ESTER FORMATION IN BREWERY FERMENTATIONS. By Hilary A. B. Peddie. (Brewing Research Foundation, Lyltel Hall, Nutfield, Redhill.

Transcription

1 J. Int. Brew., September-October, 1990, Vol. 96, pp ESTER FORMATION IN BREWERY FERMENTATIONS By Hilary A. B. Peddie (Brewing Reearch Foundation, Lyltel Hall, Nutfield, Redhill. Surrey RH1 4HY) Received 23 December 1989 Eter are econdary product produced by brewing yeat during the anaerobic metabolim of ugar and contitute one of the larget and mot important group of compound affecting beer flavour. Many eter can be formed, the mot important being ethyl acetate, ioamyl acetate, iobutyl acetate, 2-phenylethyl acetate and ethyl hexanoate. The odour threhold level for eter detection are very low. Eter are epecially important in high gravity brewing, where over production occur cauing unwanted olvent-like flavour. They are alo important in low alcohol beer production due to the low level produced, which can reult in beer with little flavour. The factor influencing eter production are reviewed, together with the way in which they can be ued to control eter ynthei. It i believed that acetate eter are yntheized by an enzyme called alcohol acetyl tranferae (AAT) which ue a ubtrate an alcohol and acetyl co-enzyme A; the latter play a central role in many intracellular reaction. However, eter can alo be yntheized by eterae enzyme working in revere. Several attempt have been made to locate the AAT enzyme and recent work ugget that it i located in either the yeat cell plama membrane or in intracellular organelle called "vacuome". Key Word: Eter, yeat, fermentation, review. Introduction Many component are reponible for determining the flavour of beer, and eter are ome of the mot important '37'38. Eter are poitive flavour determinant, i.e. they provide beer with a deirable flavour Their enory odour threhold level are low32, ranging from 0.2 ppm for ioamyl acetate to ppm for ethyl acetate8-31. Eter are yntheized within the yeat cell" and, depending on their chain length, they diffue into the urrounding medium The mot ignificant33-35 eter in beer are ethyl acetate (fruity, olvent-like), ioamyl acetate (iopentyl acetate) (pear drop), iobutyl acetate (banana), ethyl caproate (ethyl hexanoate) (apple) and 2-phenylethyl acetate (honey, fruity, flowery) The Formation of Eter Eter can be formed via a chemical condenation reaction: R'OH + RC00H-» RC00R' + H2O However, the concentration of eter found in beer i much too great to be produced in thi way when the rate of reaction and the duration of fermentation i conidered8-34. Nordtrom21 howed that eter are probably formed via a biochemical pathway, the rate of which, in Saccharomyce cereviiae (the yeat mainly ued in thee tudie), could account for the concentration of eter found in the finihed product12: RCOOH + ATP+CoASH-»RCCT RCCTSCoA + R'OH-»RCOOR' + CoASH The compound involved in eter production are ethanol or higher alcohol, fatty acid, co-enzyme A (CoASH) and an eter yntheizing enzyme which wa firt decribed by Nordtrom17. In later paper Nordtrom18-21 propoed that alcohol become eterified by reacting with fatty acid which have undergone a previou activation by combining with CoASH. He concluded that although acetyl CoA can be formed by the oxidative decarboxylation of pyruvate21 mot of the other acyl CoA compound come from acylation of CoASH by the action of acyl-coa ynthetae. It i in thee activated form that acyl-coa compound can act a the acyl donor34. A a reult of the need for activation, eter ynthei i an energy requiring proce. It i important to tre the central role played in eter ynthei by acetyl CoA, ince thi i involved in many other reaction within the yeat cell (e.g. lipid bioynthei, amino acid bioynthei, fatty acid bioynthei and the TCA