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Nonvolatile Taste Components and Antioxidant Properties of Fruiting Body and

Mycelium with High Ergothioneine Content from the Culinary-Medicinal
Golden Oyster Mushroom Pleurotus c...

Article  in  International Journal of Medicinal Mushrooms · January 2016

DOI: 10.1615/IntJMedMushrooms.v18.i8.50

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 International Journal of Medicinal Mushrooms, 18(8): 689–698 (2016)

Nonvolatile Taste Components and Antioxidant

Properties of Fruiting Body and Mycelium with High
Ergothioneine Content from the Culinary-Medicinal
Golden Oyster Mushroom Pleurotus citrinopileatus
(Agaricomycetes)
Shin-Yi Lin,1,2,3 Shih-Chang Chien,4 Sheng-Yang Wang,5 & Jeng-Leun Mau1,2,3,*
1
Department of Food Science and Biotechnology, National Chung Hsing University (NCHU), Taichung, Taiwan,
R.O.C.; 2NCHU/UCD Plant and Food Biotechnology Center, NCHU, Taichung, Taiwan, R.O.C.; 3Agricultural
Biotechnology Center, NCHU, Taiwan, R.O.C.; 4The Experimental Forest Management Office, NCHU, Taichung,
Taiwan, R.O.C.; 5Department of Forestry, NCHU, Taichung, Taiwan, R.O.C.

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

ABSTRACT: Pleurotus citrinopileatus mycelium was prepared with high ergothioneine (Hi-Ergo) content and its
proximate composition, nonvolatile taste components, and antioxidant properties were studied. The ergothioneine
contents of fruiting bodies and Hi-Ergo and regular mycelia were 3.89, 14.57, and 0.37 mg/g dry weight, respectively.
Hi-Ergo mycelium contained more dietary fiber, soluble polysaccharides, and ash but less carbohydrates, reducing
sugar, fiber, and fat than regular mycelium. However, Hi-Ergo mycelium contained the smallest amounts of total sug-
ars and polyols (47.43 mg/g dry weight). In addition, Hi-Ergo mycelium showed the most intense umami taste. On the
basis of the half-maximal effective concentration values obtained, the 70% ethanolic extract from Hi-Ergo mycelium
showed the most effective antioxidant activity, reducing power, and scavenging ability, whereas the fruiting body
showed the most effective antioxidant activity, chelating ability, and Trolox-equivalent antioxidant capacity. Overall,
Hi-Ergo mycelium could be beneficially used as a food-flavoring material or as a nutritional supplement.

KEY WORDS: 5′-nucleotides, antioxidant property, equivalent umami concentration, ergothioneine, medicinal mush-
rooms, Pleurotus citrinopileatus, soluble sugar

ABBREVIATIONS: 5′-CMP, 5′-cytosine monophosphate; 5′-GMP, 5′-guanosine monophosphate; BHA, butylated

hydroxyanisole; DF, dietary fiber; DPPH, 1,1-diphenyl-2-picrylhydrazyl; EC50, half-maximal effective concentration;
Hi-Ergo, high ergothioneine; MSG, monosodium glutamate; RS, reducing sugar; SP, soluble polysaccharide; TEAC,
Trolox-equivalent antioxidant capacity

I. INTRODUCTION the form of fruiting bodies and mycelia have been

The culinary-medicinal golden oyster mushroom,
Pleurotus citrinopileatus Singer (Pleurotaceae,
Agaricomycetes), also known as yu-huang-mo in
Chinese and nireohma in Japanese, is a popular
species in many countries, especially in Asia.1 The
production of P. citrinopileatus includes a long cul-
tivation in a plastic bag for fruiting bodies to grow,
and a short submerged fermentation for mycelium
and fermentation filtrate to develop.2 The nutritional
value and taste components of P. citrinopileatus in
thoroughly studied.3 Many reports indicate that this
mushroom possesses biological and pharmacological
activities, such as antitumor activity, antigenotoxicity,
an antihyperglycemic effect, fatigue resistance, an
immunoenhancing effect, the ability to delay aging,
and antihyperlipidemic and antioxidant effects.4–7
In addition, Chen et al.8 studied the ergothioneine
content of the fruiting bodies and mycelia of 20
edible and medicinal mushroom species and found
that ergothioneine was detected in all samples. P.

