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On the Fate of β-Myrcene during Fermentation – The Role of Stripping and Uptake of Hop Oil Components by Brewer’s Yeast in Dry-Hopped Wort and Beer

Authors:
  • Radeberger Gruppe KG

Abstract and Figures

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.
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of the Weihenstephan Scientifi c Centre of the TU Munich
of the Versuchs- und Lehranstalt für Brauerei in Berlin (VLB)
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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
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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
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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
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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
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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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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
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Received 1 September 2017, accepted 4 November 2017
... Terpenes, such as myrcene, exhibit a green/herbal character that could mask other fruitier compounds. Factors such as CO 2 scrubbing and adhesion to yeast cells (22) help explain their decrease during fermentation. Haslbeck et al. (22) show that fermentation at a lower temperature leads to less loss of volatile hydrophobic compounds, such as myrcene. ...
... Factors such as CO 2 scrubbing and adhesion to yeast cells (22) help explain their decrease during fermentation. Haslbeck et al. (22) show that fermentation at a lower temperature leads to less loss of volatile hydrophobic compounds, such as myrcene. ...
Article
Biotransformation has become a buzzword within the brewing community, with many brewers swearing by dry hopping during active fermentation to encourage it. In this review, we aim to cover the academic literature on this topic and attempt to elucidate if biotransformation is the main driving force behind the observed sensory changes of different dry hop timings or if other physical, biological, or chemical processes take a lead role. When the potential sensory impact of each biotransformation pathway is considered, we argue that only thiol release and potentially esterification (although more studies are still required to ascertain its contribution) could have a marked effect, with other causes, such as CO2 scrubbing and yeast binding, having an influence not normally recognized.
... The essential oils are transformed during brewing process as follows: first, the thermal/oxidative transformation of essential oils takes place during wort boiling. Some components of essential oils are transformed by yeasts during main fermentation (Takoi et al., 2017;Praet et al., 2012) or they get sorbed to the yeast cell surface (typical for myrcene) (Haslbeck et al., 2017). The essential oil components are extracted into a slightly alcoholic solution of young beer during dry hopping. ...
... A large part of the essential oils evaporates or is chemically altered already in the course of wort boiling. The conversions and changes in concentration of essential oils continue during fermentation by the biochemical action of the yeast or by the sorption on their surface, which is typical for instance for myrcene (Mikyška et al. 2018;Haslbeck et al., 2017;Praet et al., 2016). ...
Article
Full-text available
Characterization of varietal profiles of hop secondary metabolites in beer is of considerable importance for hop growing and brewing. This three-year study focused on pilot brewing tests of three new Saaz derived hop varieties, namely Saaz Brilliant, Saaz Shine and Saaz Comfort. The sensory profiles of kettle and kettle+dry single hopped beers were monitored. At the same time, the impact of dry hopping technology on sensory perception of final beers and changes in volatile compounds was investigated. Pilot brews (200 l) of 12% pale lager were prepared in kettle and kettle+dry variants using the new Czech varieties and traditional Saaz as a control sample. Essential oils in beers were determined by GC/MS-MS method with the aim to clarify links to the beer sensory profile of both, kettle and dry hopping mode. The descriptive sensory method of hop-derived aromas and the triangle tests were employed to determine the sensory quality of beer. The overall sensory impression of beers from all tested varieties was at least comparable to traditional Saaz, and Saaz Comfort even reached better evaluation. The profile of essential oils and hop flavours in beer was partially different, however it was not rated worse than Saaz. The kettle+dry hopped beers of new varieties were significantly different from Saaz in the triangle test, Saaz Brilliant was preferred over the Saaz. The results showed considerable potential of new varieties for the production of interesting and taste-specific beers and the enrichment of the spectrum of fine aroma varieties of Czech hops.
... When stored improperly, myrcene rapidly undergoes autoxidation to form products such as α-pinene, β-pinene, linalool, geraniol, geranial, and neral. [28,97] These oxygenated compounds are often found in beer at levels higher than their thresholds. [98] However, β-myrcene itself is essentially lost during wort boiling and fermentation or by adsorption onto the yeast. ...