cycle) Figure 1 illutrate thi. Any factor which govern production and conumption of thi compound could be crucial for eter production Mot of the work carried out on eter ha concentrated on acetate eter a they are the mot abundant32. Hence the enzyme involved, which ha a molecular weight of 220,000, ha been named alcohol acetyl tranferae (AAT) by Yohioka & Hahimoto38. However, there are many eter and each differ ent activated fatty acid may have it own pecific enzyme. In thee circumtance, if everal fatty acyl CoA compound are preent, competitive inhibition could occur and eterification of individual activated compound would be inhibited by the other Competitive inhibition of thi type i oberved in practice. In addition, ome fatty acid, which are not ubtrate for the enzyme (e.g. iovaleric acid, io-butyric acid) can alo act a inhibitor17. If everal alcohol are preent they can compete in a imilar fahion27. Not urpriingly, alcohol acetyl tranferae (AAT) i the mot tudied eter-yntheizing en zyme and o mot of the enuing dicuion refer to thi enzyme. The enzyme involved in eter breakdown are termed eterae and catalye the following reaction RCOOR1 + H2O-R"OH + RCOOH Some non-brewing yeat yntheize eter uing eterae MALTOSE 4 GLUCOSE PYRUVATE I ESTERS «ACETYL LIPIDS i AMINO ACIDS NUCLEIC ACIDS CoA TCA CYCLE Fig. 1. The central role played by acetyl CoA in yeat metabolim.

2 328 ESTER FORMATION DURING FERMENTATION [J. Int. Brew. enzyme working in revere32 and thi occur in the abence of CoASH28. One uch yeat i Hanenula anomala which can yntheize up to 400 ppm ethyl acetate in thi reaction18. H. anomala ha been hown to produce le eter in the preence of acetyl co-enzyme A than in it abence38. The normal function of eterae i to hydrolye eter and thi ha been uggeted to be important in high gravity brewing where the breakdown of eter may help to limit the eter level found in the beer in thee circumtance. However, Schemer et a/.28 have uggeted a role for eterae in eter ynthei by brewing yeat ince they found a poitive correlation between the revere activity of eterae found in different train of Sacch. cereviiae and the level of ethyl acetate and ioamyl acetate produced. One reaon why acetate eter are o abundant could be that they are hydrolyzed by eterae at a much lower rate than other eter uch a ethyl octanoate (caprylate)32 (ee Figure 2). However, ince octanoyl CoA will ony be preent at a very low concentration compared with acetyl CoA, ubtrate availability will limit the amount of ethyl octanoate which i produced. 40 ESTER CONC.[mg./lltre] Wort compoition: Thi can affect eter ynthei8-9 and i epecially important in high gravity brewing In an all-malt wort fermentation, where the carbohydrate : aimilable nitro gen (C : N) ratio i low, the yeat thrive until oxygen, the growth limiting factor, i ued up1. However, although growth ha ceaed, nitrogenou compound are abundant1-24 and meta bolite, including acetyl CoA, are till formed. Thu, within the cell there i an exce of acetyl CoA which timulate eter ynthei'. When adjunct are ued, the C : N ratio i high and o when growth ceae there i no exce of nitrogen, indeed under thee condition thi can be growth limiting24. Hence there i no exce of acetyl CoA and eter ynthei i le Yohioka and Hahimoto40 have uggeted that, in adjunct wort, yeat may attempt to aborb more nitrogen, in the form of additional peptide not normally taken up during fermentation. The oberved accumulation of unaturated fatty acid in the cell membrane under thee condition39, may encourage uptake of peptide. In addition, the accumulation of unaturated fatty acid may inhibit the eter yntheizing enzyme directly1239. The lipid content of the wort can affect the eter con centration of the final product14. It ha been hown that incorporation