Biological activity and fatty acid composition of Caesar's mushroom

Abstract

Context: Due to its pleasant aroma and flavor, Amanita caesarea (Scop.) Pers. (Amanitaceae) has been a famous macrofungus since ancient times. This species is also well known in Turkey where people consume it extensively.

Objective: Evaluation of the medicinal importance of A. caesarea for human health.

Materials and methods: Antioxidant capacity of A. caesarea was studied using the methods of a scavenging effect on 2,2-diphenyl-1-picrylhydrazyl radicals, β-carotene–linoleic acid assay, reducing power and estimation of phenolics. Chloroform, acetone and methanol extracts of A. caesarea were tested for their antimicrobial activity against four Gram-positive bacteria, five Gram-negative bacteria and one yeast by applying a micro dilution method. The fatty acids were estimated via the method of gas chromatography analysis.

Results: The scavenging effect of A. caesarea on DPPH radicals was measured as 40.91% at 0.5 mg/mL concentration, and its reducing power was 0.451 mg/mL at 1.2 mg/mL concentration. The phenolics found were catechin (32.5 mg/g), ferulic acid (7 mg/g), p-coumaric acid (6 mg/g) and cinnamic acid (6.2 mg/g). The highest minimum inhibitory concentration observed against the test microorganisms were with the acetone extract (4.8 µg/mL concentration) against Candida albicans. Thirty-seven different fatty acids were determined from A. caesarea, and oleic acid (58%) was the dominant component.

Discussion and conclusion: Amanita caesarea had a high-antioxidant and -antimicrobial activity, and it also had important essential fatty acids required for human health. According to the results, this mushroom can be recommended as a major source of natural food.

• Amanita caesarea
• gas chromatography
• Gram positive and negative bacteria
• HPLC
• micro dilution
• phenolics
• radical scavenging
• Turkey

Introduction

Fungi have been used as tea or nutritious food source in Eastern cultures for many years because of their unique scents and soft structures. Certain species of edible, inedible and poisonous mushrooms are known in terms of significant medicinal properties, and their extracts are also used for the possible treatment of a number of diseases worldwide. Some special species, such as Lentinula edodes (Berk.) Pegler (shiitake) (Marasmiaceae), Grifola frondosa (Dicks.) Gray (maitake) (Meripilaceae), Ganoderma lucidum (Curtis) P.Karst. (mannentake) (Ganodermataceae) and Cordyceps spp. (Cordycipitaceae), have a history of medicinal usage in parts of Asia. The studies have indicated that mushrooms have cardiovascular, anticancer, antiviral, antibacterial, anti-parasitic, anti-inflammatory, hepato-protective and glycemic regulatory activities (Barros et al., 2007; Jua et al., 2010; Yang et al., 2002).

Antioxidants, or the molecules that have a scavenging effect on free radicals, are known as potentially protective substances (Ramirez-Anguiano et al., 2007). This protective effect is mainly attributed to well-known antioxidants such as ascorbic acid, tocopherols and β-carotenes, but plant phenols also play an important role.

Fatty acids are important constituents of fungal cells with recognized roles as storage material, and as components of plasmalemma and cell organelle membranes. In fungi, the major fatty acids that typically occur in membrane phospholipids and storage triacylglycerols are palmitic and stearic acids, and their unsaturated derivatives palmitoleic, oleic, linoleic and linolenic acids (Suutari, 1995). Mushrooms reveal highly variable fatty acid profiles, and palmitic, oleic and linoleic acids are the most abundant fatty acids found in the members of Basidiomycetes. Interest in lipids, especially in their fatty acid composition, is currently expanding. Such data are used for physiological, chemotaxonomic and intrageneric differentiation studies of many organisms such as bacteria, algae, fungi and vascular plants. Nutritionally, linoleic and α-linolenic acids are essential for basal metabolism in humans, while long-chain polyunsaturated fatty acids (PUFA) have many beneficial effects on human health (Pedneault et al., 2006).

Amanita caesarea (Amanitaceae), commonly known as Caesar's mushroom, is a highly regarded edible mushroom. It has a distinctive orange cap, yellow gills and stem. Its value has been known ever since the time of ancient Romans. Amanita caesarea is also one of the well-known species used as food in Turkey. It grows in mixed forests of coniferous and deciduous trees. Although A. caesarea is an important food source in Turkey, there is no conclusive report available concerning its antioxidant activity, antimicrobial effects or fatty acid composition. The main objectives of this study are (i) to reveal the antioxidant activity of a methanol extract of A. caesarea, (ii) to observe the antimicrobial effects of chloroform, acetone and methanol extracts of A. caesarea against Gram-positive and Gram-negative bacteria and yeast, and (iii) to characterize the fatty acid composition of A. caesarea.

