Effects of Cultivation Techniques and Processing on

Effects of Cultivation Techniques and Processing on
Antimicrobial and Antioxidant Activities of Hericium
erinaceus (Bull.:Fr.) Pers. Extracts
Kah Hui Wong1, Vikineswary Sabaratnam1*, Noorlidah Abdullah1,
Umah Rani Kuppusamy2 and Murali Naidu3
1Institute of Biological Sciences, Faculty of Science, University of Malaya,
MY-50603 Kuala Lumpur, Malaysia
2Department of Molecular Medicine, Faculty of Medicine, University of Malaya,
MY-50603 Kuala Lumpur, Malaysia
2Department of Anatomy, Faculty of Medicine, University of Malaya,
MY-50603 Kuala Lumpur, Malaysia
Received: August 19, 2007
Accepted: April 7, 2008
Summary
Hericium erinaceus, a temperate mushroom, is currently cultivated in Malaysia. As cultivation
and processing conditions may affect the medicinal properties, antimicrobial and antioxidant
properties of locally grown H. erinaceus have been investigated. The fruitbodies
that were fresh, oven-dried or freeze-dried were extracted with methanol. Their properties
were compared to those exhibited by mycelium extract of the same mushroom. Various
extracts of H. erinaceus inhibited the growth of pathogenic bacteria but not of the tested fungus.
Mycelium extract contained the highest total phenolic content and the highest ferric
reducing antioxidant power (FRAP). The fresh fruitbody extract showed the most potent
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. However, oven-dried
fruitbody extract was excellent in reducing the extent of b-carotene bleaching. The total phenolic
content and total antioxidant activity in the oven-dried fruitbody extract was high
compared to the freeze-dried or fresh fruitbody extract. This may be due to generation and
accumulation of Maillard’s reaction products (MRPs), which are known to have antioxidant
properties. Thus, the consumption of H. erinaceus fruitbody grown in tropical conditions
may have health promoting benefits. Furthermore, the production of H. erinaceus mycelium
in submerged cultures may result in standardized antioxidant formulation for either human
nutrition or therapy. Hence, it has been shown that the processing of fruitbody and not the
cultivation conditions affects the selected bioactive properties of H. erinaceus.
Key words: Hericium erinaceus, antioxidant activity, antimicrobial activity, fruitbody, mycelium
Introduction
Medicinal properties of Hericium erinaceus (Bull.: Fr)
Pers. (also known as lion’s mane, monkey’s head, hedgehog
fungus, pom pom blanc, and yamabushitake) have
been well-known for hundreds of years in traditional
Chinese and Japanese cooking and herbal medicine to
treat various human diseases. The fruitbody is composed
of numerous constituents such as polysaccharides,
proteins, lectins, phenols, hericenones, erinacines
K.H. WONG et al.: Antimicrobial and Antioxidant Activities of H. erinaceus, Food Technol. Biotechnol. 47 (1) 47–55 (2009) 47
*Corresponding author; Phone: ++60 3 79674 349; Fax: ++60 3 79674 178; E-mail: viki@um.edu.my
and terpenoids. Some of the biological activities of these
components have also been studied (1).
A study carried out at the Third People’s Hospital
of Shanghai showed that H. erinaceus, in tablet form,
was effective in treating ulcer, inflammation and tumor
of the alimentary canal (2). The most promising activity
of H. erinaceus is the stimulation of nerve growth factor
(NGF) synthesis by hericenones from the fruitbody and
erinacines from mycelium (3). It has been shown that
aqueous extracts of the mushroom grown in tropical environment
could stimulate neurite outgrowth of the cultured
cells of the neural hybrid clone NG108-15 (4).
These findings show that H. erinaceus may have a potential
in stimulation of neurons to regrow in the treatment
of senility, Alzheimer’s disease, repairing neurological
trauma from strokes, improve muscle/motor response
pathways and cognitive function.
In recent years, multiple drug resistance in human
pathogenic microorganisms is rampant, due to indiscriminate
use of antimicrobial drugs commonly used in the
treatment of infectious diseases. In the continuous search
for new antimicrobial structures, mushrooms are of interest
to investigators (5–7). Sixty antimicrobial compounds
have been isolated from mushrooms. However,
only the compounds from microscopic fungi have been
present on the market as antibiotics until now (7,8).
Phenolic compounds are one of the most widely
distributed plant secondary products. The ability of these
compounds to act as antioxidants has been well established.
Polyphenols are multifunctional antioxidants that
act as reducing agents, hydrogen donating antioxidants
and singlet oxygen quenchers (9). Mushrooms accumulate
a variety of secondary metabolites, including phenolic
compounds, polyketides, terpenes and steroids.
Their phenolic compounds have been found to be excellent
antioxidants and synergists that are not mutagenic
(10). Phenolic antioxidants, such as variegatic acid and
diboviquinone, have been found in mushrooms (11).
Some common edible mushrooms, which are widely
consumed in Asian culture, have recently been found to
possess antioxidant activity, which was well correlated
with their total phenolic content (11). Mushrooms may
thus be a potential source of natural antioxidants for
many applications including the food industry.
Research on the medicinal value of H. erinaceus grown
in Malaysia, a tropical country, is minimal and yet to be
explored. To our knowledge, little or no information is
available on the antimicrobial and antioxidant properties
of the locally grown mushroom H. erinaceus. Cultivation
techniques and processing may affect the medicinal
properties of H. erinaceus. Therefore, the aim of the
study is to determine the antimicrobial and antioxidant
activities of fresh, freeze-dried and oven-dried fruitbody
and mycelium of H. erinaceus.
Materials and Methods
Fruiting substrates and preparation
of dried fruitbody
In Malaysia, H. erinaceus is cultivated on the medium
containing rubberwood sawdust, rice bran and
calcium carbonate in the mass ratio of 400:8:5. The inoculated
bags are placed in a well ventilated mushroom
house at (27±2) to (32±2) ・・C. About 300 g of fresh fruitbody
per 800 g of substrate per bag are harvested after
60 days of spawn run (Cheng Poh Guat, personal communication).
