Pharmaceutical Biology 2006, Vol. 44, No. 9, pp.

660–667

Antimicrobial Activities of Fruit Bodies and/or Mycelial Cultures of Some Mushroom Isolates
Mustafa Yamac ¸ 1 and Fatma Bilgili2
1

Department of Biology, Faculty of Science and Arts, Eskis ¸ ehir Osmangazi University, Eskis ¸ehir, Turkey; 2Graduate Schools of Natural and Applied Sciences, Eskis ¸ehir Osmangazi University, Eskis ¸ehir, Turkey

Abstract
In this study, we have investigated the antimicrobial activities of intracellular and=or extracellular metabolites of some mushrooms such as Amanita caesarae (Scop.: Fr.) Pers., Armillaria mellea (Vahl) P. Kumm., Chroogomphus rutilus (Schaeff.) O.K. Mill., Clavariadelphus truncatus (Quel.) ´l., Ganoderma sp., Donk., Clitocybe geotropa (Bull.) Que Ganoderma carnosum Pat., Hydnum repandum L., Hygrophorus agathosmus (Fr.) Fr., Lenzites betulina (L.) Fr., Lepista nuda (Bull.) Cooke, Leucoagaricus pudicus (Bull.) Bon, Paxillus involutus (Batsch) Fr., Polyporus arcularius (Batsch) Fr., Rhizopogon roseolus (Corda) Th.Fr., Sarcodon imbricatus (L.) P. Karst., Suillus collitinus (Fr.) O. Kuntze., Trametes versicolor (L.) Lloyd, Tricholoma auratum (Paulet) Gillet, and Tricholoma fracticum (Britzelm.) Kreisel. Antimicrobial activities of these mushroom extracts were examined on test microorganisms Escherichia coli ATCC 25922, Enterobacter aerogenes NRRL-B-3567, Salmonella typhimurium NRRL-B-4440, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis NRRL-B-4377, Bacillus subtilis NRRL-B-558, Candida albicans ATCC 10259, and Saccharomyces cerevisiae NRRL-Y-2034 by the disk diffusion and microdilution methods. The chloroform extract of Hygrophorus agathosmus and the dichloromethane extract of Suillus collitinus were the most active extracts against both yeast and bacteria. MIC values of these extracts were detected between 7.81 and 250 mg=mL for Hygrophorus agathosmus and between 31.25 and 250 mg=mL for Suillus collitinus. Antimicrobial activity was mostly static. Substances responsible for the antimicrobial activity were heat-stable. Keywords: Antimicrobial activity, macrofungi, medicinal mushroom basidiomycetes,

Introduction
Infections diseases remain one of the major threats to human health. Although a number of natural=synthetic antimicrobial agents have been isolated=developed to kill pathogenic microorganisms effectively, global antimicrobial resistance is an increasing public health problem. Therefore, novel antimicrobial agents from different biological sources are continuously sought. Many antibiotics in clinical use were isolated= developed from fungi or the order Actinomycetales. Although production of important antibiotics such as penicillin, cephalosporin, and griseofulvin by fungi is wellknown, the occurrence of antibiotics in mushrooms is less well documented for discovery of new antibiotics with different structural types. It has been known since Greek and Roman antiquity that macrofungi are used as food and medicine and thus these may be a source of new and useful bioactive compounds (Anke, 1989). Mushrooms need antibacterial and antifungal compounds to survive in their natural environment. Therefore, antimicrobial compounds could be isolated from many mushroom species and could be of benefit for humans. As a matter of fact, macrofungi produce a large number of metabolites that show antibacterial, antifungal, antiviral, antitumor, hypoglycemic, antiallergic, immunomodulating, anti-inflammatory, hypolipidemic, and hepatoprotective activity (Espenshade & Griffith, 1966; Benedict & Brady, 1972; Anke, 1989; Bobek et al., 1991; Wang et al., 1995; Kiho et al., 1996; Kim et al., 1999; Suay et al., 2000; Hatvani, 2001; Byung-Keun et al., 2002; Wasser, 2002; Yang et al., 2002; Gao et al., 2005; Lindequist et al., 2005). In early studies growing the potential of mushrooms as sources of antibiotics, performed by Anchel, Hervey