Materials and methods

Collection of A. caesarea samples

The samples of A. caesarea were collected in mixed Pinus brutia, Quercus sp. and Arbutus sp. forest, with elevation of 300 m, on 4 November 2008. The collection site was Karatepe district, Gazipaşa, Antalya Province, Turkey, and its Fungarium number is HD2234.

The species identification was performed by Hasan Hüseyin Doğan as described in the literature (Galli, 2001). A stock sample of the species was also deposited at the Fungarium of the Mushroom Application and Research Centre, Selcuk University, Konya, Turkey.

Sample preparation

The fruiting bodies of each mushroom sample were dried in a dehydrator at 37–40 °C for 5 d. The dried samples were homogenized in a household blender at the full speed until they turned into powder.

Antioxidant activities

Chemicals

β-Carotene, linoleic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and α-tocopherol were purchased from Sigma (Sigma-Aldrich, St Louis, MO). Tween 20, Folin-Ciocalteu’s phenol reagent (FCR), sodium carbonate and methanol were purchased from Merck (Darmstad, Germany). All the chemicals and reagents were of analytical grade and were obtained from either Sigma or Merck.

Preparation of the methanol extracts for testing antioxidant activities

Briefly, 100 g of dried and powdered sample was extracted by stirring in methanol at 60 °C for 6 h in a Soxhlet apparatus. The extract was then filtered through Whatman No. 4 filter paper and concentrated under vacuum at 45 °C using a rotary evaporator. The extracts were then lyophilized and stored in the dark at 4 °C till further use.

Scavenging effect on DPPH radicals

Methanol extract (1 mL) in a range of concentrations (0.03–0.5 mg/mL) was added to 1 mL DPPH radical solution in methanol (the final concentration of DPPH was 0.2 mM). The mixture was shaken vigorously and kept for 30 min in the dark, and the absorbance was then measured at 517 nm against a blank using a Hitachi U-2001 spectrophotometer (Hitachi High-Tech Co., Kyoto, Japan) (Shimada et al., 1992). BHT and BHA were employed as standard controls. Scavenging of DPPH free radicals was calculated in percentages via the following equation: where A_blank is the absorbance of the control reaction and A_sample is the absorbance of the test compound. The extract concentration providing 50% inhibition (IC₅₀) was calculated from a graph plotting % scavenged against extract concentration. Tests were performed in triplicate.

β-Carotene–linoleic acid assay

The β-carotene-linoleic acid assay was conducted as described by Taga et al. (1984). β-Carotene solution (1 mL) in chloroform (3.34 mg/mL) was pipetted into a flask containing 40 mg linoleic acid and 400 mg of Tween 20. The chloroform was then removed by means of a rotary evaporator at 40 °C for 5 min. To the resulting residue, 100 mL of oxygen passed through distilled water was added slowly, with vigorous agitation to form an emulsion. A 5 mL aliquot of this emulsion was added to a tube containing 0.2 mL of the 200 mg/mL antioxidant solution, and the absorbance was measured immediately at 470 nm against a blank, which consisted of the emulsion without β-carotene. The tubes were then placed in a water bath at 40 °C, and the absorbance was measured again at 15 min intervals.

Reducing power

Each extract (0.04–0.4 mg/mL) in methanol (2.5 mL) was mixed with 2.5 mL of 200 mM sodium phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide, and the mixture was incubated at 50 °C for 20 min. Next, 2.5 mL of 10% trichloroacetic acid (w/v) was added, and the mixture was centrifuged at 200 g for 10 min. The upper layer (2.5 mL) was mixed with 2.5 mL of deionized water and 0.5 mL of 0.1% ferric chloride. Finally, the absorbance was measured at 700 nm against a blank, using a Hitachi U-2001 spectrophotometer.

Determination of the total phenolic content

The total phenolic content of the methanol crude extract was determined by the Folin–Ciocalteu method with some modifications, according to Singleton and Rossi (1965). The total phenolic content of the samples was expressed in gallic acid equivalents, which reflects the phenolic content as the quantity of gallic acid (mg) contained in 1 g of the sample.