The fresh fruitbodies were freeze-dried at
(–53±2) ・・C or oven-dried at (50±2) ・・C for 48 h, then were
blended in Waring Commercial Blender (New Hartford,
CT, USA) and stored in airtight containers prior to assay.
Culture and mycelium production
A pure culture of H. erinaceus obtained from a mushroom
farm in Tanjung Sepat, Selangor, Malaysia was
grown on potato dextrose agar (PDA, Difco). A volume
of 50 mL of dextrose peptone yeast (DPY) liquid medium
in 250-mL Erlenmeyer flasks (12) was inoculated
with 5 mycelium discs from 8-day-old H. erinaceus and
incubated for 8 days at (26±2) ・・C and 150 rpm. After incubation,
the contents of the flasks were homogenized
aseptically in Waring Commercial Blender for 15 s. A
volume of 1 mL of 2 % (by volume) mycelium suspension
was inoculated into 49 mL of DPY medium and incubated
for 10 days at (26±2) ・・C and 150 rpm. After submerged
cultivation, the whole broth was freeze-dried for
48 h at (–50±2) ・・C.
Preparation of extracts
Biologically active substances from freeze-dried
whole broth and fresh, freeze-dried and oven-dried
fruitbodies were extracted with 100 % methanol for 24 h
at (26±2) ・・C and 150 rpm. Extracts were filtered through
Whatman no. 1 filter paper. The organic solvent in the
extracts was removed by a rotary evaporator, and the
extracts were kept in the dark at 4 ・・C for not more than
one week prior to analysis for antimicrobial and antioxidant
activities.
Antimicrobial activity
The antimicrobial activity of various H. erinaceus extracts
was tested against Gram-positive bacteria Bacillus
cereus, B. subtilis, Staphylococcus aureus, S. aureus ATCC
6538 and Enterococcus faecalis ATCC 7080; Gram-negative
bacteria Salmonella sp. ATCC 13076, S. typhimurium,
Shigella sp., S. flexneri ATCC 12022, Pseudomonas aeruginosa,
Escherichia coli ATCC 29552, E. coli strain O157 and
Plesiomonas shigelloides; and fungi Candida albicans, C. parapsilosis
and Schizosaccharomyces pombe.
Antimicrobial activity was studied by using well
diffusion method (13). Fruitbody and mycelium extracts
were dissolved in distilled water to the concentration of
200 mg/mL. Two-day-old cultures of bacteria and fungi
were cultured on nutrient agar (Oxoid) and glucose
yeast peptone agar plates, respectively. Each plate was
divided into four sections and one well in each section
was loaded with 40 mL (equivalent to 8 mg) of extract.
Distilled water was used as negative control. Chloramphenicol
(30 mg/disc) and nystatin (4 mg/well) were used
as positive controls for all tested bacteria and fungi, respectively.
Three replicate plates were prepared for each
species of tested bacteria and fungi. The plates were incubated
for 10 h at (37±2) ・・C, except for S. pombe, which
was incubated at (27±2) ・・C, and were then examined for
48 K.H. WONG et al.: Antimicrobial and Antioxidant Activities of H. erinaceus, Food Technol. Biotechnol. 47 (1) 47–55 (2009)
the presence of clear and hazy inhibition zones. The diameters
of the inhibition zones were measured and recorded
after 10 h.
Determination of total phenolic content
The concentration of phenolic compounds in the
methanol extracts of fruitbody and mycelium, expressed
as gallic acid equivalents (GAEs), was determined according
to the method described by Cheung et al. (11).
All samples were assayed in triplicate. Butylated hydroxyanisole
(BHA) was used as positive control. The gallic
acid calibration plot was obtained by plotting the absorbance
against the concentration of gallic acid ranging
from 25 to 1000 mg/L.
Ferric reducing antioxidant power (FRAP) assay
The modified FRAP assay was performed on 96-well
microplates (14,15). The working FRAP reagent was prepared
by mixing 10 volumes of 300 mmol/L of acetate
buffer, pH=3.6, with a volume of 10 mmol/L of TPTZ
(2,4,6-tripyridyl-s-triazine) in 40 mmol/L of hydrocloride
acid and with a volume of 20 mmol/L of ferric
chloride. A volume of 0.025 mL of methanol extract of
each concentration of 4 to 20 mg/mL was added to the
wells of 96-well microtiter plate. For each concentration,
the test was set up in quadruplicate. Then, 0.175 mL of
freshly prepared FRAP reagent at 37 ・・C were added to
three of the replicates, while the same volume of acetate
buffer was added to the fourth replicate (blank sample).
The plate was placed in an automated microplate reader
operated at 593 nm and the temperature was maintained
at 37 ・・C for 4 min. Absorbance values (Asample)
were measured after 4 min. Reagent blank reading, using
0.175 mL of FRAP reagent (Areagent blank), and blank sample
reading, using sample and acetate buffer (Ablank sample),
were taken. The change in absorbance [Asample– (Areagent
blank+Ablank sample)] was calculated. BHA was used as positive
control. The FeSO4・E7H2O calibration plot was obtained
by plotting the change in absorbance against 25-
to 1000-mM concentrations of FeSO4・E7H2O.
Scavenging activity on 1,1-diphenyl-2-picrylhydrazyl
(DPPH) radical
The scavenging activity of fruitbody and mycelium
extracts on DPPH radical was measured according to
the method of Cheung et al. (11). All samples were assayed
in triplicate. BHA was used as positive control.
