused, flavors produced by biological methods (bioflavors)are now more and more requested by consumers,increasingly concerned with health and environmentalproblems caused by synthetic chemicals. Bioflavors canbe extracted from plants or produced with plant cellcultures, microorganisms or isolated enzymes. This Mini-Review paper gives an overview of different systems forthe microbial production of natural flavors, either denovo, or starting with selected flavor precursor molecules.Emphasis is put on the bioflavoring of beer and thepossibilities offered by beer refermentation processes.The use of flavor precursors in combination with non-conventional or genetically modified yeasts for theproduction of new products is discussed.

Introduction

For thousands of years humans have intentionally oraccidentally used techniques to produce foods withattractive flavors. It was not realized that in mostinstances microorganisms were involved in flavor pro-duction or enhancement. New knowledge, mainly gainedin the last century of microbiology and biochemistry hasrevealed the importance of microorganisms for flavordevelopment. In consequence, much research has sincebeen focused on the possibilities of designing processesfor flavor bioproduction under well-understood andcontrolled conditions. In contrast to chemically produced

substances, flavors produced by biological methods(bioflavors) may be considered as natural flavors(Cheetham 1993). Foods containing such natural flavorsare favored by consumers, who are increasingly con-cerned with possible health issues and environmentaldamage caused by synthetic chemicals and the productionthereof. For the bioproduction of flavors, several ap-proaches are possible: extraction from plant material,plant cell cultures, enzymatic synthesis or the use ofspecific microorganisms. This review will focus onmicrobial bioflavoring with emphasis on the bioflavoringof beer through refermentation.

Flavors produced by microorganisms

Microorganisms produce an amazingly broad array ofsecondary metabolites, of which many are importantflavor compounds. In foods and beverages this may cause(unwanted) off-flavors, but there are numerous examplesof the need for microorganisms for the development ofpleasant, attractive, or requested flavors. Most traditionalfermented foods and beverages rely on a large variety ofmicroorganisms to obtain their typical characteristics. Thebest-known examples are kefir (Hall� et al. 1994),sourdough (Halm et al. 1993), soy sauce (Hamada et al.1991), dry sausages (Montel et al. 1996), etc. In thebrewing sector, a very special beer relying on a mixedculture fermentation for flavor development is the Bel-gian gueuze (Verachtert et al. 1989). However, purecultures of one specific microorganism can also beresponsible for a specific and complex flavor profile.Probably the best-known example is the use of specificstrains of the yeast Saccharomyces cerevisiae to producebeers with the required complex final flavor. Thus, theflavors produced by a pure culture are still the result of amixture of several flavor compounds. However, in somecases it is desirable to produce or modify only specificflavor substances. To introduce a single new flavorcompound or enhance its presence in foods and bever-ages, new approaches are needed. These may include

B. Vanderhaegen ()) · H. Neven · S. Coghe · K. J. Verstrepen ·G. Derdelinckx · H. VerachtertCenter for Malting and Brewing Science,Katholieke Universiteit Leuven,Kasteelpark Arenberg 22, 3001 Heverlee, Belgiume-mail: bart.vanderhaegen@agr.kuleuven.ac.beTel.: +32-16321460Fax: +32-16321576

genetic modification of a production strain, the imple-mentation in the process of additional strains producingthe required compound, or the addition to the medium ofspecific flavor precursor molecules. Finally, it might beinteresting to produce the bioflavor in a side-chainprocess operating in conditions not suited for the mainproduction process. In the following sections, the differ-ent possibilities for bioflavor production are brieflyconsidered. Subsequently, the specific possibilities forbeer refermentation are discussed.

Microbial de novo synthesis of flavor compounds

Various microorganisms are capable of producing denovo odorous compounds when they are grown in adefined medium. Examples from molds include theproduction of terpenes by Ceratocystis sp. (Collins1976) and 6-pentyl-a-pyrone by Trichoderma viridae.This lactone is characterized by a coconut-like odor. Toavoid inhibition phenomena, H�usler and M�nch (1997)proposed a continuous pervaporation system to extract thelactone from the culture medium (Fig. 1).

