Biotransformation of hop aroma terpenoids by ale and lager yeasts

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1 FEMS Yeast Research 3 (2003) 53^62 Biotransformation of hop aroma terpenoids by ale and lager yeasts Andrew J. King 1, J. Richard Dickinson Cardi School of Biosciences, Cardi University, PO Box 915, Cardi CF10 3TL, UK Received 29April 2002; received in revised form 28 June 2002; accepted 28 June 2002 First published online 12 August 2002 Abstract Terpenoids are important natural flavour compounds, which are introduced to beer via hopping. It has been shown recently that yeasts are able to biotransform some monoterpene alcohols. As a first step towards examining whether yeasts are capable of altering hop terpenoids during the brewing of beer, we investigated whether they were transformed when an ale and lager yeast were cultured in the presence of a commercially available syrup. Both yeasts transformed the monoterpene alcohols geraniol and linalool. The lager yeast also produced acetate esters of geraniol and citronellol. The major terpenoids of hop oil, however, were not biotransformed. Oxygenated terpenoids persisted much longer than the alkenes. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Saccharomyces cerevisiae; Saccharomyces bayanus; Hop aroma terpenoid 1. Introduction Terpenoids are a class of compounds derived from a common precursor, isopentyl pyrophosphate. They are found widely in nature and include monoterpenoids (C 10 ), sesquiterpenoids (C 15 ), diterpenoids (C 20 ), triterpenoids (C 30 ), carotenoids, sterols, phytols and quinones. Monoterpenoids and sesquiterpenoids are compounds with strong sensory qualities, and are found in the essential oils of many plants [1]. Consequently, they are used extensively in those industries where avour and/or fragrance are important. The structures of terpenoids relevant to brewing and their aromas are shown in Fig. 1. The aromas of terpenoids vary widely and include oral, fruity, minty and peppery. Di erent isomers of a given terpenoid can have varying aromas. For example, geraniol (Fig. 1) has a rose-like, citrus odour, whereas the cis isomer, nerol (Fig. 1), has a fresh, green odour [1]. In plants, monoterpenoids and sesquiterpenoids are produced via the 1-deoxy-D-xylulose-5-phosphate pathway [2]. Of all plants, hops (Humulus lupulus) are perhaps the species * Corresponding author. Tel.: +44 (29) ; Fax: +44 (29) address: dickinson@cardi.ac.uk (J.R. Dickinson). 1 Present address: KPMG, 8 Salisbury Square, London EC4Y 8BB, UK. which have the most complex essential oil known. Over 200 constituents have been identi ed to date [3]. In past times, hops were added to beers to impart avour (bitterness and aroma) and to act as a preservative. The preservative e ect of hops today is disputed, but in medieval times beer was probably brewed to a much lower alcohol content than today. Thus a preservative e ect seems likely. Recently, we reported the biotransformation of a number of monoterpene alcohols by Saccharomyces cerevisiae, Torulaspora delbrueckii and Kluyveromyces lactis [4]. The present work is a rst step towards examining whether yeasts may alter hop terpenoids during the brewing of beers. This is a somewhat novel perspective, because the major focus of brewing chemists for many years has been upon the wort-boiling process. Brewers worts di er greatly from the media used in most academic studies. Worts contain high concentrations of sugar ( s 10%), comprising mainly mono-, di- and trisaccharides. A typical laboratory medium may contain only 2% of a single monosaccharide. Since carbon catabolite repression is a major regulator of virtually every aspect of yeast metabolism [5], we decided to investigate whether hop terpenoids were transformed when an ale and lager yeast were cultured in the presence of a commercially available syrup at concentrations more akin to wort than academic laboratory media. Oxygen is severely limited during the brewing process. Usually, the wort is sparged prior to the addition of the yeast (pitching), and then oxygen will not be pro / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S (02)