Phenolic composition of A. caesarea

A Shimadzu 1100 series (Shimatzu Co., Kyoto, Japan) high-performance liquid chromatography (HPLC) equipped with a SIL-10AD vp autosampler and LC-10Advp pump system, a diode array detector (DAD) and an Inertsil Agilent Eclipse XDB column (240 mm × 4.60 mm, 5 μm particle size) (Inertsil Co., Boston, Billerica, MA) was utilized to analyze phenolic compounds. The mobile phase comprised of (A) 100% methanol and (B) 3% (v/v) aqueous acetic acid. HPLC separation was performed as described by Maltas and Yildiz (2011). Gallic acid, catechin, caffeic acid, p-coumaric acid, ferulic acid, cinnamic acid and quercetin (Sigma-Aldrich, St Louis, MO) were used as standards. The samples were run in triplicate.

Antimicrobial activity

Preparation of the extracts

Chloroform, acetone and methanol were used as solvents. Each powdered fungus sample (30 g) was suspended with 250 mL of chloroform in a Soxhlet apparatus for 8 h. The resultant extract was concentrated by means of a rotary evaporator at 40 °C and at 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 DMSO:PBS (1:1) at a 100 000 µg/mL concentration and 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 (Escherichia 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 a 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 microdilution method

The MIC values were evaluated in accordance with NCCLS (2008). Amanita caesarea 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 A. caesarea chloroform, acetone or methanol extract was separately dispensed into MHB-containing microplate wells (A1, A2, A3, etc.), mixed well and 1/2 dilutions were prepared. Finally, this process was repeated to generate a dilution series of each extract from 20 000 to 0.305 µg/mL.

After the dilution series of each extract was prepared, 100 µL of each bacterial suspension was added separately into each well containing the MHB and the mushroom extract mixture. The current procedure was also repeated for the yeast in different plate wells.

The absorbance of each well was measured by employing 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 minimum inhibitory concentration (MIC) values. The lowest concentration that produced an inhibitory effect was recorded as the MIC for each extract (as described by Devienne & Raddi, 2002, with some modifications). Ampicillin (100 μg/mL concentration) for bacteria and amphotericin B (50 μg/mL concentration) for yeast were utilized as positive controls. Each experiment was conducted in triplicate. Due to the lack of correlation and statistical significance between each bacterial species, statistical analysis was not performed.

Fatty acid extraction

To obtain the crude oil of the fungus, a powdered fungus sample (30 g) was suspended with 250 mL of petroleum ether in a Soxhlet apparatus for 8 h. Oil sample of 0.16–0.20 g was added to a round-bottom flask containing 4 mL of a 0.5 N methanol NaOH solution. Then, the mixture was boiled in a water bath for 10 min until saponification occurred. After saponification, 5 mL of 14% BF₃–methanol solution was added to the flask, and the mixture was boiled for 5 min. Then, the flask was shaken, and 2 mL n-heptane was added. All the extract mixtures were boiled for 1 min, and 4 mL NaCl (a saturated solution) was added. Once the extract was thoroughly mixed, it was transferred into a separating funnel, and the phases were allowed to separate for 5–10 min. The lower aqueous phase was discarded, and the upper, light-yellow-colored phase was aliquoted into phials, which were stored in a freezer until needed.

Gas chromatography analysis

Gas chromatography analysis was performed by making use of an HP 6890 model Hewlett Packard Agilent gas chromatograph with an automatic injector and a flame ionization detector. A 100 m HP-88 capillary column was employed in the analysis. The temperature of the injector block was set to 240 °C, and the detector block was set to 250 °C.

The column temperature was initially set to 160 °C for 2 min, and later increased to 185 °C at a rate of 4 °C per min. This was followed by a temperature increase by 1 °C per min to 200 °C. When the temperature reached 200 °C, the column was held at this temperature for 46.75 min. The analysis was completed in 70 min. The helium flow was set to 1 mL/min. Alltech and Accu standards were applied for identification of the fatty acid content. The results were rendered as percentage of the total fatty acids. The standard errors ranged from ±1 to 3%, and three GC analysis results were evaluated together.

Results and discussions

Antioxidant activity results

The scavenging effect on DPPH radicals

The DPPH radical scavenging effects of A. caesarea methanol extract, along with those of the BHA and BHT controls increased with increasing the concentration from 0.03 to 0.5 mg/mL (Figure 1). The scavenging values recorded at 0.03 mg/mL concentration are 0.049 mg/mL for A. caesarea, 0.045 mg/mL for BHT and 0.056 mg/mL for BHA, while the scavenging values at 0.5 mg/mL are 0.039 mg/mL for A. caesarea, 0.031 mg/mL for BHT and 0.026 mg/mL for BHA. As illustrated in Figure 1, the scavenging effect of A. caesarea extract on DPPH radicals increased with increasing concentrations.

