JOURNAL OF MEDICINAL FOOD

J Med Food 13 (2) 2010, 415–419

# Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition
DOI: 10.1089=jmf.2009.0090

Antimicrobial and Antioxidant Activities of Mycelia of 10 Wild Mushroom Species

Fatih Kalyoncu,1 Mustafa Oskay,1 Hüsniye Sağlam,2 Tuğçe Fafal Erdoğan,2 and A. Üsame Tamer1
1
Department of Biology, Faculty of Science & Arts, Celal Bayar University, Muradiye, Manisa;
and 2Department of Pharmacognosy, Faculty of Pharmacy, Ege University, Bornova, İzmir, Turkey

ABSTRACT Antimicrobial and antioxidant activities of mycelia obtained from 10 wild edible mushrooms—Armillaria
mellea, Meripilus giganteus, Morchella costata, Morchella elata, Morchella esculenta var. vulgaris, Morchella hortensis,
Morchella rotunda, Paxillus involutus, Pleurotus eryngii, and Pleurotus ostreatus—were investigated. For determination of
antimicrobial activities of these mushrooms, ethanol extracts were examined with 11 test microorganisms by the agar well
diffusion method. P. ostreatus and M. giganteus were the most active species against both bacteria and yeast. Antioxidant
properties of ethanol extracts were studied by the 1,1-diphenyl-2-picrylhydrazyl free radical scavenging method. Among the
mushroom extracts, M. elata showed the most potent radical scavenging activity. This research has shown that these 10 wild
macrofungi have potential as natural antioxidants and antibiotics.

KEY WORDS:
antimicrobial activity
antioxidant activity
mycelium
Turkey
wild mushroom

INTRODUCTION with free radicals because oxidative damage to DNA, pro-

teins, and other macromolecules accumulates with age and
M ushrooms have been used as food and food-
flavoring material in soups and sauces for centuries,
because of their unique and subtle flavor.1 As mushrooms are
has been postulated to be a major type of endogenous
damage leading to aging.5 The consumption of plant foods,
such as fruits, vegetables, red wines, and juices, provides
widely distributed all over the world, some of them have
protection against various diseases, including cancer and
long been used as traditional medicines, especially in Japan
cardio- and cerebrovascular diseases. This protection can be
and China. A number of medicinal mushrooms, such as
explained by the capacity of antioxidants in the plant foods
Ganoderma lucidum, Tremella fuciformis, and Lentinula
to scavenge free radicals, which are responsible for the
edodes, are deemed to belong to the highest class of medi-
oxidative damage of lipids, proteins, and nucleic acids.6
cines.2
Like plants, mushrooms accumulate a variety of secondary
Infectious diseases remain one of the major threats to
metabolites, including phenolic compounds, polyketides,
human health. Although numerous antibiotics have been
terpenes, and steroids. Some mushrooms have currently
used against pathogens, antimicrobial resistance is an in-
been found to possess antioxidant activity that is well cor-
creasing public health problem. The antibiotics in mush-
related with their total phenolic content. Recently, mush-
rooms are less well documented in the discovery of new
rooms are considered to be a good source of antioxidants
antimicrobial agents with different structural types. Mush-
such as variegatic acid and diboviquinone, which have been
rooms need antibacterial and antifungal compounds to sur-
found in mushrooms.7
vive in their natural environments. Therefore, antimicrobial
This experimental study is part of a program focusing on
compounds could be isolated from many mushroom species
screening of mycelia obtained from wild mushrooms col-
and could be of beneficial for humans.3 Most of the me-
lected from different parts of Turkey. Wild mushrooms are
dicinal extracts from mushrooms are different forms of
seasonal, and a particular mushroom may disappear from
polysaccharides, and all of them are strengtheners of the
the initial place of collection for a number of years, ap-
immune system with few or no side effects.4
pearing in another place beyond reach. Although there are
The degenerative diseases associated with aging include
many studies on cultivated and wild mushrooms in the
cancer, cardiovascular disease, immune system decline,
world, there is little information available about antimicro-
brain dysfunction, and cataracts. They are also associated
bial and antioxidant activities of wild edible mushrooms of
Turkey, and this is the first study on the pharmacological
Manuscript received 31 March 2009. Revision accepted 21 May 2009. statue of many of the mushrooms used in this study. Our
objective was to evaluate the antimicrobial and antioxidant
Address correspondence to: Fatih Kalyoncu, Department of Biology, Faculty of
Science & Arts, Celal Bayar University, Muradiye, Manisa, 45140 Turkey, E-mail:
properties of mycelia obtained from 10 wild mushrooms
fatihkalyoncu@hotmail.com collected from different parts of Turkey.