1521-9437/16/$35.00 © 2016 Begell House, Inc. www.begellhouse.com 689

690
citrinopileatus, P. ostreatus (Korea), P. ostreatus
(Taiwan), and P. salmoneo-stramineus contained the
largest amounts of ergothioneine (2850.7, 1829.4,
1458.4, and 1245.0 mg/kg, respectively), whereas
among mycelia, P. eryngii contained the most
(1514.6 mg/kg). It seems that species of the genus
Pleurotus contained considerably large amounts of
ergothioneine.
Ergothioneine (2-mercaptohistidine trimeth-
ylbetaine) is a naturally occurring water-soluble
amino acid that is formed in some bacteria and
nonyeast fungi, but not in animals.9 For humans,
the best-known dietary sources of ergothione-
ine are mushrooms (0.1–1 mg/g) and meat. 10,11
Ergothioneine has recently attracted attention
because of its identification as the biogenic key
substrate of the organic cation transporter OCTN1
(the SLC22A4 gene).12 OCTN1 seems to have a piv-
otal protective role in monocytes, which have been
associated as a susceptible factor in the etiopathol-
ogy of autoimmune disorders such as rheumatoid
arthritis and Crohn’s disease.13,14 Some researchers
have documented that ergothioneine is an excellent
antioxidant in vivo and a cellular protector against
oxidative damage.15–17 Ergothioneine is an effective
intrinsic anti–hydroxyl radical, anti–peroxyl radical,
and anti–peroxynitrite radical antioxidant compared
with classic molecules with antioxidant capacity,
such as reduced glutathione, uric acid, and Trolox.18
When mushrooms are used as an ingredient of
food, the proximate composition and taste compo-
nents of the mushroom mycelium may correlate
with the food product’s acceptability and are of great
importance for further application. Our objective
was to prepare P. citrinopileatus mycelium with
high ergothioneine content by adding ergothione-
ine’s precursor, histidine. Thereafter, the proximate
composition and nonvolatile taste components—
including soluble sugars and polyols, umami amino
acids, and 5′-nucleotides—of regular mycelium
(low-ergothioneine mycelium), high-ergothione-
ine (Hi-Ergo) mycelium, and fruiting bodies were
studied. The antioxidant properties, including anti-
oxidant activity, reducing power, radical scavenging
ability, and ferrous ion–chelating ability, were also
evaluated.
Lin et al.
II. MATERIALS AND METHODS
A. Fruiting Bodies and Mycelia
Fruiting bodies and mycelia of P. citrinopileatus
were obtained from Q-Yo Bio-Technology Farm,
Pusin, Chunghua, Taiwan, which is one of the major
mushroom farms producing the golden oyster mush-
room in Taiwan. The mycelium was grown on potato
dextrose agar (Difco Laboratories, Sparks, MD)
plates for 7 days at 25°C, and then was stored at 4°C.
For the production of regular mycelium, the culture
was inoculated at a rate of 5% into a 250-mL flask
containing 95 mL of liquid medium and incubated
at 25°C and 125 rpm. The liquid medium contained
20 g/L glucose, 5 g/L yeast extract (Difco), 2 g/L
(NH4)2SO4, 0.5 g/L KH2PO4, 0.5 g/L K2HPO4 and 0.5
g/L MgSO4·7H2O. After 14 days of incubation, the
mycelium was harvested and washed 5 times with
deionized water, then freeze-dried as regular myce-
lium. The precursor (4 mmol/L histidine) was added
at day 7.19 After 22 days of incubation, the mycelium
was harvested and washed 5 times with deionized
water, then freeze-dried as Hi-Ergo mycelium.
B. Proximate Analysis
The proximate composition of fruiting bodies, reg-
ular mycelia, and Hi-Ergo mycelia included ash,
fat, protein, and carbohydrate. Moisture, crude ash,
crude fat, and crude protein were determined accord-
ing to the methods described by the Association of
Official Analytical Chemists.20 The nitrogen conver-
sion factor used for calculating crude protein was
4.38.21 The carbohydrate content was calculated
by subtracting the amounts of ash, fat, and protein
from 100% of dry matter. Carbohydrates can be
subdivided into reducing sugars (RSs) and dietary
fiber (DF), which consists of soluble polysaccha-
ride (SP) and crude fiber. RSs were determined
using the 3,5-dinitrosalicylic acid method.22 Crude
