beverages
Article
Process Parameters Affecting the Synthesis of Natural
Flavors by Shiitake (Lentinula edodes) during the
Production of a Non-Alcoholic Beverage
Sibel Özdemir 1 , Doreen Heerd 1,2 , Hendrich Quitmann 2 , Yanyan Zhang 3 ,
Marco Alexander Fraatz 3 , Holger Zorn 2,3 and Peter Czermak 1,2,3,4, *
1
2
3
4
*
Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences
Mittelhessen, Wiesenstrasse 14, 35390 Giessen, Germany; sibel.oezdemir@lse.thm.de (S.O.);
Doreen.Heerd@ime.fraunhofer.de (D.H.)
Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Bioresources Project Group,
Winchesterstrasse 2, 35394 Giessen, Germany; Hendrich.Quitmann@ime.fraunhofer.de (H.Q.);
holger.zorn@uni-giessen.de (H.Z.)
Institute of Food Chemistry and Food Biotechnology, Justus-Liebig-University Giessen, Heinrich-Buff-Ring
17, 35392 Giessen, Germany; yanyan.zhang@lcb.chemie.uni-giessen.de (Y.Z.);
marco.fraatz@lcb.chemie.uni-giessen.de (M.A.F.)
Department of Chemical Engineering, Kansas State University, Durland Hall 1005,
Manhattan, KS 66506, USA
Correspondence: peter.czermak@lse.thm.de; Tel.: + 49-(0)-641-309-2551
Academic Editor: Shao Quan Liu
Received: 28 December 2016; Accepted: 23 April 2017; Published: 27 April 2017
Abstract: A novel alcohol-free beverage with a fruity, slightly sour, sweetish, fresh, and plum-like
flavor was produced by incorporating the edible mushroom shiitake (Lentinula edodes) into the
fermentation process. Shiitake pellets were used as a biocatalyst to promote the synthesis of the
fruity esters methyl 2-methylbutanoate and 2-phenylethanol from amino acids and an organic acid
present in the wort. We investigated the impact of two critical process parameters (volumetric power
input and inoculum concentration) on the morphology of, and flavor production by, the shiitake
pellets in a 1 L stirred bioreactor. Increasing the volumetric power input and biomass concentration
influenced the morphology of the pellets and promoted the production of the most important flavor
compound methyl 2-methylbutanoate in the beverage. Furthermore the worty off-flavor methional
was degraded during the cultivation in stirred bioreactor by shiitake pellets. These findings provide
useful information to facilitate the scale-up of the biotransformation and fermentation process
in bioreactors.
Keywords: shiitake; volumetric power input; inoculum concentration; natural flavor; stirred
bioreactor; non-alcoholic beverage; methyl 2-methylbutanoate
1. Introduction
The submerged cultivation of basidiomycetes, such as the shiitake mushroom (Lentinula edodes),
allows the production of valuable secondary metabolites, including pharmaceuticals, enzymes,
and natural flavor compounds [1–3]. The increasing demand for alcohol-free beverages with natural
flavors has forced breweries to look for new beverages to broaden their product portfolios. Preliminary
research has shown that it is possible to produce non-alcoholic beverages based on malt by
incorporating basidiomycetes that produce diverse natural flavors during the fermentation [4,5].
However, the complex morphology of these organisms makes the cultivation process challenging,
because the submerged fungi can grow as viscous mycelia, or as dense pellets depending on process
Beverages 2017, 3, 20; doi:10.3390/beverages3020020
www.mdpi.com/journal/beverages
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parameters. There is little information concerning the impact of these parameters on the fermentation
of mushrooms in submerged cultures for flavor production.
The volumetric power input is one of the critical process parameters in the submerged cultivation
of shear-sensitive organisms such as basidiomycetes. This process parameter can be regulated by
agitation and aeration intensity and by stirrer geometry. Agitation is required for mixing and to achieve
adequate oxygen transfer, but excessive agitation subjects the mycelia to mechanical stress and this can
influence the production of target metabolites. It is, therefore, important to achieve the optimal balance
between adequate oxygen supply and minimal mechanical stress [6,7]. Therefore, the used impeller
type is another aspect, which should be considered in the cultivation of shear-sensitive basidiomycetes.
A pitched blade impeller is used mainly for cell culture applications and achieves higher oxygen
supply and less shear forces than a Rushton impeller [8]. The amount of inoculum is another critical
process parameter that can influence the efficiency of fermentation. Many researchers have shown that
the inoculum concentration affects the morphology, mycelia biomass yields, and metabolite production
by basidiomycetes. It is important to determine the optimum biomass concentration that achieves
effective biotransformation for the production of natural flavor compounds [6,7].
Odor-active compounds such as methyl 2-methylbutanoate and 2-phenylethanol generated by
shiitake pellets contribute to the overall flavor profile of beverages. In addition, the flavor compounds
of wort (β-damascenone and methional) are degraded during the biotransformation [5]. Methional is
a typical worty off-flavor and has an odor impression of “potato-like” [9]. In contrast, β-damascenone
has a fruity odor impression and smells like pear [4,5]. We used the ability of shiitake to produce
natural flavors in order to develop a fermentation process for novel beverages. Due to the loss
of volatile flavor compounds through the aeration of medium with air, the oxygen supply of the
basidiomycete is achieved with the regulation of agitation rates. In addition to create less mechanical
forces in comparison to a cultivation with a Rushton impeller, a 3 × 45◦ pitched blade impeller
(d = 45 mm, downward pumping, Applikon, Delft, The Netherlands) is used for the cultivation of
shiitake. We investigated the impact of inoculum size, and volumetric power input under different
agitation rates, on the morphology of shiitake pellets in the wort, and the quantities of key flavor
compounds produced during cultivation. We compared different volumetric power inputs and
maximal shear rates in a stirred tank bioreactor to provide information that will facilitate the scale-up
of the fermentation process.
2. Materials and Methods
2.1. Microorganism and Media
Shiitake (Lentinula edodes) was kindly donated by the Institute of Food Chemistry and Food
Biotechnology, Justus-Liebig-University, Giessen, Germany. The fungus was subcultured every month
on standard nutritional medium with agar (see below) in Petri dishes and was incubated at 24 ◦ C until
half of the agar was overgrown with mycelia (approximately 14 days). The plates were stored at 4 ◦ C.
