Introduction

The genus Agaricus belonging to the family Agaricaceae includes about 300 different mushrooms. Most of them are edible, although poisonous species are also known [1]. The fruiting bodies of Agaricus have a fleshy cap or pileus with a dry, bare or scaly surface and a number of radiating plates or gills, the hymenophore. Agaricus species are appreciated due to their nutritional value, bioactive ingredients, antioxidant activity (including free radical scavenging) and biological effects, which makes them a valuable source of nutritional and pharmacological compounds [2,3,4]. Some species are not only ingredients of human diets, but also function as a health food used for centuries in traditional medicine. Agaricus bisporus is one of the most frequently cultivated and consumed mushrooms in the world. Agaricus brasiliensis is known as a medicinal mushroom. Agaricus arvensis is a wild growing species, although its fruiting bodies are also commercially produced. Agaricus bitorquis, Agaricus campestris and Agaricus silvaticus are edible wild growing species.

Most of the studies on the Agaricus genus concern A. bisporus and A. brasiliensis, predominantly focusing on an investigation of their bioactive compounds and therapeutic effect. Little is known about other species of Agarisus and other metabolites. Phenolics belong to bioactive compounds, although they are non-essential dietary components. Their biological function is related to free radical scavenging activity, metal chelation ability and inhibition of lipid oxidation [5, 6].

Organic acids are responsible for the taste and flavor of a mushroom and can also play a biological role owing to their antioxidant, acidifying, neuroprotective, anti-inflammatory and antimicrobial properties [7,8,9,10,11,12].

Ergosterol is a highly beneficial sterol with properties that can promote human health (antimicrobial, anticomplementary and antitumor activity) and the capacity to be transformed into vitamin D2 [13,14,15].

The aim of the study was to investigate the profile of phenolic compounds and organic acids together with the ergosterol content of different species of cultivated and wild growing Agaricus genus. The ability to scavenge DPPH radicals was also estimated. The relationship between antioxidant activity and the content of phenolic compound was also investigated.

Materials and methods

Mushroom species

Fruiting bodies of different cultivated and wild growing Agaricus species were used during the investigation (Table 1). From among cultivated mushrooms, the following species were used: seven strains of Agaricus bisporus (one brown and six white), Agaricus brasiliensis and Agaricus arvensis. Three species of wild mushrooms were picked in natural conditions from the Wielkopolska Region and identified according to Wojewoda [16]: Agaricus bitorquis, Agaricus campestris and Agaricus silvaticus.

Table 1 Species of mushroom and their source
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A. bisporus, A. arvensis and A. brasiliensis were cultivated according to the conventional method in three repetition. The substrate (a mixture of chicken manure, wheat straw and gypsum) with the mycelium was placed in plastic containers, incubated at 25 °C and 90–95% moisture content. Incubation of the substrate completely covered by mycelium with a 5-cm layer of casing soil (a mixture of chalk and Sphagnum sp. peat, v:v; 5:1) was carried out in the same conditions until the soil was completely overgrown by mycelium, after which the temperature was reduced to 16 °C for A. bisporus and A. arvensis, and to 21–22 °C for A. brasiliensis. All analysis were conducted on fruiting bodies of the first flush. The fruiting bodies of cultivated and wild growing Agaricus species were dried in an electric drier (SLW 53 STD, Pol-Eko) at 40 ± 2 °C to a constant weight and ground for 30 s in a Cutting Boll Mill 200 (RETSCH GmbH, Haan, Germany). The moisture content in dried samples was ~2%. Three representative powdered samples were used for the extraction procedure.

Chemicals

Acetonitrile with 0.01% formic (LC–MS CHROMOSOLV), ethanol (≥99.5), methanol (≥98.8%), 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin–Ciocalteu’s phenol reagent, KH2PO4 (99.5%), H3PO4 (≥85%), standards of phenolic compounds such as: p-coumaric (≥98%), gallic (≥99), protocatechuic, benzoic (≥99.5%), 2,5-dihydroxybenzoic (≥99%), 4-hydroxybenzoic (≥99%), caffeic (≥98%), chlorogenic (≥95%), vanillic (≥97%), salicylic (≥99), syringic (≥97%), ferulic (≥98%), sinapic (≥98%) and trans-cinnamic acids (≥99), rutin (≥98%), catechin (≥98%), kaempferol (≥97%), quercetin (≥98), vitexin (≥95%), luteolin (≥98%), purchased from Sigma-Aldrich (St. Louis, MO, USA). The standards of organic acids [acetic (≥99%), citric (≥99.5%), formic (≥95%), fumaric (≥99%), lactic (≥98%), maleic (≥99%), malic (≥99%), malonic (≥99%), oxalic (≥99%), and succinic (≥99%) acids] were purchased from Supelco with a certified standard grade.

