development of biomass quantification methods for
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Introduction
Measurement of mycelial weight in fungal biomass is
essential for analyzing the biological and enzymological
activities of mycelia as well as for planning the best
utilization of this mycelial component in solid-state
cultivation. Some chemical methods in which the
mycelial mass is measured through the use of fungal-
specific biochemicals have been previously reported1). The
signature phospholipid fatty acid 18:2ω6,92), ergosterol
3)
and chitin4)
have all been used as biomass markers for
mycorrhizal fungi5, 6)
. Ergosterol is a fungus-specific
lipid that is used as a marker to quantify the amount of
living fungal biomass3, 7)
by quantifying the amount of
ergosterol present in active mycelia8). However, Newell et
al. demonstrated that the fungal ergosterol concentration
is dependent upon species, growth medium, incubation
conditions, and mycelial age9)
. Therefore, in order to
evaluate the pure mycelial biomass homogeneity, it is
important to develop a measurement system that is not
affected by fluctuations in environmental factors.
We began by focusing on the system used to measure
rice koji biomass utilizing commercially available cell wall-
degrading enzymes. These methods estimate mycelial
biomass by measuring β-(1,4)-n-acetylglucosamine
(GlcNAc) produced by the degradation of chitin in the rice
koji cell walls10, 11)
. The fungal cell wall of basidiomycetes
contains chitin, which is composed of straight-chain
GlcNAc and other hemicelluloses, such as β-glucans
and mannans12)
. Basidiomycetes chitin is a septum
component and plays an important role in morphogenetic
maintenance13)
.
Artificial cultivation of Lyophyllum shimeji (Kawam.)
Hongo has been achieved in bottle cultivation using a
medium composed of barley (Hordeum vulgare) grains
and sawdust14-16)
. These reports indicated that barley
starch in sufficient amounts could be used as a carbon
source and provide factors for growth of fruiting bodies
on the medium. Ohta17)
investigated the utilization of
starch and amylose by ectomycorrhizal fungi (55 strains),
and some strains demonstrated good mycelial growth on
medium containing barley grain. These reports indicate
that some ectomycorrhizal fungi are successfully able to
utilize starch as a carbon source.
Barley contains high concentrations of starch and
mixed-linkage (1,3;1,4)-β-d-glucans in its endosperm18)
.
Starch is the main component of barley, constituting
Development of biomass quantification methods for Tricholoma matsutake mycelium in solid-state medium cultivation
Hiroki ONUMA1), Kento HARA1), Zheng-Xi ZHANG2), Norifumi SHIRASAKA2) and Yasuhisa FUKUTA2)*,†
1) Graduate School of Agriculture, Kindai University, 3327-204, Naka-machi, Nara, Nara, Japan
2) Faculty of Agriculture, Kindai University, 3327-204, Naka-machi, Nara, Nara, Japan†
Present address: Laboratory of Food Microbiological Science and Biotechnology, Division of Applied Biological Chemistry, Graduate School of Agriculture, Kindai University, 3327-204, Naka-machi, Nara, Nara, Japan
(Received 8 November 2018 / Accepted 14 December 2018)
Abstract
To develop artificial cultivation of Tricholoma matsutake, it is necessary to establish stable culture conditions under which mycelia can spread quickly. However, an advantageous solid-state culture method for this fungus has not yet been identified. We developed a solid-state culture medium using barley and vermiculite to obtain a large amount of T. matsutake mycelia in a short time, and we estimated the T. matsutake fungal biomass in this artificial medium. We determined optimal conditions for mycelial biomass quantification through measurement of n-acetylglucosamine (GlcNAc) concentration in the mycelia. For degrading dry T. matsutake mycelia, 1.0% Yatalase and 0.5% Cellulase “ONOZUKA” RS solution provided optimal degradation conditions, and 139 μg GlcNAc per 10 mg of dried mycelia was produced. Subsequently, T. matsutake Z-1, NBRC 30605, and strain No. 115 were tested and demonstrated good growth using medium with barley:vermiculite composition of 2:1 (w/w). After 35 days of cultivation, T. matsutake Z-1, NBRC 30605, and strain No. 115 produced 215.1, 254.0, and 266.7 mg biomass/flask, respectively. By both visual observation and measurement of GlcNAc content in colonized substrate block, a 2:1 barley:vermiculite composition was demonstrated to be the optimum medium for the culture of T. matsutake mycelia.
