Antimicrobial activities of some mushrooms in Turkey

Abstract

Context: Terfezia boudieri Chatin (Pezizaceae), Agaricus brunnescens Peck (Agaricaceae) and Lactarius vellereus (Fr.) Fr. (Russulaceae) are well-known species in Turkey, and are used both for food and traditional medicine.

Objective: The powdered fruit bodies of T. boudieri, A. brunnescens and L. vellereus were used to evaluate the antimicrobial activities.

Materials and methods: Chloroform, acetone and methanol extracts of T. boudieri, A. brunnescens and L. vellereus were tested for their antimicrobial activities against four Gram-positive bacteria, five Gram-negative bacteria and yeast using a micro-dilution method.

Results: The strongest minimum inhibitory concentration (MIC) value observed against the test microorganisms was with the chloroform extract of T. boudieri (MIC 2.4 µg/mL) against Streptococcus pyogenes. Maximum antimicrobial effects were observed with the acetone extracts of T. boudieri and L. vellereus (MIC 4.8 µg/mL) against Bacillus subtilis. The strongest antifungal activity was observed with the acetone extracts of T. boudieri (MIC 2.4 µg/mL) and A. brunnescens (MIC 19.5 µg/mL) against Candida albicans. The strongest MIC values for all fungal extracts were observed between 78 and 2.4 µg/mL.

Discussion and conclusion: Present results demonstrated that these three mushroom species have excellent antimicrobial and antifungal activities, and thus have great potential as a source for natural health products.

• Agaricus brunnescens
• Gram-negative bacteria
• Gram-positive bacteria
• Lactarius vellereus
• micro dilution
• Terfezia boudieri
• yeast

Introduction

Medicinal mushrooms have an established history of usage in traditional oriental therapies. Modern clinical practice in Japan, China, Korea and other Asian countries continue to rely on mushroom-derived preparations. Mushrooms have been used for many years in oriental culture as both tea and nutritional food because of their unique scents and soft structures. To the ancient Romans, fungi were “the food of the Gods”, to the early Egyptians they were “a gift from the God Osiris” while, more appropriately, the Chinese considered them “the elixir of life”. Several ancient societies dating as far back as the Palaeolithic period recognized the psychoactive, hallucinogenic properties of some mushrooms, especially Amanita muscaria (L.) Lam. (Amanitaceae) and Psilocybe (Fr.) P. Kumm. spp. (Strophariaceae), and applied them in ancient religious beliefs and practices.

Many antibiotics in clinical usage were developed from fungal and actinomycetes metabolites. During the last decades, several pathogenic microorganisms developed resistance to the available antibiotics. Infections by multidrug resistant isolates of Candida spp., Staphylococcus epidermidis, S. aureus, Streptococcus spp., Enterococcus sp. and Escherichia coli, among others, became more and more frequent stimulating the search for new antibiotics with novel mechanisms of action (Kotra & Mobashery, 1998; Morschhäuser et al., 2000; Sandven, 2000; Thomson & Moland, 2000).

Scientific and medical research on mushrooms has increased and they were published in peer-reviewed journals, especially in Japan, Korea, China and more recently in the US. These studies confirmed the medicinal efficacy of mushrooms and identified their bioactive molecules (Smith et al., 2002). Recent advances in chemical technology have allowed isolation and purification of some of the relevant compounds, especially polysaccharides, which possess strong immune-modulation and anticancer activities. Some examples of these bioactive polysaccharides isolated from mushroom fruit-bodies, submerged cultured mycelial biomass or liquid cultured broths are β-d-glucans, β-d-glucans with heterosaccharide chains of xylose, mannose, galactose or uronic acid, or β-d-glucan-protein complexes (Smith et al., 2002). One of the solutions to the antibiotic resistance problem among pathogenic bacteria is to develop new drugs from natural sources such as fungi. Many efforts have been made to discover new antimicrobials or drugs from various sources such as microorganisms, animals, plants and fungi.

In Turkey, naturally-growing mushrooms are well-known species and people collect them for food, sell in open markets or export them to the different countries. Terfezia boudieri is known as “Domalan” in Central Anatolia, Agaricus brunnescens is known as “Pembe mantar” in the Mediterranean region and Lactarius vellereus is known as “Sütlü mantar” in the Black Sea region. These fungi are believed to have some medicinal properties by the local villagers. For this purpose, T. boudieri is used to irrigate eyes and the other two fungi are believed to empower body immunity.

