Anda di halaman 1dari 6

Mushroom Biology

and
Mushroom Products
Proceedings of the Sixth International Conference on Mushroom Biology and
Mushroom Products
29th September – 3rd October, 2008
Bonn, Germany

Edited by

J.I. Lelley
GAMU GmbH, Institut für Pilzforschung,
Krefeld, Germany

and

J.A. Buswell
Institute of Edible Fungi, Shanghai Academy
of Agricultural Sciences, Shanghai, China
Proceeding of the 6th International Conference on Mushroom Biology and Mushroom Products, 2008

Antimicrobial Activities of Four Wild Mushroom Species Collected from


Turkey

Fatih Kalyoncu and Mustafa Oskay


Celal Bayar University, Faculty of Science & Arts, Department of Biology, Muradiye,
Manisa, Turkey Email: fatih.kalyoncu@bayar.edu.tr

Abstract

Four wild mushrooms, namely Meripilus giganteus, Armillaria mellea, Clitocybe


geotropa and Sparassis crispa collected from the southwest of Turkey, were tested for their
antimicrobial activities by using the agar well diffusion method. Ethanol and chloroform
extracts from the fruit bodies of these mushrooms were assayed against 10 microorganisms.
The test antibiotics penicillin G, novobiocin, nalidixic acid, amphicillin, erythromycin,
imipenem, chloramphenicol, vancomycin and nystatin were used for comparison. This
research has shown that four wild mushrooms revealed antimicrobial activities against some
Gram (+) and Gram (-) bacteria, and Candida albicans.

Keywords: Meripilus giganteus; Armillaria mellea; Clitocybe geotropa; Sparassis crispa;


Antimicrobial activity; Turkey

Introduction

Macrofungi have long been used as a valuable food source and as traditional
medicines around the world, especially in Japan and China. A number of medicinal
mushrooms, such as Ganoderma lucidium, Tremella fuciformis and Lentinula edodes, are
deemed to belong to the highest class of medicines (Wasser & Weis, 1999). Furthermore,
screening programs aimed at the discovery of new bioactive metabolites from macrofungi
have been performed (Rosa et al. 2003; Dulger et al. 2002, 2004).
In research, extracts of more than 75% of the polypore mushroom species surveyed showed
antimicrobial activity and 45% of 204 mushroom species inhibited the growth of a wide
variety of microorganisms (Suay et al. 2000).
This experimental study is part of a program focusing on screening of wild edible
mushrooms collected from the southwest region of Turkey. The antimicrobial activities of
ethanol and chloroform extracts of four wild mushrooms are reported here for the first time.

Materials and Methods

Fungi
Meripilus giganteus (Pers.) P. Karst, Armillaria mellea (Vahl) P. Kumm., Clitocybe
geotropa (Bull.) Quel. and Sparassis crispa (Wulfen) Fr. were collected from the wild during
field trips between 2006 and 2007, from the southwest of Turkey. The morphological and
ecological characteristics of the collected macrofungi were recorded and photographed in
their natural habitats. The specimens were identified according to macroscopic and
microscopic features and the related literature (Watling, 1973; Moser, 1983).

Preparation of macrofungi extracts


The dried and powdered fruit bodies of macrofungi were reduced to coarse powder.
Each sample (20 g) was extracted with 100 ml of ethanol and chloroform at room

31
temperature, with stirring, for 4 d. The extraction solvent was evaporated to dryness. Sample
solutions were prepared by dissolving the extracts in extraction solvents.

Microbial test organisms


Test microorganisms included the following bacteria: Staphylococcus aureus ATCC
6538P, Escherichia coli, Sarcina lutea ATCC 9341NA, Bacillus cereus ATCC 7064, Bacillus
subtilis ATCC 6633, Salmonella typhimurium CCM 5445, Proteus vulgaris ATCC 6897,
Enterococcus faecalis ATCC 29212, Enterobacter cloacae ATCC 13047D and the yeast,
Candida albicans ATCC 10231. Bacterial cultures were grown in Mueller-Hinton broth
(Oxoid) at 37oC for 24 h and the yeast was incubated in glucose yeast extract broth at 30oC
for 48 h. All the microorganisms were obtained from the Department of Biology, Ege
University, Izmir, Turkey.