The scavenging activity (%) on DPPH radical was calculated
by the following equation:
Scavenging activity= 1−
⎛
⎝ ⎜⎜
⎞
⎠ ⎟⎟
A
A
sample
control
・~100 /1/
The scavenging ability of fruitbody and mycelium
extracts was expressed as EC50 value (mg/mL), which is
the effective concentration at which 50 % of DPPH radicals
were scavenged. Low EC50 value indicates strong
ability of the extract to act as DPPH scavenger.
b-carotene bleaching method
The antioxidant activity of fruitbody and mycelium
extracts was determined according to the b-carotene
bleaching method described by Cheung et al. (11). All
samples were assayed in triplicate. BHA was used as
positive control. The rate of b-carotene bleaching (R)
was calculated according to the equation below:
R=
ln(A /A ) 0 t
t
/2/
where ln is natural logarithm, A0 is absorbance at time
0, At is absorbance at time t, and t is 20, 40, 60, 80, 100
or 120 min. The antioxidant activity was calculated in
terms of percentage inhibition relative to the control, using
the equation below:
Antioxidant activity=
R R
R
control sample
control
⎛ −
⎝ ⎜⎜
⎞
⎠ ⎟⎟
・~100 /3/
Statistical analysis
The antioxidant data were subjected to one-way
analysis of variance (ANOVA) and the significance of
the difference between the means was determined by
the Duncan’s multiple range tests at 95 % least significant
difference (p<0.05).
Results and Discussion
Antimicrobial activity of H. erinaceus extracts
The antimicrobial activity of H. erinaceus extracts
and standard antibiotics was quantitatively assessed by
the presence or absence of clear zones indicating strong
inhibition, and hazy (partial) inhibition zones, as given
in Table 1.
The fresh fruitbody extract inhibited the growth of
tested bacteria, and clear inhibition zones of 8 to 12 mm
in diameter against B. cereus, B. subtilis, E. faecalis, Salmonella
sp., Shigella sp. and P. shigelloides were observed.
No activity was recorded against all the tested fungi.
Hazy zones of inhibition with diameters from 13–22
mm, however, were observed against all tested bacteria
and fungi (Table 1).
The freeze-dried fruitbody extract also exhibited
clear inhibition zones of 8 to 12 mm in diameter against
B. cereus, S. aureus ATCC 6538, Salmonella sp., S. typhimurium,
Shigella sp., P. aeruginosa and P. shigelloides. Furthermore,
average hazy zones of inhibition of 13 to 27
mm in diameter against all tested bacteria by the freeze-
-dried fruitbody extract was observed. The tested fungi
were inhibited (hazy zones of 8 to 12 mm in diameter),
too.
The oven-dried fruitbody extract exhibited clear inhibition
zones of 8 to 12 mm in diameter only against B.
cereus. Furthermore, hazy inhibition zones were obtained
against all tested bacteria and fungi except for C. albicans.
Mycelium extract produced clear inhibition zones of
8 to 12 mm in diameter against B. cereus, B. subtilis, E.
faecalis, Salmonella sp., Shigella sp. and P. shigelloides. Hazy
inhibition zone was, however, demonstrated against all
tested bacteria and fungi.
The extracts inhibited four out of five Gram-positive
bacteria and five out of eight Gram-negative bacteria
tested. In general, Gram-negative bacteria had a higher
resistance towards antimicrobial agents and this was ev-
K.H. WONG et al.: Antimicrobial and Antioxidant Activities of H. erinaceus, Food Technol. Biotechnol. 47 (1) 47–55 (2009) 49
ident from the susceptibility results of S. flexneri, E. coli
and E. coli strain O157.
This observation may be explained by the differences
in the cell wall structure between Gram-positive and
Gram-negative bacteria, as the Gram-negative bacteria
possess an outer membrane and a periplasmic space,
both of which are absent from Gram-positive bacteria
(16). The outer membrane is known to present a barrier
to the penetration of numerous environmental substances,
including antibiotic molecules to the peptidoglycan
layer of the cell wall. In addition, the periplasmic space
contains enzymes which are capable of breaking down
foreign molecules introduced from the outside (17). Therefore,
the cell walls of Gram-negative bacteria, which are
more complex than of the Gram-positive bacteria, act as
a diffusional barrier making them less susceptible to the
antimicrobial agents than Gram-positive bacteria (18).
According to Okamoto et al. (19), compounds in H.
erinaceus possess antibacterial and antifungal activities
against selected pathogenic microorganisms. Two novel
and one known chlorinated orcinol derivatives were isolated
from H. erinaceus mycelium. These three compounds
exhibited antimicrobial activity against B. subtilis, Saccharomyces
cerevisiae, Verticillium dahliae and Aspergillus niger.
Thin layer chromatography (TLC) revealed that methanol
extracts of fruitbody and mycelium of H. erinaceus
contained various acidic phenol-like and neutral fatty acid-
-like compounds such as hericenones and hericerins, respectively
(8). Hericenones were effective against pathogenic
microorganisms, while hericerins showed antibacterial
activity at low concentrations against S. aureus, B.
subtilis and E. coli. To our knowledge, this is the first report
of antimicrobial activity of the extracts of variously
processed fruitbody and mycelium of H. erinaceus against
enteric bacteria, including emerging pathogens such as
P. shigelloides and E. faecalis.
Antibiotics could be harmful to beneficial microorganisms
in human gut when taken continuously. In this
study, the tested microorganisms were partially inhibited
by the extracts of H. erinaceus as indicated by the
hazy zones. Hence, when fresh fruitbody or mycelium
extracts are consumed as food or nutraceuticals, the normal
flora in the stomach such as Bifidobacterium adolescentis,
B. longum and Lactobacillus acidophilus, which protect
the intestines from the invasion by pathogens and
harmful bacteria, may not be affected (20). The extracts
however, due to their weak antimicrobial activity, have
no potential to be developed into commercial drugs for
therapy.
Total phenolic content of H. erinaceus extracts
The total phenolic content, expressed as mg of GAE
per gram of fruitbody or mycelium is shown in Table 2.
The absorbance value of the tested extract and BHA after
subtraction of negative control (y) was translated
into total phenolic content using the gallic acid calibration
plot with the following formula:
Total phenolic content=
y − 0 00009
0 0011
.
.
/4/
The total phenolic compounds of mycelium were the
highest (p<0.05) at 31.20 mg of GAE per g of mycelium,
followed by oven-dried, freeze-dried and fresh fruitbody.
Total phenolic content of BHA was (417.84±28.97) mg of
GAE per g of BHA.