Other examples of flavor-producing fungi include theyeast Kluyveromyces lactis, which forms the monoter-penes citronellol, linalool and geraniol, with a fruity androse-like odor (Drawert and Barton 1978) and Sporobo-lomyces odorus, which produces lactones such as thepeach-like smelling compounds g-decalactone (4-decano-lide) and 4-hydroxy-cis-6-dodecenoic acid g-lactone (cis-6-dodecen-4-olide) (Tahara et al. 1972, 1973).

Pyrazines are synthesized de novo mostly by bacteria(Demain et al. 1967). Pyrazines are responsible for theroasted, nutty flavor of heated foodstuff like roasted nuts,coffee and cocoa beans, baked products and meat.Biologically produced pyrazines may be of interest foraddition of roasty flavors to products obtained by newfood processing systems such as microwave cooking. Adetailed overview is given by Janssens et al. (1992).

Microbial bioconversion of selected flavor precursors

Microorganisms can be used to convert (added) specificorganic molecules of biological origin to flavor com-pounds. Although there are plenty of possibilities, fewflavors are produced on an industrial scale using thisapproach. This is due to the high costs of the processesand the low production yields—partially caused byinsufficient knowledge of the biosynthetic pathways—and the relatively high costs of suitable precursorsubstrates (Feron et al. 1996). Therefore, only compoundswith a high added value like g- and d-lactones, vanillaaroma and various esters are currently produced bybioconversion strategy.

The bioproduction of vanillin is shown in Fig. 2 and adetailed overview of the biotechnological production isgiven by Priefert et al. (2001) and Rao and Ravishankar(2000). In a first route, vanillin is synthesized from thestilbene isorhapontin, which is commonly found in sprucebark. The conversion is catalyzed by stilbene dioxygenase

Fig. 1 Production of 6-pentyl-a-pyrone by Trichoderma viri-dae

Fig. 2 Bioproduction of vanillin by different pathways

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present in Pseudomonas sp. This method has led tonumerous patents (Yoshimoto et al. 1990a, 1990b).

Alternatively, vanillin bioproduction starts with euge-nol, the main constituent of clove oil, which is aninexpensive, commercially available, raw material. Theconversion is carried out by strains of Corynebacteriumand Pseudomonas. In recent years, enzymes and corre-sponding genes responsible for the conversion of eugenolto ferulic acid and further to vanillin in Pseudomonasstrains have been characterized (Achterholt et al. 1998;Brandt et al. 2001; Gasson et al. 1998; Narbad and Gasson1998; Overhage et al. 1999a, 1999b; Priefert et al. 1997,1999). Increased insight into the biochemical and geneticproperties of these metabolic pathways offers the possi-bility of increasing the production yield and industrialapplication of this bioproduction pathway.

Important raw materials for the bioproduction offlavors and fragrances are fatty acids. Since they arederived from plant or animal oil, they are cheap andavailable in large amounts. A good example of the use offatty acids for bioflavoring is the use of the filamentousfungus Botryodiplodia theobromae, which can convert a-linolenic acid (C18:3; found in linseed oil) into jasmonicacid. This compound has a favorable, sweet-floral,jasmine-like odor (H�usler and M�nch 1997).

Flavor-active lactones, such as g-decalactone and d-decalactone, can be produced by the bioconversion ofdifferent hydroxy fatty acids. Interestingly, microorgan-isms are capable of forming optically active g-decalactone(Cheetham 1993). The bioproduction of g-decalactone isan important process since consumers prefer the naturalstereochemical pure product over the chemical product.Whereas g-decalactone has a defined stereospecificity infruit [(R)-isomer in peaches and most other fruit, (S)-isomer in mango], the chemical synthesis of this moleculeresults in a racemic mixture. A well-studied industrial

example is the microbial conversion of ricinoleic acid intog-decalactone by yeasts such as Sporidiobolus salmoni-color and Yarrowia lipolytica (Fig. 3) (Lee et al. 1998;Lin et al. 1996). The main problem for the industrialproduction of some lactones is, however, the limitedavailability of hydroxy fatty acids. For ricinoleic acid theonly source of considerable amounts is castor oil (H�uslerand M�nch 1997).

