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159 November / December 2017 (Vol. 70)
Yearbook 2006
The scientifi c organ
of the Weihenstephan Scientifi c Centre of the TU Munich
of the Versuchs- und Lehranstalt für Brauerei in Berlin (VLB)
of the Scientifi c Station for Breweries in Munich
of the Veritas laboratory in Zurich
of Doemens wba – Technikum GmbH in Graefelfi ng/Munich www.brauwissenschaft.de
BrewingScience
Monatsschrift für Brauwissenschaft
https://doi.org/10.23763/BrSc17-16haslbeck
Authors
K. Haslbeck, S. Bub, C. Schönberger, M. Zarnkow, F. Jacob and M. Coelhan
On the Fate of β-Myrcene during Fermentati-
on – The Role of Stripping and Uptake of Hop
Oil Components by Brewer’s Yeast in Dry-
Hopped Wort and Beer
Hops play a significant role in determining the aroma of beer. The essential oil of hops contains a large
number of flavor-active components. Concentrations of essential oil constituents in beer depend on factors
such as the time of hop addition in the brewing process and hop amount added. Generally, compound classes
such as mono- and sesquiterpenes do not reach the threshold concentrations in the final product, but in
dry-hopped beers after main fermentation they often do. Two factors that potentially cause decreased amounts
of terpenoids in beer were investigated. In case of the non-polar compound β-myrcene, losses due to releases
into the gas phase during standardized laboratory-scale fermentations were studied. Samples of
industrially produced all malt wort (11.5 °P) were dry-hopped at pitching with Mosaic hops. Two yeast strains
that are widespread in German beer production were used in trials, TUM 68 (S. cerevisiae) and TUM 34/70
(S. pastorianus). A method for dissolving fermentation gases in bubbling water columns was used. The hops,
SPE-water extracts and beer samples were analyzed by several chromatographic systems using two different
GC-FID, nanoLC-MS/MS, GC-MS and HS-GC-MS, respectively. Tendency was shown that higher temperatures
at primary fermentation cause increased releases of aroma compounds into the gas phase, which was
observed on model fermentations in previous studies. The reversible uptake of β-myrcene by yeast cells,
identified in separate test series, was determined as being a highly effective factor decreasing amounts in beer
systems. In bottled beers 100 million cells/ml led to decreased amounts of about 98–99 %. It was shown that
solvent systems with similar properties to beers (5 % and 10 % ethanolic solution) are inadequate for
re-dissolving compounds attached to yeasts. The absorbed amount in yeast therefore cannot contribute to
the flavor of beer. Incomplete recovered amounts of β-myrcene even in pure ethanol suspensions indicate
that there are strong bonds between yeast cells and the odor compound. Linalool, on the other hand, was not
affected by the test conditions used.
Descriptors: S. cerevisiae and S. pastorianus, Humulus lupus L., dry hopping, fermentation, beer flavor, β-myrcene and linalool
Korbinian Haslbeck, Martin Zarnkow, Fritz Jacob, Mehmet Coelhan, Tech-
nical University of Munich, Research Center Weihenstephan for Brewing
and Food Quality, Freising, Germany; Stefan Bub, Brauerei Bub GbR,
Leinburg, Germany; Christina Schönberger, Joh. Barth & Sohn GmbH &
Co. KG, Nuernberg, Germany; corresponding author: coelhan@wzw.tum.de
1 Introduction
In recent years the interest in beers with special and diverse flavors
has grown. Many brewers use newly developed raw materials such
as flavor hops, more variety in aroma intense yeast strains and
apply rediscovered traditional techniques such as dry hopping [1,
2]. Aroma compounds in beer originate from malt, hops (that are
partially transformed in process steps such as wort boiling) and
arise from the metabolic activity of brewing yeast [3, 4]. Hops play
a significant role in determining the aroma of beer and there are
a large number of popular beer types with a pleasantly enhanced
hop bouquet. Research in the field of hoppy flavor of beer focuses
on essential oil as the primary source of hop flavoring. More than
1000 different constituents are assumed in the essential oils [5].
β-Myrcene and linalool in hop essential oil were identified as
some of the most potent odorants by applying AEDA to the volatile
fraction isolated from a hop cultivar (Spalter Select) [6, 7]. In beer,
the concentrations as well as the combinations of key compounds
such as linalool (“floral”, “fruity”) [8] determine the final particular
hoppy flavor in beer [3, 9]. Roughly summarized, the type of hop
flavor can be distinguished as kettle hop or dry hop flavor. Diffe-
rences occur due to the time of addition in the brewing process.
The kettle hop flavor is formed when boiling wort in the presence of
hops. Essential oil constituents such as sesquiterpenes are partly
oxygenated and can evoke spicy flavors in beer [10]. Other volatile
compounds like monoterpenes are usually reduced to traces [1, 11,
12]. It is assumed that their generally non-polar and very volatile
character might lead to adsorption to the trub and evaporation
November / December 2017 (Vol. 70) 160
Yearbook 2006
The scientifi c organ
of the Weihenstephan Scientifi c Centre of the TU Munich
of the Versuchs- und Lehranstalt für Brauerei in Berlin (VLB)
of the Scientifi c Station for Breweries in Munich
of the Veritas laboratory in Zurich
of Doemens wba – Technikum GmbH in Graefelfi ng/Munich www.brauwissenschaft.de
BrewingScience
Monatsschrift für Brauwissenschaft
with wort steam [12]. Hopping beer after the main fermentation
can lead to monoterpene concentrations above threshold values
contributing to a particular dry hop beer flavor [1, 13]. β-Myrcene
in particular is an important component of the essential oil of hops
that is described as “herbaceous”, “resinous”, “green”, “balsamic”,
“fresh hops” and often found in dry-hopped beers [12, 14]. Losses
of monoterpenes such as β-myrcene were noted not only at wort
boiling, but also during yeast fermentation, which can be significant
[15]. Decreasing contents of linalool were also documented during
fermentation, but in much smaller amounts [16]. With regard to
their importance for beer aroma there is a high level of interest in
obtaining information on factors that may lead to the loss of these
pleasantly aromatic essential oil constituents.