50 K.H. WONG et al.: Antimicrobial and Antioxidant Activities of H. erinaceus, Food Technol. Biotechnol. 47 (1) 47–55 (2009)
Table 1. Antimicrobial activity of H. erinaceus extracts and standard antibiotics
Bacteria/Fungi
Fresh
fruitbody
Freeze-dried
fruitbody
Oven-dried
fruitbody
Mycelium
Chloramphenicol
or nystatin
Bacillus cereus +/3+* +/3+* +/3+* +/4+* 4+c
Bacillus subtilis +/3+* 4+* 4+* +/5+* 3+c
Staphylococcus aureus 3+* 3+* 3+* 5+* 4+c
Staphylococcus aureus ATCC 6538 3+* +/3+* 4+* 3+* 4+c
Enterococcus faecalis ATCC 7080 +/3+* 3+* 3+* +/3+* 3+c
Salmonella sp. ATCC 13076 +/3+* +/3+* 3+* +/3+* 5+c
Salmonella typhimurium 3+* +/3+* 3+* 4+* 4+c
Shigella sp. +/3+* +/3+* 3+* +/5+* 3+c
Shigella flexneri ATCC 12022 3+* 3+* 3+* 5+* 5+c
Pseudomonas aeruginosa 3+* +/2+* 3+* 4+* 4+c
Escherichia coli ATCC 29552 3+* 3+* 3+* 3+* 4+c
Escherichia coli strain O157 3+* 3+* 3+* 3+* 4+c
Plesiomonas shigelloides +/3+* +/4+* 4+* +/5+* 5+c
Candida albicans 2+* +* - +* 3+n
Candida parapsilosis 3+* +* +* +* 3+n
Schizosaccharomyces pombe 2+* +* +* +* 2+n
Symbols:
* hazy zone; c chloramphenicol; n nystatin; diameter of inhibition zone: + low level of activity (8 to 12 mm), 2+ 13 to 17 mm, 3+ 18 to
22 mm, 4+ 23 to 27 mm, 5+ high level of activity (28 to 32 mm), – no inhibition
Besides classical antioxidants including vitamin C, E
and b-carotene, phenolic compounds have been identified
as important antioxidants in mushrooms. Phenolic
compounds, or polyphenols, constitute one of the most
numerous and widely distributed groups of substances
in the plant kingdom. They can range from simple molecules,
such as phenolic acids, to highly polymerized
compounds, such as tannins. Flavonoids are reported to
be the most abundant polyphenols in human diets (21).
Phenolics are one of the major groups of nonessential
dietary components that have been associated with
the inhibition of atherosclerosis and cancer (22). The reasons
for recent renewed interest in phenolics is that
most phenolics possess strong antioxidant activity when
compared to vitamins C and E in vitro, and are readily
bioavailable in vivo as demonstrated by animal and human
studies (23). Phenolic compounds in plants are
powerful free radical scavengers which can inhibit lipid
peroxidation by neutralizing peroxyl radicals generated
during the oxidation of lipids (24). Since mushrooms
also possess phenolic compounds (11), it is important to
consider the effect of the total phenolic content on the
antioxidant activity of mushroom extracts.
Ferric reducing antioxidant power (FRAP) of
H. erinaceus extracts
The absorbance value of the tested extract and BHA
after subtraction of reagent blank and blank sample was
translated into FRAP value (mM of FeSO4・E7H2O equivalents)
using the FeSO4・E7H2O calibration plot with the
following formula:
FRAP value=
y + 0 0754
0 0013
.
.
/5/
There was a significant difference (p<0.05) between
the FRAP values of the different extracts (Table 2). Mycelium
extract displayed the highest total antioxidant activity
of 21.93 mmol of FeSO4・E7H2O equivalents per g of
freeze-dried mycelium. The hierarchy of ferric reducing
activity per gram of fruitbody was oven-dried fruitbody>
>freeze-dried fruitbody>fresh fruitbody (Table 2). FRAP
value of BHA was (22618.50±3637.76) mmol of FeSO4・E7H2O
equivalents per g of BHA.
Furthermore, there was a weak positive correlation
between the antioxidant activity determined by FRAP
assay and total phenolic content of the extracts of fruitbody
and mycelium (R2=0.4084), indicating the possible
role of the phenolic compounds in the antioxidant activity
of H. erinaceus (Fig. 1).
Various assays are available to evaluate antioxidant
potential in food and food-based products. In this study,
evaluation of the total antioxidant activity of the extracts
of fruitbody and mycelium of H. erinaceus was carried
out by FRAP assay. It provided a reliable method to
study the antioxidant activity of various compounds.
This method has been frequently used for a rapid evaluation
of the total antioxidant activity of various food
and beverages (25), different plant extracts containing
flavonoids (26) and dietary polyphenols and a limited
number of flavonoids in vitro (27). The reaction is nonspecific,
and any half-reaction that has a less-positive redox
potential, under reaction conditions, than the Fe3+/
Fe2+-TPTZ half-reaction will drive Fe3+-TPTZ reduction
(28). FRAP assay measures the change in absorbance at
593 nm due to the formation of a blue coloured Fe2+-
-tripyridyltriazine compound from the colourless oxidised
Fe3+ form by the action of electron donating antioxidants.