Another interesting example of aroma-active lactonesynthesis was described by Wanikawa et al. (2000). Theseauthors found that, during the maturation of whisky, adodecalactone with whisky flavor was formed through thecombined activity of lactic acid bacteria and yeast. Thebacteria synthesized the precursor hydroxy-fatty acidfrom an unsaturated fatty acid coming from yeast.

The enzymatic synthesis of flavoring compounds

The bioproduction of flavors and fragrances with isolatedmicrobial enzymes is an alternative to the use ofmicrobial cell-based processes. Isolated enzymes can beused under mild environmental conditions and often showa high specificity and stereoselectivity in the conversionof substrates. On the other hand, the use of isolatedenzymes may be restricted by high costs and low enzymestability. An additional problem for cofactor-dependentenzymes is the need to regenerate the co-enzymes.

Nevertheless, recent developments in biochemistry,biology and technology may solve most of theseproblems. New techniques have been developed for theimmobilization and stabilization of enzymes (Krishna2002; Lee and Dordick 2002; Savelli et al. 2000; van deVelde et al. 2002). Improvements in molecular andcellular biology have made it possible to construct genesfor the production of enzymes with better stability andimproved cofactor binding and regeneration (Kragl et al.1996; Lehmann and Wyss 2001; Lehmann et al. 2000;Waldo 2003). This, together with the fact that thestereochemical properties of molecules have a greatimpact on their flavor characteristic (Amoore et al.1964), has boosted the use of isolated enzymes for theproduction of enantiomerically pure compounds.

An industrial example is the production of l-menthol,which is the major constituent of peppermint oil and isused in large quantities in the flavor and fragranceindustry. Only l-menthol has the characteristic pepper-mint odor and cooling effect. The process relies onmicrobial lipases that preferentially hydrolyze l-menthylesters leaving the d-menthyl esters untouched (Fig. 4)(Schreier 1997). Enantioselective biocatalysis by a bac-terial lipase has been extensively discussed by Jaeger etal. (2002).

Bioflavoring of beer by refermentation

In beverages such as beer and wine, flavor is apredominant quality aspect for producers and consumers.

Fig. 3 Formation of the peach flavored lactone g-decalactonethrough bioconversion of castor oil

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The required flavors originate from the raw materials, butto a large part also from the yeast used for the mainfermentation. The flavor of beer can be further developedand improved by a refermentation process. Often, a yeaststrain is added to the finished beer to initiate refermen-tation in casks or bottles. In Belgium, many specialtybeers are produced in this way. In wine production, the“m�thode champ�noise” for the production of champagneis the best example of a refermentation system.

Bioflavoring of beer by refermentation with S. cerevisiae

For beer refermentation, just before bottling, sugar (5–9 g/l) and a yeast strain (105–106 cells/ml) are added. Duringrefermentation, the ethanol and the CO2 concentrationsincrease. Carbonation gives the beer a refreshing taste andcreates foam. As an oxygen scavenger, yeast makes beerless sensitive to oxygen-dependent off-flavors (Derde-linckx et al. 1992). After the main fermentation, the beeris poor in nutrients and does not contain any oxygen.Therefore, yeast metabolism during the refermentation ofbeer will be mainly fermentative. This results in an excessof reduced coenzymes NADH and NADPH. In order toregenerate these coenzymes, they are used by severalaldoketoreductases (Debourg et al. 1993, 1995; Van Ierselet al. 1997; Van Nedervelde et al. 1997, 1999). This so-called yeast reducing power may result in the reduction ofseveral aldehydes to alcohols. Off-flavors in beer areoften associated with certain aldehydes and a refermen-tation quenches these flavors since the alcohols are lessflavor active (Table 1)

During refermentation, residual amino acids in beer(about one-third to one-fifth of the concentration in wort)are further taken up. However, their uptake and desam-

ination to keto-acids are too low to account for largechanges in flavors due to the subsequent formation ofhigher alcohols and esters.