fiber was determined according to the methods of
the Association of Official Analytical Chemists.20
Thus SP content was calculated by subtracting the
amounts of RS and crude fiber from the amount of
carbohydrates.
International Journal of Medicinal Mushrooms
Taste Components and Antioxidant Properties of P. citrinopileatus Mycelium
C. Assays of Soluble Sugars and Polyols,
Free Umami Amino Acids, and
5′-Nucleotides
Soluble sugars and polyols were extracted and
analyzed as described by Tsai et al.23 Free umami
amino acids and 5′-nucleotides were extracted and
analyzed as described by Mau et al.24 and Taylor
et al., respectively.25 The high-performance liquid
chromatography system consisted of a Shimadzu
LC-10AT pump (Tokyo, Japan), a Rheodyne 7161
injector (Rohnert Park, CA), a 20-μL sample loop,
and a SISC chromatography data system (version
2.1; Scientific Information Service Corp., Taipei,
Taiwan). A Shimadzu RID-10A detector and a
Phase Sep-NH2 column (4.6 × 250 mm, 5 μm; Phase
Separation Inc., Norwalk, CT) were used for the
soluble sugar and polyol assay; the mobile phase
was acetonitrile/deionized water (85:15, v/v) at a
flow rate of 1.0 mL/min.
A Hitachi L-7485 fluorescence detector, with
fluorescence excitation at 340 nm and emission at
450 nm, and a LiChrospher 100 RP-18 column (5
μm; Merck, Darmstadt, Germany) were used for
the free amino acid assay; the mobile phases were
50 mmol/L sodium acetate (pH 5.7) containing 5%
tetrahydofuran (phase A), deionized water (phase
B), and methanol (phase C) at a flow rate of 1.2 mL/
min. The gradients were (A:B:C) 80:0:20 (v/v/v) to
33:0:67 for 0–38 minutes, 0:33:67 for 38–40 min-
utes, and 0:100:0 for 40–43 minutes.
A Hitachi L-4000 ultraviolet detector and a
LiChrospher 100 RP-18 column were used for the
5′-nucleotide assay. The mobile phase was 500 mmol/L
KH2PO4/H3PO4 (pH 4.3) at a flow rate of 1 mL/min;
ultraviolet detection was set at 254 nm. Each sugar
or polyol, umami amino acid, and 5′-nucleotide was
identified using the authentic compound and quanti-
fied by its respective calibration curve.
D. Ergosterol and Ergothioneine Assays
Ergosterol and ergothioneine of the mycelia and
fruiting bodies were extracted and analyzed accord-
ing to methods described by Liang et al.26 and Chen
et al.27 Freeze-dried powder (1 g) was added to 20 mL
Volume 18, Issue 8, 2016
691
extracting solution (10 mmol/L 1,4-dithiothreitol,
100 mmol/L betaine, and 100 mmol/L 2-mercapto-
1-methylimidazole in 70% ethanol) and vortexed for
90 seconds. After 4 mL of 10 g/L sodium dodecyl
sulfate solution was added, the mixture was cen-
trifuged at 25°C and 3000 g for 10 minutes. The
supernatant was then rotary evaporated at 40°C to 5
mL and filtered through a 0.45-μm cellulose acetate
nonsterile filter. A Shimadzu SPD-10A ultraviolet-
visible detector was used. The Li-Chrospher 100
RP-18 (4.6 × 250 mm) and Luna PFP(2) columns
(5 mm; Phenomenex, Torrance, CA) were used,
and ergosterol and ergothioneine were detected at
282 and 254 nm, respectively. The mobile phases
were isocratic methanol and 50 mmol/L sodium
phosphate buffer (3% acetonitrile and 0.1% trieth-
ylamine), 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 authentic compound.
E. Preparation of Extracts
For ethanolic extraction, a subsample (10 g) from
3 types of products was extracted with 100 mL
of 75% ethanol at 25°C at 150 rpm for 24 hours
and filtering through Whatman No. 1 filter paper.
The residue was then extracted with 2 additional
100-mL portions of ethanol, as described above.
The combined ethanolic extracts were then rotary
evaporated at 40°C to dryness. The dried extracts
were used directly for analysis of antioxidant com-
ponents or redissolved in ethanol to a concentration
of 50 mg/mL and stored at 4°C for later use.
F. Determination of Antioxidant
Properties
Antioxidant activity was determined by the con-
jugated diene method.28 The antioxidant activity
is the ability of the extracts from 3 types of prod-
ucts to inhibit the peroxidation of linoleic acid,
in which the double bond is converted into con-
jugated diene. Reducing power was determined