The standard nutritional medium (SNLH) comprised 30 g/L glucose, 4.5 g/L L-asparagine,
1.5 g/L KH2 PO4 , 1.0 g/L MgSO4 . 7H2 O, 3.0 g/L yeast extract and 1 mL trace element solution
(plus 15 g/L agar-agar for the preparation of agar medium) in distilled water. The trace element
solution comprised 5 mg/L CuSO4 . 5H2 O, 80 mg/L FeCl3 . 6H2 O, 90 mg/L MnSO4 . H2 O, and 400 mg/L
Titriplex III (EDTA). The medium was adjusted to pH 6.0 with 1 M NaOH and autoclaved at 121 ◦ C for
20 min.
2.2. Wort Production
Wort (Kölsch type, 12–13 ◦ Brix) was produced at the University of Applied Sciences (Giessen,
Germany) using a KB50 pilot-scale brewing device (Alfred Gruber GmbH, Eugendorf, Austria).
A mixture of Pilsner (80% w/w), Munich (13% w/w) ,and wheat (7% w/w) malt types were used
to produce the wort, which was passed through a 3-µm filter and a 0.25-µm sterile filter, filled into
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aluminum bottles and tyndallized twice at 80 ◦ C for 1 h before storing at −20 ◦ C. Before fermentation,
the wort was thawed and pasteurized at 80 ◦ C for 1 h and then cooled to room temperature.
2.3. Pre-Culture Cultivation
The basidiomycete Lentinula edodes was initially grown on SNLH-Agar (see Section 2.1) in Petri
dishes for 14 days and then stored in refrigerator at 4 ◦ C. An agar plug with an edge length of about
1 cm with growing mycelia from the periphery of a Petri dish was removed with a sterilized spatula
and transferred to a 500 mL shake flask filled with 200 mL standard nutritional medium. The culture
was homogenized at 9500 rpm for 15 s using an Ultra Turrax T25 homogenizer (Janke and Kunkel,
IKA Labortechnik, Staufen, Germany). The inoculated medium was then incubated on a rotary shaker
(InforsHT, Multitron Standard, Bottmingen; Switzerland) at 24 ◦ C and 150 rpm for 12 days in the dark.
2.4. Biotransformation
Fermentations were carried out in a 1 L stirred glass bioreactor with a 0.8 L working volume
(Applikon, Delft, The Netherlands) and a 3 × 45◦ pitched blade impeller (d = 45 mm, downward
pumping, Applikon, Delft, The Netherlands). The standard fermentation was set up with the following
parameter: 800 mL wort, 35 mg/L pre-culture, 150 rpm and 24 ◦ C. Parallel fermentations with different
agitation rates (100 and 250 rpm) and inoculum sizes (21 and 68 mg/L) were carried out for 72 h to
determine the effect of these process parameters on the production of natural flavors. The agitation rate,
pH, and temperature were controlled using BioXpert (Applikon, Delft, The Netherlands). Dissolved
oxygen in the fermentation process was measured in-line using an oxygen probe (Presens GmbH,
Regensburg, Germany). Samples were taken every 12 or 24 h for further analysis. Each sample for
flavor analysis was analyzed in duplicate. Samples were also tested for sterility on lysogeny broth
agar medium (5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl, and 15 g/L agar).
The volumetric power input was calculated using the following equations:
P = ρ×
N
60
3
× D5 × N p
Volumetric power input
W
m3
=
(1)
P
V
(2)
where P = power (W), ρ = the density of the medium (kg/m3 ), N = stirrer speed (s−1 ), D = impeller
diameter (m), and N p = the power number of the impeller type (1.5 for the Applikon pitched blade
impeller).
The maximal shear rate (γmax ) in the stirred bioreactor was calculated to characterize the
mechanical stress according to the following equation [10]:
γmax = 3.3N 1.5 D
ρL
µL
0.5
(3)
where N = agitation speed (s−1 ), D = impeller diameter (m), ρ L = the density of the medium (kg/m3 ),
and µ L = viscosity of the fluid (Pa·s).
2.5. Measurement of Cell Dry Weight
The dry weight of mycelial biomass was determined after recovery with a sieve (Rotilabo® , mesh
size: 0.5 mm, Carl Roth, Karlsruhe, Germany), washing with distilled water, and drying at 80 ◦ C for
24 h in aluminum cups.
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2.6. Characterization of Fungal Morphology
Mycelial pellets gathered from the pre-culture (inoculum) and fermentation were separated
using a round sieve (Rotilabo® , mesh size: 0.5 mm, Carl Roth, Karlsruhe, Germany), suspended in
a water-filled centrifuge tube and stained with 3 mL methylene blue solution (0.3 g methylene blue
(Merck, Darmstadt, Germany), 30 mL 95% ethanol, and 100 mL water) for 5 min. After staining, the
pellets were separated using a sieve (Rotilabo® , mesh size: 0.5 mm, Carl Roth, Karlsruhe, Germany)
and washed in distilled water. The stained pellets (approximately 100–200 pellets per Petri dish) were
placed in a Petri dish filled with water and scanned (CanoScan 9000F, Canon Deutschland, Krefeld,
Germany). The pellets were also carefully separated from each other with a help of tweezers to obtain
a good image for the analysis. The images were then analyzed using ImageJ v1.47. The morphology
of the pellets was characterized by their equivalent diameter and the D10, D50 and D90 value of the
particle size distribution. The equivalent diameter (deq ) was calculated according to the following
equation:
r
4A
(4)
deq =
π
where A = projected area (cm2 ).
The morphological shape parameter roundness was used to characterize the particles. Particles
with a roundness smaller than 0.5 were not considered as pellets, but rather as irregular particles such
as mycelium filaments and clumps. The shape parameter roundness was calculated by using ImageJ
v1.47 according to the following equation:
Roundness = 4 ×
[ Area]
π × [ Major axis]2
(5)
2.7. Flavor Analysis
Ten milliliters of the wort or fermented wort was transferred to a 20 mL headspace vial.
Then 100 µL thymol (TCI, Eschborn, Germany) or ethyl butyrate (Acros Organics) was added
as an internal standard and the volatile components were extracted by headspace solid phase
micro-extraction (HS-SPME) using a carboxen/polydimethylsiloxane (CAR/PDMS) fiber (Supelco,
Steinheim, Germany). The extract was then analyzed by gas chromatography tandem mass
spectrometry/olfactometry (GC-MS/MS-O) as previously described [5]. The key odor-active
compounds were detected by olfactometry, and changes in the levels of methyl 2-methylbutanoate,
2-phenylethanol, β-damascenone and methional during fermentation were expressed as relative
peak areas. The odor activity values (OAVs) for the main flavor compounds were also calculated as
previously described [5].
