Eur Food Res Technol DOI 10.1007/s00217-017-2952-9

ORIGINAL PAPER

Profle of phenolic and organic acids, antioxidant properties and ergosterol content in cultivated and wild growing species of

Monika Gąsecka1 · Zuzanna Magdziak1 · Marek Siwulski2 · Mirosław Mleczek1

Received: 31 March 2017 / Revised: 19 June 2017 / Accepted: 8 July 2017 © The Author(s) 2017. This article is an open access publication

Abstract Among the Agaricus genus, both cultivated and source of diferent bioactive compounds including phenolic wild growing species can be found. In the study, the pro- compounds and organic acids. fle of phenolic compounds and organic acids, as well as ergosterol content of diferent species of edible Agaricus, Keywords Agaricus · Phenolic acids · Organic acids · was estimated together with a comparison of their ability Antioxidant activity · Ergosterol to scavenge DPPH radicals. The investigation was car- ried out on seven strains of (one brown and six white), Agaricus blazei, , Aga- Introduction ricus bitorquis, and Agaricus silvati- cus. Before analysis, the samples were dried. Among nine The genus Agaricus belonging to the family organic acids detected in Agaricus species, oxalic, lactic includes about 300 diferent . Most of them are and succinic acids were the most abundant. The profle was edible, although poisonous species are also known [1]. The very heterogeneous with A. silvaticus, A. camperstis and fruiting bodies of Agaricus have a feshy cap or pileus with A. arvensis found to be the species richest in organic acids. a dry, bare or scaly surface and a number of radiating plates The phenolic profle revealed only phenolic acids, among or gills, the hymenophore. Agaricus species are appreci- which gallic, cafeic and ferulic were detected in all spe- ated due to their nutritional value, bioactive ingredients, cies. The dominant were gallic, trans-cinnamic and chlo- antioxidant activity (including free radical scavenging) and rogenic acids. The highest sum of phenolic acids and total biological efects, which makes them a valuable source of phenolic content was found in A. brasiliensis. Antiradical nutritional and pharmacological compounds [2–4]. Some of the extracts against DPPH radical was as follows: A. species are not only ingredients of human diets, but also bitorquis > A. arvensis > A. brasiliensis > brown A. bispo- function as a health food used for centuries in traditional rus > A. campestris > A. silvaticus > white A. bisporus. medicine. Agaricus bisporus is one of the most frequently The lowest ­EC50 value was estimated for A. brasiliensis and cultivated and consumed mushrooms in the world. Agari- A. arvensis. A correlation was confrmed between antioxi- cus brasiliensis is known as a medicinal . Agari- dant activity and phenolic acids. Ergosterol content reached cus arvensis is a wild growing species, although its fruiting the highest level in A. silvaticus and A. campestris. The bodies are also commercially produced. Agaricus bitorquis, investigation emphasizes the value of Agaricus species as a Agaricus campestris and Agaricus silvaticus are edible wild growing species. Most of the studies on the Agaricus genus concern A. * Monika Gąsecka bisporus and A. brasiliensis, predominantly focusing on an [email protected] investigation of their bioactive compounds and therapeu- 1 Department of Chemistry, Poznań University of Life tic efect. Little is known about other species of Agarisus Sciences, Poznan, Poland and other metabolites. Phenolics belong to bioactive com- 2 Department of Vegetable Crops, Poznań University of Life pounds, although they are non-essential dietary compo- Sciences, Poznan, Poland nents. Their biological function is related to free radical