In several studies on fermentations of beer worts or wine musts
it was shown that volatile compounds are partly transported to
the surface of the media by fermentative carbon dioxide and
subsequently released into the gas phase [17–19]. Besides, little
is known about the fate of compounds produced by yeast or pre-
existing odorant compounds and losses through stripping during
fermentation which is why the final flavor of the beer is not always
uniform [20]. In recent years, methods for the real-time monitoring
of stripped aroma compounds during beer fermentation were de-
veloped [17, 21]. In 2013 Haefliger and Jeckelmann determined
mass flows with 5 minutes resolution of released gases from
yeast metabolism and compounds derived from hops, including
monoterpenes, sesquiterpenes and some esters in the headspace
of wort during fermentation. The mass flows were determined
by gas chromatography mass spectrometry (GC-MS) equipped
with an automatic cryotrapping sampling system [18]. In 2014,
Keupp and Zardin observed dynamic changes in the release of
acetic acid, ethyl acetate, isobutyl acetate and isoamyl acetate
by proton-transfer-reaction mass spectrometry (PTR-MS) directly
in the headspace of fermenting wheat beer wort [19]. Real-time
monitoring of fermentation gases can provide extensive information
on the dynamics of aroma compound release that could contribute
to controlling various processing parameters with the objective of
creating the final aroma of beer.
However, real-time applications could only be achieved to date in
a laboratory-like environment at limited scales of fermentations. It
is well known when upscaling brewing batches that differences in
aroma profiles will occur and so there is a need to further develop
existing systems or to use other methods [22, 23]. In the field of
chemical engineering, different kinds of gas sampling methods
are used, which allow the subsequent analysis of gas constituents
[24, 25]. The bubbling water column is an example of when gases
become specifically dissolved in solvents. In this very flexible and
robust method, a gas stream is passed through a water column.
Gaseous substances present in small bubbles are absorbed by the
water [24, 26]. The absorption rate of a dissolved gas in bubbling
columns is determined by the density of the water, the gas mass
fraction and gas diffusivity [24]. The water of bubbling columns
containing compounds that are transferred from fermenting worts
can be used for gas chromatographic analysis.
When addressing the issue of loss of hop essential oil constituents
during fermentation, many authors believe that adsorption at the
surface of hydrophobic yeast cells [4, 27–33] and migration to the
foam layer might occur [34]. It is worth mentioning that enzymatic
cleavage of glycosidically-bound constituents and biotransforma-
tions of monoterpene alcohols such as linalool, geraniol, α-terpineol,
citronellol and nerol can affect the amounts of essential oils during
fermentation [9, 31, 33, 35]. Other hop constituents such as bitter
acids were determined in spent brewer’s yeast at reasonable
amounts depending on the hopping regime [36]. So far, the effect
of brewer’s yeast regarding the large hydrophobic surface of
yeast cells in fermenting wort and beer [4] on concentrations of
odor compounds has been little studied. These considerations
are directly connected with a pronounced hydrophobic character
of a part of the essential oil constituents [37]. There are large
differences between the solubility of a relatively polar component
such as linalool and a relatively non-polar component such as
β-myrcene in water: 10.1 ± 0.61 mmol/l and 0.22 ± 0.02 mmol/l
(measured at 25 °C by Fichan and Larroche), respectively [38].
It is assumed that this is the primary reason of the differences in
the varying levels of different aroma compounds in wort and beer,
which is an essential part of the following investigations.
In this study, a brewing trial at standardized fermentations at a
10-l laboratory-scale was conducted. Mosaic hop was added at
the pitching stage of all malt wort that was produced on an indus-
trial scale. Fermentations were achieved using the brewing yeast
strains TUM 68 (S. cerevisiae) and TUM 34/70 (S. pastorianus)
at low and high temperatures for each strain. Hop samples were
analyzed by GC-FID and nanoLC-MS/MS. Volatile compounds
in beer samples were analyzed using GC-FID, HS-GC-MS and
nanoLC-MS/MS. In this approach bubbling water columns were
used between each fermentation vessel and bung apparatus in
order to dissolve the fermentation gases in water, then extracted
by SPE and analyzed by GC-MS. In separate experiments, the
affinity of brewer’s yeast for β-myrcene and linalool, respectively,
was investigated.
2 Materials and methods
2.1 Hop raw material
Mosaic hop pellets type-90 of crop 2015 (USA) were provided by
Barth Haas (84048 Mainburg, Germany). The total essential oil in
hops was determined according to standard ASBC methods [39].
The essential oil was used for further gas chromatographic analysis.
2.2 Brewing trial
2.2.1 Dry-hopped pitching wort
Lager beer wort used for the brewing trial (Table 4, see page
164) was produced on an industrial scale (300 hl batch). The wort
was moderately kettle-hopped with Perle 16 (65 g/hl). Samples
of a batch were taken after whirlpool rest and directly inserted in
10-kg-portions into four fermenting vessels (Cornelius NC) and
subsequently cooled down in water baths until they reached pitching
temperatures. Wort and yeast samples were prepared for pitching
using climate chambers at 8 °C, 15 °C and 22 °C. Immediately prior
to pitching, a sterile nylon fiber bag containing 9.6 g Mosaic pellets
(Table 1) was added to each of the four fermentation vessels. Hop
161 November / December 2017 (Vol. 70)
Yearbook 2006
The scientifi c organ
of the Weihenstephan Scientifi c Centre of the TU Munich
of the Versuchs- und Lehranstalt für Brauerei in Berlin (VLB)
of the Scientifi c Station for Breweries in Munich
of the Veritas laboratory in Zurich
of Doemens wba – Technikum GmbH in Graefelfi ng/Munich www.brauwissenschaft.de
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bags attached to stainless steel weights using 15 cm long nylon
cords were positioned on the vessel bottom in order to prevent
floating to the surface.
