See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/236112702

Submerged Cultivation of Mycelium with High Ergothioneine Content from the

Culinary-Medicinal King Oyster Mushroom Pleurotus eryngii (Higher
Basidiomycetes) and its Composition

Article  in  International Journal of Medicinal Mushrooms · April 2013

DOI: 10.1615/IntJMedMushr.v15.i2.40 · Source: PubMed

CITATIONS READS

7 608

5 authors, including:

Jeng-Leun Mau
NCHU
171 PUBLICATIONS   9,566 CITATIONS   

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Pharmacological Properties of Antrodia cinnamomea View project

All content following this page was uploaded by Jeng-Leun Mau on 09 February 2014.

The user has requested enhancement of the downloaded file.

 International Journal of Medicinal Mushrooms, 15(2): 153–164 (2013)

Submerged Cultivation of Mycelium with High

Ergothioneine Content from the Culinary-Medicinal
King Oyster Mushroom Pleurotus eryngii (Higher
Basidiomycetes) and its Composition
Chih-Hung Liang,1 Ling-Yi Huang,2 Kung-Jui Ho,2 Shin-Yi Lin,2,3 & Jeng-Leun Mau2,3,*
1
Department of Food Science, Tunghai University, Taichung, Taiwan, Republic of China; 2Department of Food
Science and Biotechnology, Biotechnology Center, National Chung Hsing University, Taichung, Taiwan, Republic
of China; 3National Chung Hsing University–University of California, Davis Plant and Food Biotechnology Program,
Biotechnology Center, National Chung Hsing University, Taichung, Taiwan, Republic of China.

*Address all correspondence to: Jeng-Leun Mau, Department of Food Science and Biotechnology, National Chung-Hsing University, 250
Kuokuang Road, Taichung 40227, Taiwan, ROC; Tel.: +886-4-2285-4313; Fax: +886-4-2287-6211; jlmau@dragon.nchu.edu.tw.

ABSTRACT: The culinary-medicinal king oyster mushroom, Pleurotus eryngii, was used to produce mycelia with
high ergothioneine content using a one-factor-at-a-time method. The optimal culture conditions for mycelia harvested
at day 14 were 25°C, 10% inoculation rate, 2% glucose, 0.5% yeast extract, and no adjustment to the initial pH
value. With histidine or amino acid mix added, biomasses and the ergothioneine content of mycelia were higher than
those of the control. The ergothioneine content of mycelia harvested at days 16–20 were higher than that of mycelia
harvested at day 14. In addition, the ergothioneine content of mycelia from the fermentor (5.84–5.76 mg/g) was much
higher than that of mycelia from the shaken flask (4.93–5.04 mg/g). Mycelia with high ergothioneine content showed
a profile of proximate composition similar to that of regular mycelia but lost its characteristic umami taste. Overall,
mycelia high in ergothioneine could be prepared by optimal culture conditions, the addition of precursors, prolonged
harvest, and aeration in the fermentor.

KEYWORDS: ergothioneine, Pleurotus eryngii, mushroom mycelia, histidine

ABBREVIATIONS: 5′-AMP, 5′-adenosine monophosphate; 5′-GMP, 5′-guanosine monophosphate; 5′-IMP,

5′-inosine monophosphate; 5′-XMP, 5′-xanthine monophosphate; EUC, equivalent umami concentration; MSG,
monosodium glutamate; RS, reducing sugar

I. INTRODUCTION mushroom was found to have effective antioxidant

The culinary-medicinal king oyster mushroom,
Pleurotus eryngii (DC: Fr.) Quél. (Pleurotaceae,
higher Basidiomycetes), is native to southern Eu-
rope and areas of central Asia and North Africa
and is commercially available in Taiwan. This ed-
ible mushroom was introduced to Taiwan around
1993 and then became commercially available. Its
consistency, pleasant aroma, culinary qualities, and
longer shelf life make it preferable to other species
of Pleurotus.1,2
The proximate composition, volatile flavor
compounds, and nonvolatile taste components of
the king oyster mushroom had been studied.2 The
activity, reducing power, radical scavenging ability,
and chelating ability on ferrous ions.3 In addition,
the acidic glycosphingolipid from P. eryngii stimu-
lated the immune system.4 This king oyster mush-
room contained a large amount of l-ergothioneine,
a precious amino acid.5,6 Ergothioneine was found
to be an excellent antioxidant in vivo and a cellular
protector against oxidative damage.7–9 This com-
pound is also associated with autoimmune disorders
such as rheumatoid arthritis and Crohn’s disease.10,11
Furthermore, ergothioneine showed effective intrin-
sic antihydroxyl, antiperoxyl, and antiperoxynitrite
radical antioxidant activity.12
The cultivation of P. eryngii takes 1 to 2 months