... [98] However, β-myrcene itself is essentially lost during wort boiling and fermentation or by adsorption onto the yeast. [97,99] It has a low odor threshold (see Table 2) and only exceeds it in dryhopped beers. The concentration of β-myrcene in dry-hopped beer reportedly can be 500 times higher than in kettle-hopped beers. ...
Article
Hop cones (Humulus lupulus L.), or more specifically the lupulin glands, hold the reason for the specific, pleasant aroma of hops – its essential oil. The hops themselves, or the extracted oil, are used almost exclusively in beer production. The essential oil is an indispensable part of beer and is responsible for its characteristic aroma. However, hop essential oil (HEO) also has a broad range of positive effects on human health and is a potential natural pesticide that has no harmful impacts on humans. This review summarizes basic information about HEO, including its chemical composition and methods for extraction and analysis, while also providing a comprehensive overview of the contribution to beer aroma, health, and insecticide applications for this versatile essential oil.
... Another cause for significant β-myrcene losses during fermentation are higher temperatures resulting in increased release of the compound (Haslbeck et al., 2017). ...
Article
Considering the substantial amount of research that has been published in the field of hop science during the last decades, very little is known with regard to the multimodal flavour perception of hop-derived volatiles that not only contribute to the pleasant ‘hoppy’ aroma and flavour, but are also involved in other sensations of gustatory and trigeminal origin perceived in beer. The aim of this research was to further understand the sensory complexity of Magnum hop essential oil and scCO2 hop oil fractions extracted therefrom. This PhD project combined static and dynamic sensory techniques, an established gas chromatographic method, and comprehensive statistical analyses to investigate the relationship between hop volatile compounds and their sensory characteristics (quantitative and qualitative) in different matrices. The olfactory, gustatory and trigeminal differences between five hop oil fractions representing the main chemical classes of Magnum hop oil were determined in a simple model solution (4% ABV) using a newly established attribute lexicon and following a Quantitative Descriptive Analysis (QDA) approach. The fractions induced a range of different aroma and flavour sensations, which could partly be attributed to specific hop aroma compounds. The most polar compounds in the terpene alcohol fraction were suggested to be responsible for cross-modal interactions eliciting both aroma and/or taste and trigeminal sensations. A peppery tingling mouthfeel was perceived, which is assumed to be a sensation innervated by the trigeminal nerve. The terpene alcohol fraction was further categorised into monoterpene alcohols (i.a. geraniol, linalool) assumed to be mainly responsible for olfactory sensations and sesquiterpene alcohols (i.a. humulol, humulenol II) to foremost induce gustatory and tactile sensations. Further fractionation specifically targeting single compounds and compound groups (sub-fractions) that were added to a commercial lager beer base (4.5% ABV) to measure the impact of perceptual interactions between compounds and the beer matrix using a revised attribute lexicon and adjusted dosage rates. A clear cause-effect-relationship could be located between geraniol and the sweet taste perceived in the beer. Geraniol also induced a smooth bitterness, which was opposed by the harsh bitterness quality added by sesquiterpene hydrocarbons. Linalool was classified as a aroma/flavour ‘enhancer’ rather than individually contributing to the sensory profile. Significant effects on lingering mouthfeel sensations remained absent, which illustrated the need for temporal sensory assessments to adequately and holistically discriminate the samples with regard to these sensations. A Temporal Check-All-That-Apply (TCATA) by modality approach was used to assess multiple sensory characteristics of selected hop flavour products perceived simultaneously. The products contained the previously studied hop oil fractions and were combined with either iso-alpha-acids or oxidised beta acids (hulupones) in a lager base beer brewed without any hop materials. Bitter acid extracts were found to significantly affect the duration and sensory profiles of the hop flavour products in the beer suggesting a sensory interaction induced by the co-occurrence of hop aroma compounds and hop bitter stimuli. Lingering sensations (peppery tingling, astringency) were foremost found to significantly discriminate between the samples at the end of the evaluation period (>2min). Since temporal sensory data is inherently noisy, a part of this research included the examination of TCATA data pre-processing approaches using comprehensive statistical analyses. This revealed that time standardising the TCATA by modality data could not remove inter- and intra-individual variation between the panellists and thus, not improved the quality of the sensory data. This research has provided new and in-depth knowledge on the sensory properties of scCO2 hop oil fractions, sub- fractions, and key compounds extracted from Magnum hop. Moreover, different sensory characterisation strategies and tools are presented that captured the fine nuances of the sensory profiles of these hop extracts. The findings demonstrated the involvement of hop volatile compounds in sensory interaction effects causing multi-modal profiles in beer. Their ability to modify gustatory and trigeminal sensations should be considered for future developments of flavour preparations.