Vol.:(0123456789)1 3 Eur Food Res Technol scavenging activity, metal chelation ability and inhibition containers, incubated at 25 °C and 90–95% moisture con- of lipid oxidation [5, 6]. tent. Incubation of the substrate completely covered by Organic acids are responsible for the taste and favor of mycelium with a 5-cm layer of casing soil (a mixture of a mushroom and can also play a biological role owing to chalk and Sphagnum sp. peat, v:v; 5:1) was carried out in their antioxidant, acidifying, neuroprotective, anti-infam- the same conditions until the soil was completely over- matory and antimicrobial properties [7–12]. grown by mycelium, after which the temperature was Ergosterol is a highly benefcial sterol with properties reduced to 16 °C for A. bisporus and A. arvensis, and to that can promote human health (antimicrobial, anticom- 21–22 °C for A. brasiliensis. All analysis were conducted plementary and antitumor activity) and the capacity to be on fruiting bodies of the frst fush. The fruiting bodies of transformed into vitamin ­D2 [13–15]. cultivated and wild growing Agaricus species were dried in The aim of the study was to investigate the profle of an electric drier (SLW 53 STD, Pol-Eko) at 40 ± 2 °C to a phenolic compounds and organic acids together with the constant weight and ground for 30 s in a Cutting Boll Mill ergosterol content of diferent species of cultivated and wild 200 (RETSCH GmbH, Haan, Germany). The moisture con- growing Agaricus genus. The ability to scavenge DPPH tent in dried samples was ~2%. Three representative pow- radicals was also estimated. The relationship between anti- dered samples were used for the extraction procedure. oxidant activity and the content of phenolic compound was also investigated. Chemicals

Materials and methods Acetonitrile with 0.01% formic (LC–MS CHROMO- SOLV), ethanol (≥99.5), methanol (≥98.8%), 2,2-diphe- Mushroom species nyl-1-picrylhydrazyl (DPPH), Folin–Ciocalteu’s phenol reagent, ­KH2PO4 (99.5%), ­H3PO4 (≥85%), standards of Fruiting bodies of diferent cultivated and wild grow- phenolic compounds such as: p-coumaric (≥98%), gal- ing Agaricus species were used during the investigation lic (≥99), protocatechuic, benzoic (≥99.5%), 2,5-dihy- (Table 1). From among cultivated mushrooms, the follow- droxybenzoic (≥99%), 4-hydroxybenzoic (≥99%), cafeic ing species were used: seven strains of Agaricus bisporus (≥98%), chlorogenic (≥95%), vanillic (≥97%), salicylic (one brown and six white), Agaricus brasiliensis and Agari- (≥99), syringic (≥97%), ferulic (≥98%), sinapic (≥98%) cus arvensis. Three species of wild mushrooms were picked and trans-cinnamic acids (≥99), rutin (≥98%), catechin in natural conditions from the Wielkopolska Region and (≥98%), kaempferol (≥97%), quercetin (≥98), vitexin identifed according to Wojewoda [16]: Agaricus bitorquis, (≥95%), luteolin (≥98%), purchased from Sigma-Aldrich Agaricus campestris and Agaricus silvaticus. (St. Louis, MO, USA). The standards of organic acids A. bisporus, A. arvensis and A. brasiliensis were culti- [acetic (≥99%), citric (≥99.5%), formic (≥95%), fumaric vated according to the conventional method in three rep- (≥99%), lactic (≥98%), maleic (≥99%), malic (≥99%), etition. The substrate (a mixture of chicken manure, wheat malonic (≥99%), oxalic (≥99%), and succinic (≥99%) straw and gypsum) with the mycelium was placed in plastic acids] were purchased from Supelco with a certifed stand- ard grade.