Extraction

The extraction of phenolic compounds and organic acids was carried out according to the procedure of Carvajal et al. [17] with some modification. Dried and ground mushroom samples (5 g) were twice extracted with 70% ethanol by shaking in Ika KS 260 shaker (IKA-Werke GmbH & Co. Kg, Staufen, Germany) for 3 h at room temperature. After centrifugation at 3000 rpm with a Universal 320 R centrifuge (Hettich, Tuttlingen, Germany) and filtration through Whatman No. 4 paper (UK) the combined extracts were evaporated to dryness at 40 °C with a rotary vacuum evaporation Büchi Rotavapor R-205 (Flawil Switzerland) and frozen at −12 °C. The extracts were used in further analyses.

HPLC analysis of organic acids

The extracts were dissolved in 1 mL deionized water (Mili-Q, Millipore). For determination of organic acids, a Waters Alliance 2695 Chromatograph with a Waters 2996 Photodiode Array Detector (Waters Corp., Milford, MA, USA) was used. Separation was performed on a Waters Atlantis C18 column (250 mm × 4.6 mm × 5 μm), at 220 nm wavelength, with the following column conditions: 25 mM KH2PO4 (adjusted to pH 2.5 with concentrated H3PO4) and methanol as an eluent (95:5, v/v), and flow rate at 0.8 mL/min [18]. Acids were identified by the retention times of their peaks in a chromatogram and quantified by peak area comparison with standards at a known compound concentration according to the appropriate standard curve.

UPLC analysis of phenolic compounds

The extracts were dissolved in 1 mL of 80% methanol and used for the determination of phenolic compounds using an ACQUITY UPLC H-Class System equipped with a PDA eλ Detector (Waters Corp., Milford, MA, USA). A gradient elution of solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid) was applied to an Acquity UPLC BEH C18 column (2.1 mm × 150 mm, 1.7 µm, Waters). The gradient program was as follows: flow 0.4 mL/min—5% B (2 min), 5–16% B (5 min), 16% B (3 min), 16–20% B (7 min), 20–28% B (11 min) flow 0.45 mL/min—28% (1 min), 28–60% B (3 min) flow 5.0 mL/min—60–95% B (1 min), 65% B (1 min), 95–5% B (0.1 min) flow 0.4 mL/min—5% B (1.9 min). The injection volume was 2 µL. The quantification of phenolic compounds was performed using an external standard. The preferable wavelengths were 280 and 320 nm [19].

Total phenolic content

The Folin–Ciocalteu assay was used for determination of the total phenolic (TP) as previously described with some modifications [20]. The absorbance of 1 mL of methanol extract mixed with 1 mL of Folin–Ciocalteu reagent (diluted with H20; 1:1, v/v) and 3 mL of 20% Na2CO3 after 30 min incubation in darkness at room temperature was measured at 765 nm. The results were expressed as mg of gallic acid equivalent (GAE) per 100 g of dried weight (DW).

Evaluation of DPPH radical scavenging activity

The scavenging effect against the DPPH radicals was carried out according to Dong et al. [21]. Extracts (1 mL) at different concentrations (0, 2, 4, 6, 8, 10 and 12 mg/mL) were mixed with methanolic solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals (2.7 mL of 6 µmol/L), shaken and kept in the dark for 1 h. The absorbance of each samples was measured at 517 nm to determine the reduction of the DPPH. The percentage of DPPH scavenging activity was calculated according to Reis et al. [22]:

$$(\% {\text{RSA}}) \, = { [}({\text{Ac}} - A)/{\text{Ac}}] \, \times { 1}00$$

RSA is the radical scavenging activity, Ac is the absorbance of control and A is the absorbance of samples.