Following the active refermentation phase, a longstorage period in the bottle of refermented beer in contactwith the yeast may result in yeast autolysis. In its turn,autolysis can lead to the excretion in beer of intracellularcompounds as amino acids, peptides, nucleotides, fattyacids and enzymes which may affect the flavor profile(Ernest et al. 1980; Leroy et al. 1990; Masschelein 1986;Ormrod et al. 1991). For example, Horsted et al. (1998)and Neven et al. (1997) showed that the excretion ofesterases leads to a considerable change of the esterprofile of beer. Isoamyl acetate, ethyl hexanoate and ethyloctanoate are hydrolyzed. In contrast, ethyl isovalerate,ethyl isobutyrate and ethyl phenylacetate are formed.During the main fermentation, esters are synthesized byan energy-dependent mechanism but through the releaseof esterases, the ester profile in stored refermented beer isnow largely determined by the chemical equilibriumbetween acids, alcohols and esters. Ester hydrolysis mayalso depend on the molecular weight of the ester: largeresters are more rapidly broken down (Ramey and Ough1980; Schermers et al. 1976). The esterase activity foundby Neven et al. (1997) in refermenting beer was strain-dependent and top fermenting yeasts were more activethan bottom fermenting yeasts. Horsted et al. (1998)showed the extracellular esterase to have an optimalactivity at pH 4–5 and identified TIP1 as the structuralgene.

Bioflavoring by refermentation using a mixedmicrobial culture

Beer originated in Mesopotamia and Egypt about6,000 years ago. Starting with a wide variety of carbo-hydrate-rich material and non-sterile conditions, it is quitecertain that a mixed yeast-bacterial fermentation wasinvolved. Around 1850, Cagniard de Latour for the firsttime directly linked yeast with fermentation. This hy-pothesis was confirmed by Pasteur in 1860. Hansen(1842–1909) and Jorgensen (1848–1925) discovered theadvantages of using pure cultures of S. cerevisiae.Nevertheless, mixed cultures of yeast or yeast andbacteria are still exploited for the production of somespecialty beers (DeKeersmaecker 1996). The evolutionand interaction between different groups of microorgan-isms in the fermentation and the refermentation of someacidic ales in Belgium (lambic and gueuze) have been

Fig. 4 Stereoselective conver-sion of d,l-menthylacetate intol-menthol

Table 1 Reduction of some aldehydes responsible for off-flavorsin beer. The aldehyde and corresponding alcohol are given togetherwith their respective threshold and flavor

Aldehyde Alcohol

3-Methyl-butanal 3-Methyl-butanol0.6 ppm 70 ppmUnripe banana, apple, cherry Alcohol, banana, sweetish

2-Trans-nonenal 2-Trans-nonenol0.00011 ppm 0.08 ppmPapery (cardboard), oxidised,stale

Coconut, walnut, oily

Diacetyl 2,3 Butanediol0.15 ppm 4,500 ppmButterscotch Rubber, sweetish, warming

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studied intensively by Dawoud and Verachtert (1992),Martens et al. (1997), Shantha Kumara and Verachtert(1991) and Van Oevelen et al. (1977).