2 54 A.J. King, J.R. Dickinson / FEMS Yeast Research 3 (2003) 53^62 vided [6]. This limited amount of oxygen is added to satisfy the yeast cells requirement for sterols and unsaturated fatty acids, which cannot be made under anaerobic conditions [7^8]. To circumvent any metabolic consequences of oxygen limitation whilst still retaining conditions with some resemblance to a brewery fermentation, we opted to pitch aerobically grown yeast into air-saturated medium. The terpenoids selected for this study were geraniol, linalool, L-myrcene, L-caryophyllene, and K-humulene (Fig. 1). Geraniol and linalool have already been shown to undergo biotransformation under laboratory conditions. These monoterpene alcohols have also been detected in beer in a number of studies [9^12]. Additionally, linalool has been shown to be an important indicator of hop variety [13]. The monoterpene L-myrcene, and the two sesquiterpenes L-caryophyllene and K-humulene are the most abundant terpenoids found in the essential oil of hops. L-myrcene constitutes around 30% of hop oil, whereas K-humulene and L-caryophylene make up 8^ 33% and 4^22% of hop oil, respectively [3]. These terpenoids were selected as a starting point to examine whether they are transformed by brewing yeasts. 2. Materials and methods All terpenoids used in this study are commercially available from Sigma, Aldrich or Fluka, and were purchased as the purest form available. Terpenoids were introduced into the culture medium by addition from ethanolic stock solutions, prepared at a concentration of 100 mg ml 31. The exception to this was L-myrcene, which is insoluble in ethanol, and was therefore prepared in diethyl ether, also at a concentration of 100 mg ml 31. Control cultures (i.e. with no added terpenoids) contained equivalent volumes of ethanol or ether. RM-81 Fermentose syrup (74.07% maltose, 22.87% maltotriose, 1.59% sucrose, 1.35% glucose and 0.12% fructose) was obtained from Tunnel Re neries, London, UK. S. cerevisiae NCYC 1681 and Saccharomyces bayanus NCYC 1324 (National Collection of Yeast Cultures, Norwich, UK) were used as representative brewing ale and lager strains, respectively. For preliminary experiments to determine the recovery of terpenoids, S. cerevisiae prototrophic haploid strain IWD72 [4] was grown in YEPD medium which comprised 1% (w/v) yeast extract, 2% (w/v) bacteriological peptone (Difco), 2% (w/v) glucose, 0.01% (w/v) adenine and 0.01% (w/v) uracil. For all other experiments media were prepared containing RM-81 Fermentose syrup diluted with distilled water to give a speci c density in the range 1.045^ Other nutrients were provided as ammonium sulfate (5.87 g l 31 ) and Bacto yeast nitrogen base (without ammonium sulfate, 1.7 g l 31 ). Inocula for fermentations were produced by growing overnight aerobic cultures in 1000-ml conical asks containing 250 ml of medium at 30 C, with orbital shaking at 150 r.p.m. After reaching an OD 600 nm of 6^10 (approximately 6U cells ml 31 ), cells were harvested by centrifugation at 1000Ug for 10 min at room temperature. The cell pellets were then transferred to European Brewery Convention tall tubes [14] so as to give an estimated OD 600 nm of 2.0 (approximately 2U10 7 cells ml 31 ). The tall tubes (total capacity just over 1.1 l) contained 800 ml of sparged (50 min with lter-sterilised air) medium. The fermentations were at 18 C, and 50-ml samples were removed periodically under aseptic conditions. Progress of fermentation was monitored by measuring the speci c gravity of degassed wort, with a Paar DMA46 Calculating Digital Density Meter (Paar Scienti c Ltd, London, UK). Samples for gas chromatography^mass spectrometry (GC^MS) analysis were prepared following the method of Cocito et al. [15]. Cell-free medium (50 ml), spiked with 500 Wg of 2-octanol (as an internal standard) was extracted once with 25 ml of dichloromethane (DCM) and then twice with 15 ml of DCM, in an ultrasonic waterbath. The combined extracts were then dried over anhydrous magnesium sulfate, before concentration by rotary evaporation at 50 C, without vacuum. The extracts were then transferred to 2-ml GC^MS sample vials prior to analysis. Eluted peaks were identi ed and quanti ed by comparing the retention times and mass spectra with those of authentic standards. GC^MS samples were analysed with a CE Instruments (Altrincham, UK) GC800 series chromatograph coupled to a CE Instruments MD800 mass spectrometer, using electron ionisation. The chromatograph was tted with a SupelcoWax 10 column (30 m, 0.32 mm ID, 0.25 Wm lm thickness) (Supelco, Poole, UK). 