The main objective of this study was to evaluate antimicrobial properties of these three mushroom species.

Materials and methods

Collection of the fungal samples

All fungal samples were collected in 2008, and their collected areas and Fungarium numbers are as follows: T. boudieri: Karaman (Central Anatolia), Fungarium no: HD 7561. L. vellereus: Ordu (Black Sea), Fungarium no: HD 7469. A. brunnescens: Bozyazı (Mediterranean Sea), Fungarium no: HD 7567. The identification of the species was performed by Hasan Hüseyin Doğan as described in the literature (Astier, 1998; Bretienbach & Kränzlin, 1991; Kränzlin, 2005). Voucher specimens for all species were also deposited at the Fungarium of the Mushroom Application and Research Centre, Selçuk University, Konya, Turkey.

Samples preparation

The fruiting bodies of each mushroom species were dried in a dehydrator at 37–40 °C for five days. The dried samples were homogenized in a household blender at full speed until they turned into powder.

Antimicrobial activities

Preparation of the extracts

Due to their resolving effects, chloroform, acetone and methanol are better for extraction than hot water extraction methods; hence, they were generally used as the principal solvents for the extractions (Duman et al., 2003; Gao et al., 2005; Hirasawa et al., 1999; Iwalokun et al., 2007; Takazawa & Kashino, 1991; Vahidi & Namjoyan, 2004).

Chloroform, acetone and methanol can dissolve non-polar and polar compounds. Generally, extractions begin with a non-polar solvent and finish with a polar solvent. Using this method, substances that cannot be dissolved with a non-polar solvent can be dissolved by other polar solvents. Solvents may be classified according to their dielectric constants as polar (δ > 50), semi-polar (δ = 20–50) or non-polar (δ = 1–20). Non-polar solvent is chloroform (δ = 5), semi-polar is acetone (δ = 21) and more polar is methanol (δ = 33).

Each powdered fungus sample (30 g) was extracted with 250 mL of chloroform in a Soxhlet apparatus for 8 h. The resultant extract was concentrated using a rotary evaporator at 40 °C and low pressure, and the desired phase was separated from the crude extract with chloroform. Later, the residue was extracted with acetone and methanol, respectively. After extraction, all the semi-solid extracts were dried by a freeze-dryer to yield powders. The powdered extracts were dissolved in dimethyl sulfoxide (DMSO):phosphate buffered saline (PBS; 1:1) at a 100 000 µg/mL concentration. These extracts were filtered through a sterile filter (0.45 µm) and stored at 4 °C.

Test microorganisms

All the microorganisms were obtained from the Department of Biology, Faculty of Science, Selçuk University. Four Gram-positive bacteria, (Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 6633, Listeria monocytogenes type 2 NCTC 5348 and Streptococcus pyogenes ATCC 19615) and five Gram-negative bacteria (E. coli ATCC 35218, Klebsiella pneumoniae ATCC 10031, Pseudomonas aeruginosa ATCC 15442, Proteus vulgaris ATCC 7829 and Salmonella enteritidis RSHMB) were chosen as test bacteria. Candida albicans ATCC 1023 was chosen as the test yeast.

Antimicrobial assay

Brain heart infusion broth (BHIB, Oxoid) was used to cultivate the bacteria and malt extract broth (MEB, Difco) was used for the yeast. Each bacterial species obtained from stock cultures were added into 4–5 mL BHIB and incubated at 35 °C for 24 h. The bacterial cultures were prepared in the same medium at a density adjusted to 0.5 McFarland turbidity standards (10⁸ Cfu/mL), and the final concentration of each bacterial culture was adjusted to 10⁵ Cfu/mL. The yeast strain obtained from the stock culture was added into 4–5 mL MEB and incubated at 25 °C for 48 h. After incubation, the final concentration of the yeast was adjusted to 10⁴ Cfu/mL.