2.2.2 Propagation
Yeast was propagated from pure culture provided by Yeast Center of
the Research Center Weihenstephan for Brewing and Food Quality
(Freising, TU München, Germany). Isolates were inoculated from
agar slants into 70 ml of sterile wort medium in a 100-ml-Erlenmeyer
flask. The wort was made using an unhopped pilsner barley malt
extract (Weyermann GmbH & Co. KG, Bamberg, Germany). The
extract was diluted with distilled boiling water to an original gravity
of 12.0 °P to guarantee sterile conditions. Incubation in this and
the following steps took 96 hours at ambient temperature (20 °C)
and pressure. After the first incubation period yeast was transferred
to 1 l of sterile wort in a 2.5-l-glass vessel and further incubated.
Then the supernatant was decanted and yeasts were transferred to
3.5 l of sterile wort in a 5.0-l-glass vessel. After incubation, yeasts
were added to two 5.0-l-glass vessels each containing 3.5 l of
sterile wort and incubated. The incubation in two 5-l-vessels was
repeated until the desired amount of yeast for trials was reached.
Before fermentation, yeasts were softly tempered within 24 hours
until they reached pitching temperatures (8 °C, 15 °C, and 22 °C).
Yeast cell concentrations (cells/ml) were determined using a cell
counter (Nexcelom Bioscience, Lawrence, MA, USA) that was
calibrated for the corresponding yeast strains.
2.2.3 Fermentation
Laboratory-scale fermentations were per-
formed using Cornelius NC stainless steel
vessels with dimensions of 21.6 cm diameter
x 62.9 cm height (18.9 l) and sealed by caps
that were equipped with gas ports (Cornelius,
Inc., Osseo, MN, USA). Pure cultures of S.
cerevisiae TUM 68 and S. pastorianus TUM
34/70 (Research Center Weihenstephan for
Brewing and Food Quality, Freising, TU Mün-
chen, Germany) were used as representative
brewer’s top- and bottom fermenting strains,
respectively. The wort was not oxygenated.
Fermentations at different test set-ups were
achieved in single-issue approaches. The
fermentation was started by adding 30 million
cells/ml of propagated yeast TUM 34/70 to
both vessels in cooling chambers at 8 °C and
15 °C, respectively and 15 million cells/ml of
propagated yeast TUM 68 to both vessels at
15 °C or 22 °C chambers. In order to imitate
fermentation in vessels on an industrial scale,
a head pressure of 0.5 bar was applied by a
bung apparatus simulating liquid heights of
10 m (median hydrostatic pressure) [22]. The temperatures were
maintained for at least 10 days of primary fermentation. Primary
fermentation was considered complete after the specific gravity
remained constant for two consecutive days. Maturation was carried
out for three weeks at 0 °C. The beer samples were then filled in
0.5-l-portions with pilot scale bottle filler (Esau-Hueber, Schroben-
hausen, Germany) into 0.5-l-brown glass (NRW-) beer bottles under
anti-oxidizing conditions. The alcohol content, residual extract and
fermentation degree of the beers were determined from filtered
(Whatman folded filter paper, diameter: 320 mm, GE Healthcare
Europe GmbH, Freiburg, Germany) samples using a DMA 35N
(Anton-Paar GmbH, Graz, Austria). In beers, the hop essential oil
constituents were analyzed by HS-GC-MS and nanoLC-MS/MS,
fermentation by-products were measured by GC-FID.
2.2.4 Bubbling water column
Five bubbling water columns bound in series were connected to the
gas line between each fermentation vessel and a bung apparatus.
Thus fermentation gases were forced to pass five water columns
before escaping via the bung apparatus. Therefore stainless steel
containers with dimensions of 10 cm diameter × 36 cm height (2.7 l)
were filled completely with (non-carbonated) mineral water of a
single batch (ja!, REWE Group) ensuring standardized conditions,
slight pH-buffering capacities and non-hazardous handling. The
caps of each container were equipped with two gas ports, one of
which was connected with a riser pipe. Sealed containers were
hermetically connected by gas lines plugged into the ports so that
fermentation gases could escape the riser pipe at the bottom of
each container and leave the container via the gas port in the cap
(Fig. 1). The containers were placed outside of climate chambers at
room temperature (20–21 °C) during fermentation in order to ensure
equal conditions for the dissolution of the released gases [24]. After
fermentation, 1-l-samples of each column were extracted by SPE
and subsequently analyzed by GC-MS (see 2.4.3 for details of
preparation of SPE extracts and 2.4.4 for GC-MS of SPE extracts).
Table 1 Dry hopping doses for 1.5 ml hop oil/hl
Variety α-Acids
(% w/w)
Oil Content
(ml/100 g)
Dosage
(g/kg)
Mosaic 12.3 1.55 0.96
Fig. 1 Experimental set-up for the dissolution of volatiles in beer wort headspace by five
bubbling water columns connected in series
November / December 2017 (Vol. 70) 162
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The scientifi c organ
of the Weihenstephan Scientifi c Centre of the TU Munich
of the Versuchs- und Lehranstalt für Brauerei in Berlin (VLB)
of the Scientifi c Station for Breweries in Munich
of the Veritas laboratory in Zurich
of Doemens wba – Technikum GmbH in Graefelfi ng/Munich www.brauwissenschaft.de
BrewingScience
Monatsschrift für Brauwissenschaft
2.3 Concentration of aroma compounds in yeast
2.3.1 Recovery of β-myrcene and linalool in beer
A pale filtered non-alcoholic lager beer filled in 0.5-l-brown glass
NRW-bottles was used in these test series. The beer was indus-
trially produced from a comparable batch of wort and yeast strain
(TUM 34/70) as that utilized in the brewing trial. The experimen-
tal set-up, which included contact duration, temperature, slight
agitation, cell count and medium, was selected to reflect the
main fermentation. At the same time losses by outgassing were
avoided. Four different yeast counts of 1, 5, 20 and 100 million
cells/g were prepared in bottled beers by adding propagated and
washed yeast samples to opened bottles. The method of yeast
washing was based on references [36] and [40]. Briefly, 500 g of
propagated yeast suspensions adjusted to 100 million cells/g was
centrifuged in 600-ml centrifuge tubes at 4000 rpm for 10 minutes
using a Megafuge 40R (Thermo Scientific, Waltham, MA, USA).