Key words: n-Acetylglucosamine, Barley, Biomass, Solid-state cultivation, Tricholoma matsutake
Mushroom Science and Biotechnology, Vol. 26 (4) 156-163, 2019Copyright © 2019, Japanese Society of Mushroom Science and Biotechnology
*Corresponding author. E-mail: yfukuta@nara.kindai.ac.jp
Regular Paper
approximately 75% of the endosperm19)
. Some of the
recently developed barley cultivars contain starch with
a broad range of amylose content, varying between 0%
and 40%20)
. In addition, the water absorption coefficient
of barley is 73.2% to 78.2% of grain weight after 2 h of
immersion, which is 3.3 to 3.5 times higher than that of
rice21)
. Barley not only shows superior water absorption
but also has high starch content, which is useful as a
solid culture substrate in the artificial cultivation of
ectomycorrhizal mushrooms.
There are only three reports regarding the primordia
or fruiting body formation of T. matsutake in an artificial
culture system22-24)
. These studies report successful
formation of the fruiting body of T. matsutake in artificial
culturing systems using vermiculite. Tricholoma spp. can
grow slowly on a starch substrate when a small amount
of glucose is added as a starter25)
. Kusuda et al.26)
found
that 5% glucose medium inhibited the mycelial growth
of T. matsutake Z-1 and J-1 strains, whereas these strains
grew well on media with a soluble starch concentration
of up to 10% and 15%, respectively. Therefore, barley-
based medium containing vermiculite as a medium-
supporting material may be a possible new solid-state
culture method for T. matsutake.
In the present study, we determined the optimal
conditions for enzymatic chitin degradation for
quantification of T. matsutake mycelial biomass. We
also developed a solid-state culture method using media
composed of barley and vermiculite to obtain a large
amount of T. matsutake mycelia in a short time and
estimated T. matsutake fungal biomass in this artificial
cultivation medium.
Materials and methods
1. Materials
Analytical grade N-acetyl-d-glucosamine (GlcNAc)
and p-dimethylaminobenzaldehyde (DMAB) were obtained
from Nacalai Tesque, Co., Ltd. (Kyoto, Japan). Yatalase
was purchased from Takara Bio (Shiga, Japan). Cellulase
“ONOZUKA” RS and Cellulase “ONOZUKA” R-10 were
obtained from Yakult Pharmaceutical Industry Co., Ltd.
(Tokyo, Japan). Hulled barley grain was obtained from
Hakubaku (Hyogo, Japan). Vermiculite (approximately 4.0
mm particle size) was obtained from Vermitech (Gunma,
Japan). Pine-dex #1 was obtained Matsutani Chemical
Industry Co., Ltd. (Hyogo, Japan).
2. Microorganism and cultivation
In the present study, T. matsutake NBRC 30605, Z-1,
and strain No. 115 were used. The mycelia of T. matsutake
strains were cultivated in SY agar medium (2.0% soluble
starch, 0.5% yeast extract, 2.0% agar, pH 5.1) at 24℃ for 30
days.
T. matsutake strains were cultured in 100 mL
Erlenmeyer flasks containing 30 g hulled barley-
based vermiculite media [1:0, 0:1, 1:1, 2:1, and 1:2 (w/w);
approximately 60% moisture content]. Liquid components
of the media were prepared with 2% Pine-dex #1 and 0.3%
yeast extract, although the water uptake of hulled barley
was absorbed sufficiency at 4℃ for 12 h. The initial pH of
the media was adjusted to 5.1 before sterilization at 121℃
for 90 min. To cultivate the strains, 5 pieces of mycelia in
SY agar medium blocks (dimensions, 5 × 5 × 5 mm) were
inoculated to solid state media and placed in an incubation
chamber (Nippon Medical & Chemical Instruments, Osaka,
Japan) at 24℃ with 70% humidity for 24 h in the dark.
3. Preparation of samples for T. matsutake cell wall
degradation
T. matsutake NBRC 30605 mycelia were cultivated
on Potato Dextrose Broth at 24℃ for 35 days. Cultivated
mycelia were lyophilized for 24 h and homogenized
(2,800 rpm, 10 s, two cycles) using a Multi-Beads Shocker
(Yasui Kikai, Osaka, Japan). Homogenized mycelia were
washed using MilliQ water and diethyl ether and dried
by evaporation.