Determination of antimicrobial activity by the micro-dilution method

The minimum inhibitory concentration (MIC) values were evaluated in accordance to NCCLS (2008). Each mushroom extract in the stock solutions was prepared at a 20 000 µg/mL concentration in PBS:DMSO (1:1). MHB (100 µL) was dispensed into each well of a flat-bottom, 96-well microtiter plate. To prepare serial dilutions, 100 µL of the T. boudieri chloroform, acetone or methanol extract was separately dispensed into MHB-containing micro plate wells (A1, A2, A3, etc.) and mixed well. Next, 100 µL of this mixture was pipetted from the first well into a second well, generating a ½ dilution. Finally, this process was repeated to generate a dilution series of each extract from 20 000 to 0.305 µg/mL. This method was also applied for L. vellereus and A. brunnescens.

After the dilution series of each extract was prepared, 100 µL of each bacterial suspension were separately added into each well containing the MHB and the mushroom extract mixture. This procedure was also repeated for the yeast in different plate wells. The absorbance of each well was measured using an ELISA reader at 630 nm (EL × 800). After the first reading was finished, all the plates were covered and incubated at 37 °C for 24 h afterwards, the absorbance was measured again. The first absorbance was subtracted from the second absorbance, and the difference was used to calculate the MIC values. The lowest concentration that produced an inhibitory effect was recorded as the MIC for each extract (as described by Abbasoğlu et al., 1995; Devienne & Raddi, 2002). Ampicillin (100 µg/mL concentration) for bacteria and amphotericin B (50 µg/mL concentration) for yeast were used as positive controls. Each experiment was conducted in triplicate. The resolving power of the solvents used for each mushroom species may produce varied bioactive compounds in the extracts. Hence, each extract can show independent different effects on the bacteria species. Due to this independent effect, there is a lack of correlation and statistical significance between each bacterial and mushroom species, so statistical analysis was not performed.

Results and discussion

Description of the species

Ascomycota

Pezizaceae

Terfezia boudieri Chatin

Fruit body hypogeous to partially emergent at maturity, 2–6 cm in size, subglobose, turbinate, pyriform, with more or less apparent sterile base, grayish beige at first, becoming blackish brown with age, unpolished, often cracked, sometimes with deep crevices. Odor faint, not distinctive. Taste mild, pleasant, gastronomically prized. Asci inamyloid, subglobose to ovoid, pyriform, sessile or short-stipitate, 60–110 × 50–80 µm, with 4–6(−8) irregularly disposed spores, randomly arranged in fertile pockets. Ascospores globose, 21–25 µm diam. including ornament, hyaline, smooth and uniguttulate at first, then yellow and ornamented with rounded, sometimes truncated warts, up to 2 µm tall and 2 µm broad.

Basidiomycota

Agaricaceae

Agaricus brunnescens Peck

Pileus 5–10 cm across, hemispherical to convex, surface with concentric adpressed light brown, fibrillose scales on a white background. Flesh whitish, yellowing very slightly when cut, taste mild, aromatic. Lamellae pale pink to dark purple brown colored. Stipe 5–8 × 2–4 cm, cylindrical, tapered to base, white to light brown colored. Spores broadly elliptical, smooth, gray brown, 6 8 × 4–6 µm.

Russulaceae

Lactarius vellereus (Fr.) Fr.

Pileus 2.5–7(9) cm across, planoconvex to infundibuliform shaped, surface even to with indistinct fine radial wrinkles, dull and white pruinose when dry, lubricous and silky when moist. Flesh whitish, turning light grayish after a few minutes when cut, odor weak and taste acrid. Lamellae whitish to ocher with orange tint. Stipe 3–7 × 5–15 cm, cylindrical, solid when young, soon hollow, white pruinose when young, later dingy light ocher when old. Spores subglobose, 6.5–8.9 × 5.6–7 µm, ornaments projecting up to 1.5 µm, hyaline.