2.8. Measurement of the Oxygen Gradient
The upper part of a cuvette was removed and fixed to a Petri dish, and a hole was introduced in
the middle of the cuvette using a hot needle. The Petri dish was then filled with water. Different size
of pellets from pre-culture was carefully placed onto the hole to provide stability during measurement
(see Figure 1). Before measurement, the oxygen sensor was prepared by two-point calibration in
oxygen-free and air-saturated water. The oxygen profile in the shiitake pellets was measured using
a needle-type oxygen sensor (OXR50, tip diameter 50 µm; Pyro Science, Aachen, Germany) which was
driven into the pellet with a depth of 100 µm with an automatic manipulator. Measurements were
recorded using Profix software (Pyro Science GmbH, Aachen, Germany).
the middle of the cuvette using a hot needle. The Petri dish was then filled with water. Different size of pellets from pre‐culture was carefully placed onto the hole to provide stability during measurement (see Figure 1). Before measurement, the oxygen sensor was prepared by two‐point calibration in oxygen‐free and air‐saturated water. The oxygen profile in the shiitake pellets was measured using a needle‐type oxygen sensor (OXR50, tip diameter 50 μm; Pyro Science, Aachen, Beverages 2017, 3, 20
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Germany) which was driven into the pellet with a depth of 100 μm with an automatic manipulator. Measurements were recorded using Profix software (Pyro Science GmbH, Aachen, Germany). Beverages 2017, 3, 20 5 of 14 Figure 1. Holder for the measurement of the oxygen gradient in shiitake pellets. Figure 1. Holder for the measurement of the oxygen gradient in shiitake pellets.
2.9. Statistical Analysis 2.9. Statistical
Analysis Analysis
of variance (one‐way ANOVA) was used to test the significance of the factors volumetric power input and inoculum concentration on the flavor production at 72 h. A significance Analysis of variance (one-way ANOVA) was used to test the significance of the factors volumetric
level of 5% was used to run the analysis of variance. Error bars are standard errors of the mean for power input and inoculum concentration on the flavor production at 72 h. A significance level of
two measurements with two samples. 5% was used to run the analysis of variance. Error bars are standard errors of the mean for two
measurements with two samples.
3. Results and Discussion 3. Results and Discussion
3.1. Microprofiling 3.1. Microprofiling
The oxygen gradient in shiitake pellets was measured using a microelectrode oxygen sensor The oxygen gradient in shiitake pellets was measured using a microelectrode oxygen sensor and it
and it was found that the fungal cells in the center of the pellets were subjected to oxygen starvation was found that the fungal cells in the center of the pellets were subjected to oxygen starvation (Figure 2).
(Figure 2). Oxygen penetrated to a depth of approximately 0.10–0.15 cm at 84%–100% air saturation. Oxygen penetrated to a depth of approximately 0.10–0.15 cm at 84%–100% air saturation. Therefore,
Therefore, oxygen can reach deeper into pellets, which have a diameter less than 0.20 cm. The D50 oxygen
deeperused into pellets,
which have a under diameter
less than
0.20 cm.
Theand D50inoculum value of
value of can
the reach
pre‐culture in the fermentations different agitation rates the pre-culture used in the fermentations under different agitation rates and inoculum concentration
concentration was 0.22 and 0.10 cm, respectively. According to the microprofiling measurement it is obviously clear the cells in According
the center toof can become oxygen‐limited during clear
the was 0.22 and
0.10that cm, respectively.
thepellets microprofiling
measurement
it is obviously
fermentation A negative effect the flavor production was not during the that the cells process. in the center
of pellets
can on become
oxygen-limited
during
the observed fermentation
process.
fermentation of the beverage. A negative effect on the flavor production was not observed during the fermentation of the beverage.
Figure 2. Measurement of oxygen profile in shiitake pellets with different particle sizes (R = Radius of Figure 2. Measurement of oxygen profile in shiitake pellets with different particle sizes (R = Radius of
the pellet). the pellet).
Oxygen can penetrate fully into small pellets and all the cells remain active, whereas in larger Oxygen can penetrate fully into small pellets and all the cells remain active, whereas in larger
pellets the cells in the center are starved of oxygen. The reported critical pellet radius for filamentous pellets
the cells in the center are starved of oxygen. The reported critical pellet radius for filamentous
microorganisms in the literature is about 200–400 μm [11–13]. It has been shown that the particle size microorganisms
in therole literature
about 200–400
[11–13]. ItFor has been
shown
that the particle
plays an important in the is production of µm
metabolites. example, microprofiling in size
plays
an
important
role
in
the
production
of
metabolites.
For
example,
microprofiling
in
Phanerochaete chrysosporium also revealed that the oxygen concentration in the pellet declined with Phanerochaete
chrysosporium
also
revealed
that
the
oxygen
concentration
in
the
pellet
declined
with
penetration depth. The oxygen limitation in the P. chrysosporium pellets reduced the production of lignin‐degrading enzymes [14]. For the production of the ligninolytic enzyme manganese peroxidase (MnP) by Pleurotus ostreatus, mycelial pellets of 0.1–0.2 cm in diameter were necessary. However, the cultivation in shake flask with larger pellets (0.5–1.0 cm) resulted in a lower MnP production by P. ostreatus [15]. Leisola and Fiechter also reported that P. chrysosporium pellets with an average diameter of 0.1–0.2 cm produced the maximal lignin peroxidase activity (LiP) [16]. On the Beverages 2017, 3, 20
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penetration depth. The oxygen limitation in the P. chrysosporium pellets reduced the production of
lignin-degrading enzymes [14]. For the production of the ligninolytic enzyme manganese peroxidase
(MnP) by Pleurotus ostreatus, mycelial pellets of 0.1–0.2 cm in diameter were necessary. However,
the cultivation in shake flask with larger pellets (0.5–1.0 cm) resulted in a lower MnP production
by P. ostreatus [15]. Leisola and Fiechter also reported that P. chrysosporium pellets with an average
diameter of 0.1–0.2 cm produced the maximal lignin peroxidase activity (LiP) [16]. On the other hand
Beverages 2017, 3, 20 there are investigations with basidiomycetes which state that the oxygen limitation in pellets6 of 14 can
promote the secondary metabolite production [17–19].