1045-4403/13/$35.00 © 2013 Begell House, Inc. www.begellhouse.com 153

154
to produce fruiting bodies; the colonization of my-
celia in sawdust takes a long time. However, the
submerged culture requires only a short time to
produce mycelia usable on a commercial scale.
Accordingly, mushroom mycelia have become an
alternative or substitute mushroom product. Al-
though mycelia are not used directly as food, they
could be used as a food-flavoring material and
food ingredient or as a nutritional supplement. To
provide the beneficial effect of this mushroom in
the form of mycelia with a large amount of ergot-
hioneine, a solid-state fermented product has been
prepared.13 However, mycelia with a large amount
of ergothioneine in submerged culture have not
been studied.
Accordingly, our objective was to prepare
P. eryngii mycelia with high ergothioneine con-
tent by adding its precursors (histidine, cysteine,
methionine, or all three) and monitoring the bio-
mass and ergothioneine content during submerged
cultivation. As an ingredient of food, the proxi-
mate composition and taste components of mush-
room mycelia may correlate with the product’s ac-
ceptability and is of great importance for further
application. Therefore, the proximate composition,
umami amino acids, umami 5′-nucleotides, and
equivalent umami concentration (EUC) of regular
mycelia and mycelia high in ergothioneine also
were evaluated.
II. MATERIALS AND METHODS
A. Mycelia
Mycelia of P. eryngii were obtained from Q-Yo
Bio-Technology Farm, Pusin, Chunghua Country,
Taiwan, which is the major mushroom farm pro-
ducing the king oyster mushroom in Taiwan. The
mycelia were maintained on potato dextrose agar
at 25°C. For the production of regular mycelia, the
culture was inoculated at the rate of 10% into a
250-mL flask containing 100 mL of liquid medium
and incubated at 25°C and 125 rpm. The liquid me-
dium contained the following: 20 g/L of glucose, 5
g/L of yeast extract, 2 g/L of ammonium sulfate,
0.5 g/L of potassium dihydrogen phosphate, 0.5
Liang et al.
g/L of dipotassium sulfate, and 0.5 g/L of magne-
sium sulfate heptahydrate. After 14 days of incu-
bation, the mycelia were harvested and washed 5
times with deionized water, then freeze-dried as
regular mycelia.
B. Culture Conditions
The optimal culture conditions for mycelia with
the highest ergothioneine content were performed
using a one-factor-at-a-time method. The follow-
ing 6 factors were studied: (1) temperature (20, 25,
or 30°C); (2) inoculation rate (5, 10, 15, or 20%);
(3) initial pH value adjusted with 3 N hydrochloric
acid or sodium hydroxide solution (6.0, 7.0, 8.0,
9.0, or 10.0) and control (pH 6.3–6.8); (4) supple-
mented 2% carbon source (glucose [the control],
fructose, lactose, maltose, or sucrose); (5) supple-
mented 0.5% nitrogen source (yeast extract [the
control], beef extract, casamino acid, peptone, or
tryptone); and (6) 4 mM amino acid addition (none
[the control], histidine, methionine, cysteine, histi-
dine at day 7, methionine at day 7, or cysteine at day
7). After the study of 4 mM amino acid addition,
the trials of amino acid addition were performed
further as follows: (1) the addition of each precur-
sor amino acid (histidine, methionine, or cysteine)
before inoculation at a concentration of 0.1, 1.0,
4.0, 8.0, or 12.0 mM, and (2) the addition of 4 mM
histidine or amino acid mix (4 mM histidine, 1 mM
cysteine, and 1 mM methionine) at days 0, 3, 5,
7, or 9. After 14 days of incubation, mycelia were
harvested, freeze-dried, and weighed, and their er-
gothioneine content was determined.
C. Growth Curve of Submerged Culture
After the optimal culture conditions had been ob-
tained, the growth curve of mycelia in a shaken
flask was studied. The culture was inoculated at
the rate of 10% into a 250-mL flask containing 100
mL of liquid medium (as described earlier) and
incubated at 25°C and 125 rpm. At day 7, 4 mM
histidine was added. During mycelial growth in
submerged culture, an aliquot (2 mL) of the cul-
ture was drawn at 2-day intervals for the analyses
International Journal of Medicinal Mushrooms
Pleurotus Mycelium With High Ergothioneine Content
of biomass, residual sugar, and ergothioneine. In
addition, for mass production of mycelia, mechan-
ically stirred fermentation was performed in an
MG-1000SL fermentor with a 10-L capacity (7-L
working volume; Micro-Giant Technology Co.,
Taichung, Taiwan). The fermentation conditions
were set at 25°C with an aeration rate of 1 vvm and
an agitation speed of 150 rpm. An inoculum rate of
10% was added to the liquid medium. At day 7, 4
mM histidine was added. During fermentation, an
aliquot (20 mL) of the culture was drawn at 2-day
intervals for the analyses of biomass, residual sug-
ar, and ergothioneine. After 20 days of fermenta-
tion, the mycelia in the fermentor were harvested
and washed 5 times with deionized water, then
freeze dried as high-ergothioneine mycelia.
D. Chemicals
Beef extract, peptone, potato dextrose agar, tryp-
tone, and yeast extract were purchased from Difco
Laboratories (Sparks, MD, USA). 3,5-Dinitro-
salicylic acid, 5′-adenosine monophosphate (5′-
AMP), 5′-guanosine monophosphate (5′-GMP),
5′-inosine monophosphate (5′-IMP), 5′-xanthine
monophosphate (5′-XMP), amino acid standards,
ergosterol, ergothioneine, fructose, glucose, lac-
tose, maltose, sucrose, and triethylamine were pur-
chased from Sigma Chemical Co. (St. Louis, MO,
USA). Ammonium sulphate and casamino acid
were obtained from Wako Pure Chemical Indus-
tries (Osaka, Japan).
Potassium dihydrogen phosphate, dipotas-
sium hydrogen phosphate, sodium phosphate, and
acetonitrile were purchased from J. T. Baker Co.
(Philipsburg, NJ, USA). Methanol was obtained
from Tidia Co. (Fairfield, OH, USA). Hydrochlo-
ric acid and sodium hydroxide were purchased
from Union Chemical Works (Hsinchu, Taiwan).
Other chemicals used in the experiments were of
analytical grade.
E. Ergosterol and Ergothioneine Assay
Ergosterol and ergothioneine of mycelia were