During fermentation and refermentation with mixedcultures, there is a successive activity of enterobacteria,Saccharomyces yeasts, lactic acid bacteria and Bret-tanomyces yeasts. Brettanomyces species are especiallyinvolved in the typical bioflavoring of these beers.Together with pediococci, they metabolize dextrins notused by Saccharomyces and develop acidity and specialflavors (Decort et al. 1994; Kumara et al. 1993).Brettanomyces lambicus and Brettanomyces bruxellensisdominate the yeast population after the Saccharomycesphase. The impact of Brettanomyces on the beer flavor ismultiple. The presence of a cell-bound esterase changesthe ester profile (Spaepen and Verachtert 1982). Inaddition, Brettanomyces affects the beer flavor also bythe release of isovaleric acid and volatile phenols derivedfrom hydroxycinammic acids (Chatonnet et al. 1992;Licker et al. 1997) (Fig. 5). For example, the synthesis of4-ethylguiacol creates a smoky, clove-like spicy odor and4-ethylphenol has a strong smoky-like phenolic odor.Brettanomyces has frequently been studied for its effectson wine flavor (Chatonnet et al. 1995, 1997; Dominguezet al. 2002). These yeasts are suspected to grow oncellobiose released from oak barrel surfaces or added oakchips. The species, which dominate during lambicbrewing, do not have the required cellobiase and growin metal tanks as well. Nevertheless, some lambicus andbruxellensis species isolated from lambic beer havecellobiase activity (H. Verachtert, unpublished results)indicating adaptations to the special lambic media andwooden casks. Moreover, in another Belgian acidic ale(Rodenbach) Brettanomyces appears only during a tertia-ry refermentation in large wooden casks. In the produc-tion of a particular Belgian ale (a trappist beer)Brettanomyces is used together with Saccharomyces forbioflavoring. In fact, Brettanomyces is one of the mostfrequently studied non-Saccharomyces yeast for flavormodulation.

In the mixed culture fermentation of acidic ales,oxidative yeasts as Candida, Torulopsis, Hansenula,Pichia and Cryptococcus are found as film formingyeasts on the surface of the beer during its longmaturation. Such yeasts are well known in sherryproduction, but their effect on the flavor of the acid ales

has not yet been studied. The malolactic fermentation ofwines is another example of flavor modulation by a mixedculture. Yeasts and lactic acid bacteria are also present insome German and South African beers: weissbier andkaffir beer, respectively (Wackerbauer and Methner 1988,1989). Taken together, it is clear that the use ofrefermentation and mixed microbial cultures greatlyenhance the variety of the beer’s flavor profile. However,using modern techniques and scientific insights muchmore is possible, including the introduction of completelynew flavors.

Novel methods for beer bioflavoring by refermentation

The possibilities for the generation of new flavors in beerare diverse. Figure 6 shows different approaches. Route Ais the normal route leading to beer a. New flavors can beobtained by supplementing the wort with natural flavoringcompounds such as spices (route B) leading to beer b orby fermenting the wort with S. cerevisiae plus othermicroorganisms (route C), leading to beer g. By addingflavor precursors plus other microorganisms (route D),beer d is obtained. Alternatively, the new flavors can beintroduced in the primary fermented beers by refermen-

Fig. 5 Biosynthesis ofethylphenols by Brettanomycessp.

Fig. 6 Innovative scheme for bioflavoring of beer using non-conventional brewery microorganisms in combination with addedflavor precursors

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tation with other microorganisms (route E) or othermicroorganisms plus flavor precursors (route L) givingbeers e or l.

Route B is quite common for some Belgian specialtybeers characterized by a fruity aroma. Herbs are addedduring maturation or fruits are added during the fermen-tation, as in lambic brewing. Table 2 shows differentplants and parts of plants known for their flavoringproperties and parts that are used to obtain a particularflavor.