Scavenging activity of H. erinaceus extracts on
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical
H. erinaceus extracts contain active substances, including
phenolic compounds that have hydrogen-donating
activity to scavenge DPPH radical as a possible
mechanism for their antioxidant activity (Fig. 2). Scavenging
effects of the fresh, oven-dried and freeze-dried
fruitbody on DPPH radical increased up to 87.35, 87.78
and 89.24 % with the increasing concentrations of extracts
at 7, 10 and 14 mg/mL, respectively, after which
they reached a steady state (Fig. 2). The scavenging effect
of mycelium extract also increased with increasing
concentrations of the extract, but did not reach steady
state at 15 mg/mL of the extract. This implied that the
DPPH radical scavenging of the fruitbody and mycelium
extracts was dose-dependent. In this study, at 6.4
K.H. WONG et al.: Antimicrobial and Antioxidant Activities of H. erinaceus, Food Technol. Biotechnol. 47 (1) 47–55 (2009) 51
Table 2. Total phenolic content and ferric reducing antioxidant
power of H. erinaceus methanol extracts per g of fruitbody or
mycelium
Total phenolic
content
mg of GAE/g
FRAP value
mmol of FeSO4・E7H2O
equivalents/g
Oven-dried
fruitbody
(2.37±0.24)a (13.72±1.98)e
Freeze-dried
fruitbody
(0.78±0.11)ab (4.06±0.30)f
Fresh
fruitbody
(0.26±0.02)b (1.27±0.07)g
Mycelium (31.20±2.66)c (21.93±1.66)h
GAE, gallic acid equivalents; FRAP, ferric reducing antioxidant
power
Values expressed are means±SD of three measurements. Means
with different letters in the same column are significantly different
(p<0.05, ANOVA)
y=0.7342x
R2=0.4084
0.00
5.00
10.00
15.00
20.00
25.00
30.00
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
Total phenolic content/(mg of GAE per g of fruitbody or mycelium)
FRAPvalues/ mol of FeSO ·7H O
equivalents per g of fruitbody ormycelium
(m
)
Fig. 1. Correlation between the antioxidant activity determined
by ferric reducing antioxidant power (FRAP) assay and total
phenolic content of H. erinaceus extracts
mg/mL of the extract, scavenging effect of the oven-
-dried fruitbody extract was 56.84 %, compared to 63.2–
67.8 % of oven-dried H. erinaceus, G. frondosa and T. giganteum
obtained by Mau et al. (29).
EC50 values of the extracts in DPPH radical scavenging
of the fresh fruitbody, oven-dried fruitbody, freeze-
-dried fruitbody and mycelium extracts were approx. 3.75,
5.81, 8.67 and 13.67 mg/mL, respectively, whereas that
of BHA was 0.0126 mg/mL (Table 3). Freeze-dried fruitbody
and mycelium extracts were not effective in scavenging
DPPH radical. Among the tested extracts, that of
fresh fruitbody was the best DPPH scavenger with the
lowest EC50 value, followed by the extracts of oven-
-dried fruitbody, freeze-dried fruitbody and mycelium.
There was a strong negative correlation between the
scavenging ability on DPPH radical and total phenolic
content in the extracts of fruitbody and mycelium (R2=
0.5629), as shown in Fig. 3, which indicates that high
scavenging ability on DPPH radical is not due to phenolic
compounds in H. erinaceus extracts.
As compared to commercial and specialty mushrooms,
Auricularia sp. are good DPPH・E scavengers. Methanol
extracts of Auricularia mesenterica and Auricularia
polytricha showed an outstanding scavenging effect of
100 % at 1.0 mg/mL of the extract, whereas Auricularia
fuscosuccinea and Tremella fuciformis showed scavenging
effects of 95.4 % at 3.0 mg/mL of the extract, and 71.5 %
at 5.0 mg/mL of the extract, respectively (30). Auricularia
fuscosuccinea scavenged DPPH radical by 94.5 % at
0.4 mg/mL of the extract (31). Excellent scavenging effects
(96.3 to 99.1 % and 97.1 %) were also observed
with methanol extracts of Antrodia camphorata and Agaricus
brasiliensis at 2.5 mg/mL of the extract, respectively.
Scavenging effect of methanol extracts of other medicinal
mushrooms was measured at up to 0.64 mg/mL and
it was 24.6, 67.6, 74.4 and 73.5 % for Coriolus versicolor,
Ganoderma lucidum, antler-shaped Ganoderma lucidum
and Ganoderma tsugae, respectively (31).
The scavenging activity of mushroom extracts was
tested using a methanol solution of the ’stable’ free DPPH
radical. Unlike laboratory-generated free radicals such
as hydroxyl radical and superoxide anion, DPPH radical
has the advantage of being unaffected by side reactions,
such as metal ion chelation and enzyme inhibition (32).
Extracts of H. erinaceus were free radical inhibitors or
scavengers, acting possibly as primary antioxidants. The
extracts might react with free radicals, particularly peroxy
radicals, which are the major propagators of the
autoxidation chain of fat, thereby terminating the chain
reaction (29).
b-carotene bleaching activity of H. erinaceus extracts
Table 4 shows the antioxidant activity of the extracts
and BHA with the coupled oxidation of b-carotene and
linoleic acid. The antioxidant activity of BHA gradually
increased with the increasing concentration of BHA
(p<0.05). The antioxidant activity of the extracts, however,
varied with different concentration of the extract
depending on how the fruitbody was processed. Oven-
-dried and freeze-dried fruitbody extracts exhibited higher
antioxidant activities than the extracts of fresh fruitbody
and mycelium. At the concentration of 20 mg/mL of the
extract, the antioxidant activity was in the following order:
oven-dried fruitbody>freeze-dried fruitbody>fresh
fruitbody>mycelium.
It is probable that the antioxidative components in
the mushroom extracts can reduce the extent of b-carotene
destruction by neutralizing the linoleate free radical
and other free radicals formed in the system. The mechanism
of b-carotene bleaching is a free-radical-mediated
phenomenon resulting from the hydroperoxides formed
from linoleic acid. b-Carotene, in this model system, undergoes
rapid discolouration in the absence of an antioxidant.