According to Craig (1998), to evaluate antimicrobial activity MIC values should be measured from the 4th to the 16th dilutions. The antimicrobial effects of T. boudieri, L. vellereus and A. brunnescens against bacteria and yeast were measured in accordance with the following ranges (Gülay, 2002; Morales et al., 2008):

1. MIC values are lower than 100 µg/mL = antimicrobial activity is high.

2. MIC values are between 100 and 500 µg/mL = antimicrobial activity is moderate.

3. MIC values are between 500 and 1000 µg/mL = antimicrobial activity is weak.

4. MIC values are more than 1000 µg/mL = no antimicrobial effect.

In accordance with these ranges, the antimicrobial results are given in Table 1. The most effective MIC value on the test microorganisms was observed with the chloroform extract of T. boudieri (MIC 2.4 µg/mL) against S. pyogenes. Overall, the acetone extract was observed to exhibit the maximum antimicrobial effect with values generally lower than 100 µg/mL, placing it in the high activity category. The highest antifungal effect was also observed with the acetone extract of T. boudieri (MIC 2.4 µg/mL) and the acetone extract of A. brunnescens (MIC 19.5 µg/mL) against C. albicans. Other mushroom extracts have different MIC values against the test microorganisms. The weakest antimicrobial effect was observed with the chloroform extracts of T. boudieri (MIC 625 µg/mL) against all microorganisms, and this result were followed by the chloroform extract of L. vellereus (MIC 78 µg/mL) against K. pneumoniae, the acetone extract of A. brunnnescens (MIC 625 µg/mL) against B. subtilis and the methanol extract of A. brunnnescens (MIC 625 µg/mL) against E. coli.

Table 1. MIC values of the mushroom extracts (µg/mL)^a.

	T. boudieri			L. vellereus			A. brunnescens
	Chl.	Ace.	Met.	Chl.	Ace.	Met.	Chl.	Ace.	Met.
B. subtilis	625	4.8	39	39	4.8	39	78	625	78
S. aureus	625	39	39	39	39	39	39	39	156
L. monocytogenes	625	78	78	39	39	39	39	39	156
S. pyogenes	625	2.4	78	39	39	39	39	39	156
E. coli	625	39	78	39	39	39	78	78	625
K. pneumoniae	625	39	39	78	19.5	39	39	78	78
P. aeruginosa	625	39	39	39	39	39	156	39	156
P. vulgaris	625	39	39	39	39	39	39	39	78
S. enteritidis	625	39	39	39	39	39	78	78	156
C. albicans	625	2.4	39	39	39	39	39	19.5	39

The lowest MIC values are italicized.

^a<100 µg/mL = high activity, 100–500 µg/mL = moderate, 500–1000 µg/mL = weak.

Chl, chloroform; Ace, acetone and Met, methanol.

In general, acetone and methanol extracts of T. boudieri were observed to be more effective than the other mushroom extracts against bacteria and yeast. Different extracts of L. vellereus and A. brunnescens also demonstrated antimicrobial effects against the test microorganisms.

The most effective MIC concentrations of the extracts were typically measured between the 8th and 13th dilutions (MIC 78–2.4 µg/mL), and these results demonstrate that the inhibition values are lower than 100 µg/mL or between 500 and 1000 µg/mL, except those for the chloroform extract of T. boudieri and methanol extracts of A. brunnescens.

These current results confirm previous studies; according to Yoon et al. (1994), Ganoderma lucidum (Curtis) P. Karst. had a good antimicrobial effect against P. vulgaris (MIC 1.25 mg/mL) and E. coli (MIC 1.75 mg/mL) and MICs against six bacteria species were lower than 5 mg/mL. In the present study, MIC values were generally measured between 39 and 156 µg/mL, and present results are better than those of Yoon et al. (1994). Gbolagade et al. (2007) studied the antimicrobial effects of certain fungal species using disc diffusion and micro-dilution methods. They determined the MIC concentration for Marasmius jodocodo Henn. as 2.75 mg/mL against E. coli and for Tricholoma robustum (Alb. & Schwein.) Ricken as 15.75 mg/mL against M. bourlardii. Janeš et al. (2007) applied the broth micro-dilution test for screening of antibacterial activity in extracts of higher and endophytic fungi. Among the tested extracts, the three that possessed significant antibacterial activity were extracts of Amanita virosa Secr. and Cortinarius praestans (Cordier) Gillet against P. aeruginosa and Staphylococcus aureus, respectively, and an extract of endophytic fungus Truncatella hartigii (Tubeuf) Steyaert against Enterococcus faecalis and S. aureus. Quereshi et al. (2010) tested the antimicrobial activity of various solvent extracts (40 mg/mL) of G. lucidum against six bacteria species. The acetone extract exhibited maximum antibacterial activity, while the most susceptible bacterium observed was Klebsiella pneumoniae. Bala et al. (2011) investigated the antimicrobial effect of 47 different specimens from Australia, and found that water and ethanol extracts were more effective against S. aureus than E. coli, whereas a small number of hexane extracts showed better results for antimicrobial potential against E. coli at higher concentrations only. In general, a number of macrofungi in the genera Agaricus, Amanita, Boletus, Cantharellus, Fomitopsis, Hohenbuehelia, Lentinus, Ramaria and Strobilomyces showed good inhibition rates. In the present study, the lowest MIC concentration (19.5 µg/mL) was recorded with the acetone extract of L. vellereus against K. pneumoniae, and the lowest MIC concentration (39–156 µg/mL) was observed with all fungal species against P. aeruginosa except the chloroform extract of all fungi species. However, the lowest MIC concentrations (39–78 µg/mL) were observed with all fungal species against E. coli except the chloroform extract of T. boudieri and methanol extract of A. brunnescens. The lowest MIC concentration (2.4 µg/mL) was also observed with the acetone extract of T. boudieri against C. albicans and this result was followed by the acetone extract of A. brunnescens (MIC 19.5 µg/mL).