The scavenging activity was expressed as EC50 (the concentration at which the ability to scavenge DPPH˙ was 50%) and was estimated graphically.

Determination of ergosterol

Ergosterol in fruiting bodies of Agaricus species was determine by UPLC according to Perkowski et al. [23] with some modifications. Powdered mushroom samples (100 mg), 2 mL of pure methanol and 1 mL of 2 M NaOH were mixed together, irradiated twice in a microwave (2 × 15 s) and cooled. Then, 2 mL of 1 M HCl was added to the mixture. Then, samples were cooled, extracted with pentane and evaporated to dryness in a nitrogen stream. The quantification of ergosterol was done using an ACQUITY UPLC H-Class System and a PDA eλ Detector (Waters Corp., Milford, MA, USA). Identification was carried out on an ACQUITY UPLC HSS T3 C18 column (150 mm × 2.1 mm, particle size 1.8 μm) (Waters, Ireland) protected with 1.7  m ACQUITY UPLC BEH C18 VanGuard Pre-column. The injection volume was 2 µL. The flow rate of the isocratic elution of the mobile phase (mixture of methanol, acetonitrile and water; 85:10:5, v/v/v) was 0.5 mL/min. The run time of analysis was 10 min.

Statistical analysis

All analyses for each mushroom were performed in triplicate. The results are expressed as mean ± standard deviation (SD). Statistical analysis was done using STATISTICA 13.1. One-way analysis of variance ANOVA followed by post hoc Tukey’s test was conducted to estimate differences between mean values. Correlations were estimated by the Pearson correlation coefficients between scavenging ability and phenolic acids.

Results and discussion

Organic acid profile

The profile of organic acids showed that nine acids (acetic, citric, formic, fumaric, lactic, malic, malonic, oxalic and succinic) were detected in Agaricus species, although the profile was very heterogeneous (Table 2). A. silvaticus, A. camperstis and A. arvensis were the richest species in organic acids (23,566.5, 22,985.3 and 21,540.6 mg/100 g DW, respectively), while A. bitorquis and two strains (Kanmycel K2 and Sylvan A 15) of white A. bisporus were very poor in these compounds (6600.5, 6033.1 and 5798.3 mg/100 g DW, respectively). However, all the acids were detected only in A. bisporus (brown) Hollander Spawn C9. Oxalic, lactic and succinic acids were the most abundant components. Oxalic acid was detected in each of the studied Agaricus species. Lactic and succinic acids were detected in almost every sample, with the exception of Kanmycel K2 and Sylvan A15 for the first and A. brasiliensis for the second acid. The highest level of lactic and oxalic acids was found in A. brasiliensis (13,358.1 and 5467.1 mg/100 g DW, respectively), while succinic acid was present in A. bisporus Amycel 2600 (11478.4 mg/100 g DW). The other identified acids were much lower in content or were below the limit of detection.

Table 2 Content of organic acids in fruiting bodies of Agaricus species (mg/100 g DW)
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Organic acids have also been detected in other mushroom species, among which are cultivated and wild growing species with oxalic, citric, malic or fumaric as the main compounds [8, 12, 24,25,26,27]. Oxalic, malic, citric and fumaric acids have been identified in A. bisporus portobello and A. silvaticus, while A. bisporus is additionally known to contain quinic and citric acids [26]. In A. campestris only oxalic, malic and fumaric acids have been found [26]. The range of the acids in the previously mentioned species was: 4.86–19.61, 23.88–30.05, 34.62–43.23, 1.14–3.77 and 6.44 mg/g DW (486–1961, 2388–3005, 3462–4323, 114–377 and 644 mg/100 g DW) for oxalic, malic, citric, fumaric and quinic acids, respectively [26]. Carvajal et al. [17] identified acetic, alpha-ketoglutaric, citric (dominant), fumaric, malic, oxalic and trans-aconitic acids in A. brasiliensis. This profile differs considerably from the one presented in Table 2. In the present study, citric and acetic acids were not detected in A. brasiliensis, while Carvajal et al. [17] show they were dominant [11.79 and 7.57 µg/mg dried fungus (1179 and 757 mg/100 g), respectively]. Carvajal et al. [17] suggested that organic acids could have an effect on antioxidant activity (ferrous ion chelation and an ABTS scavenging assay) of mushroom extract. Due to the antioxidant activity some organic acids (e.g., citric, malic, succinic) can play a protective role in some diseases in humans [8, 9]. Oxalic acid displays antibacterial activity [27], while anti-inflammatory, neuroprotective and antimicrobial properties have been confirmed for fumaric acid [7, 28]. Malic acid is responsible for flavor, while owing to its antioxidant and antibacterial properties citric acid can extend the shelf life of mushrooms and prevent their browning [11, 12, 25, 26]. It is widely used as an acidulant in pharmaceutical and food industries due to its low toxicity [29]. Thus, organic acids have a considerable influence on the acceptability, nutrition and stability of mushrooms [30].