415
416 KALYONCU ET AL.
MATERIALS AND METHODS
Mushrooms and growth of mycelia
Mycelia obtained from 10 wild edible mushroom species
(Armillaria mellea, Meripilus giganteus, Morchella costata,
Morchella elata, Morchella esculenta var. vulgaris,
Morchella hortensis, Morchella rotunda, Paxillus involutus,
Pleurotus eryngii, and Pleurotus ostreatus) were grown at
258C in submerged liquid cultures. The liquid medium (pH
6.6) contained glucose (20 g=L), peptone (10 g=L), and
yeast extract (2 g=L). Erlenmeyer flasks that included liquid
medium were inoculated with agar plugs (potato dextrose
agar, 6 mm in diameter) covered by the mycelium.8 After 30
days of incubation in the dark, the liquid medium was fil-
tered, and the biomass was separated from the liquid.
Voucher specimens of mushroom were deposited in the
Fungarium of Microbiology, Department of Biology, Celal
Bayar University, Manisa, Turkey.
Test microorganisms and growth conditions
Test microorganisms included the bacteria Bacillus cereus
CM 99, Bacillus subtilis ATCC 6633, Enterobacter aero-
genes ATCC 13048, Enterobacter cloacae ATCC 13047D,
Enterococcus faecalis ATCC 29212, Escherichia coli ATCC
39628, Proteus vulgaris ATCC 8427, Salmonella typhimur-
ium CCM 5445, Sarcina lutea ATCC 9341NA, and Staphy-
lococcus aureus ATCC 6538P and the yeast Candida
albicans ATCC 10231. Cultures of these bacteria were grown
in Mueller-Hinton broth (Oxoid, Basingstoke, UK) at 378C
for 24 hours, and the yeast studied was incubated in glucose
yeast extract broth at 308C for 48 hours.9 Test microorgan-
isms were obtained from the culture collection of the Basic
and Industrial Microbiology Department, Faculty of Science,
Ege University, Izmir, Turkey.
Antimicrobial activity assay
The dried and powdered mycelia were reduced to coarse
powder. Two grams of each species was extracted with
20 mL of ethanol at room temperature with stirring for 3
days (125 cycles=minute). The ethanol was evaporated to
dryness after the extraction process. Sample solutions were
prepared by dissolving the extracts in ethanol (1 mL).
In vitro antimicrobial studies were carried out by the agar
well diffusion method against test microorganisms. Bac-
terial strains grown on nutrient agar at 378C for 24 hours
were suspended in a saline solution (0.85% NaCl) and ad-
justed to the turbidity of the 0.5 MacFarland standard
(106 colony-forming units=mL). In brief, a 50-mL inoculum
(containing approximately 105 bacteria=mL and 104
yeast=mL) was added to 25 mL of melted Mueller-Hinton
agar and potato dextrose agar medium cooled at 458C. This
was then poured into 90-mm diameter Petri dishes and
maintained for 1 hour at room temperature. Small wells
(6 mm in diameter) were cut in the agar plate using a cork
borer; 60 mL of extract concentration with a negative control