Assay for antimicrobial activity


The assay was conducted as described by Perez et al. (1990) with slight modification
according to the present experimental conditions. Briefly, 50 µl inoculum (containing
approximately 108 bacteria per ml and 107 yeast per ml) were added to 25 ml melted Mueller-
Hinton agar (MHA) and potato dextrose agar (PDA) medium cooled at 50 °C. This was then
poured into 90 mm diameter sterile Petri dishes, and maintained for 1 h at room temperature.
Small wells (6 mm) were cut in the agar plate using a cork borer; 60 µl of extract
concentration with a negative control (EtOH 96° and chloroform, 60 µl) were loaded in the
wells. The dishes were pre-incubated at 4 °C for 2 h to allow uniform diffusion into the agar.
After pre-incubation, for bacteria, the plates were incubated aerobically at 37°C for 24 h, and
28 °C for 48 h for yeast. The antimicrobial activity was evaluated by measuring the inhibition
zone diameter observed. In addition, commercial antibiotics [penicillin G (10 i.u.), nalidixic
acid (30 µg), novobiocin (30 µg), ampicillin (10 µg), imipenem (10 µg), erythromycin (15
µg), vancomycin (30 µg), chloramphenicol (30 µg) and nystatin (10 µg)] were used as
positive control to determine the sensitivity of the strains. These studies were performed in
triplicate.

Statistical Analysis
The mean values were statistically analyzed with the MINITAB Release 13.20
program by the general one-way (unstacked) analysis of variance (ANOVA) to find out the
most effective extracts and the most sensitive test organisms. 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 well diffusion assay.

Results and Discussion

Table 1 show the antimicrobial activities of the extracts obtained from A. mellea, C.
geotropa, M. giganteus and S. crispa. As clearly seen from Table 1, with an inhibition zone of
20 and 19 mm, the ethanol extracts of A. mellea and C. geotropa presented significant
antibacterial activity against B. subtilis and B. cereus, respectively. The ethanol extracts of A.
mellea and M. giganteus showed antiyeast activity against C. albicans, 19 and 20 mm
respectively. Also, the ethanol extract of S. crispa has antiyeast activity, with an inhibition
zone 17 mm. The ethanol extract of A. mellea was found to be active against S. lutea and P.
vulgaris, 17 and 16 mm, respectively. Similarly, the ethanol extract of C. geotropa has
antibacterial activity against P. vulgaris (16 mm).

32
Table 1. Antimicrobial activity of four wild mushrooms against bacteria and C. albicans

Test Microorganismsa
Macrofungi Dose SA EC SL BC BS STYP PV EF ECLO CA
(mg/well)
CHR 12 0* 0b 0 0 0 0 0 0 0 2
±
Armillaria mellea ETH 12 10 10 17 13 20 12 16 10 10 19
CHR 8 0 0 0 0 0 0 0 0 0 0
Clitocybe geotropa ETH 6 14 8 10 19 12 14 16 10 10 13
CHR 12 0 0 0 10± 0 0 0 14± 0 10±
Meripilus giganteus ETH 5 13 10 8 11 0 14 12 10 14± 20
±
CHR 8 0 12 0 0 0 0 0 0 0 10±
Sparassis crispa ETH 15 11 8 10± 14 10± 12 16± 8 12± 17
CHR 0 0 0 0 0 0 0 0 0 0
c ± ± ±
NC ETH 9 8 10 11 10 9 10 8 8 9
a
Microorganisms: SA - Staphylococcus aureus; EC - Escherichia coli; SL - Sarcina lutea; BC - Bacillus cereus;
BS - Bacillus subtilis; STYP - Salmonella typhimurium; PV - Proteus vulgaris; EF - Enterococcus faecalis;
ECLO - Enterobacter cloacae; CA - Candida albicans. binhibition zone diameter in mm, including well diameter
(6 mm); * mean values, n = 3; 0 - no inhibitory activity (equal to well diameter); ± partially inhibition.
c
NC - negative control, CHR – Chloroform; ETH – Ethanol 96%, 60 µl.

In a previous study ethanol was observed as the best solvent for extracting
antimicrobial substances from Lycoperdon pusilum and L. giganteum (Jonathan & Fasidi,
2003). Similarly, our results showed that ethanol was better solvent for extracting than
chloroform. Suay et al. (2000) reported that the methanol extract of C. nebularis
(Tricholomataceae) was active against S. aureus (<15 mm) and B. subtilis (>15 mm). In the
same study, simple hot-water extracts of Lepista nuda

Table 2. Inhibitory activity of selected antibiotics against test bacteria and C. albicans

