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:

157日本きのこ学会誌

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

159日本きのこ学会誌

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．

References

1) Olsson, P A: Signature fatty acids provide tools for

determining of the distribution and interactions of mycorrhizal

fungi in soil. FFMS Microbiology Ecology, 29, 303-310 (1999)

2) Olsson, P A and Johansen, A: Lipid and fatty acid composition

of hyphae and spores of arbuscular mycorrhizal fungi at

different growth stages. Mycological Research, 104, 429-434

(1996)

3) Nylund, J E and Wallander, H: Ergosterol analysis as a

means of quantifying mycorrhizal biomass. In: Norris JR, Read

DJ, Varma AK, eds. Methods in microbiology, vol.24. London,

UK: Academic Press, 77-88 (1992)

4) Ekblad, A and Näsholm, T: Determination of chitin in fungi

and mycorrhizal roots by an improved HPLC analysis of

glucosamine. Plant and Soil, 178, 29-35 (1996)

5) Wallander, H, Nilsson, L O, Hagerberg, D and Baath, E:

Estimation of the biomass and seasonal growth of external

mycelium of ectomycorrhizal fungi in the field. New

Phytologist, 151, 753-760 (2001)

6) Olsson, P A and Johansen, A: Lipid and fatty acid composition

of hyphae and spores of arbuscular mycorrhizal fungi at

different growth stages. Mycological Research, 104, 429-434

(2000)

7) Ekblad, A, Wallander, H and Näsholm, T: Chitin and ergos-

terol combined to measure total and living biomass in ectomy-

corrhizae. New Phytologist, 138, 143-149 (1998)

8) Carrillo, C, Díaz, G and Honrubia, M: Improving the

production of ectomycorrhizal fungus mycelium in a bioreactor

by measuring the ergosterol content. Engineering in Life

Sciences, 4, 43-45 (2004)

9) Newell, S Y, Miller, J D and Fallon, R D: Ergosterol content

of saltmarsh fungi: effect of growth conditions and mycelial

age. Mycologia, 79, 688-695 (1987)

10) Gomi, K, Okazaki, N, Tanaka, T, Kumagai, C, Inoue, H, Iimura,

Y and Hara, S: Estimation of mycelial weight in rice-koji with

use of fungal cell wall lytic enzyme: Studies on application of

fungal cell wall lytic enzyme produced by Oerskcvia sp. CK.

(part II) (in Japanese). Journal of The Society of Brewing, Japan,

82, 130-133 (1987)

11) Fujii, F, Ozeki, K, Kanda, A, Hamachi, M and Nunokawa,

Y: A simple method for the determination of grown mycelial

content in rice-koji using commercial cell wall lytic enzyme,

Yatalase (in Japanese). Journal of The Society of Brewing,

Japan, 87, 757-759 (1992)

12) Bartnicki-Garcia, S: Cell wall chemistry, morphogenesis, and

taxonomy of fungi. Annual Review of Microbiology, 22, 87-

108 (1968)

13) Sietsma, J H and Wessels, J G H: Evidence for covalent

linkages between chitin and beta-glucan in a fungal wall.

Journal of General Microbiology, 114, 99-108 (1979)

14) Ohta, A: Production of fruit-bodies of a mycorrhizal fungus,

Lyophyllum shimeji, in pure culture. Mycoscience, 35, 147-151

(1994)

15) Watanabe, K, Kawai, M and Obatake, Y: Fruiting body

formation of Lyophyllum shimeji in pure culture. Mokuzai

Gakkaishi, 40, 879-882 (1994)

16) Yoshida, H and Fujimoto, S: A trial cultivation of Lyophyllum

shimeji on solid media (in Japanese). Transactions of Mycology

Society of Japan, 35, 192-195 (1994)

17) Ohta, A: Ability of ectomycorrhizal fungi to utilize starch

and related substrates. Mycoscience, 38, 403-408 (1997)

18) Wood, P: Cereal β-glucans in diet and health. Journal of

Cereal Science, 46, 230-238 (2007)

19) Evers, T and Millar, S: Cereal grain structure and development:

some implications for quality. Journal of Cereal Science, 36, 261-

284 (2002)

20) Izydorczyc, M S, Storsley, J, Labossiere, D, MacGregor, A W

and Rossnagel, B G: Variation in total and soluble β- glucan

content in hulless barley: Effects of thermal, physical, and

enzymatic treatments. Journal of Agricultural Food Chemistry,

48, 982-989 (2000)

21) Takasaki, F and Ohya, C: Studies on physical and chemical

properties and taste characteristics of Barley (in Japanese).