Figure 1. DPPH radical scavenging effects of A. caesarea, BHA and BHT.

The IC₅₀ value of A. caesarea extract is higher than BHA and BHT. The IC₅₀ values were 0.7615 mg/mL for A. caesarea, 0.35519 mg/mL for BHA and 0.4119 mg/mL for BHT (Figure 2). A lower IC₅₀ value indicates a higher antioxidant activity (Pourmorad et al., 2006). The inhibition values of A. caesarea extract and the standards on the DPPH radicals at a concentration of 0.5 mg/mL were 40.91% for A. caesarea, 60.60% for BHA and 53% for BHT.

Figure 2. IC₅₀ values of A. caesarea, BHA and BHT.

In previous studies, the scavenging effects of the mushroom extracts ranged from 36% to 96% at a concentration of 5–10 mg/mL. In view of the fact that different methods and concentrations have been used in the literature, there are no standard levels for measurement of scavenging effects. Some studies can be summarized as follows. Yang et al. (2002) studied a number of commercially important mushrooms [Flammulina velutipes (Curtis) Singer (Physalacriaceae), Lentinula edodes (Berk.) Pegler (Marasmiaceae), Pleurotus ostreatus (Jacq.) P. Kumm. and P. cystidiosus O.K.Mill. (Pleurotaceae)], and they established that the scavenging effects of methanol extracts of the said mushrooms varied between 42.9% and 81.8% at 6.4 mg/mL concentration. The scavenging effect of a methanol extract from Volvariella volvacea (Bull.) Singer (Pluteaceae) was found 57.8% at 9 mg/mL concentration (Cheung et al., 2003). Mau et al. (2004) applied a 10 mg/mL concentration for their studies, which reported scavenging effects of 78.8, 79.9 and 94.1% for Termitomyces albuminosus (Berk.) R.Heim (Agaricaceae), Grifola frondosa (Dicks.) Gray (Meripilaceae) and Morchella esculenta (L.) Pers. (Morchellaceae), respectively. Additionally, Lee et al. (2008) studied the fruiting body and mycelia of Hypsizygus marmoreus (Peck) H.E.Bigelow (Lyophyllaceae) with hot water and methanol extraction. According to their results, the scavenging effects of the fruiting body and mycelia ethanol extracts, at a concentration of 5 mg/mL, were both 75.5%, while the scavenging effects of the fruiting body and mycelia hot water extracts were 36.8 and 55.5%, respectively, also at 5 mg/mL concentration. At 10 mg/mL concentration, the scavenging effect of Russula delica Fr. (Russulaceae) extract was 26% (Yaltirak et al., 2009). Compared with the inhibition values reported in the aforementioned studies, A. caesarea extract was effective at a much lower concentration. In the present study, inhibition levels by the A. caesarea extract reached 40.91% at 0.5 mg/mL concentration, while those of the BHA and BHT controls were 60.6% and 53%, respectively.

β-Carotene–linoleic acid assay

The rate of absorbance change was calculated from T₀ to 120th min and used to calculate the coefficient of oxidation prevention as a percent (%). The bottom absorbance curve was employed as a control sample. Antioxidant activity was assayed as an ability to inhibit the peroxidation of linoleic acid. The inhibition values of A. caesarea extract were determined to be higher than those of Trolox, but similar to BHT and lower than BHA. Amanita caesarea extract, BHA, BHT and Trolox exhibited 71.29, 85.14, 74.12 and 50.51% inhibition, respectively (Figure 3).

Figure 3. β-Carotene–linoleic acid assay.

According to the literature, inhibition values of mushroom extracts vary between 50% and 96% at different concentrations. The antioxidant activity of methanol extracts of young and mature Agaricus brasiliensis Fr. (Agaricaceae) specimens were evaluated with the β-carotene–linoleic assay, and they were found to inhibit oxidation with 92% at 0.2 mg/mL concentration (Soares et al., 2009). Gürsoy et al. (2009) studied six Morchella species (Morchella rotunda (Fr.) Boud., M. esculenta (L.) Pers. var. umbrina (Boud.) S. Imai, M. deliciosa Fr., M. elata Fr., M. conica Pers. and M. angusticeps Peck.), and they ascertained that M. esculenta var. umbrina and M. angusticeps were the most active species with 96.89% and 96.88%, respectively, at 4.5 mg/mL concentration. Sarikürkcü et al. (2010) observed A. caesarea, Clitocybe geotropa (Bull.) Quél. (Tricholomataceae) and Leucoagaricus pudicus (Bull.) Bon, and they discovered that L. pudicus possessed the highest oxidation level with 81.8%, followed by A. caesarea with 70.1% and by C. geotropa with 61.3% at 25.5 mg/mL concentration. According to the proposed previous study, A. caesarea has a high oxidation level (71.29%) even at a low concentration (2.284 mg/mL).