extracted and analyzed according to methods de-
Volume 15, Number 2, 2013
155
scribed previously.6,14 The high-performance liquid
chromatograph consisted of a Shimadzu LC-10AT
pump (Tokyo, Japan), a Shimadzu SPD-10A UV-
VIS detector, and an SISC chromatography data
system version 2.1 (Scientific Information Ser-
vice Corp., Taipei, Taiwan) connected to a com-
puter. The columns (4.6 × 250 mm) used were Li-
Chrospher 100 RP-18 (5 μm; Merck, Darmstadt,
Germany) and Luna PFP (2) (5 mm; Phenomenex,
Torrance, CA, USA). Ergosterol and ergothioneine
were detected at 282 and 254 nm, respectively.
The mobile phases were isocratic methanol and 50
mM sodium phosphate buffer (3% acetonitrile and
0.1% triethylamine), and the flow rates were 1.0
and 1.2 mL/min for ergosterol and ergothioneine,
respectively. Ergosterol and ergothioneine were
quantified by the calibration curve of each authen-
tic compound.
F. Proximate Analysis
The proximate composition of regular and high-
ergothioneine mycelia included ash, fat, protein,
and carbohydrate. Moisture, crude ash, crude fat,
and crude protein were determined according to
the methods of the Association of Official Ana-
lytical Chemists.15 A nitrogen conversion factor
of 4.38 was used to calculate crude protein.16 The
carbohydrate content was calculated by subtract-
ing the contents of ash, fat, and protein from 100%
of dry matter. Carbohydrates could be subdivided
into reducing sugars (RSs) and dietary fiber, which
consists of soluble polysaccharides and crude fiber.
RSs were determined using the 3,5-dinitrosalicylic
acid method.17 Crude fiber was determined accord-
ing to the methods of the AOAC.15 Thus, soluble
polysaccharide content was calculated by subtract-
ing the contents of RS and crude fiber from the car-
bohydrate content.
G. Assays of Umami Amino Acids,
5′-Nucleotides, and EUC
Free umami amino acids and umami 5′-nucleo-
tides of regular and high-ergothioneine mycelia
were extracted and analyzed according to meth-
156
ods described previously.18,19 Each umami amino
acid and 5′-nucleotide were identified using the
authentic amino acid and 5′-nucleotide, respec-
tively, and then quantified by the calibration curve
of the authentic compound. EUC (grams monoso-
dium glutamate [MSG]) per 100 g of sample was
the concentration of MSG equivalent to the umami
intensity given by the mixture of MSG and the
5′-nucleotide and was represented by the follow-
ing addition equation20:
Y = Σaibi + 1218 (Σaibi) (Σajbj),
where Y is the EUC of the mixture in terms of
grams MSG per 100 g; ai is the concentration of
each umami amino acid (grams per 100 g); aj is the
concentration of each umami 5′-nucleotide (grams
per 100 g); bi is the relative umami concentration
for each umami amino acid to MSG (glutamic
acid, 1; aspartic acid, 0.077); bj is the relative uma-
mi concentration for each umami 5′-nucleotide to
5′-IMP (5′-IMP, 1; 5′-GMP, 2.3; 5′-XMP, 0.61; and
5′-AMP, 0.18); and 1218 is a synergistic constant
based on the concentration of grams per 100 g
used.
H. Statistical Analysis
Every trial for the study of optimal culture condi-
tions was conducted in triplicate, and each assay
also was determined in triplicate. Experimental
data are expressed as mean ± standard error and
were subjected to an analysis of variance for a
completely random design to determine the Fish-
er’s least significant difference (at the level of α
= 0.05) using SAS software (SAS Institute Inc.,
Cary, NC, USA).
III. RESULTS AND DISCUSSION
A. Optimization of Culture Conditions
According to the preliminary studies, the culture
was inoculated at the rate of 10% and incubated at
25°C and 125 rpm, and the mycelia harvested at
day 14 showed the highest ergothioneine content.
Therefore, in the following one-factor-at-a-time
Liang et al.
studies, mycelia were harvested at day 14. The cul-
ture incubated at 20°C showed the most biomass
but the least ergothioneine content (Table 1). The
temperature for the highest ergothioneine content
was 25°C. The culture also was incubated at 35°C,
but no mycelial growth was found. It seems that P.
eryngii mycelia could not tolerate the incubation
temperature of 35°C. Higher inoculation rates gave
rise to higher mycelial biomass, and the biomasses
from inoculation rates of 10% and 15% were com-
parable. Therefore, the inoculation rate for further
study was 10%.
The cultures with an initial pH value of 6 and
the control (pH 6.3–6.8) showed the most biomass,
whereas the highest ergothioneine contents were
found in the cultures with initial pH values of 7
and 8 and the control (Table 1). Accordingly, the
initial pH value was not adjusted because the con-
trol showed the highest biomass and ergothioneine
content. Glucose as the carbon source showed the
largest biomass and most ergothioneine content.
Therefore, the optimal carbon source used for my-
celial growth was glucose. Three nitrogen sourc-
es—yeast extract, beef extract, and peptone—gave
rise to the highest amount of biomass, whereas
only tryptone resulted in mycelia with the highest
ergothioneine content. Considering both biomass
and ergothioneine content, yeast extract was used
as the nitrogen source in the subsequent experi-
ments.
Using a one-factor-at-a-time method for 14
days of incubation, the optimal culture conditions
for mycelia with the highest ergothioneine content
were determined as follows: temperature of 25°C,
inoculation rate of 10%, 2% glucose, 0.5% yeast
extract, and no adjustment to the initial pH value.
B. Amino Acid Addition
Askari and Melville21 mentioned that ergothio-
neine could be biosynthesized by fungi using 3
amino acids as precursors: histidine, cysteine, and
methionine. Melville et al.22 stated that histidine
contained an imidazole ring, which contributed to
the molecular structure of ergothioneine, and that
cysteine and methionine could provide sulfhydryl
International Journal of Medicinal Mushrooms
Pleurotus Mycelium With High Ergothioneine Content 157