The supernatant was replaced by deionized water and the (cen-
trifuge) tube content subsequently treated using an ARE magnetic
stir bar (VELP Scientifica, Usmate Velate, Italy) at medium stirring
speed for 10 min in order to suspend the sedimented yeast. The
washing procedure for each portion of yeast was repeated four
times. Yeast quantities for setting the desired cell concentrations
(cells/ml) were determined using a cell counter calibrated for the
corresponding yeast strain (Nexcelom Bioscience, Lawrence,
MA, USA). 50 µl of β-myrcene (0.7 g/l, tetrahydrofuran solution)
or linalool (0.7 g/l ethanol solution) was added to beers to set the
concentrations to 70 µg/l, which is within the characteristic range
for moderately dry-hopped beers [41]. Aluminum foil was inserted
between the bottle mouth and crown cap and subsequently sealed
to inhibit migration of β-myrcene into crown cork liner polymers
[42]. The prepared beer bottles were then agitated for one week
at 75 rpm (20 °C) using VKS-75 Control (Edmund Bühler GmbH,
Hechingen, Germany). A reference sample was treated the same
way but without yeast addition. The trial was conducted in triplicate.
Before gas chromatographic analysis, yeast cells were removed
from beer samples by centrifuging the entire bottle contents in 600-
ml tubes at 4000 rpm for 10 min using a Megafuge 40R (Thermo
Scientific, Waltham, MA, USA).
2.3.2 Recovery of β-myrcene and linalool from yeast
In consecutive steps, propagated amounts of yeasts TUM 68 and
TUM 34/70 were washed, then brought into contact with β-myrcene
or linalool, washed again and subsequently treated with solvents;
finally solvent extracts (SPE) were analyzed by GC-MS.
For contact with aromatics and performing the test in duplicate,
600 ml deionized water in a 1-l-SCHOTT bottle was set to 100
million cells/g using washed yeast and split equally between six
250-ml-Erlenmeyer flasks. Concentrations (cells/ml) of yeast cells
were determined using a cell counter calibrated for the correspon-
ding yeast strain (Nexcelom Bioscience, Lawrence, MA, USA). Into
three 250-ml-Erlenmeyer flasks containing 100 ml yeast-water
suspensions (100 million cells/ml) 70 µg of β-myrcene were added,
which is within a characteristic range of that particular compound
for strongly dry-hopped beers [1]. This was also done for linalool.
For the amount of 70 µg, 100 µl of the β-myrcene pure substance
solution (0.7 g/l, tetrahydrofuran-ethanol (1:1 [v/v]) solution) or 100 µl
of the linalool pure substance solution (0.7 g/l, ethanol solution)
was used. Erlenmeyer flasks were sealed with glass stoppers and
agitated at 75 rpm for 16 hours at 20 °C using VKS-75 Control
(Edmund Bühler GmbH, Hechingen, Germany). Control samples
were treated equally until this step without yeast contents and sub-
sequently extracted (SPE) and analyzed (GC-MS). After agitation
step, entire quantities of yeast-water suspensions in Erlenmeyer
flasks were washed four times as described before to remove any
Table 2 Chromatography system applications and settings
Sample type
(targeted comp.) Hop essential oil
Beer
(hop essential oil
constituents)
Beer
(fermentation by-
products)
Water SPE-extracts
(full scan volatile comp.)
System GC-FID HS-GC-MS GC-FID GC-MS
Manufacturer Perkin Elmer Shimadzu Perkin Elmer Thermo Scientific
Sampler (integrated) HS-20
10-ml vial, 5 ml sample vol.
Turbo Matrix 40 (HS)
20-ml vial, 2 ml sample vol. AS 3000
GC Clarus 580 GC-2010 Plus Clarus 580 Trace GC Ultra
MS
transfer line temp., ion
source temp.
–GCMS-QP2010 Ultra
250°C, 200°C –DSQ II
230 °C, 230 °C
Column ZB-WAX ZB-WAX INNOWAX TR-5MS
Film thickness [μm] 0.5 0.25 0.5 0.25
Length [m] 60 30 60 30
[mm] 0.25 0.25 0.32 0,25
Injection volume 2.0 μl 1 ml pressure controlled 1.0 μl
Carrier gas helium 5.0 ECD-quality helium 5.0 ECD-quality helium 5.0 ECD-quality helium 5.0 ECD-quality
Split 20 ml/min 1:5 20 ml/min 1:10
Internal standard p-cymene pulegone p-cymene pulegone
Software TotalChrom LabSolutions TotalChrom Thermo Electron
163 November / December 2017 (Vol. 70)
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of the Veritas laboratory in Zurich
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residual amounts of flavor compounds. Each of the sedimented yeast
portions was subsequently suspended with 100 ml ethanol-water
solutions in 250-ml-Erlenmeyer flasks containing 5 %, 10 % and
100 % [v/v] ethanol, respectively. Erlenmeyer flasks were sealed
with glass stoppers and agitated at 75 rpm for 3 days at 20 °C.
Then, suspensions were centrifuged at 4000 rpm for 10 min at
20 °C. Supernatants were adjusted to solutions at 5 % [v/v] ethanol
contents with distilled water in 2-l-SCHOTT bottles setting samples
at similar properties to calibration medium of SPE method. The
entire sample quantity was subsequently extracted by SPE. The
extracts were used for gas chromatographic analysis.
2.4 Analytical methods
In this study five different chromatography systems were used to
analyze volatiles in essential oil, beer, and water samples. Table
2 shows the system applications. NanoLC-MS/MS of thiols in hop
and beer samples was performed by laboratory Nyseos, sample
processing and system application according to Roland and Viel
in 2016 [43].