Table 1 Species of mushroom and their source No Species Strain Source Extraction

1 A. arvensis Mycelia M7400 Cultivated The extraction of phenolic compounds and organic acids 2 A. bisporus (brown) Hollander Spawn C9 Cultivated was carried out according to the procedure of Carvajal 3 A. bisporus (white) Sylvan 767 Cultivated et al. [17] with some modifcation. Dried and ground mush- 4 A. bisporus (white) Amycel 2600 Cultivated room samples (5 g) were twice extracted with 70% ethanol 5 A. bisporus (white) Kanmycel 3-1 Cultivated by shaking in Ika KS 260 shaker (IKA-Werke GmbH & Co. 6 A. bisporus (white) Italspawn F599 Cultivated Kg, Staufen, Germany) for 3 h at room temperature. After 7 A. bisporus (white) Kanmycel K2 Cultivated centrifugation at 3000 rpm with a Universal 320 R centri- 8 A. bisporus (white) Sylvan A15 Cultivated fuge (Hettich, Tuttlingen, Germany) and fltration through 9 A. bitorquis Wild growing Whatman No. 4 paper (UK) the combined extracts were 10 A. brasiliensis Cultivated evaporated to dryness at 40 °C with a rotary vacuum evapo- 11 A. campestris Wild growing ration Büchi Rotavapor R-205 (Flawil Switzerland) and fro- 12 A. silvaticus Wild growing zen at −12 °C. The extracts were used in further analyses.

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HPLC analysis of organic acids 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals (2.7 mL of 6 µmol/L), shaken and kept in the dark for 1 h. The absorb- The extracts were dissolved in 1 mL deionized water (Mili- ance of each samples was measured at 517 nm to determine Q, Millipore). For determination of organic acids, a Waters the reduction of the DPPH. The percentage of DPPH scav- Alliance 2695 Chromatograph with a Waters 2996 Photo- enging activity was calculated according to Reis et al. [22]: diode Array Detector (Waters Corp., Milford, MA, USA) (%RSA) = [(Ac − A)∕Ac]×100 was used. Separation was performed on a Waters Atlantis ­C column (250 mm × 4.6 mm × 5 μm), at 220 nm wave- RSA is the radical scavenging activity, Ac is the absorb- 18 A length, with the following column conditions: 25 mM ance of control and is the absorbance of samples. The scavenging activity was expressed as ­EC50 (the ­KH2PO4 (adjusted to pH 2.5 with concentrated H­ 3PO4) and methanol as an eluent (95:5, v/v), and fow rate at 0.8 mL/ concentration at which the ability to scavenge DPPH˙ was min [18]. Acids were identifed by the retention times of 50%) and was estimated graphically. their peaks in a chromatogram and quantifed by peak area comparison with standards at a known compound concen- Determination of ergosterol tration according to the appropriate standard curve. Ergosterol in fruiting bodies of Agaricus species was UPLC analysis of phenolic compounds determine by UPLC according to Perkowski et al. [23] with some modifcations. Powdered mushroom sam- The extracts were dissolved in 1 mL of 80% methanol and ples (100 mg), 2 mL of pure methanol and 1 