3.2. Effect of Volumetric Power Input and Maximum Shear Rate 3.2. Effect of Volumetric Power Input and Maximum Shear Rate
3.2.1. Morphology and Biomass Growth 3.2.1. Morphology and Biomass Growth
There is a well‐known relationship between fungal morphology and the production of target There is a well-known relationship between fungal morphology and the production of target
metabolites [6,19]. It was found that increasing the volumetric power input and maximum shear rate metabolites
[6,19].
It was found
the volumetric
power input
maximum
shearwere rate
influenced the morphology of that
the increasing
shiitake pellets. Pellet growth and and
erosion of pellets influenced the morphology of the shiitake pellets. Pellet growth and erosion of pellets were observed
observed during the fermentation process, which led to the formation of a heterogeneous culture. As during the fermentation process, which led to the formation of a heterogeneous culture. As seen
seen in Figure 3 and 4 the pellet hairs were shaved‐off by mechanical forces in the bioreactor system. in Figures
andformation 4 the pellet
were filaments shaved-offand by clumps. mechanical
forces
in the volumetric bioreactor system.
This led to 3the of hairs
mycelium With increasing power This
led
to
the
formation
of
mycelium
filaments
and
clumps.
With
increasing
volumetric
power
input,
input, the percentage of mycelium filaments and clumps in the culture were also increased. the
percentage
of
mycelium
filaments
and
clumps
in
the
culture
were
also
increased.
Especially
the
3
Especially the fermentation with the highest volumetric power input of 26.2 W/m led to the 3
fermentation with the highest volumetric power input of 26.2 W/m led to the formation of smaller
formation of smaller pellets. On the other hand the increase of volumetric power input up to a value pellets.
On the
other handthe theenlargement increase of volumetric
powerwhich input up
to areflect value of
5.7enhanced W/m3 promoted
3 promoted of 5.7 W/m
of the pellets, may the oxygen the enlargement of the pellets, which may reflect the enhanced oxygen transfer (Figure 3).
transfer (Figure 3). Figure 3. D10, D50, and D90 value of particle size distribution of pre‐culture and fermentations at Figure 3. D10, D50, and D90 value of particle size distribution of pre-culture and fermentations at
different volumetric power inputs at 72 h (a); Percentage of pellets, mycelium filaments and clumps different volumetric power inputs at 72 h (a); Percentage of pellets, mycelium filaments and clumps of
of pre‐culture and fermentations at different volumetric power inputs at 72 h (b), the points at pre-culture and fermentations at different volumetric power inputs at 72 h (b), the points at 0 W/m3
0 W/m3 volumetric power input indicate the parameter values of the pre‐culture. volumetric power input indicate the parameter values of the pre-culture.
Due to the heterogeneous distribution of pellets, the D10, D50 (median), and D90 value of the Due to the heterogeneous distribution of pellets, the D10, D50 (median), and D90 value of the
particle size distribution was used to characterize the particle size distribution of the fermentations. particle
size distribution was used to characterize the particle size distribution of the fermentations.
With increasing volumetric power input, the D50 and D90 values increased first, which indicates the With
increasing
volumetric power input, the D50 and D90 values increased first, which
indicates the
3, a decrease of the growth of pellets. However at the highest volumetric power input of 26.2 W/m
3
growthD50 of pellets.
However
at the highest
volumetric
power
input
26.2
W/m
, a decreased decrease ofwith the
values and D90 was observed. Furthermore it was found that ofthe D10 value values
D50
and
D90
was
observed.
Furthermore
it
was
found
that
the
D10
value
decreased
with
increasing volumetric power input (Figure 3). This result shows that the pellets were damaged by increasing volumetric power input (Figure 3). This result shows that the pellets were damaged by
the mechanical forces created in stirred tank bioreactor. It is generally observed that the pellet size the mechanical forces created in stirred tank bioreactor. It is generally observed that the pellet size
decreases with increasing agitation intensity, which is related to mechanical shear forces [20,21]. It decreases with increasing agitation intensity, which is related to mechanical shear forces [20,21]. It was
was reported that higher shear forces created in stirred tank bioreactor can destroy the hairiness of the fungal pellets [22]. The increase of volumetric power input through agitation rate was used in the fermentation process to increase the oxygen supply of the pellets and to avoid the loss of volatile flavor compounds by stripping via exhaust air. The increase in volumetric power input within the range we analyzed promoted the biomass productivity and the production of biomass, which may reflect better oxygen transfer to the pellets, mycelium filaments and clumps (Table 1). The biomass Beverages 2017, 3, 20
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reported that higher shear forces created in stirred tank bioreactor can destroy the hairiness of the
fungal pellets [22].
The increase of volumetric power input through agitation rate was used in the fermentation
process to increase the oxygen supply of the pellets and to avoid the loss of volatile flavor compounds
by stripping via exhaust air. The increase in volumetric power input within the range we analyzed
promoted the biomass productivity and the production of biomass, which may reflect better oxygen
transfer to the pellets, mycelium filaments and clumps (Table 1). The biomass concentration of
Pleurotus ostreatus was also improved with increasing agitation and aeration rates in a stirred tank
bioreactor [23]. On the other hand it was found that the pellet size of P. ostreatus was decreased
when the agitation rate was increased from 200 rpm to a value of 400 rpm in a stirred tank bioreactor.
Beverages 2017, 3, 20 7 of 14 It was speculated that the shear forces were sufficient to decrease the pellet stability which led to the
restriction of
growth
[24]. that the shear forces were sufficient to decrease the pellet stability bioreactor. It cell
was speculated which led to the restriction of cell growth [24]. Table 1. Kinetic parameters of L. edodes fermentation at different agitation rates in a 1 L stirred tank
bioreactor. Data were part of a design of experiment (DoE) with four repetitions of the center point
Table 1. Kinetic parameters of L. edodes fermentation at different agitation rates in a 1 L stirred tank experiment. The corresponding experimental standard deviation was below 28%.
bioreactor. Data were part of a design of experiment (DoE) with four repetitions of the center point experiment. The corresponding experimental standard deviation was below 28%. Biomass
Agitation
Max. Shear
Xstart
P/V 1
Xfinal 2
Productivity
Biomass Max. 3
RateAgitation (rpm)
Rate (1/s)
(W/m
P/V 1 )
start Xfinal(mg/L)
2 X(mg/L)
(mg/L h)
productivity shear rate 3
[W/m ] [mg/L] [mg/L] rate [rpm] [mg/L h] [1/s] 100
247
1.7
35
112
1.08
150 100 453
5.7
247 1.7 35 35
112 167
1.08 1.84
250 150 974
26.2 453 5.7 35 35
167 231
1.84 2.72
1
2
P/V: Volumetric
power
input, Xfinal35 : biomass dry231 weight at 72 h. 2.72 250 974 26.2 P/V: Volumetric power input, 2 Xfinal: biomass dry weight at 72 h. 1
(a) (b)
Figure 4. Shiitake pellet stained with methylene blue solution (a); hyphel fragments observed in Figure 4. Shiitake pellet stained with methylene blue solution (a); hyphel fragments observed in
fermentations (b). fermentations (b).