TABLE 1. Biomass and Ergothioneine Content of Pleurotus eryngii Mycelia at

Various Submerged Conditions
Ergothioneine Content
Conditions Biomass (g/L) (mg/g dry weight)
Temperature (°C)
 20 11.67 ± 0.23 a 1.19 ± 0.06 c
 25 8.03 ± 0.06 b 3.72 ± 0.16 a
 30 6.36 ± 0.09 c 1.89 ± 0.06 b
Inoculation rate (%)
 5 9.31 ± 0.09 c 1.63 ± 0.11 b
 10 9.77 ± 0.31 b 2.79 ± 0.29 a
 15 9.78 ± 0.12 b 1.03 ± 0.01 c
 20 10.53 ± 0.16 a 0.97 ± 0.03 c
Initial pH value
  6.3–6.8 (Control) 10.36 ± 0.22 a 2.07 ± 0.01 a
 6 10.37 ± 0.05 a 1.52 ± 0.02 b
 7 9.56 ± 0.18 b 2.17 ± 0.03 a
 8 8.89 ± 0.16 c 2.21 ± 0.20 a
 9 7.14 ± 0.23 d 1.76 ± 0.04 b
 10 7.78 ± 1.36 d 0.69 ± 0.05 c
Carbon source (2%)
  Glucose (control) 9.73 ± 0.17 a 3.47 ± 0.11 a
 Fructose 8.96 ± 0.04 b 3.08 ± 0.08 b
 Lactose 4.02 ± 0.29 c 1.81 ± 0.01 c
 Maltose 3.67 ± 0.15 c 1.99 ± 0.24 c
 Sucrose 3.50 ± 0.14 c 1.27 ± 0.04 d
Nitrogen source (0.5%)
  Yeast extract (control) 10.56 ± 0.29 a 2.37 ± 0.05 b
  Beef extract 10.44 ± 0.27 a 1.68 ± 0.04 d
  Casamino acid 8.11 ± 0.23 c 2.37 ± 0.04 b
 Peptone 10.44 ± 0.14 a 1.95 ± 0.11 c
 Tryptone 8.96 ± 0.36 b 2.65 ± 0.06 a
Amino acid (4 mM)
 Control 10.19 ± 0.13 b 2.28 ± 0.08 c
 Histidine 11.47 ± 0.25 a 3.46 ± 0.07 b
 Methionine 10.23 ± 0.07 b 2.01 ± 0.04 d
 Cysteine 9.83 ± 0.39 b 1.80 ± 0.08 d
  Histidine at day 7 a
11.72 ± 0.04 a 3.93 ± 0.01 a
  Methionine at day 7a 9.67 ± 0.08 b 2.35 ± 0.12 c
  Cysteine at day 7a 8.69 ± 0.23 c 2.49 ± 0.06 c
Each value is expressed as mean ± standard error (n = 3). Means with same letter
within a column of a specific condition are not significantly different (P > 0.05).
a
Each amino acid was added to the medium at day 7.

