Ethyl ester production during brewery fermentation: a review
Sofie M.G. Saerens1,2, Kevin J. Verstrepen1,2,3, Johan M. Thevelein2,
and Freddy R. Delvaux1
1Centre for Malting and Brewing Science, Department of Microbial and Molecular Systems,
Faculty of Bioscience Engineering, Katholieke Universiteit Leuven,
Kasteelpark Arenberg 22, B-3001 Leuven- Heverlee, Belgium;
2Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, Katholieke Universiteit Leuven,
and Department of Molecular Microbiology, Flemish Interuniversity Institute of Biotechnology (VIB),
Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Belgium
3FAS Center for Systems Biology, Harvard University, 7 Divinity Avenue, Cambridge MA 02138, USA
ABSTRACT
The production of volatile esters by yeast is of major industrial interest because the presence of these compounds determines the fruity aroma of fermented beverages, like beer and wine (6-8, 11, 13, 14, 23, 24, 31-33, 37-41, 50). The need to understand and control ester synthesis is driven by the fact that esters play a key role in the sensorial quality of these fermented alcoholic beverages. Especially in the brewery, problems with ester production have been encountered by the introduction of modern brewing practices, such as high-gravity brewing, the use of tall cylindroconical fermentors and the production of reduced-alcohol beers. Indeed, it is well-known that the use of worts of high specific gravity results in disproportionate amounts of esters (2, 30). In contrast to high-gravity brewing, fermentations in tall ‘Apollo’ fermentors or the production of low alcohol beers result in a reduction of ester formation, so that the produced beers lack desirable fruity tones (30). As esters are synthesized via several yeasts’ complex metabolic pathways, there is a need to gain a clear understanding of ester metabolism. One needs to take a closer look at the individual genes involved, their functions and regulatory mechanisms.
In alcoholic beverages, there are two important groups of esters: the acetate esters and the medium-chain fatty acid (MCFA) ethyl esters. For acetate ester synthesis, the genes involved have already been cloned and characterized (16, 26, 28, 34, 52, 53, 55-57). Also the biochemical pathways and the regulation of acetate ester synthesis are well-defined (10, 28). About the molecular basis of MCFA ethyl ester synthesis however, research was only recently successful (10, 45, 46). This paper reviews the current knowledge of the synthesis of MCFA ethyl esters and discusses the different factors that allow MCFA ester formation to be controlled during fermentation.
Cerevisia, 33(2) 2008
MCFA ETHYL ESTERS IN BEER
As they are responsible for the fruity character of fermented beverages, volatile esters are an important group of aroma compounds in beer. The most significant flavour-active esters in beer are acetate esters of ethanol and higher alcohols: ethyl acetate (solvent-like aroma), isoamyl acetate (banana aroma), and phenyl ethyl acetate (roses, honey); and ethyl esters of MCFAs: ethyl hexanoate (aniseed, apple-like aroma), ethyl octanoate (sour apple aroma), and ethyl decanoate (floral odour). MCFA ethyl esters are found only in trace compounds in beer, but a certain concentration of these esters is necessary for optimum aroma and flavour (14). Among these esters, only isoamyl acetate concentrations are above threshold level in most lager beers (1, 9, 32). In top fermented beers, also ethyl acetate, and ethyl hexanoate are above the threshold levels. MCFA ethyl esters also reach their threshold levels in wine (5, 18, 19, 43). As with all flavour components in beer, certain limits should not be exceeded, otherwise, a single compound or a single group of compounds may dominate and destroy the flavour balance. Moreover, the presence of different esters can have a synergistic effect on the individual flavours, which means that esters can also effect beer flavour well below their threshold level (31). The threshold levels and concentration of the MCFA ethyl esters in lager beer are given in Table 1.
Important amounts of ethyl octanoate (13 ppm after 20 months) and ethyl decanoate (4.5 ppm) are found in lambic and gueuze. Lambic and gueuze are typical Belgian beers obtained by spontaneous fermentation of wort. These beers are characterized by high concentrations of acetic and lactic acid, ethyl acetate and ethyl lactate and result from the successive development of enterobacteria, Kloeckera and Saccharomyces strains, and bacteria of the genus Pediococcus, and Brettanomyces yeasts. As ethyl decanoate is almost absent in other beers, it might be considered as a typical aroma component of lambic and gueuze (48).
Component / Threshold level (ppm) / Concentration in lager beer (ppm) / Flavour descriptionEthyl butyrate / 0.4 / No data / Papaya, butter, sweetish, apple
Ethyl hexanoate / 0.23 / 0.14 / Apple, fruity, sweetish, aniseed
Ethyl octanoate / 0.9 / 0.17 / Apple, sweetish, fruity
Ethyl decanoate / 1.5 / No data / Fatty acids, apple, solvent
Table 1: Threshold values and concentration of MCFA ethyl esters in lager beer (9, 32).