... Therefore, when β-myrcene migrates from the beer solution it can pack in the voids between the palmitoyl chains and change their orientation at the yeast/beer interface [48,[51][52][53]. The non-polar β-myrcene may attach to the non-polar surface of the yeast cells [54][55][56]. This leads to a decrease in the concentration of β-myrcene during the fermentation of young beer. ...
Article
Full-text available
The production of heavily hopped beers, such as Indian Pale Ale (IPA) styles, has been gaining momentum in recent years in the Central European markets. To this end, the dry hopping process is becoming increasingly popular, mostly in microbreweries, but also with larger manufacturers. In our research, we investigated the dissolution rate of the main volatile component of hops, β-myrcene with a modified dry hopping method. Following the primary fermentation, we applied the dry hopping process, where the weighed hops were chopped and blended into a container with 0.5 L of beer and later added to the young beer. During the dry hopping process, we determined various important parameters of the beer, and we repeated the same measurements for the bottled beer. In the first 96 h of the dry hopping process, we monitored the concentration of β-myrcene so that we managed to determine the dissolution rate constant (k = 0.1946 h-1). The β-myrcene concentration stabilizes after 44 h in the fermenter. At the same time, measurements were conducted for bitterness, pH, CO2 and alcohol content, extract and density during the process. Our experiment demonstrates that a new method of dry hopping provides a much higher concentration of β-myrcene (215 μg/L) than other methods indicated in former studies in the field. A health and safety assessment of β-myrcene was also made and we determined what the safe amount of β-myrcene ingested with IPA beer is. Our modified process was successful, we were able to determine the dissolution rate of β-myrcene, and the recommended daily intake of IPA beer with particular reference to β-myrcene.
... There was no need to truly quantify the degree or character of difference, therefore the highly valuable discrimination information that increased reliability and accuracy of the study could be obtained. (Forster, & Gahr, 2013;Haslbeck et al., 2017), additive or masking effects of hop-derived aromas in beer , or transfer rates of specific aroma compounds in different hops varieties (Hanke et al., 2008), may help to supply the insufficiency of modeling aroma profiles. ...
Article
In recent years, proprietary hops (Citra, Simcoe, and Mosaic) become the most sought-after hops among brewers due to their excellent aroma. However, they are restricted to the owners unless other growers purchase the costly licensing agreements. Many public hops are available to the growers without any additional costs, but their aroma is difficult to match to the proprietary hops. Although proprietary and public hop varieties are unique in their aroma profiles, all hops varieties contain similar volatile compounds, merely differ in the quantity of different individual compounds. The main objective of this thesis was to investigate the feasibility of matching the aroma of proprietary hops by blending a number of public hops. The aroma profiles of hops were detected by flavor-detection methods including Gas chromatogram-flame ionization detector (GC-FID), Gas chromatography-mass spectrometry (GC-MS), Headspace solid-phase microextraction/gas chromatogram–mass spectrometry (HS-SPME/GC-MS), and sensory evaluation. The modeling was achieved by applying a mathematical optimization technique – quadratic programming. For matching proprietary hop aroma, public hop pellets or hop oils were mixed with different percentages in models (e.g., proprietary hop A= x% public hop B + y% public hop C + z% public hop D). The aroma of Citra pellets was closely mimicked by 25.2 % Eureka, 33.2 % Centennial, and 36.7 % Triple Pearl. The aroma of Simcoe pellets was closely mimicked by 10.0 % Cascade, 50.0 % Us Goldings, 71.1 % Centennial, and 10.4 % Triple Pearl. The aroma of Mosaic pellets was closely mimicked by 6.5 % Eureka, 84.8 % Centennial, and 3.0 % Triple Pearl. In addition, it nearly mimicked the Citra oil with 35.2% Brewers Gold, 5.2 % Cashmere, 32 % Centennial, and 35.7 % Triple Pearl. The obtained aroma of models was validated in the beers with both the high similarity of aroma profiles (R2 > 0.90) and sensory evaluation. This research provided a novel idea on the application of mathematical optimization and flavor-detection technologies for modeling the aroma of hops. The success of this project can increase the usage of public hops and extend such an application to other flavor developments. Advisor: Changmou Xu
... Interestingly, there is a significant positive correlation between the percentage of added grape must and this descriptor, likely due to the fermentable sugars conferred. "Fruity citrus" is a descriptor mainly linked to hop addition, an ingredient rich in citrus flavour compounds (Haslbeck et al., 2017). The analysed IGAs were characterized by low bitterness and no dry-hopping (addition of hop in the late stage of production, i.e. fermentation and cold maturation), thus citrus flavour was unexpected. ...