The linoleic acid free radical, formed upon the
abstraction of a hydrogen atom from one of its diallylic
52 K.H. WONG et al.: Antimicrobial and Antioxidant Activities of H. erinaceus, Food Technol. Biotechnol. 47 (1) 47–55 (2009)
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
100.00
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
g(extract)/(mg/mL)
Scavenging effect/%
Oven-dried fruitbody
Freeze-dried fruitbody
Fresh fruitbody
Mycelium
Fig. 2. Scavenging effect of H. erinaceus extracts on 1,1,-diphenyl-
2-picrylhydrazyl (DPPH) radical. Values are expressed as
means±SD of three measurements
Table 3. Scavenging ability (EC50 values) of H. erinaceus extracts
and BHA on DPPH radical
EC50 values/(mg/mL)
Oven-dried fruitbody 5.81
Freeze-dried fruitbody 8.67
Fresh fruitbody 3.75
Mycelium 13.67
BHA 0.01261
EC50, 50 % effective concentration
y=-0.992x+87.747
R 2=0.8041
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
100.00
0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00
Total phenolic content/(mg of GAE per g of fruitbody or mycelium)
Scavenging effect/%
Fig. 3. Correlation between the antioxidant activity determined
by scavenging effect on DPPH radical and total phenolic content
in H. erinaceus extracts
methylene groups, attacks the highly unsaturated b-carotene
molecules in an effort to reacquire a hydrogen
atom. As b-carotene molecules lose their double bonds
by oxidation, the compound loses its chromophore and
characteristic orange colour, which can be monitored
spectrophotometrically (33). The presence of antioxidant
extracts can hinder the extent of b-carotene bleaching,
neutralising the linoleate-free radical and other free radicals
formed within the system (34). Hence, this forms
the basis by which mushroom extracts can be screened
for their antioxidant potential.
Effect of cultivation and processing conditions on
the selected bioactive properties of H. erinaceus
Traditionally, Hericium erinaceus prefers cool climate
such as in the northern part of Thailand, central part of
Taiwan and southeastern coast of China (35) to grow
and produce fruitbody. However, this mushroom can be
grown in tropical climates, too. Cultivation of H. erinaceus
in tropical conditions (high temperatures and humidity)
in Malaysia did not affect the production of selected
bioactive properties as shown in this study and in
a previous one (4). Mycelium biomass produced under
standard conditions in submerged fermentation, which
requires sterile handling technology and more technical
demands, is now becoming increasingly important especially
for nutraceutical and pharmaceutical production
(36). Furthermore, environmental conditions can be optimized
and controlled to ensure the quality and quantity
of active ingredients of the mycelium biomass, and subsequently
the extract.
Promising results were obtained with the methanol
extracts of fresh and freeze-dried fruitbody and mycelium,
which to varying extents inhibited the growth of
the tested microorganisms. Fungi, however, were not sensitive
to any of the tested extracts. Extracts of oven-dried
fruitbody lost their antimicrobial activity, which could be
due to inactivation via oxidation of the bioactive compounds
that were responsible for antimicrobial activity
at (50±2) ・・C. Therefore, heat-processed mushrooms may
have lower or no antimicrobial activity compared to the
corresponding fresh and freeze-dried forms. Freeze dried
fruitbody was able to maintain not only its form after
the water had been evaporated, leaving it dry and easy
to store for long periods of time, but caused minimal
damage to the bioactive compounds (37). However, for
commercial purposes, this could be an expensive processing
method.
It is well known that natural nutrients could be significantly
lost during the thermal processing due to the
fact that most of the bioactive compounds are relatively
unstable to heat. Temperature during drying and heating
process affects compound stability due to chemical
and enzymatic decomposition, or losses by volatization
or thermal decomposition. These latter have been suggested
to be the main mechanism causing the reduction
of polyphenol content (38). In some cases, however, heat
treatment caused no change or even improved the content
and activities of naturally occurring antioxidants.
Moreover, novel compounds having antioxidant property,
such as Maillard’s reaction products (MRPs), may
be formed as a result of heat treatment. Therefore, the
loss of natural antioxidants or heat labile nutrients can
be minimized by an enhancement of overall antioxidant
activity in plant food due to their various chemical changes
during heat treatment (38). The most likely explanation
for the higher total antioxidant activity in oven-
-dried fruitbody is an increased antioxidative principle
due to the generation and accumulation of antioxidant
compounds and MRPs during the heating process.
It has been reported that a prolonged heating time
(30 min) and higher heating temperature (121 ・・C) significantly
enhanced the overall antioxidant activity of Lentinula
edodes (38). This could be explained by the increased
amount of antioxidant compounds, especially
free polyphenolic compounds. Heat-treated Lentinula
edodes (38) and oven-dried fruitbody of H. erinaceus
mushroom may have increased the beneficial effects on
health associated with the increase of antioxidant activity.
However, only two mushroom varieties have been
studied so far. Further studies with different mushrooms
are needed to validate matrix effects. The formation of
phenolic compounds during the heating process might
be due to the availability of precursors of phenolic molecules
by non-enzymatic interconversion between phenolic
molecules subjected to the effects of external factors,
such as temperature. Thus, the mushroom composition
and the degree of heating could be important factors
contributing to high total polyphenol content. However,
further investigation is needed to truly explain this phenomenon.
Increased heat treatment led to development
of antioxidant activity, which has positive effects on hu-
K.H. WONG et al.: Antimicrobial and Antioxidant Activities of H. erinaceus, Food Technol. Biotechnol. 47 (1) 47–55 (2009) 53
Table 4. Antioxidant activity of H. erinaceus extracts and BHA measured by b-carotene bleaching method
g(extract)
mg/mL
Antioxidant activity
Oven-dried
fruitbody
Freeze-dried
fruitbody
Fresh
fruitbody
Mycelium BHA
4 (43.21±2.92)ap (36.67±1.16)aq (15.35±1.46)ar (14.64±3.76)ar (58.34±1.14)as
8 (47.40±2.89)bp (49.26±1.44)bp (30.00±3.05)bq (25.44±2.76)br (78.27±0.75)bs
12 (62.93±2.33)cp (53.10±2.35)cq (45.92±1.25)cr (25.91±2.45)bs (85.22±0.77)ct
16 (59.22±2.39)dp (54.87±1.51)cp (27.65±2.83)bq (30.66±3.81)cq (89.30±0.46)dr
20 (60.67±1.18)cdp (60.04±1.87)dp (38.17±2.13)gq (29.45±1.32)bcr (99.13±0.38)es
Values expressed are means±SD of three measurements. For the same extract with different concentrations, means in the same column
with different letters (a–e) were significantly different (p<0.05, ANOVA). For different extracts with the same concentration,
means in the same row with different letters (p–t) were significantly different (p<0.05, ANOVA)
man health, but browning that occurred during heating
was not desirable to consumers. Hence, a balance between
positive and negative effects should be taken into
account before simulating their formation during processing
(39).