according to the method describe by Oyaizu.29
The reducing power is the ability of the extracts
692
to form a colored complex with ferricyanide,
which is an electron acceptor. Scavenging ability
on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radi-
cals was determined using the method described
by Shimada et al.30 The scavenging ability is the
ability of the extracts to react with DPPH radi-
cals and reduce most DPPH radical molecules.
Ascorbic acid, butylated hydroxyanisole (BHA),
and α-tocopherol were used for comparison.
Chelating ability was determined according to
the method described by Dinis et al.31 Ferrous ions
play an important role as catalysts in the oxida-
tive process, leading to the formation of hydroxyl
radicals and hydroperoxide decomposition by the
Fenton reaction. Chelating ability is the ability of the
extracts to inhibit the complex formation of ferro-
zine with ferrous ions. Ethylenediaminetetraacetic
acid was used for comparison. Trolox-equivalent
antioxidant capacity (TEAC) was determined using
the method described by Arts et al.32 The value (mil-
ligrams extract per milliliter) at an half-maximal
effective concentration (EC50) is the concentration
at which the antioxidant activity was inhibited by
50%; the absorbance was 0.5 for reducing power;
DPPH radicals were scavenged by 50%, ferrous
ions were chelated by 50%, and 2,2′-azino-bis(3-
ethylbenzothiazoline-6-sulphonic acid) radicals
were scavenged by 50%. The EC50 value was
obtained by interpolation from linear regression
analysis.
G. Assays of Antioxidant Components
Total phenols and flavonoids were determined
according to the methods described by Taga et al.33
and Zhishen et al., respectively.34 The amounts of
components were calculated on the basis of the
calibration curve of the corresponding authentic
compounds; gallic acid and quercetin were used for
total phenols and flavonoids, respectively.
H. Statistical Analysis
For each product of the fruiting body and regular
and Hi-Ergo mycelia, 3 samples were prepared for
assays of every antioxidant attribute and analyses of
Lin et al.
the amount of every component. The experimental
data were expressed as mean ± SE and subjected to
an analysis of variance for a 1-way classification
design to determine the least significant difference
at the level of α = 0.05 using SAS software (SAS
Institute Inc., Cary, NC).
III. RESULTS AND DISCUSSION
A. Proximate Analysis
The amount of moisture in 3 samples was between
8.88% and 9.03% (Table 1). The protein in the
3 samples was similar. In addition, the fruiting
body had more ash and fat but less carbohydrates.
Regarding the carbohydrate content, the fruiting
body was high in DF. Among the 2 mycelia, the
Hi-Ergo mycelium contained more DF, SP, and
ash than the regular mycelium. It seems that regu-
lar and Hi-Ergo mycelia had different proximate
compositions, in addition to their ergothioneine
content. RS is an energy source and the most
was found in regular mycelium (34.95%), then
Hi-Ergo mycelium (27.01%), followed by the
fruiting body (19.45%). SP is thought to be the
biologically active component in mushrooms,35
and its amounts in the fruiting body and Hi-Ergo
mycelium were comparable and higher than that
in the regular mycelium.
B. Soluble Sugars and Polyols
Mannitol, ribose, and trehalose were found in the
3 samples, whereas arabinose and lactose were
detected only in the fruiting body (Table 2). of the
amounts of total sugars and polyols ranged from
47.43 to 92.17 mg/g dry weight (dw) and were in
the fruiting body, regular mycelium, and Hi-Ergo
mycelium (in descending order). Mannitol was high
in the fruiting body, whereas trehalose was high
in the regular mycelium. Mannitol and trehalose
are 2 major components of common mushrooms
(e.g., Agaricus bisporus), paddy straw mushrooms
(Volvariella volvacea), and oyster mushrooms
(Pleurotus spp.).24,36,37 These soluble sugars and
polyols contribute a sweet taste.38
International Journal of Medicinal Mushrooms
Taste Components and Antioxidant Properties of P. citrinopileatus Mycelium 693