2.4.1 Chromatographic analysis of essential oil
The essential oil was analyzed using a gas chromatograph con-
nected with a FID. Separation was achieved using in a ZB-WAX.
The oven was programmed at a rate of 5 °C/min from 45 °C (11 min
isotherm) to 210 °C, increased at 20 °C/min to 240 °C (8 min hold)
and at 10 °C/min to 260 °C (5 min hold).
2.4.2 Chromatographic analysis of beer
Essential oil constituents in beer samples were quantified using a
gas chromatograph that was directly connected to a mass spec-
trometer (Table 2). The system was equipped with a headspace
sampler loop system. Samples were equilibrated for 30 min at
80 °C. The temperature program of the oven was at a rate of 4
°C/min from 50 °C to 130 °C, increased at 8 °C/min to 180 °C and
at 15 °C/min to 240 °C. Samples were assessed in SIM mode.
Fermentation by-products were analyzed by GC-FID equipped
with a headspace sampler. Vials containing beer samples were
equilibrated at 60 °C for 25 min. 1 min after injection at 50 °C the
temperature was increased at 7 °C/min to 85 °C. After 1 min hold
190 °C was reached at 25 °C/min (4 min hold).
2.4.3 Preparation of SPE extracts
SPE was performed using 6-ml HR-P-cartridges filled with 500 mg
polystyrene-divinylbenzene (Chromabond, Macherey Nagel, Düren,
Germany). A vacuum port with gauge was used to control the vacuum
applied to the chamber at 0.8 bar abs. using a vacuum pump to
accelerate flow rates. Cartridges were pretreated successively with
5 ml dichloromethane, 5 ml methanol and 5 ml deionized water. Then
samples of bubbling water columns (1 l) or yeast extracts (0.1–2 l)
were increased by 50 μl of internal standard pulegone (400 mg/l,
ethanol solution) in 2-l-SCHOTT bottles and subsequently loaded
on pretreated cartridges at flow rates of approx. 15 ml/min. Loaded
cartridges were washed with 5 ml 2-% [v/v] methanol solution and
eluted twice with 4 ml dichloromethane. The eluent was collected
in 10-ml glass tubes equipped with a length gauge. The eluent was
Table 3 Contents of 35 selected aroma compounds in Mosaic
essential oil in µg/g pellet. 3-mercaptohexan-1-ol (3MH),
3-mercaptohexyl acetate (3MHA), 4-methyl-4-mercapto-
pentan-2-one (4MMP)
Mosaic
Linalool 85 ± 2.3
Geraniol 61 ± 0.1
β-Citronellol 129 ± 0.4
Menthol 5 ± 1.7
1-Octen-3-ol 16 ± 1.1
α-Pinene 14 ± 0.4
β-Pinene 56 ± 3.9
β-Myrcene 4,288 ± 67.3
α-Humulene 97 ± 7.2
Trans-β-Farnesene 5 ± 1.8
Trans-Caryophyllene 188 ± 1.4
Limonene 101 ± 4.6
γ-Terpinene 28 ± 1.2
Heptanol 3 ± 0.2
2-Octanol 23 ± 0.8
Isobutyl isobutyrate 41 ± 0.9
Geranyl acetate 12 ± 0.6
Cis-4-methyl-decenoate 1,120 ± 8.0
Methyl decanoate 3 ± 0.1
C11-Methyl ester 85 ± 22.1
Methyl hexanoate 225 ± 6.0
Neryl acetate 9 ± 1.2
β-Selinene 36 ± 10.0
Methyl nonanoate 5 ± 0.2
Methyl octanoate 11 ± 0.5
Citronellal 15 ± 0.8
β-Damascenone 14 ± 2.5
2-Decanone 61 ± 7.8
2-Nonanone 68 ± 6.0
2-Undecanone 43 ± 19.3
Carvone 67 ± 1.0
Dimethyl disulfide 3 ± 0.8
4MMP (ng/g) 22
3MH (ng/g) 54
3MHA (ng/g) 6
Sum 6,917
reduced down to a volume of about 200 μl using a fine nitrogen
stream and stored at – 20 °C until the GC-MS analysis.
2.4.4 GC-MS of SPE extracts
A gas chromatograph/mass spectrometer system was equipped
with an automatic liquid injection system. The temperature of the
GC oven was increased at a rate of 8 °C/min from 50 °C (7 min
isotherm) to 150 °C, 20 °C/min to 280 °C (5 min isotherm) and
10 °C/min to 330 °C. The samples were measured in full scan
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Table 4 General analysis data of the wort and four beers
Wort Beer
TUM 68 TUM 34/70
Ferm. 22 °C Ferm. 15 °C Ferm. 15 °C Ferm. 8 °C
Extract [°P] (residual) 11.47 3.49 3.55 3.61 3.52
Alcohol [% vol/vol] 0.00 5.66 5.66 5.75 5.77
Final fermentation
degree [%]
– 72.2 71.8 71.8 72.3
mode at the mass range 50–250 amu.
3 Results and discussion
3.1 Hop analysis
The aroma hop cultivar Mosaic is the daughter of YCR 14 Simcoe
(multi-purpose hop variety) and a Nugget (high-alpha variety) de-
rived male. This family tree explains the relatively high contents
of α-acids for a flavor variety. Table 3 lists the analysis results of
35 substances in Mosaic pellets. Mosaic, a cultivar released in
2012, shows some specific characteristics such as having poly-
functional thiols 3-mercaptohexan-1-ol (3MH), 3-mercaptohexyl
acetate (3MHA) and 4-methyl-4-mercaptopentan-2-one (4MMP).
These three thiols have been linked to several hop cultivars such
as Nelson Sauvin and Cascade by exhibiting typical blackcur-
rant bud and grapefruit notes detected by GC-olfactometry [44].