Aqueous and methanol extracts of Trametes hirsuta (Wulfen) Lloyd were tested against pathogenic fungi and bacteria. The maximum antibacterial activity of aqueous extract T. hirsuta was found against S. aureus than that of the methanol extract. The aqueous extract was found to have significant antifungal activity against Aspergillus flavus compared to the methanol extract (Sivaprakasam et al., 2011). The antibacterial effects of three mushrooms extract G. lucidum, Auricularia auricula-judae (Bull.) Quél., Pleurotus floridanus Singer were studied against S. aureus and E. coli. Auricularia auricula-judae showed significant antibacterial activity against S. aureus. Pleurotus florida showed some antibacterial activity while G. lucidum showed no antibacterial activity. None of the extracts showed any activity against E. coli (Iftekhar et al., 2011). Antimicrobial activity of Ganoderma praelongum Murrill, G. resinaceum Boud. and G. lucidum were evaluated against 30 strains of clinical isolates of methicillin-resistant and methicillin-sensitive S. aureus. Maximum activity of crude extracts was exhibited by ethyl acetate. The MIC of sesquiterpenoids extract of G. praelongum was 0.390–6.25 mg/mL. Diterpenoids and triterpenoids displayed moderate activity while polysaccharides IIIa and IIIb showed weak activity. All bacterial strains were resistant to polysaccharides I and II (Ameri et al., 2011). In the present study, the effective results were measured as 39 µg/mL for all the extracts of L. vellereus, acetone and methanol extracts of T. boudieri and chloroform and acetone extracts of A. brunnescens.

As mentioned in “Materials and methods”, the antimicrobial effects of the mushrooms are related to dielectric constants of solvents. A solvent having high dielectric constants can solve more bioactive compounds than the solvents having low dielectric constants. This indicates that the ability of polarity for the solvents. Our results showed a parallelism with the dielectric constant of the solvents. The lowest MIC values were observed for acetone and methanol extracts also indicated that the acetone extract of C. albicans and S. pyogenes are more effective for antimicrobial substance than the methanol extract.

The current results are similar or more effective than those reported in the literature. To the best of our knowledge, there were previously no reports for MIC values to the antimicrobial effects of T. boudieri, L. vellereus and A. brunnescens, and these results are reported here for the first time.

Conclusion

T. boudieri, L. vellereus and A. brunnescens showed antimicrobial effects with the different concentrations against the test microorganisms. The acetone extract of T. boudieri showed the maximum antimicrobial effect whereas chloroform extracts of T. boudieri showed minimum antimicrobial effect. Fungal species can be regarded as a source of healthy foods, contributing high of antimicrobial effects. The present results indicate that economically important and edible mushrooms display significant antimicrobial features. Therefore, these studies should be extended to other economically important and edible mushrooms.

Declaration of interest

The authors are indebted to the Foundation for Selcuk University, Scientific Research Projects Coordinating Office (SÜ-BAP-08201036 and 10701001) and TÜBİTAK (TBAG-109T584) for their financial support of this work.