Accepted: June 22, 2006 Address correspondence to: Dr. Mustafa Yamac ¸ , Department of Biology, Faculty of Science and Arts, Eskis ¸ehir Osmangazi University, 26480 Eskis ¸ehir, Turkey. Tel.: +90 222 2393750; Fax.: +90 222 2393578; E-mail: myamac@ogu.edu.tr DOI: 10.1080/13880200601006897 # 2006 Informa Healthcare

Antimicrobial activities of mushrooms and Wilkins in 1941, diverse antibiotic activity was detected in analysis of either fruiting bodies or mycelial cultures of more than 2000 fungal species (Rosa et al., 2003). It was succeeded by the isolation and identification of pleuromutilin (Kavanagh et al., 1950). This compound has served for the development of the first commercial antibiotic of Basidiomycete origin. In recent in vitro studies, Suay et al. (2000) and Rosa et al. (2003) screened Spanish and Brazilian Basidiomycetes for antimicrobial activities. Suay et al. (2000) detected diverse antibiotic activity in mycelial cultures of 204 macrofungi species. Forty-five percent of 317 isolates inhibited growth of a wide variety of microorganisms. Similarly, Rosa et al. (2003) detected 14 mushroom isolates with significant activity against one or more of the target microorganisms. Zjawiony (2004) observed that 75% of polypore fungi that have been tested show strong antimicrobial activity. In this study, antimicrobial activities of fruit bodies and=or mycelial cultures of several higher fungi have been investigated by disk diffusion and microdilution methods. We report herein the results of screening of 20 species of Basidiomycetes against two yeast and eight bacteria.

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were extracted with 200 mL of chloroform, ethyl acetate, acetone, dichloromethane, and ethanol using a Soxhlet extractor for 8 h. The extracts were filtered, and the solvents were completely evaporated to dryness at 40C using a rotary vacuum evaporator. Extract preparation from mycelial cultures Mycelial cultures of the fungi were obtained from the inner living tissues of the fruit bodies in potato dextrose agar (PDA; Merck) medium with benomyl. All of the isolates were maintained in PDA slants at 4C. Basidiomycetes mycelia were grown at 25C in submerged liquid cultures. The liquid medium (pH 6.6) contained glucose 2%, peptone 1%, and yeast extract 2% (Hatvani, 2001). One hundred milliliters of the medium in 250 mL Erlenmeyer flasks was inoculated by five 5-mm mycelial agar plugs from the PDA Petri cultures. The inoculated flasks were shaken at 150 rpm at 25C. The culture fluid was separated from pellets by filtration after 7 days of fermentation. The culture fluids were extracted threetimes with 1:1 chloroform:methanol (9:1) solution. The chloroform:methanol fraction was evaporated and the extracted material was dissolved in DMSO (Merck). The aqueous fraction was also stored. All extracts and fractions from fruiting bodies and mycelial cultures were stored at 4C until use for assaying their activities. Microorganisms In vitro antimicrobial susceptibility studies were performed using a panel of pathogenic and nonpathogenic microorganism strains. The panel consisted of Escherichia coli ATCC 25922, Enterobacter aerogenes NRRL-B-3567, Salmonella typhimurium NRRL-B-4440, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis NRRL-B-4377, Bacillus subtilis NRRL-B-558, Candida albicans ATCC 10259, and Saccharomyces cerevisiae NRRL-Y-2034. Antimicrobial activity Antimicrobial activity was determined using the disk diffusion method and the microdilution assay. Disk diffusion method Antimicrobial activities of all extracts and fractions were screened by the agar disk diffusion method. Overnight grown bacteria and yeast cultures were adjusted to 108 and 106 CFU=mL, respectively. Then, 100 mL cell suspensions was spread on the surfaces of Mueller-Hinton agar (Oxoid) for bacteria and Saburoud dextrose agar (Merck) plates for yeasts. The disks (6 mm in diameter) were impregnated with 10 mL of the extracts or fractions as 30 mg per disk and placed on the inoculated media.