TABLE 1: Proximate composition of Pleurotus citrinopileatus fruiting body and mycelia

Component Content (%)

Fruiting Body Hi-Ergo Mycelium Regular Mycelium
Moisture* 9.03 ± 0.04 A 8.88 ± 0.38 A 8.93 ± 0.12 A
Dry matter* 90.97 ± 0.04 A 91.12 ± 0.38 A 91.07 ± 0.12 A
Carbohydrate† 56.06 ± 0.60 C 59.43 ± 0.03 B 60.68 ± 0.24 A
RS‡ 19.45 ± 0.58 C 27.01 ± 0.67 B 34.95 ± 0.97 A
Dietary fiber‡ 36.61 ± 0.92 A 32.42 ± 0.65 B 25.73 ± 0.80 C
Fiber 7.74 ± 0.13 A 5.97 ± 0.22 B 8.18 ± 0.26 A
SP 28.87 ± 0.82 A 26.45 ± 0.79 A 17.55 ± 0.98 B
Crude ash 8.31 ± 0.05 A 6.50 ± 0.10 B 4.65 ± 0.04 C
Crude fat 1.68 ± 0.05 A 0.24 ± 0.01 C 0.44 ± 0.02 B
Crude protein 33.95 ± 0.73 A 33.83 ± 0.15 A 34.23 ± 0.44 A

Values are expressed as mean ± SE (n = 3). Means with different letters within a row differ significantly (P < 0.05). Hi-Ergo,
high ergothioneine; RS, reducing sugar; SP, soluble polysaccharides.
*Moisture and dry matter were presented on freeze-dried basis; other components were presented based on dry weight basis.
†
Carbohydrate content was calculated by subtracting the amounts of crude ash, fat, and protein from 100 (expressed as a
percentage of dry weight).
‡
Dietary fiber = fiber + soluble polysaccharides.

TABLE 2: Soluble sugars and polyols in Pleurotus citrinopileatus fruiting body and mycelia

Sugar or Polyol Content (mg/g Dry Weight)

Fruiting Body Hi-Ergo Mycelium Regular Mycelium
Arabinose 16.28 ± 1.07 ND ND
Lactose 4.71 ± 0.04 ND ND
Mannitol 46.57 ± 2.16 A 16.07 ± 0.31 B 11.89 ± 0.63 C
Ribose 9.33 ± 0.14 C 18.58 ± 0.66 A 12.87 ± 0.02 B
Trehalose 15.27 ± 1.27 B 12.78 ± 0.72 C 35.28 ± 1.33 A
Total 92.17 ± 1.64 A 47.43 ± 0.37 C 60.04 ± 0.68 B

Values are expressed as mean ± SE (n = 3). Means with different letters within a row differ significantly (P < 0.05). Hi-Ergo,
high ergothioneine; ND, not detected.

C. Ergosterol, Ergothioneine, Umami
Amino Acids, and 5′-Nucleotides
Hi-Ergo mycelium had the highest ergothione-
ine content (Table 3). Normally, the fruiting body
contained more ergothioneine than the regular
mycelium. With 4 mmol/L histidine added at day
7, Hi-Ergo mycelium harvested at day 22 contained
more ergothioneine than the regular mycelium har-
vested at day 14, and more than all the fruiting bodies
described by Chen et al.,8 Dubost et al.,15 Lin et al.,39
and Lo et al.40 In addition, the Hi-Ergo mycelium
harvested at day 22 contained much more ergos-
terol than the regular mycelium harvested at day
14. However, lovastatin was not detected in of the 3
samples. Regular mycelium of P. eryngii harvested

Volume 18, Issue 8, 2016

694 Lin et al.

TABLE 3: Umami amino acids, 5′-nucleotides, ergosterol, and ergothioneine in Pleurotus citrinopileatus
fruiting body and mycelia

Component Content (mg/g Dry Weight)