1 Wl of sample was injected, via a CE Instruments AS800 autosampler. The temperature programme was set up as follows: The injector port was set at 250 C. The initial temperature, 50 C for 10 min, was raised to 150 C at a rate of 4 C min 31, and then held at 150 C for 10 min. The mass spectrometer was set up to detect ions with a mass-to-charge ratio (m/z) of between 30 and Results 3.1. The recovery of terpenoids Preliminary experiments were performed to monitor the recovery of terpenoids. In these experiments cells were inoculated into YEPD medium, containing terpenoids at a concentration of 100 Wg ml 31. After 24 h the cells were harvested by centrifugation and the culture medium was ltered and extracted with DCM. Additionally, the pellets of yeast cells were washed with 5 ml of 50% (v/v) ethanol to recover any terpenoids which might have been bound to, or entered the cells [16]. Table 1 indicates the recovery of di erent compounds from each of the cultures. Geraniol was converted mainly

3 A.J. King, J.R. Dickinson / FEMS Yeast Research 3 (2003) 53^62 55 Fig. 1. The structures of terpenoids relevant to brewing and their aromas. 1: geraniol ( oral, rose-like, citrus); 2: linalool ( oral, fresh, coriander); 3: citronellol (sweet, rose-like, citrus); 4: nerol ( oral, fresh, green); 5: K-terpineol (lilac); 6: terpinen-4-ol (spicy, medicinal); 7: linalool oxide (herbaceous, medicinal); 8: nerolidol ( oral); 9: farnesol ( oral); 10: L-myrcene (medicinal); 11: K-pinene (pine); 12: L-pinene (pine); 13: limonene (sweet, spicy, citrus); 14: K-humulene (medicinal); 15: L-carophyllene (herbaceous); 16: L-carophyllene oxide (spicy). The aromas assigned to each compound are as in Bauer et al. [1]. into citronellol and nerol. Small quantities of linalool production had occurred and traces of geranyl acetate were found, indicating that ester production occurred in YEPD medium. From linalool, no products were detected. With citronellol, small quantities of citronellyl acetate were detected, providing further evidence of esterase activity. No products were detected from K-terpineol, but some of the terpenoid was recovered from the cell pellets. Hence, the recovery of the monoterpene alcohols from the medium was in all cases greater than 80% and, with the exception of K-terpineol, greater than 90%. Small amounts (10% of the geraniol supplement, less of the other monoterpene alcohols) were recovered from the cells. Recovery of the monoterpenes and sesquiterpene alkenes was much lower. With L-carophyllene, only 0.3% of the original terpenoid was recovered. A small quantity of K-humulene was recovered and slightly higher levels of the products L-carophyllene and carophyllene oxide. No L-myrcene was recovered and no products were observed to derive from it. In theory at least, there are three possible explanations for the low recoveries of these compounds. Firstly, they might have all evaporated; secondly, perhaps they could not be recovered by 50% ethanol; or, thirdly and least likely, they might have been assimilated by the yeast. These would all be irrelevant to the brewing of beers, because only those compounds which are present in the medium (i.e. the beer) will contribute to its avour. An important point to note was that there were no novel or unidenti ed compounds detected. Table 1 The recovery of terpenoids from 50-ml samples of a laboratory haploid strain grown for 24 h in YEPD medium supplemented with 100 Wg ml 31 of various terpenoids Terpenoid Residual terpenoid at 24 h (Wg ml 31 ) Products at 24 h (Wg ml 31 ) Terpenoids extracted from cells (Wg) Approximate recovery (%) 16.9nerol geraniol :1 Geraniol citronellol 23.2 nerol 2.4 linalool trace:geranyl acetate citronellol Linalool 90 none linalool :1 Citronellol citronellyl acetate citronellol :1 6.7 geraniol 60.2 nerol :1 Nerol citronellol trace:kterpineol 3.5 citronellol K-Terpineol 79.0 none 75.4 K-terpineol :1 Linalool oxide 95.7 none 40.1 linalool oxide :1 Nerolidol 79.0 none 1295 nerolidol :1 Farnesol 32.0 none 3171 farnesol :1 L-Myrcene 0.0 none none 0 N/A K-Pinene 0.0 none none 0 N/A L-Pinene 0.0 none none 0 NIA Limonene 0.0 none none 0 N/A K-Humulene 2.9caryophyllene none 3.6 N/A caryophyllene oxide L-Caryophyllene 0.3 none none 0.3 N/A Caryophyllene oxide 23.7 none caryophyllene oxide :1 Ratio of free to bound terpenoids The results are the means of duplicate experiments. The variations between determinations in all cases were 5^10%. N/A indicates not applicable in cases where either no terpenoid remained in the medium or none was extracted from the cell pellet (or both).