Tokyo Bunka Junior College Memoirs, 21, 1-9 (2004)

22) Ogawa, M and Hamada, M: Primordia formation of

Tricholoma matsutake (Ito et Imai) Sing. in pure culture,

Transactions of mycological Society of Japan, 16, 406-415 (1975)

23) Kawai, M and Ogawa, M: Studies on the artificial reproduc-

tion of Tricholoma matsutake (S. Ito et Imai) Sing. Ⅳ . Studies

on a seed culture and a trial for the cultivation on solid media

(in Japanese). Transactions of mycological Society of Japan,

17, 499-505 (1976)

24) Inaba, K, Yoshida, T, Takano, Y, Mayuzumi, Y, Mitsunaga, T

and Koshijima, T: An instance of the fruiting-body formation

of Tricholoma matsutake. Environmental Control in Biology,

33, 59-64 (1995)

25) Norkrans, B: Studies in growth and cellulolytic enzymes of

Tricholoma. Symbolae botanicae Upsalienses, 11, 1-126 (1950)

26) Kusuda, M, Ueda, M, Konishi, Y, Yamanaka, K, Terashita

and T, Miyatake, K: Effects of carbohydrate substrate on the

vegetative mycelial growth of an ectomycorrhizal mushroom,

Tricholoma matsutake, isolated from Quercus. Mycoscience, 48,

358-364 (2007)

27) Reissig, J L, Storminger, J L and Leloir, L F: A modified

colorimetric method for the estimation of N-acetylamino

sugars. Journal of Biological Chemistly, 217, 959-66 (1955)

28) Hirato, H and Kitamoto, Y: Effect of physico-chemical

characteristics of the culture substrate and nutritional condi-

tions on mycelial growth in Tricholoma matsutake (in Japanese).

Mushroom Science and Biotechnology, 2, 67-72 (1995)

29) Kitamoto, Y: Control of basidiocarp formation: I. Nutritional

environment for mycelial growth. Mushroom Science and

Biotechnology 5, 5-11 (1997)

30) Terashita, T, Kusuda, M, Matsukawa, S, Nagai, M, Yoshikawa,

K and Sakai, T: Production of extracellular amylase from

Tricholoma matsutake and its properties on starch hydrolysis

(in Japanese). Mushroom Science and Biotechnology, 8, 115-120

(2000a)

31) Terashita, T, Kitao, T, Nagai, M, Yoshikawa, K and Sakai, T:

Amylase productions during the vegetative mycelial growth

of Lyophyllum shimeji (in Japanese). Mushroom Science and

Biotechnology, 8, 115-120 (2000b)

32) Kusuda, M, Ueda, M, Konishi, Y, Matsuzawa, M, Shirasaka, N,

Nakazawa, M, Miyatake, K and Terashita, T: Characterization

162 Vol.26 No. 4

of extracellular glucoamylase from the ectomycorrhizal

mushroom Lyophyllum shimeji. Mycoscience, 45, 383-389 (2004)

33) Kusuda, M, Ueda, M, Miyatake, K and Terashita, T: Charac-

terization of the carbohydrase productions of an ectomycor-

rhizal fungus, Tricholoma matsutake. Mycoscience, 49, 291-297

(2008)

34) Abe, M, Umetsu, H, Nakai, T and Sasage, D: Regeneration

and fusion of mycelial protoplasts of Tricholoma matsutake.

Agricultural and Biological Chemistry, 46, 1955-1957 (1982)

35) Koga, D, Sueshige, N, Orikono, K, Utsumi, T, Tanaka, S,

Yamada, Y and Ide, A: Efficiency of chitinolytic enzymes in the

formation of Tricholoma matsutake protoplasts. Agricultural

and Biological Chemistry, 52, 2091-2093 (1988)

36) Matsumoto, M, Eguchi, F, Iijima, T and Higaki, M: Preparation

and regeneration of protoplasts from mycelia of Tricholoma

matsutake (in Japanese). Mokuzai Gakkaishi, 45, 413-418 (1999)

37) Beldman, G, Searle-Van Leeuwen, M, Rombouts, F and

Voragen, F: The cellulase of Trichoderma viride; Purification,

characterization and comparison of all detectable endogluca-

nases, exoglucanases and β-glucosidases. European Journal of

Biochemistry, 146, 301-308 (1985)

38) Terashita, T, Ueda, M, Yoshikawa, K, Kono, M and Shishiyama,

J: Changes in chitin and N-acetylglucosamine contents and

chitinolytic enzyme activities during the growth of Flammulina

velutipes fruit-bodies. Nippon Shokuhin Kagaku Kogaku Kaishi,

39, 827-835 (1992)

39) Yoshida, H, Sugahara, T and Hayashi, J: Changes in the

contents of carbohydrates and organic acids in fruitbodies

of Hiratake-Mushroom [Pleurotus ostreatus (Fr.) Quel] during

development and post-harvest storage (in Japanese). Nippon

Shokuhin Kogyo Gakkaishi, 34, 288-297 (1987)

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