Materials and Methods
Fungi material Twenty Basidiomycetes species were used for the screening of antimicrobial activity. The sporokarps of Amanita caesarae (Scop.: Fr.) Pers., Armillaria mellea (Vahl) P. Kumm., Chroogomphus rutilus (Schaeff.) O.K. Mill., Clavariadelphus truncatus (Quel.) Donk., Clitocybe geo´ l., Ganoderma sp., Ganoderma carnosum tropa (Bull.) Que Pat., Hydnum repandum L., Hygrophorus agathosmus (Fr.) Fr., Lenzites betulina (L.) Fr., Lepista nuda (Bull.) Cooke, Leucoagaricus pudicus (Bull.) Bon, Paxillus involutus (Batsch) Fr., Polyporus arcularius (Batsch) Fr., Rhizopogon roseolus (Corda) Th. Fr., Sarcodon imbricatus (L.) P. Karst., Suillus collitinus (Fr.) O. Kuntze, Trametes versicolor (L.) Lloyd, Tricholoma auratum (Paulet) Gillet, and Tricholoma fracticum (Britzelm.) Kreisel were collected at different locations in Eskis ¸ehir, Turkey. The specimens were identified with the help of available literature (Phillips, 1981; Moser, 1983; Breitenbach & Kra ¨ nzlin, 1986, 1991, 1995; Ellis & Ellis, 1990). Voucher specimens were deposited in the Fungiculture Laboratory at the Department of Biology, Eskis ¸ehir Osmangazi University. The extracts were prepared from their fruit bodies and=or or mycelial cultures in liquid media and then were screened for their antimicrobial activity. Extract preparation from fruit bodies The sporokarps were cut into small pieces, air-dried, and ground to fine powder. The powdered materials (10 g)

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M. Yamac ¸ and F. Bilgili of chloroform extract of Hygrophorus agathosmus and dichloromethane extract of Suillus collitinus, were charged on the plate and developed in a solvent system of chloroform:methanol:water 60:30:10 and 65:25:10, respectively. After development, fluorescent compounds were observed under UV light at 254 nm. For bioautography, semisolid Mueller-Hinton agar inoculated with Staphylococcus aureus ATCC 25923 and Candida albicans ATCC 10259 was poured onto TLC plates. After 1 h at refrigerator temperature, the plates were incubated for bacterium and yeast for 24 h at 37C and for 48 h at 30C, respectively. Data analyses Statistical analysis of the antimicrobial activities of extracts and fractions to compare with standard antibiotics was carried out using Wilcoxon signed ranks test.

The Petri dishes were allowed to stand for 2 h at 4C for diffusion of the metabolites and then incubated at 37C for 24 h for bacteria, and at 30C for 48 h for yeasts. Antimicrobial activity was determined by measuring the radius of the clear inhibition zone around each disk. The tests were carried out in triplicate. Ceftriaxone (CRO) and amphotericin B (AT) were used as positive controls for bacteria and yeasts, respectively. Solvents were also used as negative controls. Microdilution method Analysis of the minimum inhibitory concentration (MIC) was carried out according to the method described by Zgoda and Porter (2001) with some modifications. This test was carried out for chloroform extract of Hygrophorus agathosmus (530 mg) and dichloromethane extract of Suillus collitinus (500 mg). Stock solutions of the extracts were prepared in 10% dimethylsulfoxide. A 100 mL aliquot from extracts initially prepared at the concentration of 1000 mg=mL was added to first wells of 96-well microtiter plates. Then, 100 mL of the solution was used for downstream serial dilutions in consecutive wells, each already containing 100 mL of sterile distilled water. Thus, dilution series using sterile distilled water were prepared from 1000 mg=mL to 0.98 mg=mL in microtiter plates. Overnight grown microorganism suspensions in double-strength Mueller-Hinton broth were standardized to 108 CFU=mL using McFarland no. 0.5 standard solutions. A 100 mL aliquot of microbial suspension was used as inoculant for each well. Sterile distilled water (100 mL) and the medium (100 mL) served as a positive growth control. The last well-chain without microorganisms was used as the negative control. The extracts were also evaluated in a microdilution assay using tetrazolium salts to indicate microbial growth. After incubation at 37C, the lowest concentration of an extract that inhibited microbial growth was assumed as the MIC. Streptomycin and fluconazole were used as standard antimicrobial agents for bacteria and yeasts, respectively. Minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) were defined as the lowest concentration yielding negative subcultures. Heat treatment The heat stability of antimicrobial substances was evaluated by using samples at 60C for 30 min and 100C for 5 min. After heat treatment, samples were used for assay of antimicrobial activity. The antimicrobial activity of each extract was compared with that of nontreated extracts. TLC and Bioautography Plates of Silica Gel 60 F254 (Merck) were used to detect the extracts that have antimicrobial activity. The aliquots