3.2.2. Flavor Production 3.2.2. Flavor Production
Odor‐active flavor compounds have been identified in wort and fermented wort with shiitake Odor-active flavor compounds have been identified in wort and fermented wort with shiitake [4].
[4]. However, in this study key odor‐active compounds in wort and fermented wort were regarded. However, in this study key odor-active compounds in wort and fermented wort were regarded. Flavor
Flavor compounds with an odor activity value (OAV) > 1 such as methyl 2‐methylbutanoate (OAV = compounds with an odor activity value (OAV) > 1 such as methyl 2-methylbutanoate (OAV = 30),
30), 2‐phenylethanol (OAV = 1.3), β‐damascenone (OAV = 1.1), and methional (OAV = 10), were 2-phenylethanol (OAV = 1.3), β-damascenone (OAV = 1.1), and methional (OAV = 10), were chosen
chosen for the characterization of the beverage [5]. It was found that methyl 2‐methylbutanoate and for the characterization of the beverage [5]. It was found that methyl 2-methylbutanoate and
2‐phenylethanol were produced during the fermentation whereas methional was degraded (Figure 2-phenylethanol were produced during the fermentation whereas methional was degraded (Figure 5).
5). Almost 80 % of the methional was degraded during the fermentation of the beverage (Figure 5d). Almost 80% of the methional was degraded during the fermentation of the beverage (Figure 5d).
However, ß‐damascenone was not degraded significantly at all of the fermentations with different However, ß-damascenone was not degraded significantly at all of the fermentations with different
volumetric power inputs (Figure 5c) (p‐value > 0.05). Zhang et al. showed that both of the flavor volumetric power inputs (Figure 5c) (p-value > 0.05). Zhang et al. showed that both of the flavor
compounds were degraded by L. edodes pellets in shake flask fermentation [5]. compounds were degraded by L. edodes pellets in shake flask fermentation [5].
Shiitake pellets utilize the precursor 2‐methylbutanoic acid and L‐isoleucine to produce the Shiitake pellets utilize the precursor 2-methylbutanoic acid and L-isoleucine to produce the fruity
fruity ester methyl 2‐methylbutanoate. Methyl 2‐methylbutanoate has the highest odor activity ester methyl 2-methylbutanoate. Methyl 2-methylbutanoate has the highest odor activity value with
value with a fruity impression and is therefore regarded as a key quality control parameter [5]. The increase in volumetric power input up to a value of 5.7 W/m3, which was regulated with the agitation rate, enhanced the production of this fruity aroma significantly (p‐value: 0.04), perhaps reflecting the more efficient oxygen supply to the pellets, promoting biomass, and pellet growth (Figure 5a). A further increase of volumetric power input did not enhance the production of methyl 2‐methylbutanoate significantly (p‐value: 0.08). The higher shear force might restrict the further Beverages 2017, 3, 20
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a fruity impression and is therefore regarded as a key quality control parameter [5]. The increase
in volumetric power input up to a value of 5.7 W/m3 , which was regulated with the agitation rate,
enhanced the production of this fruity aroma significantly (p-value: 0.04), perhaps reflecting the more
efficient oxygen supply to the pellets, promoting biomass, and pellet growth (Figure 5a). A further
increase of volumetric power input did not enhance the production of methyl 2-methylbutanoate
significantly (p-value: 0.08). The higher shear force might restrict the further production of fruity ester,
which was also reflected in the pellet morphology. A similar finding was observed in the cultivation
of shiitake mycelia for eritadenine production. Increasing the agitation rate from 50 to 250 rpm in
a 1 L bioreactor also promoted biomass growth and eritadenine production by shiitake. The enhanced
eritadenine production was related to the formation of dispersed mycelial filaments. However, the type
of mixer used in the cultivation of shiitake was not mentioned which can influence the morphology [25].
It was shown that agitation rate affects the morphology of shiitake pellets in shake flasks. The pellet
Beverages 2017, 3, 20 8 of 14 size was increased with decreasing agitation rates when vegetative homogenized mycelium was used
as inoculum. Furthermore it was stated that the pellet formation was faster at the lower agitation
stated that the pellet formation was faster at the lower agitation rates of 50 and 100 rpm. Although ratesbiomass of 50 and
100 rpm.
Although
thewith biomass
growth agitation was promoted
increasingproduction agitation rate,
the growth was promoted increasing rate, with
the enhanced of the enhanced production of ergothioneine was related to the compact and larger pellets produced at
ergothioneine was related to the compact and larger pellets produced at the lower agitation rate of the rpm lowerin agitation
rate ofIt 50was rpmassumed in shake flasks.
It was
assumed
that the
limitation
in larger
50 shake flasks. that the oxygen limitation in oxygen
larger pellets favored the pellets
favored
the
production
of
ergothioneine
[17].
production of ergothioneine [17]. Basidiomycetes can
synthesize
2-phenylethanol
de novo
by the
of precursors
Basidiomycetes can synthesize 2‐phenylethanol de or
novo or biotransformation
by the biotransformation of such
as
asparagine
or
L
-phenylalanine
[26].