mL of 2 M used for the determination of phenolic compounds using an NaOH were mixed together, irradiated twice in a micro- ACQUITY UPLC H-Class System equipped with a PDA wave (2 × 15 s) and cooled. Then, 2 mL of 1 M HCl was eλ Detector (Waters Corp., Milford, MA, USA). A gradi- added to the mixture. Then, samples were cooled, extracted ent elution of solvent A (water with 0.1% formic acid) and with pentane and evaporated to dryness in a nitrogen solvent B (acetonitrile with 0.1% formic acid) was applied stream. The quantifcation of ergosterol was done using an to an Acquity UPLC BEH ­C18 column (2.1 mm × 150 mm, ACQUITY UPLC H-Class System and a PDA eλ Detec- 1.7 µm, Waters). The gradient program was as follows: tor (Waters Corp., Milford, MA, USA). Identifcation was fow 0.4 mL/min—5% B (2 min), 5–16% B (5 min), 16% carried out on an ACQUITY UPLC HSS T3 C­ 18 column B (3 min), 16–20% B (7 min), 20–28% B (11 min) fow (150 mm × 2.1 mm, particle size 1.8 μm) (Waters, Ireland) 0.45 mL/min—28% (1 min), 28–60% B (3 min) fow protected with 1.7 m ACQUITY UPLC BEH ­C18 Van- 5.0 mL/min—60–95% B (1 min), 65% B (1 min), 95–5% Guard Pre-column. The injection volume was 2 µL. The B (0.1 min) fow 0.4 mL/min—5% B (1.9 min). The injec- fow rate of the isocratic elution of the mobile phase (mix- tion volume was 2 µL. The quantifcation of phenolic com- ture of methanol, acetonitrile and water; 85:10:5, v/v/v) pounds was performed using an external standard. The was 0.5 mL/min. The run time of analysis was 10 min. preferable wavelengths were 280 and 320 nm [19]. Statistical analysis Total phenolic content All analyses for each mushroom were performed in tripli- The Folin–Ciocalteu assay was used for determination of cate. The results are expressed as mean ± standard devia- the total phenolic (TP) as previously described with some tion (SD). Statistical analysis was done using STATISTICA modifcations [20]. The absorbance of 1 mL of metha- 13.1. One-way analysis of variance ANOVA followed by nol extract mixed with 1 mL of Folin–Ciocalteu reagent post hoc Tukey’s test was conducted to estimate diferences (diluted with ­H20; 1:1, v/v) and 3 mL of 20% ­Na2CO3 after between mean values. Correlations were estimated by the 30 min incubation in darkness at room temperature was Pearson correlation coefcients between scavenging ability measured at 765 nm. The results were expressed as mg and phenolic acids. of gallic acid equivalent (GAE) per 100 g of dried weight (DW). Results and discussion Evaluation of DPPH radical scavenging activity Organic acid profle The scavenging efect against the DPPH radicals was carried out according to Dong et al. [21]. Extracts The profle of organic acids showed that nine acids (ace- (1 mL) at diferent concentrations (0, 2, 4, 6, 8, 10 and tic, citric, formic, fumaric, lactic, malic, malonic, oxalic 12 mg/mL) were mixed with methanolic solution of and succinic) were detected in Agaricus species, although