Results and Discussion
Antimicrobial activity of the extracts by the disk diffusion method The antimicrobial activity of fruit body and=or mycelial culture extracts of a total of 20 Basidiomycete species was studied by the disk diffusion method. Antimicrobial activity was observed in all species included in the study. The data relating to the antimicrobial activities of fruit body samples is summarized in Table 1. Overall, 80 extracts from different fruit bodies were examined. It was detected that 61 of them have antimicrobial activity against at least one of the test microorganisms employed. Most of these activities were antibacterial. Fifty-four of 61 total active extracts showed activity only toward inhibition of bacteria. The remaining seven extracts showed activity against both bacteria and fungi. In contrast, 19 extracts did not inhibit any microorganisms. It is known that Gram-negative bacteria are highly resistant to many antibiotics. Our findings are consistent with this observation. Antimicrobial activities against gram-negative bacteria were much less common than against Grampositive (28 vs. 59 active extracts, respectively). Two extracts presented a wide antimicrobial spectrum and were active against both yeast and bacteria. The dichloromethane extract of Suillus collitinus was active against all test microorganisms except P. aeruginosa ATCC 27853. On the other hand, the chloroform extract of Hygrophorus agathosmus was active against all test microorganisms except E. coli ATCC 25922, P. aeroginosa ATCC 27853, and C. albicans ATCC 10259. The activity of dichloromethane extract of S. collitinus and chloroform extract of H. agathosmus was higher than the standard antibiotics against E. aerogenes NRRL-B3567, S. typhimurium NRRL-B-4440, S. epidermidis NRRL-B-4377, B. subtilis NRRL-B-558, and S. cerevisiae

Antimicrobial activities of mushrooms
Table 1. Antimicrobial activities of fruit bodies extracts of macrofungi species. Inhibition zoneb,c Fungus species Amanita caesarea Armillaria mellea Solventa Dm As Kl Ea Dm As Et Ea Dm As Et Kl Ea Dm As Et Kl Ea Dm As Et Kl Et Kl Dm As Et Kl Ea Dm As Et Ea Dm Kl Ea As Et Kl Ea Dm As Et Kl Ea Dm As Et Ea Dm Et Ea Dm As A À À À À À À À À À þ þ À À À À À À À À À À À À À À À þ À À À À À À þ À À À À À þ À þ þ þ À À À À À þþ À À À À B À À À À À À À þ À þ À À À À À À À À À À À þ À þþþ À À À À À À À À À þ þ þ þ þ À À À À À þ À À À À À þþ À þþ À À C À À À À À À À À À þ þ À À À À À À À À À À À À þþþ þ þ þ À À À À À À þ À À À þ À À À À À þ À À À þ þþ þþ À À À À D À À À À À À À À À þ À À À À À À À À À À À À À À À À À À À À À À À À À À À À þ þ þ þ þ À À À À À À À À À À þ E À þ þ þ þ þ þ À À þ þ þ À þ þ þ þ þ þ À þ þ À þ þ À þ À À À þ À À þ À À À À þ þ þ þ þ þ À À À À À þþþ þ þ þ þ F À À À À À À À À À À À À À À À À þ þ þ þ þ þ À þþþ þ þ þ À þ À þ þ À þ þ À À À À À À À À þ À À þ þ þþ þþ À À À À G þ À À À À À À À þ þ þ À þ À À À À À À À À þ þ þþþ À þ þ þ þ þ þ þ þ þ þ þ þ þ À À À À À þ þ þ þ À þþ À À À À H À À À À À À À À À À À À À À À À À À À À À À À À À À À À À À À À þ þ À À À À À À À À À À À À À À À þþ À À À À I