Fruiting Body Hi-Ergo Mycelium Regular Mycelium
Ergosterol 5.74 ± 0.22 B 10.22 ± 0.53 A 0.62 ± 0.01 C
Ergothioneine 3.89 ± 0.19 B 14.57 ± 0.51 A 0.37 ± 0.03 C
l-Aspartic acid 1.64 ± 0.01 A 0.37 ± <0.01 B 0.33 ±<0.01 C
l-Glutamic acid 6.35 ± 0.38 A 3.80 ± 0.21 C 4.59 ±0.04 B
5′-AMP 0.58 ± 0.01 B 0.91 ± 0.02 A 0.89 ± 0.01 A
5′-CMP 3.81 ± 0.03 B 19.38 ± 1.66 A 1.18 ± 0.03 C
5′-GMP 0.94 ± <0.01 B 4.14 ± 0.22 A 0.48 ± 0.02 C
5′-IMP 0.06 ± 0.01 ND ND
5′-UMP 2.14 ± 0.02 A ND 1.01 ± 0.02 B
5′-XMP 0.39 ± 0.01 ND ND

Values are expressed as mean ± SE (n = 3). Means with different letters within a row differ significantly (P < 0.05). AMP,
adenosine monophosphate; CMP, cytosine monophosphate; GMP, guanosine monophosphate; Hi-Ergo, high ergothioneine;
IMP, inosine monophosphate; ND, not detected; UMP, uridine monophosphate; XMP, xanthosine monophosphate.

at day 14 contained 1.68 mg ergothioneine/g dw,41
much more than the regular mycelium (Table 3).
With 4 mmol/L histidine added at day 7, the Hi-Ergo
mycelium of P. eryngii harvested at day 20 con-
tained more ergothioneine (5.76 mg/g dw),41 much
less than the Hi-Ergo mycelium (Table 3). It seems
that the P. citrinopileatus mycelium contained less
ergothioneine than the P. eryngii mycelium. After
manipulating the cultivation conditions, the P. citri-
nopileatus mycelium could become a rich source of
ergothioneine.
The fruiting body contained more umami
amino acids than the Hi-Ergo and regular mycelia
(Table 3). The Hi-Ergo mycelium contained larger
amounts of 5′-cytosine monophosphate (5′-CMP)
and 5′-guanosine monophosphate (5′-GMP) than
the fruiting body and regular mycelium. These com-
pounds were metabolites from the autolysis of the
mycelium during prolonged incubation. 5′-CMP is
not an umami 5′-nucleotide, whereas 5′-GMP is an
umami 5′-nucleotide, which gives a typical mush-
room taste.42 The umami taste, or palatable taste, is
the characteristic taste of monosodium glutamate
(MSG) and 5′-nucleotides.42 Using the equation
from a sensory evaluation,42 equivalent umami
concentration, which is the concentration of MSG
equivalent to the umami intensity of that given by
the mixture of MSG and a 5′-nucleotide, was calcu-
lated to be 268, 451, and 71 g MSG/100 g dw for the
fruiting body, Hi-Ergo mycelium, and regular myce-
lium, respectively. As food-flavoring materials and
food ingredients, or as nutritional supplements, the
amounts of umami amino acids and 5′-nucleotides
and the equivalent umami concentration of Hi-Ergo
mycelia are important to consumers.
D. Extraction Yield
The yields of the 70% ethanolic extracts were (in
descending order) 23.68% for the Hi-Ergo myce-
lium, 22.84% for the fruiting body, and 9.77% for the
regular mycelium. The higher yield of the Hi-Ergo
mycelium might be the result of more metabolites
released during autolysis of the mycelium during
prolonged incubation. Usually, the yields of the
water extracts were higher than those of the etha-
nolic extracts, with those from fruiting body being
the highest.41 Among the ethanolic, cold water, and

International Journal of Medicinal Mushrooms

Taste Components and Antioxidant Properties of P. citrinopileatus Mycelium 695

TABLE 4: EC50 values of antioxidant properties of the 70% ethanolic extracts from Pleurotus citrinopileatus
fruiting body and mycelia

Property EC50 Value* (mg/mL Extract)