Flavor compounds extracted from added plant materialcan have a direct effect on beer flavor. For example, theterpenoid alcohol linalool present in coriander (4 g/kg)gives a fresh, floral, sweet aroma with a threshold of100 ppb. S. cerevisiae can esterify linalool to linalylacetate with lavender flavor. However, some extractedmolecules like glycosides, alcohols, fatty acids andamino-acids (Erbe and Bruckner 2000) are only flavorprecursors. Table 3 shows some precursor moleculesfound in plant material. The selection of appropriatemicroorganisms for refermentation could allow to trans-form the precursor molecules into flavoring compoundsand creates the possibility of fully exploiting the flavoringpotential of added herbs or fruits.

Yeasts with alcohol oxidase activity (Harder andBrooke 1989) may oxidize alcohols formed during theprimary fermentation into aldehydes with special flavors.However, for alcohols found as aglycon in glycosides, aglycosidase is also required. The effects on wine flavorafter the hydrolysis of grape must glycosides by someyeasts has been studied by Gueguen et al. (1996) andWinterhalter and Skouroumounis (1997). In beer, the

glycosides of herbs, spices or fruits commonly used forspecialty beers can be hydrolyzed by refermentation withyeasts possessing glycosidase or both glycosidase andalcohol oxidase activities. In this way, linalool can beformed from glycosides present in citrus peel. To exploitthe presence of both alcohol oxidase and glycosidase insome yeasts, Van den Bremt et al. (2001) studied thetransformation of the glycoside salicin directly to salicylaldehyde by the yeast Candida methanolovescens (Fig. 7).It was found that the required enzymes were repressed byglucose and mutants had to be selected. After refermen-tation of a non-alcoholic type of beer, a salicyl-aldehyde-linked almond flavor could be detected. The conversionyield was very low and it was related to problems of non-efficient oxygen transport to the site in the cells wheresalicyl alcohol is oxidized to the aldehyde. Oxygenationof a fermented beer is, however, contrary to final qualityrequirements. The experiments demonstrated that, undersuch conditions, route L (Fig. 6) should be applied ononly part of the beer.

Genetically modified yeastswith improved flavoring characteristics

The development of gene technology has opened up thepossibility of engineering an organism’s metabolism andthus flavor production. Over the last 20 years, manyexamples of metabolic engineering have been described.In this paragraph, a few characteristic examples of theflavor improvement of brewer’s yeast are described. Formore elaborate reviews, see Dequin (2001), Pretorius(2000), Pretorius and Van der Westhuizen (1991) andVerstrepen et al. (2001a).

Table 2 Plant parts used for beer flavoring

Plant part Examples

Herb OreganoRoot Bergamot, ginger, licoriceLeaf Fig, mint, teaBark Cinnamon, hopResinFlower Hop, mintFruit Anise, star anise, pepper, vanillaBerry Cherry, aniseNutBeanSeed Nutmeg, coriander, seeds of paradisePeel Citrus/bitter/sweet oranges, grapefruit

Fig. 7 Conversion of salicin into salicyl alcohol and further into thealmond flavored salicyl aldehyde

Table 3 Precursor molecules present in fruit and herbs used in beer production and their enzymatic conversion to flavor compounds

Plant Precursor molecule Flavor molecule Enzyme

Sour cherry Amygdalin Benzaldehyde b-Glucosidase+ mandelonitrilelyase

(Schmid andGrosch 1986)Prunasin

Raspberry 4-(40-Hydroxyphenyl)-3-butanone40-b-d-glucopyranoside

4-(40-Hydroxyphenyl)- 3-butanone(Raspberry ketone)

b-Glucosidase (Pabst et al.1990)