1 3 Eur Food Res Technol the profle was very heterogeneous (Table 2). A. sil- on the acceptability, nutrition and stability of mushrooms vaticus, A. camperstis and A. arvensis were the rich- [30]. est species in organic acids (23,566.5, 22,985.3 and The individual profle of phenolic compounds in 21,540.6 mg/100 g DW, respectively), while A. bitorquis mg/100 g DW is presented in Table 3. The investigation and two strains (Kanmycel K2 and Sylvan A 15) of white reveals that only nine phenolic acids were detected in the A. bisporus were very poor in these compounds (6600.5, analyzed species of Agaricus. Flavonoids were not found. 6033.1 and 5798.3 mg/100 g DW, respectively). However, The results are in accordance with Gil-Ramírez et al. all the acids were detected only in A. bisporus (brown) Hol- [31], who claims that favonoids are not present in mush- lander Spawn C9. Oxalic, lactic and succinic acids were rooms because of a lack of enzymes necessary for their the most abundant components. Oxalic acid was detected synthesis. However, mushrooms are able to absorb them in each of the studied Agaricus species. Lactic and suc- from substrate or plants, from which they form mycor- cinic acids were detected in almost every sample, with the rhizae [31] and thus some favonoids have been detected exception of Kanmycel K2 and Sylvan A15 for the frst [32, 33] The most homogeneous profle was represented and A. brasiliensis for the second acid. The highest level for brown and white A. bisporus. Gallic, cafeic and feru- of lactic and oxalic acids was found in A. brasiliensis lic acids were detected in all the species. Generally, the (13,358.1 and 5467.1 mg/100 g DW, respectively), while dominant was gallic acid (5.5–9.2 mg/100 g DW). Further- succinic acid was present in A. bisporus Amycel 2600 more, trans-cinnamic and chlorogenic acids were quanti- (11478.4 mg/100 g DW). The other identifed acids were fed at a higher level than the other compounds (4.6–9.4 much lower in content or were below the limit of detection. and 5.3–6.7 mg/100 g DW, respectively). A. brasiliensis Organic acids have also been detected in other mush- was the richest in phenolic acid content containing all the room species, among which are cultivated and wild grow- analyzed acids (33.9 mg/100 g DW), while the wild grow- ing species with oxalic, citric, malic or fumaric as the main ing species A. campestris and A. silvaticus were found to compounds [8, 12, 24–27]. Oxalic, malic, citric and fuma- be poorest in phenolic acids (11.3 and 18.2 mg/100 g DW, ric acids have been identifed in A. bisporus portobello respectively). Although the phenolic compound composi- and A. silvaticus, while A. bisporus is additionally known tion of many mushroom species is well known, for some to contain quinic and citric acids [26]. In A. campestris Agaricus species (e.g., A. arvensis, A. bitorquis A. campes- only oxalic, malic and fumaric acids have been found [26]. tris, A. silvaticus), it is not yet well recognized. The range of the acids in the previously mentioned species Gallic and syringic (dominant) acids and pyrogallol was: 4.86–19.61, 23.88–30.05, 34.62–43.23, 1.14–3.77 in fruiting bodies of A. brasiliensis were identifed by and 6.44 mg/g DW (486–1961, 2388–3005, 3462–4323, Carvajal et al. [17]. In the present study, the total con- 114–377 and 644 mg/100 g DW) for oxalic, malic, citric, tent of phenolic acids contained in brown A. bisporus fumaric and quinic acids, respectively [26]. Carvajal et al. (31.8 mg/100 g DW) was comparable to that of A. brasil- [17] identifed acetic, alpha-ketoglutaric, citric (dominant), iensis (33.9 mg/100 g DW), although syringic acid was fumaric, malic, oxalic and trans-aconitic acids in A. bra- not quantifed in the frst mentioned species. The total siliensis. This profle difers considerably from the one content of phenolic acids in all strains of white A. bispo- presented in Table 2. In the present study, citric and acetic rus was similar (24.8–26.0 mg/100 g DW). The exception acids were not detected in A. brasiliensis, while Carvajal was Sylvan 767, which had a distinctly higher level of the et al. [17] show they were dominant [11.79 and 7.57 µg/ compounds (30.6 mg/100 g DW). The profle of phenolic mg dried (1179 and 757 mg/100 g), respectively]. acids and their content in A. bisporus in the present study Carvajal et al. [17] suggested that organic acids could have is very similar to the fndings of Palacios et al. [32], who an efect on antioxidant activity (ferrous ion chelation and also confrmed cafeic, chlorogenic, p-coumaric, feru- an ABTS scavenging assay) of mushroom extract. Due to lic, gallic, p-hydroxybenzoic, and protocatechuic acids, the antioxidant activity some organic acids (e.g., citric, as well as homogentistic acid, myricetin and pyrogallol in malic, succinic) can play a protective role in some diseases the species. While Kim et al. [34] quantifed gallic, proto- in humans [8, 9]. Oxalic acid displays antibacterial activ- catechuic, myricetin and pyrogallol. Other investigations ity [27], while anti-infammatory, neuroprotective and anti- have revealed only cinnamic acid in white and brown A. microbial properties have been confrmed for fumaric acid bisporus [35]. Reis et al. [22] demonstrated only gal- [7, 28]. Malic acid is responsible for favor, while owing lic, p-coumaric and cinnamic acids in white A. bisporus to its antioxidant and antibacterial properties citric acid and cinnamic acid in brown A. bisporus. The fndings can extend the shelf life of mushrooms and prevent their show that the level of the phenolic acids in A. bisporus browning [11, 12, 25, 26]. It is widely used as an acidulant was as follows: cafeic (15.54 µg/g DW; 1.554 mg/100 g in pharmaceutical and food industries due to its low toxic- DW), chlorogenic (63.73 µg/g DW; 6.373 mg/100 g DW), ity [29]. Thus, organic acids have a considerable infuence p-coumaric (2.31–10.38 µg/g DW; 0.231–1.038 mg/100 g

