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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: firstname.lastname@example.org 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 MuellerHinton 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).
Table 1. Antimicrobial activity of four wild mushrooms against bacteria and C. albicans
Test Microorganismsa Macrofungi Dose (mg/well) CHR Armillaria mellea ETH CHR Clitocybe geotropa ETH CHR Meripilus giganteus ETH CHR Sparassis crispa
SA 0* 10 0 14 0 13 0 11 0 9
EC 0b 10 0 8 0 10 12 8 0 8
12 12 8 6 12 5 8 15
0 17 0 10 0 8 0 10± 0 10
0 13 0 19 10± 11 0 14 0 11
0 20 0 12 0 0 0 10± 0 10
0 12 0 14 0 14 0 12 0 9
0 16 0 16 0 12 0 16± 0 10
0 10 0 10 14± 10 0 8 0 8
0 10 0 10 0 14± 0 12± 0 8
2 19 0 13 10± 20 10± 17 0 9
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* S. aureus ATCC 6538P E. coli S. lutea ATCC 9341NA B. cereus ATCC 7064 B. subtilis ATCC 6633 S. typhimurium CCM 5445 NAa 20b 26 10R 28 32 6
NV 32 6
AMP 15 R 6
P 24 6
ERY IPM VA CLH NYS 20 12
34 30 40 36 34 30
20 26 30 28 30 40
ND ND ND ND ND ND
28 25 13
26 6R 10 6
20 R 10 R 8
26 26 15 28
15 15 6
Table 2. continued from page 33
P. vulgaris ATCC 6897 E. faecalis ATCC 29212 E. cloacae ATCC 13047D C. albicans ATCC 10231
12 R 30 12
26 28 22 ND
10± R 16 15 6
10± R 24 12 6
20 30 15 6
22 40 30 ND
22 16 16 6
15 30 26 ND
ND ND 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.
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.
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