and methyl groups, respectively. Therefore, the optimal conditions described above.
culture was added with 4 mM of the respective With histidine added at days 0 and 7, the
precursor at day 0 or 7 and incubated under the culture showed higher biomass and ergothioneine

Volume 15, Number 2, 2013

 158 Liang et al.

content than the control (Table 1). The biomasses

of mycelia with histidine added at days 0 and 7
were similar, but the ergothioneine content of my-
celia was higher when histidine was added at day
7. It seems that the addition of methionine had no
effect on the enhancement of the biomass and er-
Ergothioneine (mg/g)

gothioneine content of mycelia, whereas cysteine

addition showed an adverse effect on the mycelial
biomass. The culture with methionine or cysteine
added at day 0 showed less ergothioneine content,
whereas those with methionine or cysteine added
at day 7 showed more ergothioneine content.
Lee et al.23 studied the enhancement of ergot-
hioneine production in Ganoderma neo-japonicum
mycelia by adding methionine to the medium and
found that at 16 mM methionine, ergothioneine
content was accumulated but mycelial growth was
inhibited. However, at 4 mM methionine, the er-
gothioneine content of mycelia was effectively in-
creased.23 Therefore, the amount of various amino
Ergothioneine (mg/g)

acids added at day 7 was studied.

With histidine added at day 7, the mycelial
biomasses increased from 11.39 to 13.97 g/L for
0.1–12 mM histidine, higher than the control (no
addition) of 10.19 g/L (Fig. 1A). The ergothioneine
contents of mycelia with histidine added at day 7
were 2.71–3.42 mg/g dry weight, with a peak at 4
mM. With cysteine added at day 7, the biomasses
of mycelia with 0.1–1 mM cysteine were 9.90–
10.08 g/L, similar to that of the control, but the
mycelial biomasses decreased as larger amounts
(4–12 mM) of cysteine were added (Fig. 1B). In
addition, the biomasses and ergothioneine contents
of mycelia with methionine added at day 7 were in
Ergothioneine (mg/g)

the range of 7.89–8.92 g/L and 2.45–2.84 mg/g, re-

FIGURE 1. Biomass and ergothioneine content of Pleu-
rotus eryngii mycelia in submerged cultivation with the
addition of histidine (A), cysteine (B), and methionine
(C) at various concentrations.
spectively (Fig. 1C). It is obvious that the optimal
concentration of added histidine was 4 mM; higher
concentrations resulted in a larger biomass but less
ergothioneine content. The addition of methionine
and cysteine alone did not increase the ergothio-
neine content but did decrease the biomass, reveal-
ing that histidine was a key precursor responsible
for higher mycelial biomass and ergothioneine
content.
The biomasses of mycelia with 4 mM histidine
added at days 3–9 were 10.19–10.85 g/L, higher

International Journal of Medicinal Mushrooms

Pleurotus Mycelium With High Ergothioneine Content 159

Ergothioneine (mg/g)
Ergothioneine (mg/g)

FIGURE 2. Biomass and ergothioneine content of Pleurotus eryngii mycelia in submerged cultivation with the ad-
dition of 4 mM histidine (A) and amino acid mix (B) at various days. Amino acid mix consisted of 4 mM histidine, 1
mM cysteine, and 1 mM methionine.