4. Effects of cell wall-degrading enzymes on T. matsutake
dry mycelia
Yatalase, Cellulase “ONOZUKA” R-10 and Cellulase
“ONOZUKA” RS were added to dry mycelia of T.
matsutake (10 mg/mL) at a final concentration of 1.0% (w/v)
in 50 mM sodium phosphate buffer (pH 7.0). Enzyme
solutions were filtered using cellulose acetate filters (0.45
μm; Merck Millipore, Darmstadt, Germany) prior to use.
Enzyme digestions were carried out at 37℃ with shaking
at 1,500 rpm. Following enzyme digestion, samples were
centrifuged at 10,000 × g at 4℃ for 3 min. The GlcNAc
concentration of supernatants was measured using
methods described by Reissig27)
.
5. Quantification of T. matsutake mycelial content on
solid-state medium
To measure T. matsutake mycelial biomass on solid-
state media, each medium cultivated with T. matsutake
was lyophilized for 1 - 2 days and dried samples were
powdered to homogeneity using a Multi-Beads Shocker
(Yasui Kikai) to achieve a particle size of approximately
0.2 μm. A 2 g aliquot from the total lyophilized sample
was suspended in 10 mL of 50 mM sodium phosphate
buffer (pH 7.0) and centrifuged (10,000 × g at 4℃ for
5 min). The pellet was then recovered and washed by this
method an additional three times11)
. The washed samples
were resuspended in 10 mL of 50 mM sodium phosphate
buffer (pH 7.0) containing 100 mg of Yatalase and 50
mg Cellulase “ONOZUKA” RS, and enzymatic digestion
was carried out at 37℃ for 60 min with shaking. After
digestion, the samples were centrifuged at 10,000 × g at
4℃ for 3 min. The GlcNAc concentration was estimated
using methods reported by Reissig27)
; after the color
development reaction, samples were centrifuged at 4℃
and 4,000 × g for 10 min. The resulting supernatant
was measured at 585 nm, and GlcNAc content and myc-
elial biomass were calculated using the equations below.
Solid-state medium GlcNAc content and mycelial weight
in dry matter were calculated as follows:
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MUSHROOM SCIENCE AND BIOTECHNOLOGY
GlcNAc (μg/2 g dry matter)
= (As - Ab) × F × V
Mycelial weight in dry matter (mg/flask)
= (As - Ab) × F × V × DM/CR
where As is the absorbance of the sample; Ab is the
absorbance of the blank; F is the slope of the standard
curve (μg GlcNAc per unit absorbance); V is the volume
(here 10 mL) of the analyzed samples; DM is the total
volume of dry matter content per flask (g); CR is the
conversion ratio of GlcNAc concentration per mycelial
dry weight (here 14.1 μg/mg dry mycelia).
Results
1. Effects of cell wall-degrading enzymes on GlcNAc
production in T. matsutake mycelia
As shown in Fig. 1, three cell wall-degrading enzymes
were used to compare the amount of GlcNAc liberated
from dried T. matsutake mycelia. Under experimental
conditions, it was demonstrated that a mixture of 1.0%
Yatalase and 1.0% Cellulase “ONOZUKA” RS produced
the highest GlcNAc yield from dry mycelia (139.2
μg/10 mg dry mycelia). Cellulase “ONOZUKA” R-10 had
lower enzymatic activity (relative activity: 12% of the
activity observed from 1.0% Yatalase and 1.0% Cellulase
“ONOZUKA” RS mixture) and yielded lower GlcNAc
concentrations, even in a mixture with 1.0% Yatalase.
Based on these results, Yatalase and Cellulase
“ONOZUKA” RS were used in the following studies. In
order to determine the optimal enzyme concentration
and reaction time, the amounts of Yatalase and Cellulase
“ONOZUKA” RS were varied against 10 mg of T.
matsutake dry mycelia, and the amount of liberated
GlcNAc at varying time points was measured (Fig. 2). The
amount of GlcNAc produced from 10 mg of T. matsutake
dry mycelia reached a maximum after 1 h of enzymatic
digestion, and the highest GlcNAc levels were obtained
using 1.0% Yatalase and 0.5% Cellulase “ONOZUKA” RS
mixture, and 1.0% Yatalase and 1.0% Cellulase “ONOZUKA”
RS mixture, yielding 138.2 and 136.9 μg, respectively.