The individual profile of phenolic compounds in mg/100 g DW is presented in Table 3. The investigation reveals that only nine phenolic acids were detected in the analyzed species of Agaricus. Flavonoids were not found. The results are in accordance with Gil-Ramírez et al. [31], who claims that flavonoids are not present in mushrooms because of a lack of enzymes necessary for their synthesis. However, mushrooms are able to absorb them from substrate or plants, from which they form mycorrhizae [31] and thus some flavonoids have been detected [32, 33] The most homogeneous profile was represented for brown and white A. bisporus. Gallic, caffeic and ferulic acids were detected in all the species. Generally, the dominant was gallic acid (5.5–9.2 mg/100 g DW). Furthermore, trans-cinnamic and chlorogenic acids were quantified at a higher level than the other compounds (4.6–9.4 and 5.3–6.7 mg/100 g DW, respectively). A. brasiliensis was the richest in phenolic acid content containing all the analyzed acids (33.9 mg/100 g DW), while the wild growing species A. campestris and A. silvaticus were found to be poorest in phenolic acids (11.3 and 18.2 mg/100 g DW, respectively). Although the phenolic compound composition of many mushroom species is well known, for some Agaricus species (e.g., A. arvensis, A. bitorquis A. campestris, A. silvaticus), it is not yet well recognized.

Table 3 Content of phenolic acids in fruiting bodies of Agaricus species (mg/100 g DW)
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Gallic and syringic (dominant) acids and pyrogallol in fruiting bodies of A. brasiliensis were identified by Carvajal et al. [17]. In the present study, the total content of phenolic acids contained in brown A. bisporus (31.8 mg/100 g DW) was comparable to that of A. brasiliensis (33.9 mg/100 g DW), although syringic acid was not quantified in the first mentioned species. The total content of phenolic acids in all strains of white A. bisporus was similar (24.8–26.0 mg/100 g DW). The exception was Sylvan 767, which had a distinctly higher level of the compounds (30.6 mg/100 g DW). The profile of phenolic acids and their content in A. bisporus in the present study is very similar to the findings of Palacios et al. [32], who also confirmed caffeic, chlorogenic, p-coumaric, ferulic, gallic, p-hydroxybenzoic, and protocatechuic acids, as well as homogentistic acid, myricetin and pyrogallol in the species. While Kim et al. [34] quantified gallic, protocatechuic, myricetin and pyrogallol. Other investigations have revealed only cinnamic acid in white and brown A. bisporus [35]. Reis et al. [22] demonstrated only gallic, p-coumaric and cinnamic acids in white A. bisporus and cinnamic acid in brown A. bisporus. The findings show that the level of the phenolic acids in A. bisporus was as follows: caffeic (15.54 µg/g DW; 1.554 mg/100 g DW), chlorogenic (63.73 µg/g DW; 6.373 mg/100 g DW), p-coumaric (2.31–10.38 µg/g DW; 0.231–1.038 mg/100 g DW), ferulic (16.37 µg/g DW; 1.637 mg/100 g DW), gallic (16–94.9 µg/g DW; 1.6–9.49 mg/100 g DW), p-hydroxybenzoic (15.39 µg/g DW; 1.539 mg/100 g DW), homogentistic (3444.3 µg/g DW; 344.43 mg/100 g DW), protocatechuic (6.21–32 µg/g DW; 0.621–3.2 mg/100 g DW) and cinnamic acids (0.38–149 µg/g DW; 0.038–14.9 mg/100 g DW) [22, 32, 33].