β-Myrcene was determined in hop oil at a level of 62 %. That is
slightly above a common value with regard to variety data sheet
(47-53 %) [45]. The linalool content, which is often used as one of
the primary markers for hop aroma in beer [8], was measured at
a typical share of essential oil such as 1.2 % [1]. Esters such as
cis-4-methyl-decenoate and methyl hexanoate were determined at
relatively high contents [41], 16.2 and 3.3 %, respectively, possibly
contributing to the fruity character of the pelletized hop samples [1].
3.2 Brewing trial
3.2.1 Wort and beer analysis
The wort and the brewed beers were analyzed comprehensively
(Table 4). Similar levels of residual extracts (real), alcohol contents
and final fermentation degrees (real) indicate to good comparability
of four brews.
Table 5 shows the values of 40 analyzed aroma components in
the pitching wort before dry hopping and brewed beers. These
include 30 hop-derived aroma compounds. Increased amounts
in beers are due to the dry hopping of the pitching wort that was
moderately kettle hopped. The threshold value of linalool was
exceeded in all beers (33.4 ± 0.8 – 36.4 ± 0.9 µg/l). In the case of
geraniol, threshold value was also achieved, though there was a
great difference between yeast strains. Contents in TUM 68-beers
were recorded at 72.8 ± 0.2 µg/l (22 °C) and 69.2 ± 3.3 µg/l (15 °C)
whereas levels in TUM 34/70-beers were determined at 54.3 ±
0.8 µg/l (15 °C) and 30.9 ± 0.9 µg/l (8 °C). Deviations might be
caused by the cleavage of geranyl glycoside and the release of
the corresponding geraniol [9, 35]. Furthermore, differences in
degradations of geraniol by biotransformations might have occurred
[9, 31, 33]. Mono- and sesquiterpenes such as α- and β-pinene,
β-caryophyllene, α-humulene, β-famesene were generally deter-
mined at trace amounts and below threshold levels, among these
the highest contents of β-myrcene were determined at 15.9 ± 2.6
µg/l in TUM 68-beer (22 °C) and 6.9 ± 0.8 µg/l in TUM 34/70-beer
(8 °C). Regarding thiols, in case of 4MMP (blackcurrant, muscat-like,
fruity) and 3MH (fruity, catty, thiol-like) threshold levels at 10–50
and 55 ng/l [46] were achieved in all beers (30–40 ng, 350–450 ng).
Ten important flavor compounds produced by yeast metabolism
were analyzed by GC-FID (Table 5). A variation in the production
of fermentation by-products for both yeast strains was determined.
Top-fermenting TUM 68 showed higher amounts of alcohols such
as i-butanol and amyl alcohols and esters such as isoamyl acetate
(“fruity”, “banana”;), a key-compound for top-fermented wheat beers
[20], compared with bottom-fermented beers, +42 mg/l, +10 mg/l,
+0.9 mg/l (higher temperature attempts), respectively.
3.2.2 Fermentation gas analysis
Figure 2 shows the levels of hop-derived compound β-myrcene
and the three products of yeast metabolism, isoamyl acetate, ethyl
hexanoate and styrene dissolved in the water of bubbling columns
after the main fermentation. This points to stripping of the com-
pounds mentioned above during standardized conditions which
were inspired by large-scale beer fermentations [22]. Releases
of aroma compounds have been determined before by several
authors using real-time monitoring methods [17–19]. β-Myrcene
was measured in column position number 1 (Fig. 1) at levels of
about 256–280 µg/l. In the subsequent column positions 2 to 5,
228–268 µg/l, 223–276 µg/l, 207–265 µg/l, respectively, decreasing
quantities were detected. This was attributed to the depletion of
β-myrcene from the fermentation gases. The highest dissolved
amounts in columns were determined at 22 °C fermentation tem-
perature and the lowest at 8 °C regardless of column position. In
this study, tendencies towards higher released amounts at primary
fermentation are probably attributed to the increased volatility of
aroma compounds as proposed by Schneiderbanger and Hutzler
[20]. They determined increased releases of aroma compounds into
the gas phase at higher temperatures from water systems when
simulating beer fermentations [20]. Considering the fact that ethyl
hexanoate was only detected in column position 1 and isoamyla-
cetate only in positions 1–3, it becomes clear that the test set-up in
its present form is highly suitable for dissolving hydrophilic aroma
compounds [38, 52] in bubbling water columns. Nonetheless, no
linalool, which is equally highly water soluble, could be detected