In
shiitake,
2-phenylethanol
can
be
produced
from
precursors such as asparagine or L‐phenylalanine [26]. In shiitake, 2‐phenylethanol can be produced L -phenylalanine
in the wort [5]. Figure 5b shows the production of 2-phenylethanol, which has
from L‐phenylalanine in the wort [5]. Figure 5b shows the production of 2‐phenylethanol, which has a rose-like
odor. The
was not not significantly
a rose‐like odor. The result
result shows
shows that
that the
the production
production of
of 2-phenylethanol
2‐phenylethanol was significantly influenced
by
increasing
volumetric
power
input
(p-value:
0.86).
influenced by increasing volumetric power input (p‐value: 0.86). Figure 5. Production of (a) methyl‐2‐methylbutanoate, (b) 2‐phenylethanol, and degradation of (c) Figure 5. Production of (a) methyl-2-methylbutanoate, (b) 2-phenylethanol, and degradation of
β‐damascenone, (d) methional by shiitake under different volumetric power inputs. Vol.: Volumetric. (c) β-damascenone, (d) methional by shiitake under different volumetric power inputs. Vol.: Volumetric.
Error bars are standard errors of the mean for two samples. Error bars are standard errors of the mean for two samples.
We observed no difference in the degradation of β‐damascenone and methional among the We observed
nopower difference
in the
degradation
β-damascenone
methional
the
different volumetric inputs (p‐value > 0.05), of
(Figure 5c,d). The and
degradation of among
methional different volumetric
power inputs
(p-value
> 0.05),
(Figure
5c,d). The
degradationfor of the methional
improves the taste attributes of the beverage because methional is responsible strong potato‐like taste. Therefore, the fermented beverage has a milder and more pleasant taste than the wort. Shiitake can secrete many different catabolic enzymes [2,27], one of which may degrade methional. The milder taste of the fermented beverage was confirmed by a tasting. Fermentation of the wort by the shiitake mushroom produced a fruity, slightly sour, sweetish, fresh, and plum‐like taste. Beverages 2017, 3, 20
9 of 15
improves the taste attributes of the beverage because methional is responsible for the strong potato-like
taste. Therefore, the fermented beverage has a milder and more pleasant taste than the wort. Shiitake
can secrete many different catabolic enzymes [2,27], one of which may degrade methional. The milder
taste of the fermented beverage was confirmed by a tasting. Fermentation of the wort by the shiitake
mushroom produced a fruity, slightly sour, sweetish, fresh, and plum-like taste.
The agitation rate has also been shown to boost the synthesis of fungal metabolites due to the
efficient mixing and improved oxygen transfer, e.g., a higher agitation rate (400 rpm) promoted mycelial
growth and EPS production by Paecilomyces japonica in a 5-L stirred tank bioreactor [28]. The production
of EPS by Tricholoma matsutake was also enhanced at a higher agitation rate although cell growth
was less efficient [29]. Furthermore, exopolysaccharide (EPS) production by Pycnoporus sanguines
and Grifola frondosa was more efficient in a stirred tank bioreactor than an air-lift bioreactor [22,30].
The degradation of cyanide by Ganoderma lucidum, Polyporus arcularius and Schizophyllum commune in
shake flasks was enhanced by increasing the agitation rate to 100 rpm, whereas degradation remained
stable at higher rates [31]. On the other hand mycelial growth and EPS production by G. frondosa was
more efficient at the lowest agitation rate in shake flask cultures [32].
In contrast to the studies listed above, there are many others showing that mechanical stress
damages cells and inhibits product formation. For example, Ganoderma lucidum is a shear-sensitive
basidiomycet and 300 rpm was reported as a critical impeller speed in a 10 L stirred tank bioreactor
mixed by three Rushton turbines [33]. It was also shown that the cell growth and the intracellular
polysaccharide production of this basidiomycet in a stirred tank bioreactor were inhibited by higher
shear stress [34]. The morphology of Cordyceps militaris was significantly affected by agitation intensity,
and mild agitation (150 rpm) promoted EPS production in a 5 L stirred tank bioreactor. Smaller and
less compact pellets were formed at the highest agitation rate of 300 rpm [20]. Mild agitation rates
are preferable for laccase production by Dichomitus squalens and Pleurotus ostreatus in stirred tank
bioreactors [21,23]. The laccase production by Panus tigrinus on olive mill wastewater-based media
was influenced negatively when the stirrer speed was increased [35]. The production of ligninolytic
enzymes by D. squalens in a 3 L stirred tank bioreactor enhanced when the agitation intensity increased
from 175 rpm (P/V: 20 W/m3 ) to 250 rpm (P/V: 80 W/m3 ). However, a further increase to an agitation
intensity of 350 rpm (P/V: 180 W/m3 ) led to a decrease in enzyme production [21]. Therefore,
bioreactor systems with lower shear environment are sometimes preferable for the fungal metabolites.
The production of a manganese-dependent peroxidase (MnP) by D. squalens was promoted when
the fungus was cultivated in a bubble column reactor, which has lower shear effects than a stirred
tank bioreactor [21]. Phenolic compounds in olive mill wastewater were also degraded more rapidly
by Panus tigrinus in a bubble column bioreactor reflecting the production of larger amounts of the
enzymes laccase and Mn (II) peroxidase [36]. The production of biomass and EPS by Tremella fuciformis
was also enhanced in an air-lift bioreactor compared to a stirred tank bioreactor [37]. An alternative
disposable bag bioreactor system for the submerged cultivation of shear-sensitive basidiomycetes was
shown to promote mycelial growth and enzyme activity [38].
3.3. Effect of Inoculum Concentration
3.3.1. Morphology and Biomass Growth
To increase the number of pellets and cell density in the medium, biotransformation was carried
out in a glass bioreactor with increasing amounts of inoculum. Oxygen consumption, which is a good
indicator of cell activity, increased as the inoculum concentration increased (see Figure 6b). The values
of D10, D50, and D90 show that there is an increase in the pellet diameter compared to pre-culture,
which indicates pellet growth. The D50 value increased from 0.10 to 0.24 cm in both biotransformation
reactions (21 mg/L and 35 mg/L inoculum) and decreased to 0.18 cm in the fermentation with the
highest initial inoculum concentration (Figure 6a). Compared to the fermentations with an inoculum
concentration of 21 mg/L and 35 mg/L smaller pellets were obtained in the fermentation (at 72 h) with
Beverages 2017, 3, 20
10 of 15
an inoculum concentration of 68 mg/L (Figure 6a). When the inoculum concentration was increased to
68 mg/L, the dissolved oxygen concentration in the medium decreased to fewer than 5% air saturation
after 12 h, which also reduced the biomass productivity and the final biomass concentration (Figure 6b
and Table 2).
