FEMS Yeast Research 3 (2003) 53^62

www.fems-microbiology.org

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 29 April 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.
+ 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
(C10 ), sesquiterpenoids (C15 ), diterpenoids (C20 ), triterpe-
noids (C30 ), carotenoids, sterols, phytols and quinones.
Monoterpenoids and sesquiterpenoids are compounds
with strong sensory qualities, and are found in the essen-
tial oils of many plants [1]. Consequently, they are used
extensively in those industries where £avour and/or fra-
grance are important. The structures of terpenoids rele-
vant 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) 2087-5762;
Fax : +44 (29) 2087-4305.
E-mail address : dickinson@cardi¡.ac.uk (J.R. Dickinson).
1
Present address: KPMG, 8 Salisbury Square, London EC4Y 8BB,
UK.
1567-1356 / 02 / $22.00 + 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII : S 1 5 6 7 - 1 3 5 6 ( 0 2 ) 0 0 1 4 1 - 1
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 (bitter-
ness and aroma) and to act as a preservative. The preser-
vative 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 num-
ber 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 metabo-
lism [5], we decided to investigate whether hop terpenoids
were transformed when an ale and lager yeast were cul-
tured in the presence of a commercially available syrup at
concentrations more akin to wort than academic labora-
tory 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-

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54 A.J. King, J.R. Dickinson / FEMS Yeast Research 3 (2003) 53^62
vided [6]. This limited amount of oxygen is added to sat-
isfy the yeast cells’ requirement for sterols and unsaturated
fatty acids, which cannot be made under anaerobic con-
ditions [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 condi-
tions. These monoterpene alcohols have also been detected
in beer in a number of studies [9^12]. Additionally, lina-
lool 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 terpe-
noids 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 avail-
able 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 so-
lutions, prepared at a concentration of 100 mg ml31 . 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 ml31 . Control cultures
(i.e. with no added terpenoids) contained equivalent vol-
umes 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, Nor-
wich, UK) were used as representative brewing ale and
lager strains, respectively. For preliminary experiments
to determine the recovery of terpenoids, S. cerevisiae pro-
totrophic 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 experi-
ments media were prepared containing RM-81 Fermentose
syrup diluted with distilled water to give a speci¢c density
in the range 1.045^1.050. Other nutrients were provided as
ammonium sulfate (5.87 g l31 ) and Bacto yeast nitrogen
base (without ammonium sulfate, 1.7 g l31 ). Inocula for
fermentations were produced by growing overnight aero-
bic cultures in 1000-ml conical £asks containing 250 ml of
medium at 30‡C, with orbital shaking at 150 r.p.m. After
FEMSYR 1510 13-2-03
reaching an OD600 nm of 6^10 (approximately 6U107 -108
cells ml31 ), 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 OD600 nm of 2.0
(approximately 2U107 cells ml31 ). The tall tubes (total
capacity just over 1.1 l) contained 800 ml of sparged
(50 min with ¢lter-sterilised air) medium. The fermenta-
tions were at 18‡C, and 50-ml samples were removed peri-
odically under aseptic conditions. Progress of fermentation
was monitored by measuring the speci¢c gravity of de-
gassed 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 ro-
tary 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 chroma-
tograph 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 min31 , 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
400.
3. Results
3.1. The recovery of terpenoids
Preliminary experiments were performed to monitor the
recovery of terpenoids. In these experiments cells were in-
oculated into YEPD medium, containing terpenoids at a
concentration of 100 Wg ml31 . 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
 A.J. King, J.R. Dickinson / FEMS Yeast Research 3 (2003) 53^62 55

into citronellol and nerol. Small quantities of linalool pro-

Fig. 1. The structures of terpenoids relevant to brewing and their aro-
mas. 1: geraniol (£oral, rose-like, citrus) ; 2: linalool (£oral, fresh, cor-
iander); 3: citronellol (sweet, rose-like, citrus); 4: nerol (£oral, fresh,
green); 5: K-terpineol (lilac); 6: terpinen-4-ol (spicy, medicinal) ; 7: lina-
lool oxide (herbaceous, medicinal) ; 8: nerolidol (£oral); 9: farnesol (£o-
ral); 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].
duction 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 de-
tected, 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 al-
kenes 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 possi-
ble explanations for the low recoveries of these com-
pounds. 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 ml31 of var-
ious terpenoids
Terpenoid Residual terpenoid at Products at Terpenoids extracted from Approximate recovery Ratio of free to
24 h (Wg ml31 ) 24 h (Wg ml31 ) cells (Wg) (%) bound terpenoids
16.9 nerol 202.2 geraniol 100 10.9:1
Geraniol 37.9 36.7 citronellol 23.2 nerol
2.4 linalool trace:geranyl 194.8 citronellol
acetate
Linalool 90 none 145.0 linalool 94 31.3:1
Citronellol 83.3 5.5 citronellyl acetate 181.7 citronellol 92 28.7:1
6.7 geraniol 60.2 nerol 93 69.1:1
Nerol 79.6 5.3 citronellol trace :K- 3.5 citronellol
terpineol
K-Terpineol 79.0 none 75.4 K-terpineol 80.5 52.4:1
Linalool oxide 95.7 none 40.1 linalool oxide 97 119.3:1
Nerolidol 79.0 none 1295 nerolidol 100 3.1:1
Farnesol 32.0 none 3171 farnesol 95 0.5:1
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 2.9 caryophyllene none 3.6 N/A
0.2 0.5 caryophyllene oxide
L-Caryophyllene 0.3 none none 0.3 N/A
Caryophyllene oxide 23.7 none 540.6 caryophyllene oxide 35 2.2:1
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).