MCFA ETHYL ESTER BIOSYNTHESIS IN YEAST
Aroma-active esters are formed intracellular by fermenting yeast cells. Being lipid soluble, MCFA ethyl esters can diffuse through the cellular membrane into the fermenting medium. Unlike acetate ester excretion, which is rapid and complete, the transfer of MCFA ethyl esters to the fermenting medium decreases with increasing chain length, from 100% for ethyl hexanoate, to 54-68% for ethyl octanoate, to 8-17% for ethyl decanoate. Longer chain fatty acid ethyl esters all remain in the yeast cell (39). Distribution of MCFA ethyl esters between yeast and beer is also influenced by the type of yeast used, with a higher proportion of the esters formed remaining in the cells of lager yeast (Saccharomyces pastorianus). Moreover, the distribution of MCFA ethyl esters between yeast cells and medium is temperature-dependent: more of each ester is retained at lower temperatures (50).
Ester biosynthesis
Synthesis of esters requires two substrates: alcohol and carboxylic acid. Esters can be formed via a chemical reaction but the reaction is too slow to account for the amount of ester present in beer. In 1962, Nordström demonstrated that esters are formed via an intracellular process catalyzed by an acyltransferase or ‘ester synthase’ (35). The reaction requires energy provided by the thioester linkage of the acyl-coenzyme A (CoA) co-substrate (Fig. 1). The most abundant acyl-CoA is acetyl-CoA, which can be formed either by oxidative decarboxylation of pyruvate or by direct activation of acetate with ATP. The majority of acetyl-CoA is formed by the oxidative decarboxylation of pyruvate, while most of the other acyl-CoAs are generated by the acylation of free CoA catalyzed by acyl-CoA synthase (fatty acid metabolism).
Enzymes involved in MCFA ethyl ester formation
MCFA ethyl esters are the product of an enzyme-catalyzed condensation reaction between an acyl-CoA component and ethanol. The formation of the majority of the MCFA ethyl esters in yeast is catalyzed by two acyl-CoA:ethanol O-acyltransferases (AEATases), Eeb1 and Eht1 (46). According to Saerens et al. (2006), the levels of ethyl butanoate, ethyl hexanoate, ethyl octanoate and ethyl decanoate produced during fermentation with an eeb1Δ strain were reduced in comparison with those produced by the wild type strain by respectively 36%, 88%, 45% and 40%. Compared to the eeb1Δ strain, deletion of EHT1 did not affect the production of ethyl butanoate and ethyl decanoate, and resulted in only minor decreases in ethyl hexanoate formation (36%) and ethyl octanoate formation (20%). A double deletion strain eht1Δ eeb1Δ produced similar levels of ethyl butanoate, ethyl hexanoate and ethyl decanoate as the eeb1Δ single deletion strain, and a lower level of ethyl octanoate, indicating that Eht1 plays only a minor role in MCFA ethyl ester synthesis, while Eeb1 is the most important enzyme for MCFA ethyl ester synthesis (Table 2). On the other hand, although the double deletion of EHT1 and EEB1 caused a pronounced drop in the production of all MCFA ethyl esters, only the production of ethyl hexanoate was virtually eliminated. Hence, yeast cells must contain one or more additional enzymes responsible for MCFA ethyl ester synthesis. In the case of ethyl octanoate and ethyl decanoate production, additional deletion of YMR210w in the eht1Δ eeb1Δ strain produced a further drop in their level. Also in the case of ethyl butanoate, one would expect the existence of one or more additional enzymes that can support its synthesis. On the other hand, chemical synthesis of ethyl butanoate might also occur.
In addition to deletion analysis, Saerens et al. also evaluated Eht1 and Eeb1 for intrinsic AEATase and esterase activity in vitro (46). Their results obtained with purified GST-Eht1 and GST-Eeb1 fusion proteins clearly indicate that these proteins display enzymatic activity both for the synthesis and the hydrolysis of MCFA ethyl esters. Of course, the combined presence of MCFA ethyl ester synthase and esterase activity in the Eht1 and Eeb1 proteins raises questions as to the precise regulation of the balance between MCFA ethyl ester synthesis and hydrolysis in vivo by Eht1 and Eeb1.
Figure 1: Chemical and biochemical synthesis of esters
Compound / wt / eht1Δ / eeb1Δ / ymr210wΔ / eht1Δ eeb1Δ / eht1Δymr210wΔ / eeb1Δ
ymr210wΔ / eht1Δ
eeb1Δ
ymr210wΔ
Ethyl butanoate / 1.00 / 0.97 / 0.64 / 1.06 / 0.70 / 0.91 / 0.55 / 0.54
Ethyl hexanoate / 1.00 / 0.64 / 0.12 / 0.95 / 0.08 / 0.84 / 0.08 / 0.05
Ethyl octanoate / 1.00 / 0.80 / 0.55 / 0.89 / 0.28 / 0.85 / 0.24 / 0.10
Ethyl decanoate / 1.00 / 1.18 / 0.60 / 1.11 / 0.56 / 0.90 / 0.20 / 0.07
Table 2: Ethyl ester production in eht1Δ, eeb1Δ, ymr210wΔ single and multiple mutants (46). Gas chromatographic measurement of ethyl butanoate, ethyl hexanoate, ethyl octanoate and ethyl decanoate produced by the wild type (wt) and the deletion strains eht1Δ, eeb1Δ, ymr210wΔ, eht1Δ eeb1Δ, eht1Δ ymr210wΔ, eeb1Δ ymr210wΔ, and eht1Δ eeb1Δ ymr210wΔ after 96 h of fermentation. Standard deviations were typically 10% and did not exceed 20%.