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Table 5 Contents of 40 selected aroma compounds in pitching wort (before dry hopping) and four beers
Wort
TUM 68 TUM 34/70 Threshold*
Ferm. 22 °C Ferm. 15 °C Ferm. 15 °C Ferm. 8 °C
Linalool [µg/l] 3.6 ± 0.33 33.4 ± 0.80 36.1 ± 1.15 36.4 ± 0.87 35.1 ± 1.01 5, 27, 80
Geraniol [µg/l] 2.7 ± 0.41 72.8 ± 0.17 69.2 ± 3.32 54.3 ± 0.82 30.9 ± 0.92 36
Citronellol [µg/l] nd 1.2 ± 0.10 1.8 ± 0.29 1.2 ± 0.05 1.7 ± 0.03 5
α-Terpineol [µg/l] nd 0.6 ± 0.23 0.2 ± 0.07 2.8 ± 0.15 0.2 ± 0.07 300, 2000
Nerol [µg/l] nd 7.7 ± 0.25 7.6 ± 0.44 6.9 ± 0.23 5.2 ± 0.10 50, 1200
1-Octen-3-ol [µg/l] nd nd nd 2.4 ± 0.04 1.9 ± 0.04 10**
α-Pinene [µg/l] nd nd nd nd nd 2.5-62
β-Pinene [µg/l] nd nd nd nd nd 140
β-Myrcene [µg/l] 0.3 ± 0.12 12.1 ± 1.12 15.9 ± 2.58 7.4 ± 0.81 6.9 ± 0.85 30, 1000
α-Humulene [µg/l] nd 0.6 ± 0.10 1.2 ± 0.41 0.4 ± 0.04 0.4 ± 0.05 800
β-Famesene [µg/l] nd 0.5 ± 0.18 nd nd nd 2000
β-Caryophyllene [µg/l] nd 0.2 ± 0.06 0.3 ± 0.12 nd nd 450
1-Heptanol [µg/l] 5.0 ± 0.82 6.2 ± 0.24 5.7 ± 0.28 4.9 ± 0.28 4.5 ± 0.22 1000
Isobutyl isobutyrate [µg/l] 0.5 ± 0.11 6.6 ± 0.37 8.0 ± 0.25 8.3 ± 0.27 8.5 ± 0.30 n/a
Methyl hexanoate [µg/l] 0.5 ± 0.20 24.7 ± 0.92 23.7 ± 15.82 33.0 ± 1.50 37.1 ± 1.25 n/a
Methyl heptanoate [µg/l] nd nd nd nd nd n/a
Methyl octanoate [µg/l] nd 0.7 ± 0.11 0.7 ± 0.10 0.8 ± 0.09 0.7 ± 0.05 n/a
Methyl nonatoate [µg/l] nd 0.2 ± 0.15 nd nd nd n/a
Methyl decanoate [µg/l] nd 0.4 ± 0.15 0.2 ± 0.01 0.2 ± 0.01 0.2 ± 0.01 n/a
4-Methyl-decenoate [µg/l] 5.5 ± 0.90 0.4 ± 0.01 0.4 ± 0.04 0.4 ± 0.05 0.4 ± 0.02 n/a
Geranyl acetate [µg/l] nd 2.3 ± 0.07 2.1 ± 0.21 2.2 ± 0.04 1.8 ± 0.03 9, 460
Citronellal [µg/l] nd 0.4 ± 0.02 0.4 ± 0.01 0.4 ± 0.02 0.4 ± 0.01 n/a
2-Undecanone [µg/l] nd 2.1 ± 0.17 1.4 ± 0.47 2.6 ± 0.11 2.1 ± 0.03 400
2-Dodecanone [µg/l] nd 0.6 ± 0.05 0.4 ± 0.01 0.5 ± 0.01 0.4 ± 0.03 n/a
Neryl acetate [µg/l] nd 1.1 ± 0.22 0.9 ± 0.11 0.9 ± 0.10 0.9 ± 0.06 n/a
2-Nonanone [µg/l] nd 2.4 ± 0.09 2.5 ± 0.09 3.1 ± 0.09 2.7 ± 0.08 200
2-Tridecanone [µg/l] nd 0.8 ± 0.05 0.7 ± 0.02 0.7 ± 0.01 0.7 ± 0.01 100
4-methyl-4-mercaptopentan-2-one
(4MMP) [ng/l] nd 31 37 40 40 10, 50
3-mercaptohexan-1-ol
(3MH) [ng/l] 29 399 398 475 359 55
3-mercaptohexyl acetate
(3MHA) [ng/l] nd 4 8 10 4 9
Acetaldehyde [mg/l] 0.88 ± 0.078 4.51 ± 0.003 2.09 ± 0.007 2.90 ± 0.170 3.21 ± 0.127 5, 25
Ethyl formiate [mg/l] 0.79 ± 0.021 0.77 ± 0.057 0.75 ± 0.049 0.59 ± 0.014 0.81 ± 0.007 150
Ethyl acetate [mg/l] nd 32.84 ± 0.09 31.56 ± 0.849 34.14 ± 1.450 27.27 ± 0.092 30
Ethyl propionate [mg/l] nd 0.23 ± 0.004 0.21 ± 0.007 0.22 ± 0.003 0.24 ± 0.002 150
n-Propanol [mg/l] 0.14 ± 0.004 19.65 ± 0.580 17.30 ± 0.417 13.16 ± 0.049 12.34 ± 0.191 2, 50
Ethyl butanoate [mg/l] nd 0.10 ± 0.007 0.12 ± 0.007 0.09 ± 0.001 0.12 ± 0.007 0.3
i-Butanol [mg/l] 0.23 ± 0.003 51.89 ± 1.280 52.59 ± 1.365 10.93 ± 6.859 11.29 ± 0.156 200
Isoamyl acetate [mg/l] nd 1.77 ± 0.021 2.23 ± 0.035 1.40 ± 0.078 1.30 ± 0.021 1.6
Amyl alcohol [mg/l] 0.48 ± 0.032 75.24 ± 0.792 78.71 ± 0.877 67.64 ± 0.940 59.92 ± 0.361 70
Ethyl hexanoate [mg/l] 0.04 ± 0.007 0.10 ± 0.003 0.13 ± 0.004 0.13 ± 0.007 0.12 ± 0.004 0.2
* Odor thresholds in beer found in the literature [3, 16, 46-51]; n/a if not available; ** determined in ethanolic solution
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(Table 6), which is attributed to the excellent dissolution of this
substance in young beer and was not released.
The experimental set-up can be modified for further investigation into
non-polar compounds using a higher number of bubbling columns
or other solvents with higher capacities to dissolve compounds
like β-myrcene since columns at position 5 contained β-myrcene
in the range 207–265 µg/l. It is most likely that the compound was
still present in outgoing gases although no solubility limits were
reached in the water of bubbling columns for any of the analyzed
compounds (Table 6) [38, 52]. However, using the method of five
bubbling water columns in its present form, tendencies towards
differences in releases of β-myrcene between fermentation
temperatures were observed. Furthermore, this was achieved in
simultaneous fermentation approaches with different yeast strains
at uniform yeast vitalities, yeast viabilities and wort characteristics,
which is the basis of acknowledged methods for characterization
of fermentations in brewing research [2, 22].