1 3 Eur Food Res Technol a b c b b b b a a d d d 5798.3 6600.5 6033.1 10,021.0 12,632.6 23,566.5 13,715.2 21,549.6 13,878.0 14,987.1 22,985.3 18,986.2 Total con - Total tent of the acids ± 707.6 a ± 248.9 ± 96.3 ± 148.4 ± 271.3 ± 259.3 ± 141.1 ± 253.1 ± 60.0 ± 366.6 h ± 415.1 f e e d cd gh fg c b 5298.9 3951.9 5351.7 11,478.4 8213.2 2727.3 2242.2 7987.2 3313.9 nd 8993.3 9977.3 Succinic ± 244.1 ± 153.3 ± 87.8 ± 151.2 ± 38.9 ± 200.4 ± 342.3 ± 142.1 ± 1.21 ± 58.9 ± 14.2 ± 18.9 b e d e d e a c e h gh fg 3764.3 1928.8 2886.1 1259.2 377.4 2023.2 1317.9 713.8 1427.6 114.6 1024.8 5467.1 Oxalic ± 294.3 ± 18.2 ± 10.9 ± 32.9 ± 12.2 a ± 17.4 ± 18.1 cd cd bc cd b d ± 4.1 d 137.2 nd 111.2 nd 1128.4 216.4 nd 367.2 nd Malonic 123.6 72.2 530.6 ± 29.4 ± 79.8 ± 31.2 b a c ± 1.9 ± 1.9 ± 4.2 d d d nd 45.3 161.0 nd 397.9 68.0 nd 37.9 Malic 929.0 nd nd nd ± 234.3 ± 893.2 ± 2032.3 ± 1373.9 ab a b ab ± 177.3 ± 165.4 ± 207.2 ± 160.2 ± 349.8 ± 271.9 de c de de d d nd 6806.6 1259.3 3091.3 10,246.1 11,599.1 Lactic nd 12,095.2 1853.2 2889.7 1693.4 13,358.1 ± 30.4 ± 5.7 ± 1.2 ± 0.9 a ± 2.1 b bc cd cd ± 0.3 ± 1.1 ± 0.4 ± 0.1 ± 1.1 d d d d d Fumaric 10.6 nd 8.5 10.6 nd 2.9 242.6 4.3 49.2 4.9 35.3 1.1 ± 168.9 ± 280.1 ± 18.1 ± 17.4 a b ± 35.1 cd c cd ± 3.1 ± 1.9 d d Formic 2431.0 362.1 126.3 nd 37.6 nd nd nd nd 181.4 1635.1 20.8 ± 30.9 ± 22.4 ± 27.1 ± 28.7 ± 25.8 ± 18.7 c b e d f a ± 3.1 f Citric nd nd 198.0 590.4 nd 259.9 nd nd 460.9 39.7 396.4 317.2 ± 41.2 ± 18.3 ± 17.2 a ± 52.3 ± 12.2 ± 7.1 c b cd cd ± 5.0 de e Organic acids (mg/100 g DW) Organic Acetic 65.8 1028.2 nd nd nd 170.0 615.1 153.9 nd 139.8 nd 95.8 - - species (mg/100 g DW) acids in fruiting bodies of Agaricus Content of organic (white), Sylvan (white), Sylvan A15 cel 3-1 cel K2 (brown), Hol - (brown), lander Spawn C9 (white), Sylvan 767 (white) Amycel 2600 (white), Kanmy (white), Ital - F599 spawn (white), Kanmy Mycelia M7400 Mycelia 2 Table A. bisporus A. bitorquis test in the Tukey’s to at p ≤ 0.05 according same columns mean diferences letters diferent by followed Values detected nd not Samples A. bisporus A. bisporus A. bisporus A. bisporus A. bisporus A. bisporus A. brasiliensis A. campestris A. silvaticus A. arvensis,