Volume 15, Number 2, 2013

160
than the control (no addition) of 9.45 g/L (Fig. 2A).
The ergothioneine contents of mycelia with his-
tidine added at days 3–7 (2.84–3.10 mg/g) were
similar, higher than those of mycelia with histidine
added at day 9 (2.55 mg/g) and the control (1.68
mg/g). It seems that with histidine added at days
3–7, the biomass and ergothioneine content of my-
celia were significantly higher. Furthermore, with
an amino acid mix added at days 0–9, the mycelial
biomasses were 9.60–10.68 g/L, similar to the bio-
masses with 4 mM histidine added at days 3–9 and
higher than the control (Fig. 2B). It is surprising that
the ergothioneine contents of mycelia with amino
acid mix added at days 0–7 were 3.71–4.70 mg/g,
higher than that with amino acid mix added at day
9 (2.85 mg/g) and with histidine added at days 3–7.
The ergothioneine contents of mycelia with the ad-
dition of amino acid mix were (in descending order)
4.70 mg/g (day 5) > 4.28 mg/g (day 3) > 3.77 mg/g
(day 0) ~ 3.71 mg/g (day 7) > 2.85 mg/g (day 9).
Using the ergothioneine content of the control
as 100% (1.68 mg/g), that of mycelia with 4 mM
histidine added at day 7 was 185% (3.10 mg/g);
that of mycelia with amino acid mix added at day 5
was 280% (4.70 mg/g). Since amino acid mix con-
tained 3 precursors, higher ergothioneine content
was expected. In addition, Lee et al.23 found that
the addition of 2 mM histidine, 2 mM cysteine, and
2 mM methionine resulted in higher ergothioneine
content in G. neo-japonicum mycelia than the ad-
dition of a single amino acid. It is concluded that
higher ergothioneine content in mycelia could be
achieved by means of optimal culture conditions
and the addition of precursors.
C. Submerged Growth Curve
In the shaken flask, as a result of mycelial growth
the residual sugar content decreased fast from day
2 to day 8 (20.00 to 1.68 g/L) and then depleted
at day 12 (<0.01 g/L) (Fig. 3A). The biomasses
reached a plateau of 12.30–12.54 g/L at days 4–8
and then decreased constantly to 8.61 g/L at day
20. This phenomenon might be due to the autolysis
that occurred in the center of mycelial pellets, as
described by Humfeld and Sugihara.24 The ergothi-
Liang et al.
oneine accumulated in mycelia to reach a plateau
of 4.93–5.04 mg/g at days 16–20. Ergothioneine
seemed to be a secondary metabolite; its accumu-
lation was not affected by mycelial autolysis and
peaked several days after the maximum biomass
was reached.6 With optimal culture conditions and
prolonged incubation, the ergothioneine content
of mycelia harvested at days 16–20 could be even
higher than that of mycelia harvested at day 14.
In the fermentor, as a result of mycelial growth
the RS content decreased fast from day 2 to day
10 (20.00–1.45 g/L) and then depleted at day 14
(<0.01 g/L) (Fig. 3B). The biomass reached a peak
of 12.60 g/L at day 10 and then decreased constant-
ly to 10.35 g/L at day 20. Similarly, the autolysis
of mycelial pellets occurred but was less evident.
It is surprising that ergothioneine accumulated to
reach a plateau of 5.84–5.76 mg/g at days 18–20,
higher than that in the shaken flask. The additional
aeration was the main difference between the fer-
mentor and the shaken flask. Additional aeration
might be the reason for lesser autolysis of mycelia
and higher ergothioneine content in the fermentor.
The mycelia harvested from the fermentor at day
20 were used as high-ergothioneine mycelia during
further study.
Before the study of optimal culture conditions
using a one-factor-at-a-time method, the highest
ergothioneine content was found in mycelia har-
vested at day 14. Using the optimal culture con-
ditions and the addition of 4 mM histidine at day
7, the highest ergothioneine content was found in
mycelia harvested at days 18–20. If amino acid
mix was added at day 5, the ergothioneine content
in mycelia harvested at days 18–20 would be high-
er than 5.84–5.76 mg/g. To maximize the ergothio-
neine content of mycelia in the scale-up fermentor,
the submerged culture was incubated under the op-
timal culture conditions described above with the
addition of 4 mM histidine at day 7, and then my-
celia were harvested at day 20.
D. Proximate Analysis
The moisture content of regular mycelia was higher
than that of high-ergothioneine mycelia (Table 2).
International Journal of Medicinal Mushrooms
Pleurotus Mycelium With High Ergothioneine Content 161

FIGURE 3. Biomass, residual sugar, and ergothioneine content of Pleurotus eryngii mycelia in submerged cultiva-
tion (A) and in a 10-L fermentor under the conditions of 25°C, 1 vvm, 150 rpm, and 4 mM histidine (B).

Volume 15, Number 2, 2013

162 Liang et al.

TABLE 2. Proximate Composition of Mycelia and High-ergothioneine Mycelia of

Pleurotus eryngii
Content (%, dry weight)
Component Regular Mycelia High-Ergothioneine Mycelia
Moisture 7.63 ± 0.03 a 6.78 ± 0.04 b
Dry matter 92.37 ± 0.03 b 93.22 ± 0.04 a
Carbohydrate 68.61 ± 0.81 a 66.80 ± 0.63 b
RS 25.27 ± 0.17 b 30.90 ± 0.88 a
SP 24.88 ± 0.17 b 25.53 ± 0.38 a
Fiber 18.46 ± 0.60 a 10.37 ± 0.06 b
DF 43.34 ± 0.56 a 35.90 ± 0.32 b
Crude ash 3.48 ± 0.03 b 3.80 ± 0.05 a
Crude fat 4.16 ± 0.25 b 4.68 ± 0.12 a
Crude protein 23.75 ± 0.11 b 24.72 ± 0.44 a
Moisture and dry matter were presented on freeze-dried weight basis; others were presented on
a dry weight basis. Each value is expressed as mean ± standard error (n = 3). Means with same
letter within a row are not significantly different (P > 0.05).
RS, reducing sugar; SP, soluble polysaccharide; DF, dietary fiber = SPs + fiber.

Regular mycelia contained higher mounts of car- E. Contents of Ergosterol, Ergothioneine,

bohydrates, fiber, and dietary fiber, whereas high-
ergothioneine mycelia contained higher amounts of
RS, soluble polysaccharides, ash, fat, and protein.
However, the 2 kinds of mycelia showed a similar
profile in proximate composition, with some dif-
ferences. In other words, these 2 kinds of mycelia
provided similar energy and nutrient sources.
Umami Amino Acids, Umami
5′-Nucleotides, and EUC
High-ergothioneine mycelia contained higher (3.4-
fold) ergothioneine contents than regular mycelia
as expected (Table 3). In addition, high-ergothio-
neine mycelia harvested at day 20 contained more
ergosterol than regular mycelia harvested at day 14.