Other tested Agaricus species contained a lower content of total phenolic acids. The content of phenolic acid in A. bitorquis (23.1 mg/100 g DW) and A. arvensis (23.3 mg/100 g DW) was similar and the mushrooms did not show any evidence of p-hydroxybenzoic, syringic, p-coumaric (only A. bitorquis) or protocatechuic (only A. arvensis). The poorest species in phenolic compounds were wild growing A. campestris (11.3 mg/100 g DW) and A. silvaticus (18.2 mg/100 g DW). Only gallic, caffeic, p-hydroxybenzoic, p-coumaric, ferulic acids were quantified in both the species. Additionally, syringic and trans-cinnamic acids were detected in A. silvaticus.

The results concerning A. campestris differ from the profile presented by Woldegiorgis et al. [33], who reported only p-coumaric (10.9 µg/g DW; 1.09 mg/100 g DW), ferulic (20.3 µg/g DW; 2.03 mg/100 g DW), gallic (561.9 µg/g DW; 56.19 mg/100 g DW) and p-hydroxybenzoic (38.7 µg/g DW; 3.87 mg/100 g DW) acids and myricetin (7.08 µg/g DW; 0.708 mg/100 g DW) for the species. However, while the level of p-coumaric and ferulic acids was comparable to the results obtained in this investigation, the content of gallic and p-hydroxybenzoic acids was higher.

The range of total phenolic content was very wide from 132.7 to 1154.7 mg GAE/100 g DW (Table 4). Cultivated A. brasiliensis proved to be richest in phenolics. The present investigation showed that brown A. bisporus strain Hollander Spawn C9 was characterized by a higher content of phenolics than the white variety. Brown A. bisporus strain Hollander Spawn C9, white A. bisporus strain Kanmycel 3-1, A. arvensis, A. bitorquis, A. brasiliensis, A. campestris and A. silvaticus had TP at the level >600 mg GAE/100 g DW, while other strains of white A. bisporus (besides Kanmycel 3-1) had a lower content of TP (<600 mg GAE/100 g DW). In other studies TP in ethanolic extract of white A. bisporus ranged from <3 up to 10 mg/g DW (<300–1000 mg GAE/100 g DW) [32, 36,37,38]. Brown A. bisporus contained up to 10.65 mg/g (1065 mg GAE/100 g) [36]. In studies of A. brasiliensis cultivated in Brazil TP in ethanolic extract ranged from 8.5 to 12.50 µg/mg DW (850–1250 mg GAE/100 g DW) [17, 37], although Mazzutti et al. [39] found TP to be between 12.6 and 74 mg/g extract (1260–7400 mg/100 g extract), depending on the solvent used for extraction. The present study is consistent with the investigation of Gan et al. [37], which indicated that A. brasiliensis was richer in phenolics than A. bisporus. Öztürk et al. [40] studying TP in A. bitorquis estimated the content at 13.06–37.94 µg/mg extract (1306–3794 mg/100 g extract) depending on the solvent used for extraction. Woldegiorgis et al. [33] showed that TP in A. campestris was 14.6 mg/g extract (1460 mg/100 g extract). Phenolic compounds are one of the main groups of antioxidants in mushrooms [41, 42]. Therefore, the antioxidant activity of the obtained extract was measured.