of elevated level of unaturated fatty acid namely oleic, linoleic and linolenic acid, into the wort reult in a decreae in eter ynthei Thi may be due to a change caued to the phyical tate of the membrane following incorporation of thee fatty acid or may be due to a direct effect upon the ynthetic enzyme Figure 3 how the change in activity of the eter yntheizing enzyme in relation to time of fermentation and the effect of addition of variou aturated and unaturated fatty acid to the wort. There i no change in the activity of AAT in the preence of aturated fatty acid uch 30 AAT ACTIVITY[mU/10g yeat] 4.0 r TIME [hour] Fig. 2. The hydrolyi of different eter by eterae enzyme. Variou eter were incubated with intact yeat and ethanol at ph 4 for the time indicated. D: ethyl acetate, O: ioamyl acetate, X: phenylethyl acetate, A: ethyl caprylate. (redrawn from ref. 32). It i clear from the above dicuion that the prime function of eterae i till not known. In Sacch. cereviiae it i uncertain whether their main role i in eter hydrolyi or eter ynthei. Factor Influencing Eter Synthei Many factor have a ignificant effect on eter production by Sacch. cereviiae during fermentation: Yeat train: The train of yeat choen i important ince each yeat train produce it own characteritic eter profile and ome yeat train can produce much greater amount of eter than other TIME [day] Fig. 3. The effect of the degree of aturation of membrane fatty acid on alcohol acetyl tranferae (AAT) activity. Variou fatty acid were incubated with intact yeat cell in a ynthetic medium for the time indicated and the alcohol acetyl tranferae activity mea ured. D: no addition, O: tearic acid, A: palmitic add, O: oleic acid, V: linoleic acid, X: linolenic acid, (redrawn from ref. 39). 10

3 Vol. 96,1990] ESTER FORMATION DURINO FERMENTATION 329 a tearic or palmitic acid when compared to the control (i.e. no addition). Upon addition of unaturated fatty acid uch a oleic, linoleic or linolenic acid there i a great reduction in the activity of AAT reulting in a reduction in the eter levelij5j9. With the exception of linoleic acid", mot unaturated fatty acid promote yeat growth during fermentation1, and o there i a reduced availability of CoASH for eter ynthei12. If the membrane i enriched with linoleic acid it eem to prevent timulation of the ynthei of eter35, even though the intracellular level of CoA increae and that of acetyl CoA remain imilar to the level in control cell34. Wort-aeration: Thi reult in increaed yeat growth8 ince oxygen i eential for key reaction in the bioynthei of terol and unaturated fatty acid3-6, which are needed for yeat growth. Increaed yeat growth create additional demand for acetyl CoA for that purpoe and o the availability of acetyl CoA for eter ynthei i reduced; even a low rate of aeration during fermentation can trongly inhibit eter formationij.ii.i4.l7jfj7 Preure: Report on the effect of preure on eter formation are omewhat inconitent Nordtedt et al.16 and Miedaner et al.15 have uggeted that, when preure i in creaed, acetate eter ynthei i reduced. However when preure i increaed, yeat growth i alo reduced and o there hould be a high availability of acetyl CoA for eter ynthei and yet eter ynthei i decreaed. Thi i in contrat to the effect of limiting oxygen, when yeat growth aociated pro cee are reduced and acetyl CoA become available for eter ynthei which increae. Poada et a/ have oberved that increaed preure caue a decreae in ethyl acetate level but an increae in thoe of ioamyl acetate and 2-phenylethyl acetate. In general, however, it eem that application of preure reult in inhibition of eter ynthei and decreaed yeat growth"12. Pitching rate: Maule14 oberved that when pitching rate i increaed four-fold, eter ynthei i reduced. However, for modet