Fruiting Body Hi-Ergo Mycelium Regular Mycelium
Antioxidant activity 0.09 ± <0.01 B 0.08 ± <0.01 B 0.24 ± 0.01 A
Reducing power 1.95 ± 0.01 A 0.80 ± <0.01 C 1.69 ± 0.01 B
Scavenging ability 0.71 ± 0.01 B 0.29 ± <0.01 C 2.32 ± 0.10 A
Chelating ability 0.29 ± <0.01 C 0.44 ± 0.01 B 1.28 ± 0.05 A
TEAC 3.14 ± 0.03 C 3.32 ± 0.03 B 5.38 ± 0.03 A

Values are expressed as mean ± SE (n = 3). Means with different letters within a row differ significantly (P < 0.05). Hi-Ergo,
high ergothioneine; TEAC, Trolox-equivalent antioxidant capacity.
*The absorbance was 0.5 for reducing power; the 1,1-diphenyl-2-picrylhydrazyl radicals were scavenged by 50%; the 2,2′-azino-
bis(3-ethylbenzothiazoline-6-sulphonic acid) radicals were scavenged by 50%; and ferrous ions were chelated by 50%. The effective
concentration at which the antioxidant activity was 50% (EC50) was obtained by interpolation from linear regression analysis.

hot water extracts prepared from P. citrinopileatus
fruiting body, mycelium, and fermentation filtrate,
the yield of cold water extracts from the fruiting
body and mycelium was the highest.2 The water
extracts contained more water-soluble substances,
including SP, which could be precipitated from
aqueous suspension using ethanol.43 The extract was
prepared using 70% ethanol in order to decrease the
amount of SP.
E. EC50 Values and Antioxidant Properties
The antioxidant properties assayed herein are sum-
marized in Table 4; EC50 values (milligrams dw of
various extracts per milliliter) were calculated for
comparison. The effectiveness of the antioxidant
properties inversely correlated with their EC50 val-
ues. With regard to antioxidant activity determined
using the conjugated diene method, EC50 values of
the 3 samples ranged from 0.08 to 0.24 mg/mL. The
extracts from the fruiting body and the Hi-Ergo myce-
lium were comparably effective and more effective
than that from the regular mycelium. However, the
EC50 values of BHA and α-tocopherol were both <0.1
mg/mL. The EC50 values of the extracts from the
fruiting body and Hi-Ergo mycelium were 0.08–0.09
mg/mL, indicating that both extracts had excellent
antioxidant activity.
With regard to the reducing power, the EC50 val-
ues were 0.80 to 1.95 mg/mL, and the extract from
the Hi-Ergo mycelium was much more effective than
those from the regular mycelium and fruiting body.
However, the EC50 values of ascorbic acid, BHA,
and α-tocopherol were 0.15, 0.05, and 0.39 mg/
mL, respectively. With regard to the DPPH radical
scavenging ability, EC50 values ranged from 0.29 to
2.32 mg/mL. Similarly, the extract from the Hi-Ergo
mycelium was more effective than those from the
regular mycelium and fruiting body. However, the
EC50 values of BHA and α-tocopherol were both
<0.1 mg/mL, whereas that of ascorbic acid was
14.99 mg/mL. With regard to the ferrous ion–che-
lating ability, EC50 values ranged from 0.29 to 1.28
mg/mL. The fruiting body was most effective at
chelating ferrous ions, followed by the Hi-Ergo
mycelium then the regular mycelium. However, the
EC50 value of ethylenediaminetetraacetic acid was
<0.1 mg/mL. With regard to the TEAC, the EC50
values ranged from 3.14 to 5.38 mg/mL. Similarly,
The fruiting body was most effective in relation to
the TEAC, followed by the Hi-Ergo mycelium then
the regular mycelium.
Overall, the Hi-Ergo mycelium showed the
most effective antioxidant activity, reducing power,
and scavenging ability, whereas the fruiting body
showed the most effective antioxidant activity,