(ethanol, 60 mL) was loaded in the wells. The dishes were
preincubated at 48C for 2 hours to allow uniform diffusion
into the agar. After preincubation, for bacteria the plates
were incubated at 378C for 24 hours; and for yeast, 308C for
48 hours was used.9 The antimicrobial activity was evalu-
ated by measuring the inhibition zone diameter observed. In
addition, commercial antibiotics (penicillin G [10 IU], na-
lidixic acid [30 mg], novobiocin [30 mg], and nystatin
[10 mg]) were used as positive controls to determine the
sensitivity of the strains.10 All experiments were performed
in triplicate.
Antioxidant activity assay
The hydrogen atom or electron donation abilities of the
extracts were measured from the bleaching of the purple-
colored methanol solution of 1,1-diphenyl-2-picrylhydrazyl
(DPPH). This spectrophotometric assay uses the stable radical
DPPH as a reagent.11 One thousand microliters of a 1 mg=mL
concentration of the extracts in ethanol was added to 4 mL of
0.004% methanol solution of DPPH. After a 30-minute in-
cubation period at room temperature, the absorbance was read
against a blank at 517 nm. Inhibition of free radical by DPPH
in percentage (I%) was calculated as follows:
I% ¼ ([Ablank  Asample ]=Ablank ) · 100
where Ablankis the absorbance of the control reaction
and Asample is the absorbance of the test compound. a-
Tocopherol (TOC) was used for comparison. Tests were
carried out in triplicate.
Statistical analysis
The mean values were statistically analyzed with the
Minitab Release 13.20 program (Minitab, Inc., State Col-
lege, PA, USA) by the general one-way (unstacked) analysis
of variance to find out the most effective extracts and the
most sensitive test microorganisms. Percentage similarity of
microorganisms in relation to their susceptibility to the
mushroom extracts was analyzed by the multivariate cluster
analysis according to the data obtained from the well dif-
fusion assay.
RESULTS AND DISCUSSION
Antimicrobial activity of mycelia
The antimicrobial activity of mycelium culture extracts of
wild mushroom species was studied by the agar well dif-
fusion method. All extracts were tested against 10 species of
bacteria and one yeast. Antimicrobial activity was observed
in all mushroom species included in the study. The data
relating to the antimicrobial activities of samples are sum-
marized in Table 1. Overall, 10 ethanol extracts from dif-
ferent mycelia were examined; antibacterial and antiyeast
activities were detected in seven of these samples.
According to results of the antimicrobial screening assay,
some of the mushrooms studied are potentially a rich source
of antimicrobial agents, but many of the mycelia have weak
activities. The most active species were P. ostreatus and
M. giganteus, which showed broad-spectrum antimicrobial
 WILD EDIBLE MUSHROOM BIOLOGICAL ACTIVITIES 417