Standard antibiotics
Tested microorganisms* NAa NV AMP P ERY IPM VA CLH NYS
S. aureus ATCC 6538P 20b 32 15 R 24 20 34 12 20 ND
R R R ±R R
E. coli 26 6 6 6 12 30 6 26 ND
S. lutea ATCC 9341NA 10R 28 26 20 R 26 40 15 30 ND
B. cereus ATCC 7064 28 25 6R 10 R 26 36 15 28 ND
R R ±R R
B. subtilis ATCC 6633 32 13 10 8 15 34 6 30 ND
R R R
S. typhimurium CCM 5445 6 40 6 6 28 30 28 40 ND

33
Table 2. continued from page 33
P. vulgaris ATCC 6897 12 R 26 10± R 10± R 20 22 22 15 ND
E. faecalis ATCC 29212 30 28 16 24 30 40 16 30 ND
±R R R
E. cloacae ATCC 13047D 12 22 15 12 15 30 16 26 ND
C. albicans ATCC 10231 ND ND 6 6 6 ND 6 ND 22
*Bacteria tested in MHA medium, yeast in PDA. aNA - Nalidixic acid (30 µg/disc); NV - Novobiocin (30
µg/disc); AMP - Ampicillin (10 µg/disc); P - Penicilline G (10 i.u./disc); ERY - Erythromycin (15 µg/disc); IPM
- Imipenem (10 µg/disc); VA - Vancomycin (30 µg/disc); CHL - Chloramphenicol (30 µg/disc); NYS - Nystatin
(10 µg/disc). bDiameter of inhibition zone in mm, including disc diameter (6 mm); Rresistant; ± partially
inhibition; 6 - no activity; ND - not determined.

Figure 1. Mean values of microorganisms in relation to their susceptibility to the


mushroom extracts.
*Means are indicated by solid circles. See Table 1 for abbreviations of test microorganisms.

(Tricholomataceae) were found to be active against C. albicans. However, this study


observed activity against C. albicans from the ethanol extracts of all mushrooms.
Sensitivity of test strains was, in decreasing order: C. albicans > B. cereus > P.
vulgaris > E. faecalis > S. typhimurium > S. aureus > E. coli > Enterobacter cloacae >
Sarcina lutea > B. subtilis (Fig. 1). Figure 2 summarizes the similarity of microorganisms in
relation to their susceptibility to the mushroom extracts.
These results confirm that bioactive components of any macrofungi may differ in their
solubility depending on the extractive solvents. Although further investigations are clearly
necessary to clarify and identify the bioactive constituents we believe that our results
presented herein may be a contribution for other researchers, who would carry out further
studies on the antimicrobial activity of macrofungi.

34
Figure 2. Similarity (%) of microorganisms in relation to their susceptibility to the
mushroom extracts. * See Table 1 for abbreviations of test microorganisms.

Acknowledgements

We wish to express our profound gratitude to the Scientific and Technological Research
Council of Turkey (TUBITAK, Accessing code number: TBAG-107T668) for financial
support.

References

Dulger B, Gonuz A, Gucin F. 2004. Antimicrobial activity of the macrofungus


Cantharellus cibarius. Pakistan J. Biol. Sci., 7, 1535-1539.
Dulger B, Yilmaz F, Gucin F. 2002. Antimicrobial activity of some Lactarius species.
Pharmaceut. Biol., 40, 304-306.
Jonathan SG, Fasidi IO. 2003. Antimicrobial activities of two Nigerian edible
macrofungi, Lycoperdon pusilum and L. giganteum. African J. Biomed. Res., 6, 85-90
Moser M. 1983. Keys to Agarics and Boleti. Gustav Fischer, London.
Perez C, Pauli M, Bazerque P. 1990 An antibiotic assay by the agar-well diffusion
method. Acta Biologica et Medica Experimentalis, 15, 113–115.
Rosa LH, Machado KMG, Jacob CC, Capelari M, Rosa CA, Zani CL. 2003.
Screening of Brazilian Basidiomycetes for antimicrobial activity. Memorıas
Do Instıtuto Oswaldo Cruz, 98, 967-974.
Suay I, Arenal F, Asenio F. 2000. Screening of basidiomycetes for antimicrobial
activities. Antonie van Leeuwenhoek, 78, 129-139.
Wasser SP, Weis AL. 1999. Medicinal properties of substances occurring in higher
Basidiomycetes mushrooms: current perspectives. Int. J. Med. Mushr. 1, 31-62.
Watling R. 1973. Identification of the larger fungi. Hulton Educational Publication
Ltd. Buckinghamshire. UK.

35