Volume 18, Issue 8, 2016

696
TABLE 5: Ergothioneine, flavonoid, and total phenol contents of 70% ethanolic extracts from Pleurotus
citrinopileatus fruiting body and mycelia
Compound
Fruiting Body
Ergothioneine 17.04 ± 0.66 B
Flavonoid 32.09 ± 0.85 A
Total phenols 35.76 ± 0.14 B
Values are expressed as mean ± SE (n = 3). Means with different letters within a row differ significantly (P < 0.05). Hi-Ergo,
high ergothioneine.
chelating ability, and TEAC. The ethanolic and hot
water extracts from the Hi-Ergo P. eryngii myce-
lium showed the most effective antioxidant activity,
reducing power, and scavenging ability.41 It seems
that the Hi-Ergo mycelium not only contained a
large amount of ergothioneine but also showed more
effective antioxidant properties over the regular
mycelium.
F. Total Phenol and Flavonoid Contents
The 70% ethanolic extract from the Hi-Ergo myce-
lium contained the largest amount of total phenols
and the smallest amount of flavonoids, whereas
that from the fruiting body contained the largest
amount of flavonoids (Table 5). The extracts also
contained ergothioneine, which was fully extracted
and concentrated, with recovery percentages of
99.95–101.6%. The extract from the Hi-Ergo myce-
lium contained the largest amount of ergothioneine.
It seems that 70% ethanol was an effective solvent
to extract and concentrate ergothioneine.
Phenolic compounds are one of the most widely
distributed plant secondary products and are known
to be effective antioxidants.15 Tsai et al.44 found
that amounts of total antioxidant components were
moderately to highly associated (r = 0.64–0.91)
with antioxidant properties. Furthermore, Dubost
et al.15 found a high correlation between oxygen
radical absorbance capacity and polyphenols (r =
0.93). It seems that total phenols in extracts were
responsible for the extracts’ effective antioxidant
properties. Therefore, the largest amount of total
Lin et al.
Content (mg/g Extract)
Hi-Ergo Mycelium Regular Mycelium
61.53 ± 1.51 A 3.84 ± 0.31 C
12.12 ± 0.43 C 29.10 ± 1.02 B
60.65 ± 1.26 A 30.38 ± 0.13 C
phenols in the extract from the Hi-Ergo mycelium
might explain its effective antioxidant properties.
Dubost et al.15 also found a moderate correlation
between antioxidant capacities and ergothioneine
(r = 0.60–71). Tepwong et al.45 showed a relatively
high positive correlation between ergothioneine
content and reducing power (R2 = 0.73) in crude
extracts from several mycelium samples. Using
high-resolution mass spectrometry and online flow
injection analysis of DPPH radical scavenging abil-
ity, ergothioneine was confirmed to be the most
potent antioxidant in the shiitake mycelial extract
and had a relatively higher positive correlation (R2
= 0.86).45 Therefore, the large amount of ergothione-
ine in the extract from the Hi-Ergo mycelium might
account for its effective antioxidant properties.
IV. CONCLUSIONS
In this research, Hi-Ergo mycelium was prepared
and compared with regular mycelium and fruiting
body to determine their proximate composition,
nonvolatile taste components, and antioxidant
properties. The Hi-Ergo mycelium contained the
largest amount of ergothioneine, as well as more
DF, SP, and ash contents, but it contained fewer
carbohydrates and less RS, fiber, and fat than the
regular mycelium. However, the Hi-Ergo mycelium
contained the smallest amount of total sugars and
polyols. The fruiting body contained more umami
amino acids, whereas the Hi-Ergo mycelium con-
tained more 5′-CMP and 5′-GMP. In addition, the
Hi-Ergo mycelium showed the most intense umami
International Journal of Medicinal Mushrooms
Taste Components and Antioxidant Properties of P. citrinopileatus Mycelium
taste. On the basis of the EC50 values obtained, the
extract from the Hi-Ergo mycelium showed the
most effective antioxidant activity, reducing power,
and scavenging ability, whereas that from fruiting
body showed the most effective antioxidant activity,
chelating ability, and TEAC. Overall, the Hi-Ergo
mycelium not only contained a large amount of
ergothioneine but also gave a stronger umami taste
and had more effective antioxidant properties.
Accordingly, the Hi-Ergo mycelium of P. citrinopi-
leatus could be beneficially used as a food-flavoring
material or as a nutritional supplement.
ACKNOWLEDGMENTS
This study was supported by the Ministry of Science
and Technology, Taiwan, Republic of China (NSC-
103-2911-I-005-301, NSC-102-2911-I-005-301)
and the Ministry of Education, Taiwan, R.O.C.,
under the ATU plan.
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