increae of pitchng rate (i.e. two-fold) only the con centration of ethyl acetate i affected8. Supended olid: The preence of trub in the wort at pitching can influence eter production. Trub i of variable compoition but often contain zinc and lipid, both of which are timu latory to yeat growth. Schuter et a/.29 have uggeted that trub timulate yeat growth becaue of the preence of thee nutri ent. A a reult of accelerated yeat growth, eter ynthei i reduced ince availability of acetyl CoA for thi purpoe i reduced. Siebert et al.30 have oberved that other olid uch a activated carbon and diatomaceou earth can alo caue a reduction in eter ynthei, mot probably by a direct phyical effect. The upended olid may timulate carbon dioxide removal from the fermentation veel a they create a larger urface area from which carbon dioxide can be evolved. Thi phenomenon i important to yeat ince carbon dioxide can act a an inhibitor of yeat growth16. By reducing carbon dioxide level the olid timulate yeat growth and hence le acetyl CoA i available for eter production, and conequently eter level are reduced30. Temperature: When temperature i elevated, the concentration of eter produced during the fermentation i alo in creaed5-21". For example, a 15*C temperature increae from IO C to 25'C can reult in a 75% increae in eter concen tration8. Thi increae in temperature may make the membrane more fluid and o affect the activity of the AAT enzyme, if it i membrane bound. An increae in membrane fluidity may allow more eter to diffue into the medium. AAT i alo known to be untable at higher temperature4. However, the higher tem perature may imply increae enzyme activity. Engan and Aubert9 have hown that the effect of temperature differ with both eter type and temperature range. Timing of Eter Synthei Thurton34 ha uggeted that during fermentation there are two induction of eter ynthei. At the beginning of ferment ation, eter ynthei i very low due to the high metabolic demand for acetyl CoA for yeat growth At thi time oxygen and acetyl CoA are rapidly conumed in production of unaturated fatty acid and terol. Immediately following thi an equilibrium i etablihed between acetyl CoA conumption for fatty acid and terol ynthei and for eter production. Thi repreent the firt induction of eter ynthei and occur after about 8 hour of fermentation. When fatty acid and terol ynthei finally top there i a peak in cellular acetyl CoA level and alo in the acetyl charge (the ratio between [acctyl-coaj and [acetyl CoA + CoASH]) and at thi point the econd induction of eter ynthei occur. Thi happen about the midpoint of fermentation (between 20 and 30 h) and i rela tively hort-lived However, it doe contribute igni ficantly to the overall eter level. At the point of maximum pecific rate of ethyl acetate ynthei about 80% of the CoASH i in the acetyl form34. The Reaon For Eter Synthei by Yeat Cell Eter could imply be overpill product from ugar meta bolim during fermentation and may be of no advantage to the yeat cell, imply leaving the cell by diffuion. However, there may be a reaon for eter production. Fatty acid with a chain length between C8-CI431 are toxic to yeat and exhibit trong anti-microbial activity; and if they happen to be unaturated thi intenifie the effect. Eter may be formed to remove, from the yeat cell, thee toxic fatty acid Eter of horter chain fatty acid (e.g. C2-C6) would be produced by the ame detoxification procee. Another reaon for eter formation could be to reduce the acetyl charge, a it i eential for the yeat cell to maintain a balance between acetyl CoA and CoASH34. Location Of Eter Syntheizing Enzyme Yohioka and Hahimoto18 believe that the AAT enzyme i bound to the plama membrane. They have attempted to iolate the plama membrane and then aay eter