TABLE 3. Contents of Ergosterol, Ergothioneine, Umami Amino Acids, Umami 5′-Nucleotides, and
Equivalent Umami Concentration of Mycelia and High-Ergothioneine Mycelia of Pleurotus eryngii
Content
Regular Mycelia High-Ergothioneine Mycelia
Ergosterol (%, dry weight) 2.06 ± 0.17 b 2.38 ± 0.04 a
Ergothioneine (%, dry weight) 1.68 ± 0.11 b 5.76 ± 0.07 a
l-Aspartic acid (mg/g dry weight) 2.99 ± 0.30 a 0.68 ± 0.04 b
l-Glutamic acid (mg/g dry weight) 3.58 ± 0.08 a 0.30 ± 0.01 b
5′-AMP (mg/g weight) 1.24 ± 0.06 a 0.41 ± 0.01 b
5′-GMP (mg/g weight) 0.89 ± 0.04 a 0.29 ± 0.04 b
5′-IMP (mg/g weight) 0.82 ± 0.08 a 0.40 ± 0.01 b
5′-XMP (mg/g weight) ND 1.56 ± 0.03
EUC (g MSG/100 g dry weight) 144 ± 5 a 9±1b
Each value is expressed as mean ± standard error (n = 3). Means with same letter within a row are
not significantly different (P > 0.05).
5′-AMP, 5′-adenosine monophosphate; 5′-GMP, 5′-guanosine monophosphate; 5′-IMP, 5′-inosine
monophosphate; 5′-XMP, 5′-xanthosine monophosphate; EUC, equivalent umami concentration; MSG,
monosodium glutamate; ND, not detected.