4 56 A.J. King, J.R. Dickinson / FEMS Yeast Research 3 (2003) 53^62 Fig. 2. Progress of fermentations with S. cerevisiae NCYC 1681 and S. bayanus NCYC 1324, in the presence of 10 Wg ml 31 of terpenoids as shown in the key. The fermentations were carried out at 18 C. The results shown are the means of three speci c density calculations, which varied no more than 2% E ects of terpenoids on the progress of fermentation All the terpenoids were supplemented in the commercial syrup-based medium at a concentration of 10 Wg ml 31: These concentrations were much higher than those which would actually be present in a hopped wort, but were convenient for analysis where only 50-ml samples were extracted. The progress of the fermentations is shown in Fig. 2. None of the terpenoids had an e ect on the rate of attenuation of the medium (compared to the control cultures with no added terpenoid), indicating that at these concentrations, they are not toxic to the yeasts. However, it is worth commenting that these fermentations were quite slow compared to typical beer fermentations, which would have been completed within 15 days Biotransformation of geraniol We have previously shown that monoterpenoids do not undergo spontaneous transformation and that S. cerevisiae does not produce these compounds [4]. In the present study, parallel control experiments were conducted in which the terpenoids of interest were incubated in the absence of either yeast strain. No conversion products were observed in these controls. In addition, we did not detect any such compounds in cultures which contained yeast but no added terpenoid. Hence, the compounds detected in cultures which were inoculated with a yeast and supplemented with a terpenoid must be the genuine products of yeast biochemical activity. Figs. 3 and 4 show the products formed from geraniol. In the presence of both the ale and lager strains, a range of products were detected. In both cases, citronellol was the most abundant product, reaching a concentration of over 1.5 Wg ml 31 in 4 days with the ale yeast (NCYC 1681), and 1.4 Wg ml 31 with the lager yeast (NCYC 1324). However, with the lager yeast it declined to around 1.0 Wg ml 31 between 7 and 15 days. Linalool was the terpene alcohol with the next highest concentration. In both cases, production of linalool occurred at a steady rate for the rst 11 days, and then declined. The concentration reached around 0.75 Wg ml 31 for the ale yeast, and 0.45 Wg ml 31 for the lager yeast. Nerol production also followed a similar pattern in both yeasts, with the peak concentration reaching about 0.25 Wg ml 31 in the 2^4-day period. There was then a steady decline in nerol concentration, to around 0.15 Wg ml 31 at 15 days. Steady production of small quantities of K-terpineol was also observed with the ale and lager yeasts, reaching concentrations of around 1.2 Wg ml 31 and 0.9 Wg ml 31, respectively. With the lager yeast, ester formation also occurred. The esters detected were geranyl acetate, and citronellyl acetate. Geranyl acetate reached a concentration of just over 0.3 Wg ml 31 after 4 days, and then declined to 0.1 Wg ml 31 by 15 days. Citronellyl acetate concentrations were higher: reaching 0.5 Wg ml 31 after 4 days, and then remaining fairly constant. However, this terpenoid was not observed until the second day of the experiment. Overall, around 3 Wg ml 31 of terpenoids could be detected in both fermentations after 15 days, although the concentrations were still declining slowly Biotransformation of linalool Figs. 5 and 6 show biotransformation products formed from linalool by S. cerevisiae NCYC 1681 and S. bayanus NCYC From linalool, fewer products were detected. In both cases, K-terpineol was formed steadily, and reached a concentration of over 0.4 Wg ml 31 after 15 days