3.3 Uptake of aroma compounds
by yeast
3.3.1 Recovery of β-myrcene and linalool
in beer
Figure 3 shows concentrations of β-myrcene
and linalool in defined media after contact
with yeast strains TUM 68 and TUM 34/70
under particular storage conditions to imitate
conditions during main fermentations. In
2003, King and Dickinson assumed that rising
alcohol contents had an effect on the concen-
trations of terpenoids during fermentation,
enabling more of the terpenoids to dissolve
[31]. With this background, a non-alcoholic
beer was used in this trial as contact media
and the impact of different alcohol contents
was tested in separate test series (3.3.2).
Amounts of β-myrcene in the beer were de-
creased depending on cell concentrations. At
the highest counts (100 million cells/g) only
traces like 0.5 ± 0.2 µg/l (TUM 68) and 1.0
± 0.2 µg/l (TUM 34/70) remained in beers,
corresponding to decreases of 99.0 % (TUM
68) and 98.0 % (TUM 34/70) compared with
contents in control tests that were not increa-
sed by yeasts (47.9 ± 2.5 µg/l). It is assumed
that the non-polar substance β-myrcene
was attached to the non-polar surface of the yeast cells [4, 31],
which were separated from the samples by centrifugation. Linalool
amounts in beers were not noticeably affected by the presence
of yeast and were determined at comparable concentrations to
the control samples. Linalool is relatively soluble in hydrophilic
solutions like beer [38] and therefore not effectively influenced by
non-polar particles such as yeast cells".
3.3.2 Recovery of β-myrcene and linalool from yeast cells
Table 7 shows amounts of β-myrcene recovered from yeasts cells
that were previously in contact with a defined quantity of β-myrcene
at yeast counts of about 100 million cells/ml. Recovery (%) was
calculated using determined amounts in solvents such as 8.2 µg
(TUM 68) and 8.1 µg (TUM 34/70) and quantified amounts in
control samples at 46.0 µg.
Using the relatively hydrophobic solvent in
this test series pure ethanol compared to
aqueous solutions, 17.2 % from yeasts TUM
68 and 16.8 % from TUM 34/70 were reco-
vered of the spent β-myrcene. At ethanol
contents of 5 % and 10 %, β-myrcene was
not recovered from yeasts. This is consi-
stent with the results of a previous study,
in which 50 % ethanol solution failed to
dissolve a measurable amount of terpenoids
possibly concentrated in yeast pellets [31].
Fig. 2 Contents of four compounds in bubbling water columns originating from the
headspace of wort during fermentation in µg/l; TUM 68, 22 °C; TUM 68,
15 °C; TUM 34/70, 15 °C; TUM 34/70, 8 °C
Table 6 Solubility limits [38, 52] and maximum recovery of released aroma compounds
in water of bubbling columns in mg/l; nd = not detected
Solubility limit in water Maximum contents determined
Linalool 1556 nd
β-Myrcene 4.0–30.0 0.280
Ethyl hexanoate 630–650 0.117
Isoamyl acetate 2000 0.956
Styrene 160-300 0.122
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It is probable that pure ethanol was still unsuitable to completely
recover β-myrcene from yeast when comparing clearly higher
losses of β-myrcene about 99.0 % (TUM 68) respectively 98.0 %
(TUM 34/70) in beer-yeast suspensions (3.3.1). We assume that
unrecovered amounts of β-myrcene (about approx. 80 %) are still
concentrated in yeast. Considering both trials regarding concen-
trations of aroma compounds in brewer’s yeast it is concluded
that solvents with similar polarity to beer systems (5–10 % [v/v]
ethanol) are insufficient to re-dissolve compounds attached to
yeasts such as β-myrcene. Therefore, the uptaken amounts are
unable to contribute to the aroma of beer. Linalool was used at
the same contents as β-myrcene, but was not recovered. This
confirms good solubility of linalool in hydrophilic solvents such as
beer and no indication that it is uptaken by yeast cells. The present
method could be adapted especially for the analysis of non-polar
flavorings by using solvents such as hexane or dichloromethane.
4 Conclusion/Summary
Standardized fermentations of dry-hopped worts and two separate
test series showed very large losses of β-myrcene during beer
fermentation and two principal causes were identified. With the
help of the bubbling water column used for these brewing tests,
releases of aroma compounds into the gas phase were confirmed
[17–19]. In addition, higher fermentation temperatures resulted in
a tendency to increase the release of flavor compounds as iden-
tified in studies on model fermentations [20]. It was shown that
the method used is very suitable for determining released volatile
compounds from the headspace during fermentations; nonetheless
no linalool could be detected, which is attributed to the excellent
dissolution of this substance in beer. Using water as a solvent in
bubbling columns proved to be unsuitable to
determine absolute stripped-off amounts of
non-polar compounds such as β-myrcene.
However, there were great advantages in
the flexibility and robustness of the method,
although the experimental series was carried
out in a single-issue experiment. In a separate
test series, the uptake of β-myrcene by yeast
cells was determined as it was assumed by
several authors [4, 27-33]. In a test set-up
that prevented evaporation, 99.0 % (TUM
68) and 98.0 % (TUM 34/70) of β-myrcene
was absorbed by yeast at cell counts (100
million cells/ml) occurring during fermenta-
tions. Furthermore, it is highly probable that
beer is an inappropriate solvent for dissolving
β-myrcene uptaken by yeast with the conse-
quence that these quantities do not contribute
to the flavor of beer. The level of linalool, on
the other hand, could not be detected as
being affected by yeast in these experiments.
These results can help to shape the flavor of
strongly kettle- or dry-hopped beers in a more
targeted way, especially for hydrophobic flavor
compounds such as monoterpenes.
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The scientifi c organ
of the Weihenstephan Scientifi c Centre of the TU Munich
of the Versuchs- und Lehranstalt für Brauerei in Berlin (VLB)
of the Scientifi c Station for Breweries in Munich
of the Veritas laboratory in Zurich
of Doemens wba – Technikum GmbH in Graefelfi ng/Munich www.brauwissenschaft.de
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Monatsschrift für Brauwissenschaft
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Received 1 September 2017, accepted 4 November 2017