Article
In the last few decades, the beer industry experienced a revolutionary moment. Italy is also part of this scenario, with many microbreweries opened in the last few decades. The merging of beer and wine has led to the development of a new kind of fermented beverage, nowadays internationally known as Italian Grape Ale (IGA). The aim of this work was the physicochemical, volatile, and sensory profile characterization of 22 commercial samples. IGAs was complex and heterogeneous derived from the possibility to choose the percentage of added grape must, the process step of the addition (boiling, whirlpool, primary fermentation), different tank materials (steel, cement, wood), and different types of fermentation (bottom, top, spontaneous). However, some distinctive characteristics emerged, such as high ethanol content, low bitterness and low pH. A high percentage of added grape must can enhance these features, although it had a negative effect on foam stability. Concerning the sensory profile, maturation in wooden barrels associated with spontaneous fermentation (the "brett" character increases the vinous sensation) was the best strategy to get the flavour in between wine and beer. Taken together, these data will support the brewery to plan and make this new kind of fermented beverage.
... In brewing, β-myrcene is one of the most potent aromatic flavour components of hop essential oils and in all analysed hop varieties is considered the most odour-active volatile (9,10). Myrcene largely determines the "green hop aroma" in beer and is a primary substance in dry hopped beers (11), with a "herbaceous, resinous, green, balsamic, fresh hop" like odour (12,13). It is also the major constituent of hop essential oil and can constitute as much as 70% of the essential oil by volume (14). ...
Article
Full-text available
Myrcene (β-myrcene) is an abundant monoterpene which occurs as a major constituent in many plant species, including hops and cannabis. It is a popular flavouring and aroma agent (food additive) used in the manufacture of food and beverages. This review aims to report on the occurrence, biological and toxicological profile of β-myrcene. The main reported biological properties of β-myrcene—anxiolytic, antioxidant, anti-ageing, anti-inflammatory, analgesic properties—are discussed, with the mechanisms of activity. Here we also discuss recent data regarding the safety of β-myrcene. Overall, β-myrcene has shown promising health benefits in many animal studies. However, studies conducted in humans is lacking. In the future, there is potential for the formulation and production of non-alcoholic beers, functional foods and drinks, and cannabis extracts (low in THC) rich in β-myrcene.
... During wort boiling, thermal and oxidative transformation of essential oils takes place. Then, during the main fermentation, the components of essential oils are transformed by yeast (Praet et al., 2012;Takoi et al., 2017) and the sorption of some essential oils, especially ß-myrcene to yeast cells takes place (Haslbeck et al., 2017). In dry hopping, the essential oil components are extracted into a mildly alcoholic solution of young beer. ...