5 A.J. King, J.R. Dickinson / FEMS Yeast Research 3 (2003) 53^62 57 Fig. 3. Biotransformation of geraniol by ale yeast S. cerevisiae NCYC Geraniol was fed at a concentration of 10 Wg ml 31. Samples of the medium were taken over a 15-day period and then analysed by GC^MS. The levels of (a) substrate and (b^e) product are shown, along with the total concentration of terpenoids detected (f). The results shown are the averages of two independent injections. The results varied by no more than 5%. and still appeared to be increasing. Both yeasts also formed very small quantities of geraniol and nerol. These were rst detected after 4 days with the ale yeast, and after 2 days with the lager yeast. Geraniol concentrations reached 0.08 Wg ml 31 and nerol concentrations were just below 0.05 Wg ml 31. The total levels of terpenoids decreased to around 3.5 Wg ml 31 after 15 days, in both cases Fate of L-myrcene, L-caryophyllene and K-humulene With experiments where L-myrcene, L-caryophyllene or K-humulene were added to the medium, no products were detected. In the case of myrcene, none of the original terpenoid could be detected either. Fig. 7 shows the levels of L-caryophyllene and K-humulene detected over the 15- day period. The initial levels of the terpenoids detected in the medium were much lower than the amounts added. This was probably due to the relative insolubility of these terpenoids. With L-caryophyllene, the initial detected concentrations were 0.38 Wg ml 31 for NCYC 1681 and 0.78 Wg ml 31 for NCYC After 24 h, these levels had decreased to around 0.05 Wg ml 31 in both cases. However, these concentrations rose, peaking at 4 days in the case of NCYC 1681 and 7 days in the case of NCYC We believe this to be due to the increased alcohol concentrations of the partially fermented medium, allowing more of the terpenoids to dissolve, and to di erential adsorption and desorption onto yeast cell components, re ecting changes in the composition of the cells at di erent stages of the fermentation. By 15 days, no L-caryophyllene was detected in either the ale or lager yeast fermentations. With K-humulene, the initial concentrations recorded were 0.2 Wg ml 31 and 0.9 Wg ml 31 respectively for NCYC 1681 and NCYC These levels then decreased

6 58 A.J. King, J.R. Dickinson / FEMS Yeast Research 3 (2003) 53^62 Fig. 4. Biotransformation of geraniol by lager yeast S. bayanus NCYC Geraniol was fed at a concentration of 10 Wg ml 31. Samples of the medium were taken over a 15-day period and then analysed by GC^MS. The levels of (a) substrate and (b^g) product are shown, along with the total concentration of terpenoids detected (h). The values are the means of duplicate determinations which varied by no more than 5%. rapidly, and K-humulene could not be detected after 11 days in the case of NCYC 1681, and 15 days in the case of NCYC Discussion We have shown that brewing yeasts have the ability to transform terpenoids found in hops under simulated brewing conditions. Geraniol was converted mainly into citronellol; linalool and nerol were also detected. This linalool and nerol was further converted into K-terpineol. The lager yeast S. bayanus NCYC 1324 also produced terpenoid esters ^ both geranyl and citronellyl acetate. It is not known whether citronellyl acetate was formed from esteri cation of citronellol, reduction of geranyl acetate, or via

7 A.J. King, J.R. Dickinson / FEMS Yeast Research 3 (2003) 53^62 59 Fig. 5. Biotransformation of linalool by ale yeast S. cerevisiae NCYC Linalool was fed at a concentration of 10 Wg ml 31. Samples of the medium were taken over a 15-day period and then analysed by GC^MS. The levels of (a) substrate and (b^d) product are shown, along with the total concentration of terpenoids detected (e). The values are the means of duplicate determinations which varied by no more than 5%. both mechanisms (Fig. 8). Moir et al. [12] have detected geranyl and citronellyl esters at higher concentrations in late-hopped lagers than in lagers hopped at other stages in the brewing process. Whilst esters of terpene alcohols have previously been found in hop oils, this study has shown that yeasts may also contribute to their presence in beer via esteri cation during the brewing process. The formation of terpenoid esters by the lager strain but not by the ale strain is clearly a re ection of the genetic (and consequent biochemical) di erences between these organisms. Whilst our knowledge of terpenoid transformation pathways in the two yeasts remains incomplete, we can only speculate whether this arises from (say) the possession of a single gene for an additional ester synthase or a larger number of genes corresponding to (a) new pathway(s). In this context it should be noted that the fermentations were carried out at 18 C. It could be argued that this temperature was too high for the lager yeast and that a more appropriate temperature would have been (say) 12 C. Whilst this may be true, the higher temperature has clearly not prevented the expression of any activities in lager yeast that are present in the ale yeast. Instead, the contrary was observed. The appearance of small quantities of geraniol and nerol in the linalool supplementation experiments indicates that some biotransformation reactions are reversible (i.e. geraniol to linalool, nerol to linalool). These terpenoids appeared midway through the fermentations where the