Beverages 2017, 3, 20 10 of 14 Figure 6. (A) D10, D50, and D90 value of particle size distribution of pre‐culture and fermentations at Figure 6. (a) D10, D50, and D90 value of particle size distribution of pre-culture and fermentations
different initial inoculum concentration at 72 h (volumetric power input: 1.7 W/m33, temperature: at different initial inoculum concentration at 72 h (volumetric power input: 1.7 W/m , temperature:
3 volumetric power inputs indicates the pre‐culture); (B) oxygen profile 24 °C, the points at 0 W/m
24 ◦ C, the points at 0 W/m3 volumetric power inputs indicates the pre-culture); (b) oxygen profile
during the fermentation at the initial inoculum concentration of 21, 35, and 68 mg/L. during the fermentation at the initial inoculum concentration of 21, 35, and 68 mg/L.
Table 2. Kinetic parameters of L. edodes fermentation at different initial inoculum concentrations in a Table 2. Kinetic parameters of L. edodes fermentation at different initial inoculum concentrations in a 1 L
1 L stirred tank bioreactor. Data were part of a DoE with four repetitions of the center point stirred tank bioreactor. Data were part of a DoE with four repetitions of the center point experiment.
experiment. The corresponding experimental standard deviation was below 14%. The corresponding experimental standard deviation was below 14%.
Inoculum Inoculum Volume
volume [ml] (mL)
4040 8080 160
160 1
Initial BDW 1 1
Initial BDW
[mg/L] (mg/L)
21 21
35 35
68
68 Biomass Xfinal 2 Biomass
Productivity
Xfinal 2
productivity [mg/L] (mg/L
h)
(mg/L)
[mg/L h] 175 2.14 175
2.14
225 2.63 225
2.63
205
1.90
205 1.90 1 BDW: biomass dry weight, 22 X
biomass dry weight at 72 h.
BDW: biomass dry weight, Xfinal
final:: biomass dry weight at 72 h. 3.3.2. Flavor Production 3.3.2. Flavor Production
The increase in inoculum concentration accelerated and enhanced the production of the fruity The increase in inoculum concentration accelerated and enhanced the production of the fruity
flavor compound methyl 2‐methylbutanoate significantly (p‐value: 0.02) (Figure 7a). This reflected flavor compound methyl 2-methylbutanoate significantly (p-value: 0.02) (Figure 7a). This reflected the
the increasing number of active cells which contributed to the biotransformation. Although the increasing number of active cells which contributed to the biotransformation. Although the highest
highest inoculum concentration resulted in the lowest biomass productivity (Table 2), it also resulted inoculum concentration resulted in the lowest biomass productivity (Table 2), it also resulted in the
in the highest concentration of the fruity ester perhaps reflecting the combined effect of the smaller highest concentration of the fruity ester perhaps reflecting the combined effect of the smaller pellet
pellet size and the larger number of pellets. At the end of the fermentation with the highest inoculum size and the larger number of pellets. At the end of the fermentation with the highest inoculum
concentration 4331 pellets were counted, whereas only 705 and 1570 pellets were counted for the concentration 4331 pellets were counted, whereas only 705 and 1570 pellets were counted for the
fermentations with an initial biomass concentration of 21 and 35 mg/L, respectively. The results fermentations with an initial biomass concentration of 21 and 35 mg/L, respectively. The results
showed that the shiitake pellets can even produce the fruity ester at a low dissolved oxygen showed that the shiitake pellets can even produce the fruity ester at a low dissolved oxygen
concentration (<5% air saturation) after 12 h (Figure 6b and 7a). Therefore, it is crucial to elucidate concentration (<5% air saturation) after 12 h (Figures 6b and 7a). Therefore, it is crucial to elucidate
the biochemical pathway of the production of methyl 2‐methylbutanoate by Lentinula edodes to the biochemical pathway of the production of methyl 2-methylbutanoate by Lentinula edodes to
understand this observed effect. With an isotopic labelling experiment it was confirmed that methyl understand this observed effect. With an isotopic labelling experiment it was confirmed that methyl
2‐methylbutanoate can be produced by biotransformation of 2‐methylbutanoic acid and L‐isoleucine 2-methylbutanoate can be produced by biotransformation of 2-methylbutanoic acid and L-isoleucine by
by L. edodes, and 2‐methylbutanoic acid was assumed to be an intermediate in the biochemical L. edodes, and 2-methylbutanoic acid was assumed to be an intermediate in the biochemical pathway of
pathway of methyl 2‐methylbutanoate [5]. A similar pathway in the production of this fruity ester methyl 2-methylbutanoate [5]. A similar pathway in the production of this fruity ester was observed in
was observed in strawberry and apple, when they were fed with the amino acid L‐isoleucine [39,40] strawberry and apple, when they were fed with the amino acid L-isoleucine [39,40] It was also reported
It was also reported that methyl branched fruity esters in apple can be produced from the precursor that methyl branched fruity esters in apple can be produced from the precursor ethyl tiglate [41],
ethyl tiglate [41], but there is no information about the effect of oxygen concentration on the fruity flavor production by L. edodes in the literature. It can be speculated that the metabolism of L. edodes may be shifted more to metabolite production. The accumulation of 2‐phenylethanol was also influenced significantly by the increase in inoculum concentration (p‐value: 0.001), but did not increase further in the biotransformation inoculated with 68 mg/L shiitake pellets (Figure 7b). The degradation of methional and Beverages 2017, 3, 20
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but there is no information about the effect of oxygen concentration on the fruity flavor production by
L. edodes in the literature. It can be speculated that the metabolism of L. edodes may be shifted more to
metabolite production.
The accumulation of 2-phenylethanol was also influenced significantly by the increase in inoculum
concentration (p-value: 0.001), but did not increase further in the biotransformation inoculated
with 68 mg/L shiitake pellets (Figure 7b). The degradation of methional and β-damascenone was
Beverages 2017, 3, 20 11 of 14 not influenced significantly by the inoculum concentration (p-value > 0.05). However, the results
showed that methional and ß-damascenone were degraded significantly during the fermentation
during the fermentation (p‐value < 0.05) (Figure 7c,d). The same findings were observed during the (p-value < 0.05) (Figure 7c,d). The same findings were observed during the fermentation of this
fermentation of this beverage by shiitake pellets in shake flasks [5]. beverage by shiitake pellets in shake flasks [5].