Reducing power

The reducing power of A. caesarea extract demonstrated a parallelism with its increased concentration (Figure 4). Namely, a higher absorbance indicates a higher reducing power. The highest reducing power was observed with BHA (2.021), followed by BHT (1.869) and A. caesarea (0.36) at 0.075–1.2 mg/mL concentration.

Figure 4. Reducing power of A. caesarea and synthetic antioxidants.

Yang et al. (2002) observed that the reducing power of mushrooms in their study exceeded 1.28 at 40 mg/mL concentration, with the reducing power of each species ordered from the highest to the lowest, as follows: Pleurotus ostreatus = P. cystidiosus > Lentinula edodes > F. velutipes. According to Elmastaş et al. (2007), the reducing power of R. delica and Verpa conica (O.F. Müll.) Sw. (Morchellaceae) extracts were 1.32 and 1.22, respectively, at 200 µg/mL concentration. At 20 mg/mL concentration, the reducing powers of A. caesarea, C. geotropa and L. pudicus were 1.5, 1.2 and 1.3 mg/mL, respectively (Sarikürkcü et al., 2010). In the current study, the reducing power of A. caesarea was 0.451 at 1.2 mg/mL concentration. In comparison with the previous studies described above, A. caesarea possessed a high reducing power at a low concentration.

Total phenolic content

The total phenolic content of the samples is expressed as milligram of gallic acid per gram equivalent of dry mushroom. The amount of phenolic compounds in the methanol extract of A. caesarea was determined to be 0.642 mg/g gallic acid equivalents. A number of common edible mushrooms, which are widely consumed in Asian cultures, have been found to possess antioxidant activities that are well correlated with their total phenolic content (Cheung et al., 2003).

Phenolic compounds of A. caesarea

Some selected phenolic acids and flavonoids of the extract were separated and compared with authentic standards by means of reverse-phase HPLC for identification. The phenolic composition of A. caesarea has been reported here for the first time (Table 1).

Table 1. Phenolic compounds of A. caesarea.

Phenolic compounds	Composition (mg/g)
1. Caffeic acid	ND
2. Catechin	32.5
3. Cinnamic acid	4.6
4. Ferulic acid	7
5. Gallic acid	ND
6. p-Coumaric acid	6
7. Quercetin	ND

Seven components of the methanol extract of A. caesarea were analyzed, and four components were identified: catechin, p-coumaric acid, ferulic acid and cinnamic acid. There were substantial qualitative and quantitative differences in the components of the extracts. Catechin was the predominant phenolic compound with a value of 32.5 mg/g, followed by ferulic acid with 7 mg/g, p-coumaric acid with 6 mg/g, and cinnamic acid with 4.6 mg/g. Nonetheless, the extract did not contain any flavonoids, such as quercetin, caffeic acid and gallic acid.

Antioxidant, antimicrobial, anti-allergy and anticancer effects of catechin have been reported in a number of earlier studies (Kondo et al., 2000; Shimamura et al., 2007). Catechin was found in R. delica with 5.33 mg/g by Yaltirak et al. (2009). In the present study, it was found in high concentration (32.5 mg/g). The high antimicrobial activity shown by A. caesarea may be due to the presence of catechin.

Ferulic acid, like many other phenols, is an antioxidant in vitro in the sense that it is reactive toward free radicals, such as reactive oxygen species (ROS). ROS and free radicals are implicated in DNA damage, cancer and accelerated cell aging. Ferulic acid may also have a direct antitumor activity against breast cancer and liver cancer, and it may have pro-apoptotic effects in cancer cells, thereby leading to their destruction. Ferulic acid may be effective in preventing cancer induced by exposure to carcinogenic compounds (Kampa et al., 2004).