yntheizing enzyme activity; the cell lyate prepared were alo aayed for enzyme activity. Table I quote the reult obtained. The fraction claimed to be the plama membrane had the greatet pecific enzyme activity3". However, it i not certain that thi fraction wa actually plama membrane ince no aay wa carried out for a marker enzyme repreentative of plama membrane, uch a vanadate enitive ATPae38. TABLE I. Alcohol Acetyl Tranferae activity in a cell membrane preparation and in cell lyate (data from ref. 38) Fraction Precipitate after 3rd lyi (Cell membrane fraction) Supernatant after 1t lyi Supernatant after 2nd lyi Supernatant after 3rd lyi Specific enzyme activity (mil) In contrat to thi, Howard and Anderon'10 reult uggeted that the AAT i located in ubcellular fraction which ediment at 105,000 x g when a 3000 * g upernatant i centrifuged. Their data i in agreement with recent work by Malcorp and Dufour13 who believe that the plama membrane ha little or no AAT activity and that the activity reide in cellular "vacuome". The latter work entailed fractionating an ale yeat homogenate by differential centrifugation. Certain marker en zyme were choen which were repreentative of the different cellular organalle a follow: Plama membrane PM ATPae (oligomycin inenitive) Mitochondrial membrane Mitochondrial ATPae (oligomycin enitive)

4 330 ESTER FORMATION DURING FERMENTATION [J. Int. Brew. ALOOHOL AOETYL TRANSFERASE PM ATPoae MTOCHONDHIAL ATPB88 < V > Si V y 8UCROSE DENSITY (g/ml) ALPHA MANNOSIDA8E ALKALINE PKOSPHATASE BETA QLUOOSIDASE. I..I ll SUCROSE DENSITY (g/ml] 8U0RO8E DENSITY [g/ml] Fig. 4. Sucroe denity gradient enzyme profile of a 12,000 * g pellet obtained from an ale yeat homogenate. The diflerent enzyme activitie were ued a indicator of different cellular fraction (ee text), (redrawn from ref. 13). Vacuolar membrane alpha mannoidae Vacuolar compartment alkaline phophatae Secretory veicle beta glucoidae The fraction which pelleted at 12,000 x g during differential centrifugation wa layered onto a ucroe gradient and ub jected to further centrifugation. The reult obtained are hown in Figure 4. Malcorp and Dufour'3 could not aociate the AAT with any one particular marker enzyme when comparing the enzyme profile. Aociation of AAT with the mitochondrial membrane wa ruled out by iopycnic centrifugation a it profile wa very different from the AAT profile. A plama membrane location wa ruled out on the ground of mag neium effect on the plama membrane during differential centrifugation which caued a hift in the oligomycin inenitive ATPae enzyme profile; the AAT profile, under imilar con dition wa unaffected. A a reult, Malcorp and Dufour13 ugget that AAT i aociated with ubcellular tructure of varying ize collectively known a "vacuome" which are widely ditributed within the yeat cell. Concluion In ummary, there exit controvery a to the location of the eter yntheizing enzyme in yeat cell and there i ome diagreement about the more important factor affecting eter ynthei. From the 1960' until the early 1980' it wa felt that factor affecting availability of acetyl-coa were the mot important, ince any reduction in the availability of thi com pound reult in a decreae in eter ynthei1-8-"-'1' However, work performed by Yohioka and Hahimoto between *0-4' ugget that factor which change the phyical tate of the plama membrane may alo be of im portance ince when the lipid compoition of the membrane i changed, the activity of the eter yntheizing enzyme i altered. The mot recent work by Malcorp and Dufour in ugget that the enzyme activity i not plama membrane bound at all and that the eter yntheizing enzyme i located within cellular vacuome. However, it i likely that no ingle factor control eter ynthei; both direct phyical and bio chemical effect on AAT and ubtrate availability could be of equal importance. Clearly more experimentation i required to clarify thee point if the mechanim involved in eter ynthei are to be fully elucidated. Acknowledgement: I would like to thank John Hammond and Jill Calderbank for advice and help with writing thi review, and the Director of the Brewing Reearch Foundation for granting permiion for publication. Reference 1. Anderon, R. G. & Kirop, B. H. Journal of the Intitute of Brewing. 