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Chroogomphus rutilus

Clitocybe geotropa

Ganoderma carnosum

Hydnum repandum Hygrophorus agathosmus

Lenzites betulina

Leucoagaricus pudicus Paxillus involutus

Rhizopogon roseolus

Sarcodon imbricatus

Suillus collitinus

Trametes versicolor

À À À À À À À À À À À À þþ À À À À À À À À À À þþþ À À À À À À À À þþ þ À À À À À À À À À À À À À À þþ þþþþ À À À þ (Continued)

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Table 1. Continued.

M. Yamac ¸ and F. Bilgili

Inhibition zoneb,c Fungus species Tricholoma auratum Solventa Kl Ea Dm As Et Kl As CRO-30 AT-30 A þ À À À À À À 10 þþþ NT B À À À À À À À 12 þ NT C À À À À À À À 12 þ NT D À þ À À À þ þ 10 þ NT E þ þ þ þ þ þ À 39 þþþ NT F þ þ þ þ þ À À 25 þ NT G þþ þ þ þ þ À À 31 þ NT H À À À À À À À 3 NT þþþ I À À À À À À À 6 NT þþ

Tricholoma fracticum Total active number Standard antibioticsd
a b

Kl, chloroform, Ea, ethyl acetate; Dm, dichloromethane; As, acetone; Et, ethanol. A, Escherichia coli (ATCC 25922); B, Enterobacter aerogenes (NRRL-B-3567); C, Salmonella typhimurium (NRRL-B-4440); D, Pseudomonas aeruginosa (ATCC 27853); E, Staphylococcus aureus (ATCC 25923); F, Staphylococcus epidermidis (NRRL-B-4377); G, Bacillus subtilis (NRRL-B-558); H, Candida albicans (ATCC 10259); I, Saccharomyces cerevisiae (NRRL-Y-2034). c Activities were classified according to the diameter of the inhibition zones around the disks containing 30 mg=disk dry matter or standard antibiotic. þ , < 10 mm; þ þ , 10–15 mm; þ þ þ , 15–20 mm, þ þ þ þ , > 20 mm; À , without activity; NT, not tested. d CRO-30, ceftriaxone; AT-30, amphotericin B; (30 mg=disk).

NRRL-Y-2034 (Table 1). The activity of dichloromethane extract of S. collitinus was the same with positive control (ceftriaxone) against S. aureus ATCC 25923. The differences of recorded proportions of antimicrobial activity between these two extracts and standard antibiotics were not statistically significant (p  0.05). The results of the current study lend weight to those from earlier reports that showed that Basidiomycetes produce a great variety of antimicrobial activities. For instance, it is known that fruit bodies of several Lactarius (Bergendorf & Sterner, 1988; Anke et al., 1989), Fomitopsis (Keller et al., 1996), Boletus (Lee et al., 1999), and Cortinarius (Nicholas et al., 2001) contain antimicrobial substances. All of 20 extracts obtained from the mycelial cultures of Basidiomycetes isolates presented antimicrobial activity against one or more of the target microorganisms (Table 2). Activities were generally weak. However, the aqueous and organic extracts of Clavariadelphus truncatus and Trametes versicolor cultures presented wider antibacterial properties. Interestingly, these two isolates were especially active against Gram-negative bacteria. Their activities were comparable with that from positive control, ceftriaxone. Some of the results reported in this study are consistent with those from earlier studies (Suay et al., 2000; Rosa et al., 2003). For instance, it was found that extracts from mycelial cultures of Lepista nuda, Polyporus arcularius, and several Ganoderma species have antibacterial activity (Suay et al., 2000). Rosa et al. (2003) found high antimicrobial activity from several Basidiomycetes species against bacteria and yeasts. The intra-specific genetic differences have already been observed (Suay et al., 2000). The production of distinct