Coumarin derivatives are substances important for human health. They have anti-thrombotic, anti-inflammatory and vasodilatory effects, coupled by antiviral and antimicrobial activities. Coumarin was also found to inhibit C. albicans in vitro. As a group, coumarins have been found to stimulate macrophages, which could have an indirect negative effect on infections. “More specifically, coumarin has been used to prevent recurrences of cold sores caused by HSV-1 in humans” (Cowan, 1999).

Cinnamic acids are common representatives of a wide group of phenylpropane-derived compounds that are in the highest oxidation state. Cinnamic acids are effective against viruses, bacteria and fungi (Cowan, 1999).

Antimicrobial results

According to Craig (1998), in order to evaluate antimicrobial activity, MIC values ought to be measured from the 4th through the 16th dilutions. The antimicrobial effects of A. caesarea against bacteria and yeast were measured in compliance 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 μg/mL and 500 μg/mL = antimicrobial activity is moderate.

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

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

Pursuant to the quoted ranges, the antimicrobial results are provided in Table 2. Amanita caesarea exhibited different antimicrobial effects in various concentrations against each test microorganism. The maximum inhibitory effect on the test microorganisms was observed with an acetone extract (MIC value, 4.8 µg/mL in the 13th dilution) against C. albicans. Overall, the methanol extract was observed to display the maximum antimicrobial effect with values generally lower than 100 µg/mL, placing it in the high-activity category. The microorganisms inhibited at this level were B. subtilis, S. aureus, L. monocytogenes, C. albicans, K. pneumoniae, P. aeruginosa, P. vulgaris and S. enteritidis (MIC values, 39 µg/mL in the 9th dilution). The effects of the methanol extract against S. pyogenes and E. coli were highly and moderately active (MIC values, 78 and 156 µg/mL in the 6th and 7th dilutions, respectively). The MIC values of chloroform and acetone extracts were almost identical with the MIC values of 39 µg/mL. The lowest antimicrobial effect was noted in the chloroform extract against K. pneumoniae and acetone extract against E. coli (MIC values, 312.5 µg/mL in the 6th dilution).

Table 2. MIC values of A. caesarea extracts (μg/mL)^a.

Microorganisms	Chloroform	Acetone	Methanol
B. subtilis	39	39	39
S. aureus	39	39	39
L. monocytogenes	39	78	39
S. pyogenes	39	78	78
C. albicans	39	4.8	39
E. coli	39	312.5	156
K. pneumoniae	312.5	39	39
P. aeruginosa	39	39	39
P. vulgaris	39	39	39
S. enteritidis	39	39	39

^a<100 μg/mL = High, 100–500 μg/mL = Moderate and 500–1000 μg/mL = Low.

In general, methanol and acetone extracts of A. caesarea were more effective than chloroform extracts against bacteria and yeast. The most effective MIC concentrations of the extracts were typically measured between the 6th and 9th dilutions (MIC values, 312.5–39 µg/mL), and these results demonstrated that the inhibition values were lower than 100 µg/mL or between 500 and 1000 µg/mL.