Therefore, these results indicate that the optimal amounts
of Yatalase and Cellulase “ONOZUKA” RS were 1.0% and
0.5% final concentration, with optimal incubation at 37℃
for 1 h. In addition, a standard curve of GlcNAc generated
by degradation of T. matsutake mycelia under these
conditions is shown in Fig. 3. The upper limit of detection
was 15 mg, and the lower limit was 0.1 mg.
2. Effects of culture substrate on enzymatic digestion
and GlcNAc production
To determine whether the solid-state substrate
inhibited the enzyme reaction or the Reissig reaction27)
,
various culture substrates were added at 10% (w/v) to
1 and 10 mg/mL of dried T. matsutake mycelia, and
enzymatic digestion was performed using the enzyme
158 Vol.26 No. 4
Fig. 1. Comparison with GlcNAc production of cell wall degradation
enzymes.
1: 1.0% Yatalase, 2: 1.0% Cellulase “ONOZUKA” R-10, 3:
1.0% Cellulase “ONOZUKA” RS, 4: 1.0% Yatalase and 1.0%
Cellulase “ONOZUKA” R-10, 5: 1.0% Yatalase and 1.0% Cellulase
“ONOZUKA” RS, and 6: 1.0% Cellulase “ONOZUKA” R-10 and
1.0% Cellulase “ONOZUKA” RS. T. matsutake cells were digested
with 1.0% (w/v) enzymes at 37℃ for 1 h. Released GlcNAc
content was measured using methods reported by Reissig et al.27).
Results are given as the means ± SD.
Fig. 2. Effect of cell wall-degrading enzyme volume and incubation
time on released GlcNAc.
Error bars indicate standard deviation (± SD) of experiments
performed in triplicate.
Fig. 3. Standard curve showing the relationships between GlcNAc
concentration and T. matsutake mycelia dry weight.
composition determined above. When barley, rice bran,
and wheat bran were used as the substrate, a precipitate
formed after the Reissig reaction. However, after the
color development reaction, the precipitates were
removed by centrifugation and the absorbance value was
the same as that of the control. As shown in Table 1, the
production of GlcNAc remained within 5% of the control
in all solid-state media substrates. This result indicates
that the enzymatic digestion reaction and the Reissig
reaction were not inhibited by the solid-state media
substrates used to culture basidiomycete fungi.
3. Time course of T. matsutake mycelia biomass content
cultivated on various solid-state media
Fig. 4 shows the vegetative mycelial biomass of three
T. matsutake strains grown on five different ratios of
rolled barley and vermiculite media, as converted from
the measured values of GlcNAc content. Mycelial biomass
was measured every 7 days for a total of 35 days. Of the
media tested, rolled barley produced the fastest vegetative
mycelial growth, followed by rolled barley and vermiculite
with a mixed weight ratio of 2:1. Mycelial biomass of
T. matsutake Z-1, NBRC 30605, and strain No. 115 grown
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MUSHROOM SCIENCE AND BIOTECHNOLOGY
Table 1. GlcNAc by solid substrate additive
SubstrateGlcNAc
(μg/1 mg mycelium)GlcNAc
(μg/10 mg mycelium)
Control 14.1 ( 100%) 139.6 ( 100%)
10% Barley 13.8 (97.9%) 135.3 (96.9%)
10% Vermiculite 14.0 (99.3%) 139.3 (99.8%)
5% Barley and 5% Vermiculite 14.1 ( 100%) 138.3 (99.3%)
10% Sawdust from hardwood 13.6 (96.5%) 137.9 (98.7%)
10% Sawdust from softwood 13.8 (97.9%) 134.6 (96.4%)
10% Rice bran 13.7 (97.2%) 138.1 (98.9%)
10% Wheat bran 13.5 (95.7%) 135.3 (96.9%)
Enzyme solution was added to a final concentration of 1.0% Yatalase and 0.5% Cellulase “ONOZUKA” RS, and the total volume was adjusted to 10 mL. The enzyme Reactions were carried out at 37℃, 1,500 rpm for 1 h.
Fig. 4. Time course of three cultivated strains of Tricholoma matsutake grown on solid-state media.