International Journal of Medicinal Mushrooms

Pleurotus Mycelium With High Ergothioneine Content
The contents of umami amino acids and umami 5′-
AMP, 5′-GMP, and 5′-IMP of high-ergothioneine
mycelia were lower than those of regular mycelia.
However, high-ergothioneine mycelia contained
a higher amount of 5′-XMP. Unfortunately, EUC
of regular mycelia was much higher than that of
high-ergothioneine mycelia using the equation de-
rived from sensory evaluation.20 It seems that the
prolonged harvest caused the mycelia to lose its
characteristic umami taste.
As a food-flavoring material and food ingre-
dient or as a nutritional supplement, the contents
of umami amino acids and umami 5′-nucleotides
and EUC of mycelia were important to consum-
ers. However, the high-ergothioneine mycelia did
not show higher content of umami components or
higher EUC than regular mycelia. To use the high-
ergothioneine mycelia as a food-flavoring material
and food ingredient, the umami taste, brought by
nonvolatile compounds, would be another area of
investigation.
IV. CONCLUSIONS
The submerged culture was studied to produce P.
eryngii mycelia with high ergothioneine content
using a one-factor-at-a-time method. The optimal
culture conditions for mycelia harvested at day 14
were temperature of 25°C, inoculation rate of 10%,
2% glucose, 0.5%yeast extract, and no adjustment
to the initial pH value. With the histidine or amino
acid mix added, the biomasses and ergothioneine
content of mycelia were higher than those of the
control produced under optimal culture conditions.
With optimal culture conditions and prolonged in-
cubation, the ergothioneine content of mycelia har-
vested at days 16–20 could be even higher than that
of mycelia harvested at day 14. The ergothioneine
content of mycelia from the fermentor was much
higher than that of mycelia from the shaken flask.
Regular mycelia and high-ergothioneine mycelia
had a proximate composition. However, high-er-
gothioneine mycelia did not show its characteristic
umami taste. It is concluded that high-ergothioneine
mycelia could be prepared by means of optimal
culture conditions, the addition of precursors, pro-
Volume 15, Number 2, 2013
163
longed harvest, and the aeration in the fermentor.
ACKNOWLEDGMENTS
This study was supported by the National Sci-
ence Council, Republic of China (grant nos. NSC
97-2313-B-005-028-MY3 and NSC 101-2911-I-
005-301). The authors thank Mr. Shih-Wen Fang,
Q-Yo Bio-Technology Farm, for providing mush-
rooms.
REFERENCES
1. Chang ST, Wasser SP. The role of culionary-medicinal
msuhrooms on human welfare with pyramid model for
human health. Int J Med Mushr. 2012;14:95–134.
2. Mau JL, Lin YP, Chen PT, Wu YH, Peng JT. Flavor com-
pounds in king oyster mushrooms Pleurotus eryngii. J
Agric Food Chem. 1998;46:4587–91.
3. Tsai SY, Huang SJ, Lo SH, Mau JL. Antioxidant prop-
erties of several culinary-medicinal mushrooms during
postharvest storage. Int J Med Mushr. 2008;10:245–53.
4. Nozaki H, Itonori S, Sugita M, Nakamura K, Ohba K,
Suzuki A, Kushi Y. Mushroom acidic glycosphingo-
lipid induction of cytokine secretion from murine T
cells and proliferation of NK1.1 alpha/beta TCR-double
positive cells in vitro. Biochem Biophys Res Comm.
2008;373:435–39.
5. Dubost NJ, Beelman RB, Peterson D, Royse DJ. Identi-
fication and quantification of ergothioneine in cultivated
mushrooms by liquid chromatography–mass spectros-
copy. Int J Med Mushr. 2006;8:215–22.
6. Chen SY, Ho KJ, Liang CH, Tsai CH, Huang LY, Mau JL.
Preparation of culinary-medicinal king oyster mushroom
Pleurotus eryngii-fermented products with high ergothio-
neine content and their taste quality. Int J Med Mushr.
2012;14:85–93.
7. Aruoma OI, Spencer JPE, Mahmood N. Protection
against oxidative damage and cell death by the natu-
ral antioxidant ergothioneine. Food Chem Toxicol.
1999;37:1043–53.
8. Dubost NJ, Ou B, Beelman RB. Quantification of poly-
phenols and ergothioneine in cultivated mushrooms and
correlation to total antioxidant capacity. Food Chem.
2007;105:727–35.
9. Hartman PE. Ergothioneine as an antioxidant. Method
Enzymol. 1990;186:310–18.
10. Peltekova VD, Wintle RF, Rubin LA, Amos CI, Huang
Q, Gu X, Newman B, Oene MV, Cescon D, Greenberg
G, Griffiths AM, George-Hyslop PHS, Siminovitch
KA. Functional variants of OCTN cation transporter
genes are associated with Crohn disease. Nat Genet.
 164
2004;36:471–75.
11. Tokuhiro S, Yamada R, Chang X, Suzuki A, Kochi Y,
Sawada T, Suzuki M, Nagasaki M, Ohtsuki M, Ono M,
Furukawa H, Nagashima M, Yoshino S, Mabuchi A,
Sekine A, Saito S, Takahashi A, Tsunoda T, Nakamura
Y, Yamamoto K. An intronic SNP in a RUNX1 binding
site of SLC22A4, encoding an organic cation transport-
er, is associated with rheumatoid arthritis. Nat Genet.
2003;35:341–48.
12. Franzoni F, Colognato R, Galetta F, Laurenza I, Barsotti
M, Di Stefano R, Bocchetti R, Regoli F, Carpi A, Balba-
rini A, Migliore L, Santoro G. An in vitro study on the
free radical scavenging capacity of ergothioneine: com-
parison with reduced glutathione, uric acid and trolox.
Biomed Pharmacother. 2006;60:453–57.
13. Chen SY, Ho KJ, Hsieh YJ, Wang LT, Mau JL. Contents
of lovastatin, γ-aminobutyric acid and ergothioneine in
mushroom fruiting bodies and mycelia. LWT Food Sci
Technol. 2012;47:274–78.
14. Liang CH, Huang SJ, Tsai SY, Lee YL, Kuo HC, Wu
TP, Jian SY, Huang WL, Mau JL. Preparation of novel
culinary-medicinal mushroom products using solid-state
fermentation and their taste quality. Int J Med Mushr.
2009;11:141–56.
15. Association of Official Analytical Chemists. Official
Methods of Analysis. 15th ed. Washington (DC): AOAC;
1990.
16. Crisan EV, Sands A. Edible mushrooms: nutritional val-
View publication stats
Liang et al.
ue. In: Chang ST, Hayes WA, editors. The biology and
cultivation of edible mushrooms. New York: Academic
Press; 1978. p. 137–65.
17. James CS. Analytical chemistry of foods. London: Chap-
man & Hall; 1995. p. 124–25.
18. Mau JL, Chyau CC, Li JY, Tseng YH. Flavor compounds
in straw mushrooms Volvariella volvacea harvested
at different stages of maturity. J Agric Food Chem.
1997;45:4726–29.
19. Taylor MW, Hershey RA, Levine RA, Coy K, Olivelle
S. Improved method of resolving nucleotides by reverse-
phase high performance liquid chromatography. J Chro-
matogr. 1981;219:133–39.
20. Yamaguchi S, Yoshikawa T, Ikeda S, Ninomiya T. Mea-
surement of the relative taste intensity of some α-amino
acid and 5′-nucleotides. J Food Sci. 1971;36:846–49.
21. Askari A, Melville DB. The reaction sequence in ergo-
thioneine biosynthesis: hercynine as an intermediate. J
Biol Chem. 1962;237:1615–18.
22. Melville DB, Eich S, Ludwig ML. The biosynthesis of
ergothioneine. J Biol Chem. 1956;224:871–77.
23. Lee WY, Park EJ, Ahn JK. Supplementation of methio-
nine enhanced the ergothioneine accumulation in the
Ganoderma neo-japonicum mycelia. Appl Biochem Bio-
technol. 2009;158:213–21.
24. Humfeld H, Sugihara TF. The nutrient requirement of
Agaricus campestris growth in submerged culture. My-
cologia. 1952;44:605–20.
International Journal of Medicinal Mushrooms