The current results were verified by previous studies. According to Yoon et al. (1994), G. lucidum had a good antimicrobial effect against Proteus vulgaris (MIC, 1.25 mg/mL) and Escherichia coli (MIC, 1.75 mg/mL), and six species of bacteria had MIC values larger than 5 mg/mL. Within the study, MIC values were generally measured between 78 and 312.5 µg/mL, and the present results are better than those of Yoon et al. (1994). Gbolagade et al. (2007) studied the antimicrobial effects of certain fungal species by the employment of micro dilution methods, and they established that the MIC concentration of Marasmius jodocodo Henn. (Marasmiaceae) was 2.75 mg/mL against E. coli, while T. robustus was 15.75 mg/mL against M. bourlardii. Janeš et al. (2007) applied the broth micro dilution test for screening of antibacterial activity on the extracts of higher and endophytic fungi. Among the tested extracts, three significant antibacterial activities were identified from the extracts of Amanita virosa Secr. (Amanitaceae) and Cortinarius praestans (Cordier) Gillet (Cortinariaceae) against P. aeruginosa and Staphylococcus aureus, respectively, and the extract of endophytic fungus Truncatella hartigii (Tubeuf) Steyaert (Amphisphaeriaceae) against Enterococcus faecalis and S. aureus. Quereshi et al. (2010) tested the antimicrobial activity of various solvent extracts of G. lucidum (40 mg/mL concentration) against six species of bacteria. Acetone extract showed the maximum antibacterial activity, whereas the most susceptible bacterium recorded was Klebsiella pneumoniae. Bala et al. (2011) investigated the antimicrobial effect of 47 different specimens from Australia, and they ascertained that water and ethanol extracts were more effective against S. aureus than E. coli, whereas an inconsiderable number of hexane extracts showed better results for their potential antimicrobial effect against E. coli at higher concentration. In general, a number of macrofungi from the genera Agaricus, Amanita, Boletus, Cantharellus, Fomitopsis, Hohenbuehelia, Lentinus, Ramaria and Strobilomyces demonstrated good inhibition rates. Aqueous and methanol extracts of Trametes hirsuta (Wulfen) Lloyd (Polyporaceae) were tested against pathogenic fungi and bacteria. Maximum antibacterial activity of the aqueous extract of T. hirsuta was found against S. aureus compared to that of the methanol extract. Significant antifungal activity of the aqueous extract was found against Aspergillus flavus compared to that of the methanol extract (Sivaprakasam et al., 2011). The antibacterial effects of the extract from three mushrooms G. lucidum, Auricularia auricula (L.) Underw. (Auriculariaceae) and Pleurotus floridanus Singer (Pleurotaceae) were studied against S. aureus and E. coli. Auricularia auricula displayed significant antibacterial activity against S. aureus. P. floridanus showed some antibacterial activity, while G. lucidum did not demonstrate any antibacterial activity. None of the extracts exhibited 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. The maximum activity of crude extracts was exhibited by ethyl acetate. The MIC of sesquiterpenoid extracts of G. praelongum was 0.390–6.25 mg/mL. Diterpenoids and triterpenoids displayed a moderate activity, while polysaccharides IIIa and IIIb showed a weak activity. All bacterial strains were resistant to polysaccharides I and II (Ameri et al., 2011).

The current results are similar or more effective than those reported in the literature. To the best of our knowledge, there have previously been no reports on the antimicrobial effects of A. caesarea, and these results have been reported here for the first time.

Fatty acids

Saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) of A. caesarea were analyzed by gas chromatography (Table 3). A total of 37 fatty acids were established. These fatty acids varied in the length from C6 to C24. The largest component of the total fatty acid was identified as C18:1 ω 9 (oleic acid). In addition, MUFA, measured as 59.07% of the total fatty acid composition, were more abundant than SFA (21.46%) and PUFA (19.48%).

Table 3. Percentage of fatty acids of A. caesarea.

Carbon numbers	A. caesarea	Common and systematic names
C 6:0	0.01 ± 0.01	Caproic acid
C 8:0	0.01 ± 0.01	Caprylic acid
C 10:0	0.02 ± 0.01	Capric acid
C 11:0	0.03 ± 0.01	Undecylic acid
C 12:0	0.03 ± 0.01	Lauric acid
C 13:0	0.01 ± 0.01	Tridecylic acid
C 14:0	0.17 ± 0.01	Myristic acid
C 15:0	0.19 ± 0.01	Pentadecylic acid
C 16:0	15 ± 0.04	Palmitic acid
C 17:0	0.04 ± 0.01	Margaric acid
C 18:0	6.09 ± 0.01	Stearic acid
C 20:0	0.07 ± 0.04	Eicosanoic acid
C 21:0	0.20 ± 0.01	Heneicosanoic acid
C 22:0	0.01 ± 0.01	Docosanoic acid
C 24:0	0.01 ± 0.01	Tetracosanoic acid
∑ SFA*	21.46 ± 0.01
C 14:1n5	0.03 ± 0.01	Myristoleic acid
C 15:1n5	0.01 ± 0.01	Pentadecenoic acid
C 16:1n7	0.63 ± 0.01	Palmitoleic acid
C 17:1n8	0.13 ± 0.01	9-Heptadecanoic acid
C 18:1n9	58 ± 0.06	Oleic acid
C 20:1n9	0.20 ± 0.04	11-Eicosenoic acid
C 22:1n9	0.01 ± 0.01	13-Docosanoic acid
C 24:1n9	0.01 ± 0.01	15-Tetracosenoic acid
∑ MUFA	59.07 ± 0.02
C 18:2n6	19.02 ± 0.02	Linoleic acid
C 18:3n6	0.06 ± 0.01	6-9-12 Octadecatrienoic acid
C 18:3n3	0.03 ± 0.01	Linolenic acid
C 20:2n6	0.01 ± 0.01	11,14-Eicosadienoic acid
C 20:3n6	0.01 ± 0.01	8,11,14-Eicosatrieonic acid
C 20:3n3	0.01 ± 0.01	Eicosatrienoic acid
C 20:4n6	0.12 ± 0.01	5-8-11-14 Eicosatetraeonic acid
C 20:5n3	0.10 ± 0.01	5-8-11-14-17 Eicosapentaenoic acid
C 22:2n6	0.05 ± 0.01	cis-13,16-Docosadienoic acid
C 22:3n3	0.01 ± 0.01	13-16-19 Docosatrienoic acid
C 22:4n6	0.03 ± 0.01	7-10-13-16 Docosatetraenoic acid
C 22:5n6	0.01 ± 0.01	4-7-10-13-16 Docosapentaenoic acid
C 22:5n3	0.01 ± 0.01	7-10-13-16-19 Docosapentaenoic acid
C 22:6n3	0.03 ± 0.01	4-7-10-13-16-19 Docosahexaenoic acid
∑PUFA^a	19.48 ± 0.03