Each bar indicates media composition as follows: black bar, 2:1 weight ratio of rolled barley and vermiculite medium;
diagonal stripe, 1:1 weight ratio of rolled barley and vermiculite medium; gray bar, 1:2 weight ratio of rolled barley and
vermiculite medium; dotted bar, rolled barley medium; and open bar, vermiculite medium. Values of T. matsutake mycelial
biomass on solid-state media were converted according to the GlcNAc content in media. Error bars indicate standard
deviations (± SD) of experiments performed in replicates of five.
on 2:1 rolled barley and vermiculite medium for 35 days
was estimated to be 215.1, 254.0, and 266.7 mg biomass
per flask, respectively. In contrast, mycelial biomass
grown on media composed of a 1:1 weight ratio and 1:2
weight ratio of rolled barley to vermiculite medium were
46% to 67% of that obtained for the strains grown on the
2:1 medium. Especially for the NBRC 30605 strain, the
differences between biomass obtained when grown on
rolled barley to vermiculite medium with a weight ratio
of 2:1 versus that grown on media with a ratio of 1:1
(129.8 mg biomass per flask) and 1:2 (109.4 mg biomass
per flask) were significant, at 1.96 and 2.32 times higher,
respectively, than for the 2:1 medium. As shown in Fig. 5,
for the cultivation of T. matsutake strain No. 115, the solid-
state medium composed of barley and vermiculite mixed
at a weight ratio of 2:1 allowed the T. matsutake mycelia
to spread not only across the surface but also throughout
the whole of the medium. Both visual observation
(Fig. 5) and measurement of GlcNAc concentration (Fig. 4)
demonstrated that a 2:1 weight ratio of rolled barley
to vermiculite was the optimum medium condition for
mycelial growth of T. matsutake.
Discussion
In order for a fungal species to form fruiting bodies
(mushrooms), large amounts of mycelia are needed either
to store the nutrients for the growth of the fruiting
bodies or to transport the nutrients28)
. Kitamoto et
al.29)
mentioned that to produce edible mushrooms by
microbial cultivation, spreading a sufficient amount of
mycelia in the culture medium is a prerequisite for the
formation of fruiting bodies. L. shimeji, which is able
to form fruiting bodies in artificial culture conditions,
shows good growth and apparent starch-degradation
160 Vol.26 No. 4
Fig. 5. Cultivated Tricholoma matsutake strain No. 115 grown on solid-state media.
A, vermiculite medium; B, rolled barley medium; C, 1:2 weight ratio of rolled barley and vermiculite medium, D; 1:1 weight
ratio of rolled barley and vermiculite medium and E, 2:1 weight ratio of rolled barley and vermiculite medium. Media were
cultured in 100 mL Erlenmeyer flasks at 24℃ and 70% humidity for 35 days.
ability compared to T. matsutake when cultivated on a
starch substrate30-33)
. In the present study, we examined a
method to estimate the mycelial biomass of T. matsutake
by measuring GlcNAc, which is a degradation product
of chitin. In addition, we established a solid-state
culture system using vermiculite and rolled barley as an
effective supporting material for artificial cultivation of
T. matsutake.
First, we determined the optimal reaction conditions
for enzymatic chitin degradation in T. matsutake mycelia.
Cellulase “ONOZUKA” R-10, which is used for the
preparation of T. matsutake protoplasts34-36)
, exhibited no
effect on GlcNAc production. In contrast, by combining
Cellulase “ONOZUKA” RS with Yatalase, the GlcNAc
production was increased about 1.4 times compared to
Yatalase alone. Cellulase “ONOZUKA” RS contains a
multi-component enzyme system with high cellulose
digestion activity (about three times higher hemicellulase
activity than Cellulase “ONOZUKA” R-10)37)
. Therefore, it
was suggested that chitin in the cell walls of T. matsutake
would degrade more efficiently if degradation of
hemicellulose was targeted. Chitin content in the mycelia
of T. matsutake, Flammulina velutipes38)
, and Pleurotus
ostreatus39)
were 1.3% - 1.5%, 1.0% - 1.5%, and 3.5% -
5.0%, respectively. Because other edible mushrooms
also contain chitin, mycelial biomass is considered to be
measurable by the same methods. In this case, enzymatic
mycelial digestion was not affected by the components of
the media. As shown in Table 1, results from the present
study suggest that quantification by enzymatic digestion
could be applied to other edible mushrooms because the
various medium components have no effect on GlcNAc
production.