Conclusions
It has been concluded from the results of this investigation
that the extracts of H. erinaceus are weak inhibitors
of bacterial and fungal growth. However, bacteria
were found to be more sensitive to the bioactive compounds
compared to fungi. Furthermore, the inhibition
was dependent on the type of the tested extracts and the
oven-dried fruitbody extract did not have antimicrobial
activity.
The use of different methods in antioxidant activity
assessment is necessary. The present study showed that
no single testing method was sufficient to estimate the
antioxidant activity of a sample. The combination of
three methods applied in this study gave valuable information
to evaluate the antioxidant activity of H. erinaceus
and could be recommended for other similar investigations.
The most widely used methods for measuring
antioxidant activity are those that involve the generation
of radical species, where the presence of antioxidants
caused the reduction or removal of radicals.
Heat treatment of fruitbody liberated phenolic compounds
and increased generation of Maillard’s reaction
products (MRPs), and thus increased the amounts of active
compounds present in the extract. Oven drying of
mushrooms for storage at predetermined temperature
may be a process to concentrate the antioxidant activity
of fresh mushrooms. Therefore, oven-dried fruitbody
could be used as food or incorporated as food ingredient
in determined amounts to replace artificial antioxidants
as possible protective agents in human diets to reduce
oxidative damage.
In this study, it was shown that the processing of
fruitbody and not the cultivation conditions affected the
selected bioactive properties of H. erinaceus. Full investigation
on the protocol of drying is also necessary for this
mushroom when cultivated on a large scale. However,
whether such extracts will act as effective therapeutic
agents remains to be investigated. Prior to the application
of these extracts by the nutraceutical/pharmaceutical
industry, the identification of the active compounds
and the study of mechanisms of actions are highly recommended.
Acknowledgements
The authors would like to thank the Ministry of Science,
Technology and Environment, Malaysia (MOSTE)
for grant 01-02-03-1000, University of Malaya for the research
grant F0170/2004A, Mrs. Cheng Poh Guat of Vita
Agrotech, a mushroom farm in Tanjung Sepat, Selangor,
Malaysia for the continuous supply of fresh Hericium erinaceus
mushroom, Prof. Dr. Thong Kwai Lin and Assoc.
Prof. Dr. Noni Ajam of the Faculty of Science for providing
bacterial cultures, and Prof. Dr. Ng Kwee Peng of
the Faculty of Medicine for providing fungal cultures
used in this study.
References
1. T. Mizuno, Bioactive substances in Hericium erinaceus (Bull.:
Fr.) Pers. (Yamabushitake), and its medicinal utilization, Int.
J. Med. Mushr. 1 (1999) 105–119.
2. G.L. Chen: Studies on the Cultivation and Medicinal Efficacy
of Hericium erinaceus, The Edible Fungus Research Institute
of The Shanghai Academy of Agricultural Science, Shanghai,
PR China (1992).
3. H. Kawagishi, S. Furukawa, C. Zhuang, R. Yunoki, The inducer
of the synthesis of nerve growth factor from lion’s
mane (Hericium erinaceus), Explore, 11 (2002).
4. K.H. Wong, V. Sabaratnam, N. Abdullah, M. Naidu, R. Keynes,
Activity of aqueous extracts of lion’s mane mushroom
Hericium erinaceus (Bull.: Fr.) Pers. (Aphyllophoromycetideae)
on the neural cell line NG108-15, Int. J. Med. Mushr.
9 (2007) 57–65.
5. A. Smania Jr., F.D. Monache, C. Loguercio-Leite, E.F. Albino
Smania, A. Lehmkuhl Gerber, Antimicrobial activity
of basidiomycetes, Int. J. Med. Mushr. 3 (2001) 87.
6. T. Anke, F. Oberwinkler, W. Steglich, G. Hofle, The striatins
– New antibiotics from the basidiomycete Cyathus striatus
(Huds. ex Pers.) Willd., J. Antibiot. (Tokyo), 30 (1977) 221–
225.
7. U. Lindequist, T.H.J. Niedermeyer, W.D. Julich, The pharmacological
potential of mushrooms, Evid. Based Complement
Altern. Med. 2 (2005) 285–299.
8. D.M. Kim, C.W. Pyun, H.G. Ko, W.M. Park, Isolation of
antimicrobial substances from Hericium erinaceum, Mycobiology,
28 (2000) 33–38.
9. C.A. Rice-Evans, N.J. Miller, G. Paganga, Structure–antioxidant
activity relationships of flavonoids and phenolic acids,
Free Radic. Biol. Med. 20 (1996) 933–956.
10. Y. Ishikawa, K. Morimoto, T. Hamasaki, Flavoglaucin, a
metabolite of Eurotium chevalieri, its antioxidation and synergism
with tocopherol, J. Am. Oil Chem. Soc. 61 (1984)
1864–1868.
11. L.M. Cheung, P.C.K. Cheung, V.E.C. Ooi, Antioxidant activity
and total phenolics of edible mushroom extracts, Food
Chem. 81 (2003) 249–255.
12. A.P. Gryganski, E.F. Solomko, B. Kirchhoff, Mycelium
growth of medicinal mushroom Hericium erinaceus (Bull.:
Fr.) Pers. in pure culture, Int. J. Med. Mushr. 1 (1999) 81–87.
13. Standard Protocol of Institute of Biological Sciences, Faculty
of Science, University of Malaya, Kuala Lumpur, Malaysia
(2004).
14. S.P. Griffin, R. Bhagooli, Measuring antioxidant potential
in corals using the FRAP assay, J. Exp. Mar. Biol. Ecol. 302
(2004) 201–211.
15. O. Firuzi, A. Lacanna, R. Petrucci, G. Marrosu, L. Saso,
Evaluation of the antioxidant activity of flavonoids by ’ferric
reducing antioxidant power’ assay and cyclic voltammetry,
Biochim. Biophys. Acta, 1721 (2005) 174–184.