Table 4 Total phenolic content, antioxidant activity and ergosterol content of Agaricus species
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Antioxidant activity was estimated according to a commonly used method for testing the ability to scavenge DPPH radicals. The results are presented in Table 4. The EC50 values ranged from 0.8 to 3.2 mg/mL depending on the species of mushrooms. The lower values of EC50 indicate better scavenging ability. The cultivated species A. brasiliensis and A. arvensis were distinguished by the lowest value of 0.8 mg/mL, respectively. Among different A. bisporus strains, the greatest scavenging ability was recorded for brown Agaricus, which was comparable to the wild growing form. All white strains of A. bisporus had significantly higher EC50 values than other tested Agaricus species. The EC50 for A. brasiliensis has confirmed that it possesses great ability to scavenge DPPH radicals, ranging from 0.084 to <3 mg/mL [17, 37, 39, 43]. The EC50 value of A. bisporus was between 2.77 and 4.57 mg/mL, depending on the solvent [37]. The percentage of inhibition of DPPH˙ (%RSA) at a concentration of 12 mg/mL was between 62.7 and 91.3%. The order of inhibition was as follows: A. bitorquis > A. arvensis > A. brasiliensis > brown A. bisporus > A. campestris > A. silvaticus > white A. bisporus. Phenolic compounds are known to be highly effective antioxidants and previous research has indicated the correlation between antioxidant properties and the content of phenolics in mushrooms [34, 37, 43]. The present investigation confirmed the strong negative correlation between EC50 value and TP (r = −0.82). Additionally, a very strong positive correlation was found between antioxidant activity expressed as % of inhibition of DPPH radical with TP (r = 0.81). This shows the possibly antioxidative function of phenolic compounds acting as scavengers of free radicals. However, the EC50 value was only weakly correlated with caffeic acid (r = −0.34) and moderately correlated with syringic acid (r = −0.452) and protocatechuic acid (r = 0.44). Moreover, TP was correlated only with syringic acid (r = 0.62). Therefore, it could be assumed that antioxidant properties may also result from the presence of other substances in the ethanolic extract and their synergic effect with phenolics, particularly since the Folin–Ciocalteu assay can be overestimated by ascorbic acid, sugars and some amino acids such as: tryptophan or tyrosine [32]. The significant correlation between scavenging activity and TP has also been confirmed by other studies on different species of mushrooms [5, 34, 44]. The authors point out that the antioxidant properties of phenolics could be associated with the ability to donate a hydrogen to scavenging DPPH˙ [5, 36].

The pattern of the sterol profile shows that ergosterol is the main compound synthesized by wild growing and cultivated mushrooms [45, 46]. Because of its antimicrobial, antitumor and anticomplementary activities, ergosterol and its peroxidation derivatives are currently under frequent investigation [47,48,49]. To our knowledge, most research on ergosterol in Agaricus species concerns white and brown A. bisporus [47, 50, 51]. There are no reports of prior investigations on ergosterol in other than Agaricus bisporus species such as: A. arvensis A. bitorquis, A. brasiliensis, A. campestris and A. silvaticus. The level of ergosterol in the studied Agaricus species ranged from 1.1 to 45.8 mg/100 g DM (Table 4).

Wild growing A. silvaticus and A. campestris were the most abundant in ergosterol (45.8 and 42.4 mg/100 g DM, respectively). The content of ergosterol in other species was lower, even several times than in Agaricus species mentioned above. The present investigation revealed significant variety in the content of ergosterol, not only among different species of Agaricus. Huge variation in the content of ergosterol was observed among different strains of A. bisporus. Ergosterol content in white A. bisporus ranged from 1.1 to 36.1 mg/100 g DW. Strain Kanmycel 3-1 contained the highest content of ergosterol in comparison with other white A. bisporus, while Sylvan A15 contained the lowest content of ergosterol among all analyzed species. The findings clearly indicate lower levels of ergosterol in comparison to other studies showing that ergosterol content in A. bisporus ranged from 4.6 to 6.4 mg/g DW (460–640 mg/100 g DW) [15, 51] and more than 50 mg/100 g fresh weight [45]. However, the obtained results are in accordance with other studies that have found that the total ergosterol content in brown A. bisporus was significantly higher than in white A. bisporus [45, 50, 51]. The concentration of ergosterol was confirmed to be different depending on the type of fungus [15, 51]. However, other factors are also able to determine the ergosterol content, e.g., growing and storage temperature and UV light, which can transform sterol into vitamin D [15, 51, 52].

In conclusion, to our knowledge the presented study is probably the first report to investigate a range of Agaricus species, (not only A. bisporus) including wild growing species, on such a wide scale.

The results show the tested cultivated and wild-growing Agaricus species to be a source of different bioactive compounds including phenolic compounds and low-molecular weight organic acids, but they are poor in ergosterol. Furthermore, the extracts revealed that antioxidant activity correlated with total phenolic content and with some phenolic acids, indicating the role of the compounds in protection against oxidative damage. The investigation pointed to a potentially increased value of Agaricus species as food compounds or nutraceuticals.