Next, the T. matsutake mycelial biomass in various
solid-state media was determined under established
conditions. Of the media tested, rolled barley yielded the
fastest vegetative mycelial growth, followed by a mixture
with a weight ratio of 2:1 of rolled barley and vermiculite.
Kusuda et al.33)
tested the vegetative mycelial growth of
18 strains of T. matsutake on a PMML (Partly Modified
Matsutake Liquid) medium at 24℃ for 80 days and found
that among the T. matsutake strains tested, T. matsutake
produced the maximum weight of mycelia with an
average of 140.5 mg per flask. In contrast, we obtained
estimated mycelial biomass of T. matsutake Z-1, NBRC
30605 and strain no. 115 of 215.1, 254.0, and 266.7 mg
biomass per flask, respectively, after 35 days of cultivation
on a 2:1 weight ratio of rolled barley to vermiculite media.
These results suggest that T. matsutake could spread a
large amount of mycelia throughout the medium and
achieve stable growth in about 30 days on a solid medium
in which rolled barley and vermiculite were mixed at a
weight ratio of 2:1. These results also demonstrate that
it is possible to estimate the total amount of mycelial
biomass by measuring GlcNAc concentration following
enzymatic digestion of mycelia.
Fruiting bodies were reported to form when T.
matsutake was cultivated in sterilized soils supplemented
with nutrients, but they did not develop into mature
fruiting bodies22, 23)
. Inaba et al.24)
reported the artificial
cultivation of T. matsutake Z-1 strain at 24℃ for 120
days using culture bottles measuring 140 × 140 × 180
mm with induction of fruiting body development by
transferring bottles to 18℃. However, cultivation methods
on a larger scale are needed.
In the present study, it was demonstrated that solid-
state cultivation using rolled barley and vermiculite is
able to support rapid mycelial growth. Similar cultivation
methods are effective for the culture of L. shimeji,
which can form fruiting bodies saprophytically14-16)
.
In future studies, it will be necessary to evaluate not
only the scalability of this cultivation method but
also the quantity of the T. matsutake mycelial biomass
produced. Furthermore, by manipulating other variables
(temperature, chemical substances, and other stimuli)
for fruiting-body development, it may be possible to
successfully culture T. matsutake fruiting bodies under
artificial growth conditions.
Acknowledgement This work was supported by a
grant from the Strategic Research Foundation, Grant-
aided Project for Private Universities from Ministry of
Education, Culture, Sport, Science, and Technology (C),
S1512004, 2015-2017 MEXT, Japan.
和 文 摘 要
固体培地培養におけるマツタケ菌糸体
バイオマス定量法の開発
大沼広宜1)・原 健人1)・張正熙2)・白坂憲章2)・福田泰久2)*
1) 近畿大学大学院農学研究科
〒631-8505 奈良県奈良市中町 3327-204
2) 近畿大学農学部
〒631-8505 奈良県奈良市中町 3327-204
マツタケの人工栽培おいて短期間で菌糸体を蔓延させる
ための培地条件の構築が必要不可欠であるが,有利な菌床培
養法が確立されておらず,今日までに子実体発生のために必
要な菌床の作成に至っていない.本研究では,押麦とバーミ
キュライトを用いた固体培養系における最適なマツタケ菌
糸体培養法を検討した.また,菌糸体細胞壁中のキチン分解
産物である n-アセチルグルコサミン (GlcNAc) 指標とした固
体培地中のマツタケ菌糸体バイオマス量の測定法を確立し,
培地中の菌糸体バイオマス量を評価した.マツタケ乾燥菌糸
体の分解における酵素反応条件としては 1% Yatalase と 0.5
% Cellulase “ONOZUKA” RS 溶液が最適であった.続いて,
マツタケ Z-1, NBRC 30605, No. 115 株を供試し,押麦 : バー
161日本きのこ学会誌
MUSHROOM SCIENCE AND BIOTECHNOLOGY
ミキュライト= 2:1 (w/w) の培養条件において良好な生
育を示した.培養 35 日目において,それぞれ 215.1, 254.0,
266.7 mg biomass/flask に達した.また,押麦 : バーミキュ
ライト= 2:1 (w/w) が最適な培地条件であることが目視お
よび GlcNAc 定量値においても証明された.
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