^aSFA: saturated fatty acid, MUFA: mono unsaturated fatty acid, PUFA: polyunsaturated fatty acid.

The most abundant fatty acid recorded in A. caesarea was oleic acid (58.6%), followed by linoleic acid (19.02%), palmitic acid (14.59%) and stearic acid (6.09%). The said four most abundant fatty acids constituted 97.76% of the total fatty acid pool.

The main fatty acid components of Lactarius deliciosus (L.) Gray (Russulaceae), Sarcodon imbricatus (L.) P. Karst.(Bankeraceae) and Tricholoma portentosum (Fr.) Quél. (Tricholomataceae) consisted of MUFA, while PUFA were the most abundant components of Agaricus arvensis Schaeff. (Agaricaceae) and Leucopaxillus giganteus (Sowerby) Singer (Tricholomataceae) (Barros et al., 2007). Unsaturated fatty acids were found at higher concentrations than saturated fatty acids in the total fatty acids of the mushrooms analyzed by Mauger et al. (2003). In the present study, the high MUFA content of A. caesarea (59.07%) is consistent with these results.

Palmitic acid is the most common saturated fatty acid in plants and animals. This is the primary fatty acid from which other longer fatty acids are synthesized. Palmitic acid is not found in a free form in nature like other fatty acids and the level of palmitic acid in A. caesarea is relatively high (15%).

The most common MUFA is oleic acid. This acid is utilized in soap-making, wax production, medicine, and textile and leather industries. Oleic acid may hinder the progression of adrenoleukodystrophy, a fatal disease that affects the brain and adrenal glands. Oleic acid may be responsible for the hypertensive (blood pressure-reducing) effects of olive oil. Adverse effects have also been documented; however, both oleic and monounsaturated fatty acid levels in the membranes of red blood cells have been associated with an increased risk of breast cancer. The oleic acid content in A. caesarea was measured as 58%, a useful level for dietary purposes. In the current study, trans fatty acid isomers were not found.

The fatty acid composition of Agaricus bisporus (J.E. Lange) Imbach, A. campestris L., Coprinus comatus (O.F. Müll.) Pers. (Agaricaceae), Boletus edulis Bull. (Boletaceae), P. ostreatus, Oudemansiella radicata (Relhan) Singer and Armilleria mellea (Vahl) P. Kumm. (Physalacriaceae) was studied, and the amount of unsaturated fatty acids present was higher than that of saturated fatty acids. Linoleic acid was determined to be common in all mushroom species. In addition, palmitic acid, oleic acid, stearic acid and arachidic acid were the most abundant fatty acids identified in a study of various fungi (Yilmaz et al., 2006). Oleic acid and linoleic fatty acids of A. caesarea were the most abundant fatty acids. Palmitic and stearic acid were the next most predominant components of A. caesarea. Linoleic acid, an essential fatty acid, comprised 19.02% of A. caesarea in the total fatty acids.

Conclusions

The antioxidant and antimicrobial effects of A. caesarea were measured, and its unsaturated fatty acid composition was revealed to be relatively high. Fungal species can be a source of healthy food, contributing high levels of antioxidants, antimicrobial effects and unsaturated fatty acids.

The present results indicate that economically important and edible mushrooms demonstrate significant antioxidant and antimicrobial features, and they are a good source of fatty acids. Therefore, these studies should be expanded to other economically important and edible mushrooms.

Declaration of interest

The authors are indebted to the Foundation TUBİTAK (TBAG/109T584) and the Scientific Research Projects (BAP) Coordinating Office (BAP/09201151) at Selçuk University for their financial support of the current work.