Hop Isovaleric acid Ethyl isovalerate Esterase

Coriander Linalool Linalyl acetate Ester synthase

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Ester profiles

Volatile aroma-active esters, such as ethyl acetate,isoamyl acetate, ethyl caproate and ethyl caprylate, areresponsible for the fruity flavors of fermented beverages(Meilgaard 2001; Nyk�nen 1986). However, especially inthe modern beer industry, where high-gravity worts andcylindroconical fermentation vessels are used, the esterbalance is often disturbed (Anderson and Kirsop 1974;Meilgaard 2001; Palmer and Rennie 1974; Younis andStewart 1999). Ester formation by S. cerevisiae is quite acomplex process. Firstly, two different co-substrates areneeded for ester synthesis: alcohols and acids. In yeast,the acid co-substrates are activated fatty acids, and thealcohols are either ethanol or more complex fuselalcohols, which are derived from amino acid metabolism(Calderbank and Hammond 1994). Secondly, the forma-tion of volatile esters requires enzymatic catalysis anddifferent groups of esters are formed by different estersynthases (Yoshioka and Hashimoto 1981, 1983, 1984).Molecular research has enabled the cloning and charac-terization of four different genes that encode ester-synthesising enzymes: ATF1, Lg-ATF1, ATF2 andEHT1 (Dufour et al. 2002; Fujii et al. 1994, 1996;Minetoki et al. 1993; Nagasawa et al. 1998; Yoshimoto etal. 1999). It is however expected that S. cerevisiaepossesses even more, as yet unknown, ester synthesisgenes (Mason and Dufour 2000). Some fermentationparameters can be adapted in order to exert a certaininfluence on ester formation rates (for a review, seeVerstrepen et al. 2003a), but in general the complexnature of ester synthesis makes it difficult to get a tightgrip on esters in industrial fermentations. Therefore, muchresearch has focused on the use of mutants and geneti-cally modified yeasts with altered ester productionprofiles.

One way to alter the ester production is to change thesynthesis rate of a certain fusel alcohol, which is in turnresponsible for an increase in the corresponding esterlevel. This method was successfully used by Hirata et al.(1992). To increase isoamyl acetate levels, they intro-duced extra copies of the LEU4 gene into the S. cerevisiaegenome. This results in an increased production ofisoamyl alcohol and its corresponding acetate ester,isoamyl acetate. A comparable Saccharomyces uvarummutant was isolated by Lee et al. (1995). The mutants,which have an altered regulation pattern of amino acidmetabolism, produce more isoamyl acetate and phenyl-ethyl acetate.

However, since this method also disturbs amino acidmetabolism and fusel alcohol levels, it is not an optimalprocedure with which to alter the ester levels. Analternative approach is the overexpression of one or moreof the ester synthesis genes. Experiments have shown thatoverexpression of the ATF1 gene results in a strongincrease in the levels of ethyl acetate, isoamyl acetate and2-phenylethyl acetate (Fujii et al. 1994; Lilly et al. 2000;Verstrepen et al. 2003b; Verstrepen et al. MS submitted).Using a combination of these mutant strains and the wild-

type yeast should make it possible to really modify theester concentration in beer to a desired profile.

Reduction of diacetyl levels

Another organoleptic compound of major importance isdiacetyl. Diacetyl, with its mostly undesired butter-liketaste, is an indirect by-product of yeast valine synthesis(Fig. 8). A variety of genetic strategies have beendeveloped to minimize diacetyl levels in green beer.One way to achieve this is by introducing a gene encodingalpha acetolactate decarboxylase (ALDC). This enzymecatalyzes the conversion of the diacetyl precursor,acetolactate, to acetoin, which has a far higher organo-leptic threshold value than diacetyl. ALDC is found inseveral bacteria including Lactococcus lactis and Aceto-bacter species, which are currently used for foodproduction (Goelling and Stahl 1988). The ALDC genederived from Acetobacter aceti ssp. xylinum has beenbrought under the transcriptional control of the constitu-tive yeast PGK promoter and introduced into the S.cerevisiae genome. The mutant strain has been used inpilot-scale brewing trials and proved to consistentlyproduce beers of good quality that were comparable tothe beers produced by the wild-type parental strain,except for drastically reduced diacetyl levels (Yamano etal. 1994a, 1994b, 1994c, 1995).