Table 1. Antimicrobial Activity Results of Mycelia Extracts and Inhibitory Activity

of Some Standard Antibiotics Against Test Microorganisms
Inhibition zone (mm)a

EC SA SL ST CA EF PV ECL BS BC EAb
M. elata 0 8 0 8 8 0 0 0 0 0 0
M. esculenta var. vulgaris 0 8 8 8 10 0 0 0 0 0 0
M. hortensis 8 0 0 8 8 0 0 0 0 0 0
P. ostreatus 18 24 30 12 12 0 8 20 12 16 8
M. giganteus 12 20 26 10 10 10 8 14 10 16 8
A. mellea 8 0 8 10 8 0 0 0 0 0 0
P. eryngii 8 8 0 0 10 0 0 0 0 0 0
M. costata 8 0 8 8 14 0 0 0 0 0 0
P. involutus 12 8 8 10 15 0 0 12 10k 0 8
M. rotunda 0 0 0 0 12 0 0 0 0 0 0
Nalidixic acid (30 mg=disk) 26 20 10R 6R 6 30 12R 12kR 32 28 26
Novobiocin (30 mg=disk) 6R 32 28 40 6 28 26 22 13R 25 17R
Penicillin G (10 IU=disk) 6R 24 20R 6R 6 24 10kR 12R 8kR 10R 6R
Nystatin (10 mg=disk) ND ND ND ND 22 ND ND ND ND ND ND
NC (60 mL of 96% ethanol) 0 8 8 8 8 0 0 0 0 0 0
Data are mean values (n ¼ 3). Values of 0 and 6 indicate no inhibitory activity.k, partial inhibition; ND, not determined;R, resistant; NC, negative control. EC,
E. coli; SA, S. aureus; SL, S. lutea; ST, S. typhimurium; CA, C. albicans; EF, E. faecalis; PV, P. vulgaris; BS, B. subtilis; BC, B. cereus; EA, E. aerogenes; ECL,
E. cloacae.
a
Inhibition zone diameter (in mm), not including well diameter (6 mm) and if equal to negative control inhibitions or under recorded as 0, but including disk
diameter (6 mm) for standard antibiotics.
b
Bacteria were tested in Mueller-Hinton agar medium; yeast was tested in potato dextrose agar.

activity against Gram-positive and Gram-negative bacteria, known that several macrofungi such as Ganoderma,16
whereas the least active species were M. elata and M. hor- Lentinus,8 Pleurotus,13 and Stereum17 produce bioactive
tensis. P. involutus and M. costata demonstrated antiyeast metabolites in culture. However, it is also apparent from this
activity against C. albicans. Extract of M. rotunda only study that the biological activity of most species has not
showed antiyeast activity against C. albicans and not the previously been studied.
other organisms tested.
The maximum antibacterial effect in tested macrofungi Antioxidant activity of mycelia
was shown by ethanol extracts of P. ostreatus against S.
The ethanolic extracts of mycelia were subjected to
lutea as 30 mm. A large inhibition zone diameter against C.
screening for their possible antioxidant activity. The DPPH
albicans was obtained by P. involutus (15 mm) (Table 1).
free radical scavenging method was used for the analysis.
Sensitivity of test strains was, in decreasing order, C.
albicans > S. lutea > E. coli > S. aureus > S. typhimurium
> E. cloacae > B. subtilis > B. cereus > E. aerogenes > P.
vulgaris > E. faecalis (Fig. 1). Figure 2 summarizes the
similarity of microorganisms in relation to their suscepti-
bility to the mushroom extracts.
Some of the results reported in this study are consistent
with those from earlier studies.3,12,13 For instance, it was
found that extracts from mycelial cultures of Lepista nuda
and several Ganoderma species have antibacterial activity.12
Rosa et al.13 found high antimicrobial activity from several
Basidiomycetes species (Agrocybe perfecta, Hexagonia
hydnoides, Irpex lacteus, Nothopanus hygrophanus, Pycno-
porus sanguineus, and Tyromyces duracinus) against bacteria
and yeasts. Also, Yamaç and Bilgili3 reported that Clavar-
iadelphus truncatus has wider antibacterial properties.
Intraspecific genetic differences have already been ob-
served.12 The production of distinct secondary metabolites
FIG. 1. Mean values of microorganisms in relation to their sus-
by cospecific isolates in fungi has been reported in the lit- ceptibility to the mushroom extracts. The bar indicates SE. *Means
erature.14,15 Thus, it is important to keep and screen for are indicated by solid circles. EC, E. coli; SA, S. aureus; SL, S. lutea;
antimicrobial activity of different samples=isolates of the ST, S. typhimurium; CA, C. albicans; EF, E. faecalis; PV, P. vulgaris;
same species of the Basidiomycetes in the collections. It is BS, B. subtilis; BC, B. cereus; EA, E. aerogenes; ECL, E. cloacae.
418 KALYONCU ET AL.