secondary metabolites by co-specific isolates in fungi was reported in the literature (Mo aez ¨ ller et al., 1996; Pel et al., 1998, Ishikawa et al., 2001a). Thus, it is important to keep and screen for antimicrobial activity of different samples=isolates of the same species of the Basidiomycetes in the collections. It is known that several fungal species such as Hericium (Okamoto et al., 1993), Ganoderma (Kawagishi et al., 1997), Hebeloma (Wichlacz et al., 1999), Lentinus (Hirasawa et al., 1999; Hatvani, 2001), Flammulina (Ishikawa et al., 2000; 2001b) and Stereum (Omolo et al., 2002) produce bioactive metabolites in culture. However, it is also apparent from this study that the biological activity of most species has not previously been studied. Antimicrobial activity of the extracts by the microdilution method The MIC values were studied for the microorganisms sensitive to the extracts in the disk diffusion method. MIC values of Hygrophorus agathosmus extracts were lower than those of Suillus collitinus. The chloroform extract of Hygrophorus agathosmus showed MIC between 7.81 and 250 mg=mL, while the dichloromethane extract of Suillus collitinus showed inhibition between at 31.25 and 250 mg=mL concentration. The highest antibacterial activity was seen in the extract from Hygrophorus agathosmus against Staphylococcus epidermidis and Bacillus subtilis at 7.81 mg=mL concentration (Table 3). The standard reference antibiotic streptomycin was effective against these two bacteria at a higher concentration (15.62 mg=mL). MIC value against Staphylococcus aureus in Hygrophorus agathosmus was similar to

Antimicrobial activities of mushrooms
Table 2. Antimicrobial activities of fractions from mycelial cultures of macrofungi isolatesa. Inhibition zone Fungus species and isolate code Clavariadelphus truncatus T-192 Ganoderma sp.T-99 Ganoderma carnosum M-88 Lenzites betulina S-2 Lepista nuda T-373 Leucoagaricus pudicus M-20 Polyporus arcularius T-438 Rhizopogon roseolus T-21 Trametes versicolor M-96 Tricholoma auratum T-174 Total active number
a

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Fraction Aq O Aq O Aq O Aq O Aq O Aq O Aq O Aq O Aq O Aq O

A þ þ þ þ þ þ À þ À À À þ þ þ þ þ þ þ þ þ 16

B þ þ À À À À À À À À À þ À À À À þ þ À þ 6

C þ þ À À À À À À þ þ À þ þ þ À À þ þ À À 9

D À À À À À À À À À À À À À À À À À À À À À

E þ þ þ À þ þ þ À þ þ þ þ þ þ þ À þ þ þ þ 17

F À À À À À À À À À À À À À À À À þ þ À À 2

G þ þ þ þ À À þ þ À À þ þ þ þ À À À þ À À 11

H À À À À À À À À À À À À À À À À À À À À À

I À À À À À À À À À À À À À À À À À À À À À

All abbreviations used are the same as those of Table 1: in addition, Aq, aqueous fraction; O, organic fraction.

that of reference antibiotic streptomycin (15.62 mg=mL). We also noted that for both fungi, the Gram-positive bacteria were more sensitive to extracts. All the extracts tested showed an exclusively static activity against test microorganisms, except Bacillus subtilis and Staphylococcus aureus. Minimum bactericidal concentration (MBC) of the chloroform extract of Hygrophorus agathosmus against S. aureus and B. subtilis were

determined as 125 mg=mL and 500 mg=mL, respectively. MBC value of dichloromethane extract of Suillus collitinus was 1000 mg=mL (Table 3). Influence of heat treatment Mushrooms have been used traditionally for thousands of years by treating with wet-heating. Therefore, heat