8 60 A.J. King, J.R. Dickinson / FEMS Yeast Research 3 (2003) 53^62 Fig. 6. Biotransformation of linalool by lager yeast S. bayanus NCYC Linalool was fed at a concentration of 10 Wg ml 31. Samples of the medium were taken over a 15-day period and then analysed by GC^MS. The levels of (a) substrate and (b^d) product are shown, along with the total concentration of terpenoids detected (e). The values are the means of duplicate determinations which varied by no more than 5%. yeasts external environment would have changed. For example, there would have been lower levels of utilisable nutrients, more ethanol, and no remaining oxygen. Intracellular stores of sterols would also be depleted at this time. Any one of, or a combination of these factors may lead to the conditions where these biotransformation reactions operate in the reverse direction. Most of the terpene alcohols were lost from the fermentations within the rst few days of the fermentations, and after that, the decreases occurred at much lower and steadier rates. This indicates that the loss of terpenoids was associated with the increase in yeast biomass. In the Fermentose-based medium no transformation products were detected from the terpene alkenes; the actual added terpenoids themselves were present at very low concentrations and undetectable by the end of the fermentations. This was very slightly di erent from the preliminary experiment in YEPD medium where, after 24 h, K-humulene gave rise to small amounts of L-carophyllene and carophyllene oxide. It should be noted, however, that the addition of L-carophyllene to YEPD medium resulted in none of that compound being recovered and no products being formed from it. Likewise, there was no biotransformation and only 35% recovery when carophyllene oxide was added to YEPD medium. Hence, there are no real contradictions between our di erent experiments. Indeed, a number of other studies have highlighted the fact that terpene alkenes are not present at high concentrations in nished beer, if at all [9^12,17]. Our results are in agreement with these studies. The relative insolubility of these compounds does not favour their presence in the nal product. Oxygenated derivatives may however contribute to hop aroma in beer. Although the terpene alkenes were present initially at much lower concentrations than the terpene alcohols, decreases in the observed concentrations again occurred much more rapidly in the early part of the

9 A.J. King, J.R. Dickinson / FEMS Yeast Research 3 (2003) 53^62 61 Fig. 7. Fate of L-caryophyllene (a and c) and K-humulene (b and d) when incubated with S. cerevisiae NCYC 1681 (a and b) and S. bayanus NCYC 1324 (c and d). Both terpenoids were added at an initial concentration of 10 Wg ml 31. Samples of the medium were taken over a 15-day period and then analysed by GC^MS. The values are the means of duplicate determinations which varied by no more than 5%. fermentation. This may also have been due to binding to the yeast biomass. The evidence presented in this study suggests that oxygenated terpenoids are much more likely to remain in beer than alkenes. A number of workers have analysed oxygenated products of hops which form during hop storage. These studies have involved rstly subjecting humulene epoxides and caryophyllene epoxide (which form during hop storage) to hydroxylation and then using Fig. 8. Potential routes for the formation of citronellyl acetate by S. bayanus NCYC 1324.