Another way to reduce diacetyl levels is by loweringthe activity of alpha acetolactate synthase and thusreducing alpha acetolactate levels. This has been accom-plished by screening yeast populations for resistance tothe herbicide sulpho meturon methyl (SMM). Thissubstance inhibits acetolactate synthase, but some muta-tions of the alpha acetolactate-encoding ILV2 gene areknown to bring about resistance to SMM (Casey et al.1988). These mutant forms of the synthase are less activethan the wild-type form, so that the SMM-resistant mutantstrains also produce less alpha acetolactate and thus lessdiacetyl. A similar way to obtain lower intracellular levelsof alpha acetolactate is by lowering the activity of the

Fig. 8 Diacetyl production during wort fermentations

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ILV2 gene. This has been accomplished by transformingyeast with a part of the ILV2 gene in such a way that themutants produced antisense ILV2 mRNA. As expected,the mutants produced less diacetyl, but they were unableto ferment wort efficiently (Hammond 1995). The last andperhaps most elegant way of reducing diacetyl formationis by increasing the flux through the valine biosynthesispathway. In this way high concentrations of intermediaryproducts such as alpha acetolactate are avoided. This highflux has been accomplished by overexpressing the ILV3and ILV5 genes: the resulting mutants indeed showed astrong reduction in diacetyl production, especially whenonly ILV5 was overexpressed (Villa et al. 1995).

Sulfur components

During fermentation, yeast cells produce some importantsulfur components such as hydrogen sulfide (H2S) andsulfur dioxide (SO2). The former component is mostlyunwanted, as it is responsible for a “rotten-egg” smell,while some SO2 is desirable in beer as it acts as anantioxidant and also stabilizes the reactive carbonylcomponents that are responsible for off-flavors in maturebeer. Again, several mutants which produce less H2S ormore SO2 have been constructed (Hammond 1995).

Taken together, it is clear that genetic modificationoffers staggering opportunities to control and adapt theflavor production of micro-organisms such as brewer’syeast. The main advantage of genetic modification is ofcourse the high selectivity with which one property can bechanged. Furthermore, when genetic modification is usedin a thoughtful way, compliance with the regulations onthe use of genetically-modified organisms (GMO) in foodproduction is in most cases not a major problem.However, today’s increasingly negative public percep-tion, enhanced by recent scandals and problems in thefood industry, hamper the wide exploitation of genetical-ly-modified organisms for food production. Self-cloningorganisms, which do not contain any DNA sequencesderived from other species and are therefore not subjectedto all GMO regulations, may be easier to introduce inindustrial food production (de Vos 1999; Verstrepen et al.2001a, 2001b).

Concluding remarks

The biotechnological production of flavors is becomingmore and more important, not only because of consumerpreference for natural products, but also because biofla-voring may yield better economic benefits compared toplant extracts. The enormous diversity of plants makes itpossible to obtain low-cost natural flavor precursors,which can be transformed to costly flavor compoundswith microorganisms or microbial enzymes. Advances inmolecular biology, microbial biochemistry and proteinchemistry will increase the interest in this approach. In

addition, the de novo synthesis of flavoring compoundsby microorganisms is being investigated.

For brewers, it becomes interesting to study theintroduction of new flavors in beers and produce inno-vative specialty beers with high added value. In practice,this can be realized through refermentation processesusing special yeasts. These can be used to produceflavoring molecules de novo or to transform flavorprecursor molecules present in added herbs and fruits.The addition of herbs or fruits during the main fermen-tation is already frequently used in Belgium. The use ofnon-conventional yeasts and herbs and fruits with onlyflavor precursor molecules will certainly expand the rangeof possibilities for beer bioflavoring. Furthermore, evenbigger possibilities for flavor modulation of beer arisewith the use of genetically modified yeasts, either duringthe main fermentation or during refermentation. Complexmetabolic engineering using genetic techniques can boostthe efficiency of existing de novo synthesis and precursorconversion pathways and may even allow the productionof completely new flavors by brewers’ yeast.

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