1974,80, Andrew, D. A. Brewer Guardian. 1984,113(2), Arie, V. & Kirop, B. H. Journal ofthe Intitute ofbrewing, 1977, 83, Ahida, S., Ichikawa, E, Suginami, K. & Imayau, S. Agricultural and Biological Chemitry. 1987,51, Caey, G. P., Chen. E. C. H. & Ingledew, W. M. Journal of the American Society of Brewing Chemit. 1985,43, David, M. H. & Kirop, B. H. Journal of the Intitute of Brewing. 1973,79, Engan, S. Journal of the Intitute of Brewing, 1970,76,

5 Vol. 96, 1990] ESTER FORMATION DURING FERMENTATION Engan, S. Brewer Diget, 1974,49(11), 4<M8. 9. Engan, S. & Aubert, O. European Brewing Convention, Proceed ing of the 16th Congre, Amterdam, 1977, Howard, D. & Anderon, R. G. Journal of the Intitute of Brewing, 1976,82, Kirop,B. H. Brewer Guardian. 1977,106(5), MacDonald, J., Reeve, P. T. V., Ruddleden, J. D. & While, F. H. Progre In Indutrial Microbiology, Ed. Buhell, M. E. Elevier; Amterdam, 1984, Vol. 19, Malcorp, Ph. & Dufour, J. P. Brewing Convention Proceeding of the 21t European Congre, Madrid, 1987, Maule, D. R. J. Journal of the Intitute of Brewing, 1967, 73, Miedaner, H., Narzi, L. & Woerner, G. Brauwienchafl, 1974, 27, Nordtedt, C. Bengton, A, Bennet, P., Lindtrom, I. < T. European Brewing Convention, Proceeding of the 15th Congre, Nice. 1975, Nordtrom, K. Journal of the Intitute of Brewing, 1962, 68, Nordtrom, K. Journal of the Intitute of Brewing. 1963, 69, Nordtrom, K. Journal ofthe Intitute ofbrewing. 1964,70, Nordtrom, K. Journal of the Intitute of Brewing. 1964, 70, Nordtrom, K. Journal of the Intitute of Brewing, 1964, 70, Nykanen, L. & Nykanen, I. Journal of the Intitute of Brewing, 1977,83, Nyk5nen, L., Nykanen, I. & Suomalainen, H. Journal of the Intitute of Brewing, 1977,83, Pfiterer, E. & Stewart, G. European Brewing Convention, Proceed ing of the 15th Congre, Nice. 1975, , 25. Poada, J., Candela, J., Calero, G., Almenar, J. & Martin, S. Europen Brewing Convention. Proceeding of the 16th Congre, Amterdam Poada, J., European Brewing Convention, Monograph V. Ferment ation and Storage Sympoium. Zoeterwoude. 1978, Rainbow, C. in "The Yeat", ed Roe, A. H. & Harrion, J. S. Academic Pre, London and New York, 1970, Schermer, F. H., Duflu, J. H. & Macleod, A. M. Journal of the Intitute of Brewing. 1976,82, Schiler, D. O., Ruocco, J. J. & Mabee, M. S. Journal of the American Society of Brewing Chemit, 1982,40, Siebert, K. J., Blum, P. H., Wik, T. J., Stenroo, L. E. & Anklam, W. J. Technical Quarterly Mater Brewer Aociation of the America. 1986,23, Suomalainen, H. Journal of the Intitute of Brewing. 1971, 77, Suomalainen, H. Journal of the Intitute of Brewing, 1981, 87, Thurton, P. A., Quain, D. E. & Tubb, R. S. European Brewing Convention, Proceeding of the 18th Congre, Copenhagen, 1981, Thurton, P. A. Ph.D. Thei. Univerity of Bath, Thurton, P. A., Taylor, R. & Ahvenainen, J. Journal of the Intitute of Brewing. 1981,87, Wainwright, T. Brewer Guardian. 1983,112(8), White, F. H. & Portno, A. D. European Brewing Convention, Proceeding of the 17th Congre, Berlin, 1979, Yohioka, K. & Hahimoto, N. Agricultural and Biological Chemitry, 1981,45, Yohioka, K. & Hahimoto, N. Agricultural and Biological Chemitry, , Yohioka, K. & Hahimoto, N. Agricultural and Biological Chemitry. 1984,48, Yohioka, K. & Hahimoto, N. Agricultural and Biological Chemitry, 1984,48,