Table 3. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) of sporokarps extracts of Hygrophorus agathosmus and Suillus collitinus and reference antibiotics against a range of microorganisms. MIC (MBC or MFC) mg=mLa Organism Escherichia coli (ATCC 25922) Enterobacter aerogenes (NRRL-B-3567) Salmonella typhimurium (NRRL-B-4440) Pseudomonas aeruginosa (ATCC 27853) Staphylococcus aureus (ATCC 25923) Staphylococcus epidermidis (NRRL-B-4377) Bacillus subtilis (NRRL-B-558) Candida albicans (ATCC 10259) Saccharomyces cerevisiae (NRRL-Y-2034)
a b

1 — 15.62 ( > 1000) 15.62 ( > 1000) — 15.62 (125) 7.81 ( > 1000) 7.81 (500) — 250 ( > 1000)

2 250 ( > 1000) 125 ( > 1000) 125 ( > 1000) — 31.25 ( > 1000) 62.5 ( > 1000) 62.5 (1000) 250 ( > 1000) 250 ( > 1000)

Standard antibiotics 3.90b 7.81b 7.81b — 15.62b 15.62b 15.62b 62.5c 62.5c

1, chloroform extract of Hygrophorus agathosmus; 2, dichloromethane extract of Suillus collitinus. Streptomycin. c Fluconazole.

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Table 4. The effects of heating on antimicrobial activities of active extracts. Relative antimicrobial activity (%) Extract 1 Temperature (C) None 60 100 None 60 100 Treatment time (min) 30 5 30 5 A 100 83 82 0 0 0 B 100 90 100 100 67 72 C 100 97 97 100 76 70 D 0 0 0 0 0 0 E 100 87 87 100 100 100 F 100 92 92 100 65 55 G 100 100 100 100 100 100 H 100 54 0 0 0 0 I 100 62 62 100 53 0

2

All abbreviations used are the same as those of Table 1: in addition, 1, chloroform extract of Hygrophorus agathosmus; 2, dichloromethane extract of Suillus collitinus.

a

stability of the extracts from mushrooms is an important factor for their biological activity. In this study, almost no change was observed in the antibacterial activity against all bacteria with a heat-treated chloroform extract of Hygrophorus agathosmus, except E. coli ATCC 25922 and yeasts (Table 4). A 20%, 40%, and 45% reduction of the antimicrobial activity with 60C heating treatment was observed against E. coli ATCC 25922, S. cerevisiae NRRL-Y-2034, and C. albicans ATCC 10259, respectively. The extract completely inactivated against C. albicans ATCC 10259 with heat treatment at 100C for 5 min. The dichloromethane extract of Suillus collitinus was more sensitive to heat treatment than H. agathosmus. With heat treatment, this extract lost its antimicrobial activities between 47% and 100%, although against S. aureus ATCC 25923 and B. subtilis NRRL-B-558, the antibacterial activity was not affected. The heating of both active extracts at 60C for 30 min and 100C for 5 min did not cause a loss of antimicrobial activity against B. subtilis NRRL-B-558. TLC and Bioautography In the bioautography sudies on TLC plates, an active spot with inhibition activity against S. aureus ATCC 25923 (Rf 0.76) was observed for the chloroform extract of H. agathosmus. In the dichloromethane extract of S. collitinus, two different active spots were detected. The first one (Rf 0.07) was active only against S. aureus ATCC 25923, the second one (Rf 0.95) was active against both S. aureus ATCC 25923 and C. albicans ATCC 10259. Thus, two different antibacterial substances and a substance that not only has antibacterial but also antifungal activity were detected in the extract of S. collitinus. In conclusion, most of the Basidiomycetes species studied exhibited wide antimicrobial activity. Especially, the chloroform extract of Hygrophorus agathosmus and the dichloromethane extract of Suillus collitinus presented prominent antimicrobial activity. Minimum inhibitory concentrations of chloroform extract of Hygrophorus agathosmus against bacteria were lower than or similar to that of reference antibiotic

streptomycin. Moreover, these extracts can be accepted as heat-stable. The results of the current study may suggest that bioactive and structurally diverse fungal metabolite(s) could be used for the development of valuable pharmaceuticals and isolated from these and other Basidiomycetes species.

Acknowledgments
The authors thank Hasan Ko ¸ i and Gu ¨ stekc ¸ ¨ l Erginbas for their kind help.

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