HOW TO CONTROL MCFA ETHYL ESTER FORMATION
The rate of MCFA ethyl ester formation is dependent on two factors: the concentration of the two substrates (the acyl-CoA component and ethanol) and the total activity of the enzymes involved in the synthesis and hydrolysis of the MCFA ethyl esters. Hence, all parameters that influence substrate concentrations or enzyme activity will affect MCFA ethyl ester production.
Factors affecting enzyme activity
An important factor to understand the influence of fermentation parameters on the production of MCFA ethyl esters is the total activity of the enzymes involved in the synthesis of MCFA ethyl esters. Today, two enzymes have been reported to be important for MCFA ethyl ester synthesis, namely Eht1 and Eeb1. However, little information is available on the regulating mechanism of these enzymes and the corresponding genes. For EEB1, two transcription factors are known to be involved in the transcriptional regulation of this gene, Oaf1 and Pip2 (22). Oaf1 and Pip2 are key components in the pathway by which several S. cerevisiae genes encoding peroxisomal proteins are activated in the presence of fatty acid such as oleate. The two proteins form a complex and bind to an upstream activating sequence in the form of a heterodimer (21, 44). DNA sequences to which this heterodimer binds contain palindromic CGG triplets separated by a 15- to 18-nucleotide spacer. This sequence is present in the promoter region of several genes encoding peroxisomal proteins, and it has been termed the oleate response element (ORE) (12, 15). Also the promoter of EEB1 contains such an ORE (Fig. 2). Karpichev et al. (1998) have showed that EEB1 expression is induced by oleate and regulated by Oaf1 and Pip2 in a similar fashion to genes encoding peroxisomal β-oxidation enzymes (22).
Figure 2: ORE sequence in the promotor of EEB1, from position -276 to position -255 (22).
The expression of EEB1 is also influenced by the inositol concentration in the growth medium. Inositol is the precursor for phospholipid synthesis in yeast. Phospholipids are the key structural elements of membrane-bounded organelles and play important roles in signalling and membrane trafficking pathways. In the yeast Saccharomyces cerevisiae, the transcription of many genes encoding enzymes of phospholipid biosynthesis is repressed in cells grown in the presence of the phospholipid precursor inositol. Because the effect of inositol on the expression of other genes was largely unexplored, Jesch et al. (2005) used a genome-wide approach using cDNA micro array technology to profile the changes in the expression of all genes in yeast that respond to the exogenous presence of inositol (20). Their results indicate that inositol is the major effector of Ino2p-Ino4p- and unfolded protein response (UPR)-targeted gene expression. One of the genes affected by inositol was EEB1. The expression of EEB1 was repressed by inositol, but in an Ino2-Ino4 and UPR pathway independent manner. Hence, why and how inositol represses EEB1 expression remains unknown.
Results with overexpression of EHT1 of wine yeast showed a slight increase in MCFA ethyl ester production. Lilly et al. (2006) used the EHT1 allele of wine yeast (VIN13) and overexpression of this EHT1 allele in the VIN13 yeast slightly increased the concentration of all of the esters, with the highest increases in the concentrations of ethyl decanoate, ethyl hexanoate and ethyl octanoate (25). The effect of increased concentrations of ethyl decanoate, ethyl hexanoate (apple aroma) and ethyl octanoate was evident in the sensory analysis, where EHT1 overexpression resulted in a significantly enhanced apple aroma of the wine. However, overexpression of the EHT1 or EEB1 allele of a laboratory strain or an industrial ale strain does not increase the production of MCFA ethyl esters (45, 46). Even when additional substrate is added to the fermenting medium, there is no difference in MCFA ethyl ester concentration between a wild type yeast strain and the EHT1 or EEB1 overexpression strains of both a laboratory and an ale yeast strain. As already mentioned, both Eht1 and Eeb1 do also posses esterase activity, besides the synthase activity for MCFA ethyl esters. This extra activity can be an explanation why overexpression of the genes does not enhance MCFA ethyl ester formation. As enhancing enzyme activity by overexpression of the ester synthesis genes only slightly affects ethyl ester production, the enzyme activity appears not to be the limiting factor for ethyl ester production.
Factors affecting substrate concentration