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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 ml31 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%.

3.2. 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 ml31:
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 cul-
tures 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.
3.3. 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 de-
tected in cultures which were inoculated with a yeast and
supplemented with a terpenoid must be the genuine prod-
ucts 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 de-
tected. In both cases, citronellol was the most abundant
product, reaching a concentration of over 1.5 Wg ml31 in
4 days with the ale yeast (NCYC 1681), and 1.4 Wg ml31
with the lager yeast (NCYC 1324). However, with the
lager yeast it declined to around 1.0 Wg ml31 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
ml31 for the ale yeast, and 0.45 Wg ml31 for the lager
yeast. Nerol production also followed a similar pattern
in both yeasts, with the peak concentration reaching about
0.25 Wg ml31 in the 2^4-day period. There was then a
steady decline in nerol concentration, to around 0.15 Wg
ml31 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 ml31
and 0.9 Wg ml31 , 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 ml31 after 4 days, and
then declined to 0.1 Wg ml31 by 15 days. Citronellyl ace-
tate concentrations were higher: reaching 0.5 Wg ml31
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 ml31 of terpenoids
could be detected in both fermentations after 15 days,
although the concentrations were still declining slowly.
3.4. Biotransformation of linalool
Figs. 5 and 6 show biotransformation products formed
from linalool by S. cerevisiae NCYC 1681 and S. bayanus
NCYC 1324. From linalool, fewer products were detected.
In both cases, K-terpineol was formed steadily, and
reached a concentration of over 0.4 Wg ml31 after 15 days

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 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 1681. Geraniol was ‘fed’ at a concentration of 10 Wg ml31 . Samples of the me-
dium 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 con-
centration 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 ml31 and nerol concentrations were just
below 0.05 Wg ml31 . The total levels of terpenoids de-
creased to around 3.5 Wg ml31 after 15 days, in both cases.
3.5. 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 con-
centrations were 0.38 Wg ml31 for NCYC 1681 and 0.78 Wg
ml31 for NCYC 1324. After 24 h, these levels had de-
creased to around 0.05 Wg ml31 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 1324. We
believe this to be due to the increased alcohol concentra-
tions 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 ml31 and 0.9 Wg ml31 respectively for
NCYC 1681 and NCYC 1324. These levels then decreased

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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 1324. Geraniol was ‘fed’ at a concentration of 10 Wg ml31 . Samples of the me-
dium 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 con-
centration 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 1324.
4. Discussion
We have shown that brewing yeasts have the ability to
transform terpenoids found in hops under simulated brew-
ing conditions. Geraniol was converted mainly into citro-
nellol ; linalool and nerol were also detected. This linalool
and nerol was further converted into K-terpineol. The la-
ger yeast S. bayanus NCYC 1324 also produced terpenoid
esters ^ both geranyl and citronellyl acetate. It is not
known whether citronellyl acetate was formed from ester-
i¢cation of citronellol, reduction of geranyl acetate, or via

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 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 1681. Linalool was ‘fed’ at a concentration of 10 Wg ml31 . Samples of the me-
dium 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 forma-
tion of terpenoid esters by the lager strain but not by the
ale strain is clearly a re£ection of the genetic (and conse-
quent biochemical) di¡erences between these organisms.
Whilst our knowledge of terpenoid transformation path-
ways 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 temper-
ature 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 ne-
rol 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

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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 1324. Linalool was ‘fed’ at a concentration of 10 Wg ml31 . Samples of the me-
dium 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 ex-
ample, there would have been lower levels of utilisable
nutrients, more ethanol, and no remaining oxygen. Intra-
cellular 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 re-
actions operate in the reverse direction. Most of the ter-
pene 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 ac-
tual added terpenoids themselves were present at very low
concentrations and undetectable by the end of the fermen-
tations. This was very slightly di¡erent from the prelimi-
nary 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 prod-
ucts being formed from it. Likewise, there was no bio-
transformation and only 35% recovery when carophyllene
oxide was added to YEPD medium. Hence, there are no
real contradictions between our di¡erent experiments. In-
deed, 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 agree-
ment 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

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 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 ml31 . 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 analysed oxygenated products of hops which form during
the yeast biomass. The evidence presented in this study hop storage. These studies have involved ¢rstly subjecting
suggests that oxygenated terpenoids are much more likely humulene epoxides and caryophyllene epoxide (which
to remain in beer than alkenes. A number of workers have form during hop storage) to hydroxylation and then using

Fig. 8. Potential routes for the formation of citronellyl acetate by S. bayanus NCYC 1324.

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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 prop-
erties in beer for the ¢rst time [20,21]. Many of these had
sensory thresholds in the ppb range. It would be interest-
ing to determine the fate of these compounds in the pres-
ence 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 me-
dia. Nitrogen catabolite repression is also a major regula-
tory phenomenon in yeast [22] and it could be argued that
ammonium sulfate at 5.87 g l31 (as used in the experi-
ments described above) is not a perfect model for the
nitrogen sources present in wort. However, a recent exami-
nation 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 student-
ship with Whitbread plc (grant number 96/A3/F/0232).
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