16. A. Basile, S. Sorbo, S. Giordano, A. Lavitola, R. Castaldo
Cobianchi, Antibacterial activity in Pleurochaete squarrosa
extract (Bryophyta), Int. J. Antimicrob. Agents, 10 (1998)
169–172.
17. C.F. Duffy, R.F. Power, Antioxidant and antimicrobial properties
of some Chinese plant extracts, Int. J. Antimicrob.
Agents, 17 (2001) 527–529.
18. A. Nostro, M.P. Germano, V. D’Angelo, A. Marino, M.A.
Cannatelli, Extraction methods and bioautography for
evaluation of medicinal plant antimicrobial activity, Lett.
Appl. Microbiol. 30 (2000) 379–384.
19. K. Okamoto, A. Shimada, R. Shirai, H. Sakamoto, S. Yoshida,
F. Ojima, Y. Ishiguro, T. Sakai, H. Kawagishi, Antimicrobial
chlorinated orcinol derivatives from mycelia of Hericium
erinaceum, Phytochemistry, 34 (1993) 1445–1446.
54 K.H. WONG et al.: Antimicrobial and Antioxidant Activities of H. erinaceus, Food Technol. Biotechnol. 47 (1) 47–55 (2009)
20. I. Mitsuhara, Y. Nakajima, S. Natori, T. Mitsuoka, Y. Ohashi,
In vitro growth inhibition of human intestinal bacteria
by sarcotoxin IA, an insect bactericidal peptide, Biotechnol.
Lett. 23 (2001) 569–573.
21. M.M. Cowan, Plant products as antimicrobial agents, Clin.
Microbiol. Rev. 12 (1999) 564–582.
22. P.L. Teissedre, E.N. Frankel, A.L. Waterhouse, H. Peleg, J.B.
German, Inhibition of in vitro human LDL oxidation by
phenolic antioxidants from grapes and wines, J. Sci. Food
Agric. 70 (1996) 55–61.
23. C. Guo, J. Yang, J. Wei, Y. Li, J. Xu, Y. Jiang, Antioxidant
activities of peel, pulp and seed fractions of common fruits
as determined by FRAP assay, Nutr. Res. 23 (2003) 1719–
1726.
24. F. Shahidi, U.N. Wanasundara, R. Amarowicz, Natural antioxidants
from low-pungency mustard flour, Food Res. Int.
27 (1994) 489–493.
25. I.F.F. Benzie, Y.T. Szeto, Total antioxidant capacity of teas
by the ferric reducing/antioxidant power assay, J. Agric.
Food. Chem. 47 (1999) 633–636.
26. A. Luximon-Ramma, T. Bahorun, M.A. Soobrattee, O.I. Aruoma,
Antioxidant activities of phenolic, proanthocyanidin,
and flavonoid components in extracts of Cassia fistula, J.
Agric. Food. Chem. 50 (2002) 5042–5047.
27. R. Pulido, L. Bravo, F. Saura-Calixto, Antioxidant activity
of dietary polyphenols as determined by a modified ferric
reducing/antioxidant power assay, J. Agric. Food. Chem. 48
(2000) 3396–3402.
28. I.F.F. Benzie, J.J. Strain, The ferric reducing ability of plasma
(FRAP) as a measure of ’antioxidant power’: The
FRAP assay, Anal. Biochem. 239 (1996) 70–76.
29. J.L. Mau, H.C. Lin, S.F. Song, Antioxidant properties of several
specialty mushrooms, Food Res. Int. 35 (2002) 519–
526.
30. J.L. Mau, G.R. Chao, K.T. Wu, Antioxidant properties of
methanolic extracts from several ear mushrooms, J. Agric.
Food. Chem. 49 (2001) 5461–5467.
31. J.H. Yang, H.C. Lin, J.L. Mau, Antioxidant properties of several
commercial mushrooms, Food Chem. 77 (2002) 229–235.
32. R. Amarowicz, R.B. Pegg, P. Rahimi-Moghaddam, B. Barl,
J.A. Weil, Free-radical scavenging capacity and antioxidant
activity of selected plant species from the Canadian prairies,
Food Chem. 84 (2004) 551–562.
33. G.K. Jayaprakasha, R.P. Singh, K.K. Sakariah, Antioxidant
activity of grape seed (Vitis vinifera) extracts on peroxidation
models in vitro, Food Chem. 73 (2001) 285–290.
34. K.P. Suja, A. Jayalekshmy, C. Arumughan, Antioxidant activity
of sesame cake extract, Food Chem. 91 (2005) 213–219.
35. T. Tapingkae: Monkey Head Mushroom (Hericium erinaceus
Bull. ex Fr. Pers.) Growing in Thailand (2004) (https://
www.mushworld.com/oversea/view.asp?cata_id=5178&vid=6598).
36. J.E. Smith, N.J. Rowan, R. Sullivan, Medicinal mushrooms:
A rapidly developing area of biotechnology for cancer therapy
and other bioactivities, Biotechnol. Lett. 24 (2002)
1839–1845.
37. V. Sabaratnam, N. Abdullah, K.H. Wong, Y.S. Kho, M. Naidu,
U.R. Kuppusamy: Mushrooms in Healthy Diet: Fresh
or Processed?. In: Current Topics on Bioprocesses in Food Industry,
Vol. II, A. Koutinas, A. Pandey, C. Larroche (Eds.),
Asiatech Publishers, New Delhi, India (2008) pp. 474–480.
38. Y. Choi, S.M. Lee, J. Chun, H.B. Lee, J. Lee, Influence of
heat treatment on the antioxidant activities and polyphenolic
compounds of Shiitake (Lentinus edodes) mushroom,
Food Chem. 99 (2006) 381–387.
39. N. Turkmen, F. Sari, E.S. Poyrazoglu, Y.S. Velioglu, Effects
of prolonged heating on antioxidant activity and colour of
honey, Food Chem. 95 (2006) 653–657.