Article
Full-text available
The specification of varietal profiles of secondary metabolites of hops in beer is rather important for hop growing and beer brewing. Chemical and sensory profiles of beers hopped with the varieties of Saaz Special and the traditional Saaz semiearly red bine hops (Saaz) were compared in three-year pilot brews (200 L). Single kettle hopped beers and single kettle + dry hopped beers were prepared. The overall sensory impression of the Saaz Special beers was comparable to Saaz hops. The profile of essential oils and hop aromas/flavours in the beer was partially different, but the hop aroma did not receive a worse rating than Saaz. The Saaz Special dry hopped beers were clearly distinguished from Saaz in triangle tests, but no preference was given to either variety. Based on the achieved results, the Saaz Special variety was listed among varieties recommended for the production of the beer with the Protected Geographical Indication of Czech Beer thanks to the achieved results.
Article
Besides providing bitterness to beer, hops also impart a whole range of aromas, such as herbal, spice, floral, citrus, fruity and pine to this beverage. Although hops are usually added in relatively small amounts, they have a significant impact on the sensory characteristics of the product. Raw hop aroma significantly differs from the aroma resulting from its addition to the beer. The final aroma of the beer arises from substances in the malt, hops, other additives, and yeast metabolism. The biochemical transformation of hop compounds by yeast has become more and more popular in recent years. Knowledge of this process may allow more precise control over the final sensory characteristics of the beverage. The article describes the chemical composition of hops and discusses the influence of the hopping regime on the concentration of volatile compounds in the finished product. Moreover, the article describes the biotransformation of hop-derived compounds by traditionally used Saccharomyces cerevisiae yeast, as well as less commonly used non- Saccharomyces yeast. The paper outlines the current state of knowledge on biotransformation of hop-derived hydrocarbons, terpenoids, esters, sulfur compounds and glycosidically bound aroma precursors.
Article
Full-text available
Brewing yeast plays a pivotal role in determining the flavor and quality of beer. Different process techniques and fermentation conditions can interact with each yeast strain to create a wide variety of different flavor profiles. The craft beer movement encourages brewers to use more and more aroma-intense ale strains to create special, innovative beers. Breweries either maintain individual brewing strains or they order yeast strains from yeast strain providers or culture collections. To ensure a reliable level of quality and product stability it is necessary to genetically classify the strains involved. The origin of a strain is often unclear, and genetic drift or population drift over time cannot be excluded. Some isolates represent very close strains or the same strain. Whether two yeast strains are the same, similar or different, this does not provide any information on their phenotypic (brewing) properties. To determine these properties, genetic and phenotypic characterization methods were used, which distinguished brewing yeasts and determined their suitability and application potential for brewing. The five yeast strains Saccharomyces cerevisiae TUM 210, 211, 213, 506, 511 were characterized using a broad spectrum of genetic and phenotypic methods with a focus on brewing properties and sensorial performance. Sequencing ribosomal genes and spacer regions revealed that the strains belong to Saccharomyces cerevisiae and showed some polymorphisms. DNA fingerprinting techniques demonstrated that all strains were genetically different. Phenotypic characterization revealed that the brewing properties (e.g. fermentation performance, sugar utilization, amino acid utilization, cell growth, flocculation behavior, change in pH value, phenolic off-flavor, fermentation by-products, sulfur dioxide) and the sensorial characteristics of each strain were unique. The developed yeast characterization platform using special 2 l fermentation vessels is a broadly based, standardized tool to find the right yeast strain for distinct brewing aims.
Article
Full-text available
Brewing yeast plays a pivotal role in determining the flavor and quality of beer. Therefore, it is crucial that reliable and practical information be obtained regarding the characteristics of individual strains. This paper presents a comparison of six commercially available bottom fermenting yeast strains. TUM 34/70 served as the standard against which these yeast strains were compared, since it is the most widely used bottom fermenting yeast strain in the world and is also the most comprehensively described scientifically. A fermentation plant consisting of 27 fermentors was developed and tested with this goal in mind. This plant was designed to allow fermentation to be controlled according to a set of variables which included temperature, aeration, cell count and pressure. For the purpose of carrying out fermentation trials, these fermentors provide conditions comparable to those found in large industrial tanks. In three separate sets of trials, the following attributes were measured at regular intervals during fermentation and after lagering: the drop in specific gravity and in pH, CO2 production, FAN reduction, residual maltotriose content, total yeast cell count, the number of cells in suspension, the surface charge of the cells, turbidity, sedimentation and the foam stability of the unfiltered beer, as well as the formation and degradation of vicinal diketones, fusel alcohols, esters, aldehydes and SO2. Finally, a proper organoleptic description comparing the unfiltered products was performed after lagering. In doing so, the strains were tested under conditions as close as possible to those present in actual brewing operations and thereby exhibited, in part, significant differences in their behavior. Moreover, new insights have been gained which had yet to be fully described scientifically. These include the change in the surface charge on the yeast cells and the absorption rate of certain amino acids during fermentation, which occur independent of a specific yeast strain.