10 62 A.J. King, J.R. Dickinson / FEMS Yeast Research 3 (2003) 53^62 GC^MS and nuclear magnetic resonance to determine the structures of the products [18,19]. Only by concentrating large volumes of beer to small aliquots was it possible to determine a number of terpenoids and their sensory properties in beer for the rst time [20,21]. Many of these had sensory thresholds in the ppb range. It would be interesting to determine the fate of these compounds in the presence of yeast. Since carbon catabolite repression is a major regulator of yeast metabolism [5], the idea behind this study was to examine the transformation of hop terpenoids in media with high concentrations of sugars which more closely resemble the spectrum of sugars in commercial media than the (usually) single sugar of academic laboratory media. Nitrogen catabolite repression is also a major regulatory phenomenon in yeast [22] and it could be argued that ammonium sulfate at 5.87 g l 31 (as used in the experiments described above) is not a perfect model for the nitrogen sources present in wort. However, a recent examination of this proposition showed that in minimal medium the e ects of altering the carbon source were much greater than altering the levels of the nitrogen source [23]. Acknowledgements This project was sponsored by a BBSRC CASE studentship with Whitbread plc (grant number 96/A3/F/0232). References [1] Bauer, K., Garbe, D. and Surburg, H. (1990) Common Fragrance and Flavor Materials: Preparation and Uses, 2nd edn. VCH Publishers, New York. [2] Eisenreich, W., Sagner, S., Zenk, M.H. and Bacher, A. (1997) Monoterpenoid essential oils are not of mevalanoid origin. Tetrahedron Lett. 38, 3889^3892. [3] Sharpe, F.R. and Laws, D.R.J. (1981) The essential oil of hops ^ A review. J. Inst. Brew. 87, 96^107. [4] King, A. and Dickinson, J.R. (2000) Biotransformation of monoterpene alcohols by Saccharomyces cerevisiae, Torulspora delbrueckii and Kluyveromyces lactis. Yeast 16, 499^506. [5] Dickinson, J.R. (1999) Carbon metabolism. In: The Metabolism and Molecular Physiology of Saccharomyces cerevisiae (Dickinson, J.R. and Schweizer, M, Eds.), pp. 23^55. Taylor and Francis, London, Philadelphia, PA. [6] Hough, J.S., Briggs, D.E. and Stevens, R. (1971) Malting and Brewing Science, 1st edn. Chapman and Hall, London. [7] Andreasen, A.A. and Stier, T.J.B. (1953) Anaerobic nutrition of Saccharomyces cerevisiae. I. Ergosterol requirement for growth in a de- ned medium. J. Cell. Comp. Physiol. 41, 23^26. [8] Andreasen, A.A. and Stier, T.J.B. (1954) Anaerobic nutrition of Saccharomyces cerevisiae. II. Unsaturated fatty acid requirement for growth in a de ned medium. J. Cell. Comp. Physiol. 43, 271^281. [9] Peacock, V.E. and Deinzer, M.L. (1981) Chemistry of hop aroma in beer. J. Am. Soc. Brew. Chem. 39, 136^141. [10] Peacock, V.E. and Dienzer, M.L. (1988) Fate of hop oil components in beer. J. Am. Soc. Brew. Chem. 48, 104^107. [11] Peacock, V.E., Deinzer, M.L., Likens, S.T., Nickerson, G.B. and McGill, L.A. (1981) Floral hop aroma in beer. J. Agric. Food Chem. 29, 1265^1269. [12] Moir, M., Seaton, J.C. and Suggett, A. (1983) Hop avour manipulation in brewing. EBC Congress, pp. 63^70. [13] Stucky, G.J. and McDaniel, M.R. (1997) Raw hop aroma qualities by trained panel free-choice pro ling. J. Am. Soc. Brew. Chem. 55, 65^72. [14] Cook, A.H. (1963) Yeast and the work of the Yeast Group. EBC Congress, pp. 477^486. [15] Cocito, C., Gaetano, G. and Del ni, C. (1995) Rapid extraction of aroma compounds in must and wine by means of ultrasound. Food Chem. 52, 311^320. [16] Bishop, J.P.R., Nelson, G. and Lamb, J. (1998) Microencapsulation in yeast cells. J. Microencapsul. 15, 761^773. [17] Deinzer, M.D. and Yang, X. (1994) Hop aroma character impact compounds found in beer ^ methods of formation of individual compounds. EBC Symp. on Hops, Zoeterwoude, The Netherlands, pp. 181^197. European Brewery Convention, Monogr. XXII. [18] Yang, X. and Deinzer, M.L. (1992) Hydrolysis and reversible isomerization of humulene epoxides II and III. J. Org. Chem. 57, 4717^ [19] Yang, X. and Deinzer, M. (1994) Hydrolysis and rearrangement reactions of caryophyllene oxide. J. Nat. Prod. 57, 514^517. [20] Yang, X., Lederer, C., McDaniel, M. and Deinzer, M. (1993a) Chemical analysis and sensory evaluation of hydrolysis products of humulene epoxides II and III. J. Agric. Food Chem. 41, 1300^1304. [21] Yang, X., Lederer, C., McDaniel, M. and Deinzer, M. (1993b) Hydrolysis products of caryophyllene oxide in hops and beer. J. Agric. Food Chem 41, 2082^2085. [22] Dickinson, J.R. (1999) Nitrogen metabolism. In: The Metabolism and Molecular Physiology of Saccharomyces cerevisiae (Dickinson, J.R. and Schweizer, M., Eds.), pp. 57^77. Taylor and Francis, London, Philadelphia, PA. [23] King, A.J. (2000) Yeast: Terpenoid biotransformation, toxicity and potential for production, PhD thesis. Cardi University, Cardi, UK.