Similarity (%) mycelia of Antrodia camphorata and Agaricus blazei

51.13
67.42
83.71
100.00
EC EA BS PV EF BC ELC ST CA SA SL
Microorganismsa
FIG. 2. Similarity (%) of microorganisms in relation to their sus-
ceptibility to the mushroom extracts. aEC,E. coli; SA, S. aureus; SL,
S. lutea; ST, S. typhimurium; CA, C. albicans; EF, E. faecalis; PV,
P. vulgaris; BS, B. subtilis; BC, B. cereus; EA, E. aerogenes; ECL,
E. cloacae.
The model of scavenging the stable DPPH radical is a
widely used method to evaluate antioxidant activities in
a relatively short time compared with other methods. DPPH,
a stable free radical, has a characteristic absorption at
517 nm. As antioxidants donate protons to these radicals, the
absorption decreases. The decrease in absorption is taken as
a measure of the extent of radical scavenging. Free radical
scavenging values of mycelial extracts as percentages are
shown in Table 2.
It can be seen that the mycelium extracts prepared by
ethanol exhibited varying degrees of scavenging capacities.
M. elata showed the strongest radical scavenging effect
(59.22%) in the case of 1 mg=mL. This activity was fol-
lowed by M. giganteus (43.12%) and M. costata (27.32%),
respectively (Table 2). The lowest scavenging activity was
exhibited by A. mellea (2.85%). However, the scavenging
effect was 91.6% at 0.5 mg=mL for TOC. At 1 mg, M. elata
ethanol extract has an equivalent inhibition value of 6.50 mg
of TOC. The TOC equivalent inhibition values of all
mushroom extracts are shown in Table 2. The scavenging
effect of TOC is higher than that of all extracts of mushroom
species. In previous studies, the same situations were re-
ported.18,19 Huang20 found that the methanolic extracts from
scavenged DPPH radicals by 97.1% and 98.8% at 5 mg=mL,
respectively. At 10 mg=mL, the methanolic extracts from
mycelia of Agrocybe cylindracea and Ganoderma tsugae
scavenged DPPH radicals by 91.4% and 95.6%, respec-
tively.21 According to Mau et al.,18 scavenging effects of
Termitomyces albuminosus, Grifola frondosa, and M. es-
culenta mycelia at 10 mg=mL were 78.8%, 79.4%, and
94.1%, respectively. Lee et al.22 reported that the ethanolic
extract of Hypsizigus marmoreus mycelium showed scav-
enging ability (75.5%) at 5 mg=mL.
These results revealed that ethanolic extracts of mush-
rooms were free radical scavengers, acting possibly as pri-
mary antioxidant. Ethanolic extracts from wild mushrooms
might react with free radicals, which are the major initiator
of the autooxidation chain of fat, thereby terminating the
chain reaction.23,24
According to the results of this study, it is clearly indi-
cated that the ethanolic extracts of some mushroom species
have significant antioxidant and antimicrobial activity
in vitro. Moreover, macrofungi can be used as a good source
of natural antibiotics and antioxidants and as a possible food
supplement. The spectrum of detected biological activities
of mushrooms is very broad. Necessary for a use as a drug,
food supplement, or other purpose is the continuous pro-
duction of mycelium in high amounts and in a standardized
quality. According to Chang,25 mycelial products are the
‘‘wave of the future’’ because they ensure standardized
quality and year-round production.
As far as our literature survey could ascertain, there is no
information about the mycelia of mushroom species except
for Pleurotus spp. presented here. Further works could be
done on the isolation and purification of the biological active
components from the crude extracts of mycelia of wild
mushroom species for showing their mode of action.
ACKNOWLEDGMENTS
The authors wish to express their profound gratitude to
the Scientific and Technological Research Council of Tur-
key (accessing code number TBAG-107T668) for financial
support.

AUTHOR DISCLOSURE STATEMENT

Table 2. Scavenging Capacity of the Mycelial Extracts The authors report no conflicts of interest. The authors
and Equivalent Inhibition Values of TOC
alone are responsible for the content and writing of this article.
Equivalent inhibition
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