Article
In this study beers that were dry hopped in three different batch sizes were compared. The hop-oil concentrations were measured and possible aroma-active hop volatiles were identified in the hop pellets as well as in the beers. The analytical results were correlated with the tasting results from a trained tasting panel. Mass transfer rates were not comparable between the different scales, and significant differences could be detected by the tasters. The beers dry hopped on an industrial scale are not comparable in flavor and odor to those dry hopped on a laboratory scale.
Article
Since ten years, many studies conducted on beers showed an important impact of polyfunctionnal thiols of the aroma profiles. Among them, three thiols responsible for blackcurrant bud, passion fruit, citrus and rhubarb notes have been intensively studied: the 4-methyl-4-mercaptopentan-2-one (4MMP), the 3-mercaptohexan-1-ol (3MH) and its corresponding acetate. Their origin was very complex in beers since they probably came from odorless precursors present either in hops or in malts. Our work focused on the formal identification of thiol precursors in hops and on their quantification. By using pure synthetic standards and mass spectrometry characterization, we formally identified for the first time the occurrence of glutathionylated conjugates of 4MMP and 3MH and the cysteinylated conjugate of 4MMP in hops. First quantification results obtained on 10 hop varieties, showed that 3MH conjugates were more ubiquitous than 4MMP ones. Conjugates of 3MH occurred at very high level until 20 mg.kg-1 in Cascade hop, which was considerably higher than concentrations found in grapes. Then, we compared the proportion of bound and free thiol fractions and we demonstrated that more than 99 % of 3MH occurred as precursors in hops. On the contrary, free 4MMP fraction represented the most important source of 4MMP in hops.
Article
Absorption of hop volatiles by crown cork liner polymers and can coatings was investigated in beer during storage. All hop volatiles measured were prone to migrate into the closures and the absorption kinetic was demonstrated to fit well Fick's 2(nd) law of diffusion for a plane sheet. The extent and rate of diffusion was significantly dissimilar and was greatly dependent on the nature of the volatile. Diffusion coefficients in cm(2)/day ranged from 1.32×e(-5) (limonene) to 0.26×e(-5) (α-humulene). The maximum amounts absorbed into the material at equilibrium were in the order limonene > α-humulene > t-caryophyllene > myrcene > linalool > α-terpineol > geraniol. Applying low-density polyethylene (LDPE) liners with oxygen scavenging functionality, oxygen barrier liners made up from high-density polyethylene (HDPE), or liner polymers from a different manufacturer had no significant effect on the composition of hop volatiles in beers after prolonged storage of 55 days; however, significantly higher amounts of myrcene and limonene were found in the oxygen barrier-type crown cork while all other closures behaved similarly. Can coatings were demonstrated to absorb hop volatiles in a similar pattern as crown corks but to a lesser extent. Consequently, significantly higher percentages of myrcene were found in the beers.
Article
Within the scope of this assignment it could be shown that the chirale distribution of Linalool in aroma hops starting with raw hops up to all conventional hop products stays almost stable at 94 % R-Linalool. In beer considerably lower amounts of R-Linalool are found. A racemisation during the brewing process is thought to be the cause. Hereby the degree of racemisation obviously depends considerably on the type of hopping. In the course of beer staling racemisation continues with the overall amount of Linalool remaining constant. As a result a flavour loss is detectable also by organoleptic testing. A main factor for the transformation from (R)-Linalool into (S)-Linalool during beer staling may be the pH.- Bitter compounds undergo changes during beer staling that are reflected in an organoleptical and analytical decline of the intensity and quality of the beer bitterness.
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