Figure 7. Production of (a) Figure
7. Production ofmethyl‐2‐methylbutanoate, (b) 2‐phenylethanol, and degradation of (c) (a) methyl-2-methylbutanoate, (b) 2-phenylethanol, and degradation
ß‐damascenone and (d) methional by shiitake under under
different inoculum concentrations. BDW: of (c) ß-damascenone
and (d) methional
by shiitake
different
inoculum
concentrations.
BDW: biomass dry weight. Error bars are standard errors of the mean for two samples.
biomass dry weight. Error bars are standard errors of the mean for two samples. Homogenized vegetative mycelia are generally used to test the impact of inoculum size on cell Homogenized vegetative mycelia are generally used to test the impact of inoculum size on cell
morphology
and the impact of this parameter on metabolite production. Berovic et al. showed that
morphology and the impact of this parameter on metabolite production. Berovic et al. showed that the inoculum concentration affected the biomass growth of Ganoderma lucidum in a 10 L stirred tank
the inoculum concentration affected the biomass growth of Ganoderma lucidum in a 10 L stirred tank bioreactor [33]. Vegetative inoculum of G. lucidum (six-day old shake flask culture) in concentrations of
bioreactor [33]. Vegetative inoculum of G. lucidum (six‐day old shake flask culture) in concentrations 14%, 17%, and 20% (wet weight) was used for the investigation. The maximal biomass concentration
of 14%, 17%, and 20% (wet weight) was used for the investigation. The maximal biomass was obtained in the cultivation with an inoculum concentration of 17%. However, the highest inoculum
concentration was obtained in the cultivation with an inoculum concentration of 17%. However, the concentration (20%) led to the decrease of biomass growth probably due to the substrate limitation in
highest inoculum concentration (20%) led to the decrease of biomass growth probably due to the the culture. In addition, the inoculum concentration greatly affected the pellet size and the production
substrate limitation in the culture. In addition, the inoculum concentration greatly affected the pellet of polysaccharide and ganoderic acid in shake flasks by Ganoderma lucidum. The maximal cell
size and the production of polysaccharide and ganoderic acid in shake flasks by Ganoderma lucidum. concentration of G. lucidum was promoted when the inoculum concentration was increased from
The maximal cell concentration of G. lucidum was promoted when the inoculum concentration was 70 to 330 mg dry weight/L. A further increase in inoculum concentration (670 mg dry weight/L) led to
increased from 70 to 330 mg dry weight/L. A further increase in inoculum concentration (670 mg dry the decrease of the maximal cell concentration. At the lowest inoculum concentration 91% of the pellets
in the culture
hadthe a diameter
larger
0.16
cm. On the
contrary,
most of the pellets
were
smallerinoculum than
weight/L) led to decrease of than
the maximal cell concentration. At the lowest 0.12 cm when the cultivation was carried out with the highest inoculum concentration. Polysaccharide
concentration 91% of the pellets in the culture had a diameter larger than 0.16 cm. On the contrary, most of the pellets were smaller than 0.12 cm when the cultivation was carried out with the highest inoculum concentration. Polysaccharide production was enhanced by high concentrations of inoculum, which led to the formation of small pellets, whereas ganoderic acid production was promoted by lower inoculum concentrations which led to the formation of large pellets. The Beverages 2017, 3, 20
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production was enhanced by high concentrations of inoculum, which led to the formation of small
pellets, whereas ganoderic acid production was promoted by lower inoculum concentrations which
led to the formation of large pellets. The limitation of nutrients in large pellets, thus, appears to favor
ganoderic acid production [19]. As reported by Yang and Liau, an increase in inoculum concentration
promoted the mycelium yield but decreased the pellet size of G. lucidum [42]. Lin and Yang also
stated that a linear correlation exists between inoculum concentration and mycelia yield. However,
the mycelia growth of Agaricus blazei Murrill was not significantly influenced when the cultivation was
done at an inoculum concentration of higher than 40 mg/L [43]. Laccase production by P. ostreatus
is promoted by a biomass concentration of up to 1.38 g/L (wet weight of mycelia) immobilized
on polyurethane foam cubes, but a further increase in biomass concentration reduces the yield of
this enzyme [44]. Laccase production by P. ostreatus was also enhanced by the inoculum size when
increased numbers of fungal disks were used as the inoculums [45]. The biomass concentration of
G. frondosa was significantly higher at an inoculum ratio of 4% than the inoculum ratios of 2% and 6%.
A higher biomass concentration did not increase the secretion of EPS by G. frondosa in shake flask
cultures, but the smaller and hairier pellets formed at an inoculation rate of 2% favored EPS production,
suggesting the outer layer of the pellet regulates the synthesis of EPS [32]. Other investigations with
basidiomycetes also showed that a too low and a too high inoculum concentration decreased the
mycelia growth and metabolite production [30,46,47].
4. Conclusions
We investigated volumetric power input and inoculum concentration as critical process
parameters affecting the production of natural flavor compounds by shiitake pellets. The production
of methyl 2-methylbutanoate, which has the highest odor activity value and generates the fruity
flavor of the beverage, was promoted by the better mixing and growth conditions. Furthermore,
the results showed that the production of the fruity ester can be enhanced by the regulation of
inoculum concentration. The findings highlight the importance of process parameters in the cultivation
of basidiomycetes in a stirred tank bioreactor for the production of natural flavors. Our future work
will focus on the scale-up of the fermentation process and its effect on the metabolic pathways leading
to methyl 2-methylbutanoate and 2-phenylethanol.
Acknowledgments: This project (HA project No. 305/11-50) is funded in the framework of Hessen Modell
Projekte, financed with funds of LOEWE—Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer
Exzellenz, Förderlinie 3: KMU-Verbundvorhaben (State Offensive for the Development of Scientific and Economic
Excellence). We gratefully thank Richard M. Twyman for the English editing of the manuscript. We also thank Jan
Zitzmann for drawing the Figure 1.
Author Contributions: Sibel Özdemir did the experimental work, research, and wrote the paper. Doreen Heerd
critically reviewed the paper and helped in preparing the manuscript. Hendrich Quitmann gave advices in the
experimental design. Yanyan Zhang and Marco Alexander Fraatz conducted the flavor analysis of the samples
and analyzed the flavor data. Holger Zorn and Peter Czermak reviewed the paper and gave advice in preparing
the manuscript. Peter Czermak gave advice in the experimental design and is the head of the research program.
Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the
decision to publish the results.
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