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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-hydroxy- d b b c c c cd d a f e benzoic (15.39 µg/g DW; 1.539 mg/100 g DW), homogen- 26.0 11.3 18.2 24.8 23.3 31.8 30.6 25.9 33.9 25.7 23.1 Total con - Total tent of the acids 25.9 tistic (3444.3 µg/g DW; 344.43 mg/100 g DW), protocat- echuic (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 ± 0.1 ± 0.1 ± 0.0

± 0.0 ± 0.2 ± 0.0 ± 0.1 DW) [22, 32, 33]. ± 0.1 ± 0.1 ab cd ab a abc bcd d bcd cd Other tested Agaricus species contained a lower con- Protocatechuic nd 1.8 1.9 nd 1.6 1.9 2.0 1.9 1.7 1.8 nd 1.7 tent 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 ± 0.1 ± 0.1 ± 0.2 ± 0.1 ± 0.2 ± 0.3 ± 0.1 ± 0.2 ± 0.1 ± 0.3 p ± 0.2 not show any evidence of -hydroxybenzoic, syringic, cde b a cd bc de f ef bcd cde g p A. bitorquis -cinnamic trans nd 6.7 7.7 6.9 9.4 7.3 6.1 6.4 7.2 7.0 4.6 6.9 -coumaric (only ) or protocatechuic (only A. arvensis). The poorest species in phenolic compounds were wild growing A. campestris (11.3 mg/100 g DW) ± 0.1 ± 0.2 a b and A. silvaticus (18.2 mg/100 g DW). Only gallic, cafeic, Syringic nd 3.4 nd 5.6 nd nd nd nd nd nd nd nd p-hydroxybenzoic, p-coumaric, ferulic acids were quanti- fed in both the species. Additionally, syringic and trans-

± 0.1 ± 0.3 ± 0.1 ± 0.1 ± 0.0 ± 0.1 cinnamic acids were detected in A. silvaticus. ± 0.1 ± 0.1 ± 0.3 ± 0.1 a d cd bc e cd cd cd cd b The results concerning A. campestris difer from the Chlorogenic 6.7 6.2 5.7 5.8 5.6 nd nd 5.3 4.6 5.4 5.7 5.4 profle presented by Woldegiorgis et al. [33], who reported only p-coumaric (10.9 µg/g DW; 1.09 mg/100 g DW), feru- ± 0.1 ± 0.1 ± 0.2 ± 0.1 ± 0.2 ± 0.1 ± 0.1 ± 0.1 ± 0.1 ± 0.0 lic (20.3 µg/g DW; 2.03 mg/100 g DW), gallic (561.9 µg/g ± 0.1 ± 0.2 ab cde cde bcde cde cde de bc de bcd a e DW; 56.19 mg/100 g DW) and p-hydroxybenzoic Ferulic 1.1 1.2 1.3 1.2 1.2 1.4 1.6 1.5 1.2 1.8 1.1 1.0 (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. How- ± 0.1 ± 0.1 ± 0.1 ± 0.1 ± 0.1 ± 0.1 ± 0.0 ± 0.0 ± 0.0 ± 0.1 ± 0.1 ever, while the level of p-coumaric and ferulic acids was a bc de de fg fg g fg g cd ab p -coumaric 1.6 nd 1.3 0.8 0.7 0.7 1.5 1.1 1.0 1.0 0.6 0.5 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). Cul- tivated A. brasiliensis proved to be richest in phenolics. ± 0.2 ± 0.1 ± 0.1 ± 0.1 ± 0.1 ± 0.1 ± 0.1 ± 0.1 ± 0.1 ± 0.2 b cd cd d d cd d bc a b The present investigation showed that brown A. bisporus p -hydroxybenzoic 1.7 1.3 1.0 1.1 1.3 1.2 1.5 2.8 nd 1.3 nd 1.8 strain Hollander Spawn C9 was characterized by a higher content of phenolics than the white variety. Brown A. ± 0.1 ± 0.2 ± 0.1 ± 0.1 ± 0.1 ± 0.1 ± 0.1 ± 0.2 ± 0.1 ± 0.2 ± 0.0 ± 0.0 bisporus strain Hollander Spawn C9, white A. bisporus cd b bc d d d d d bc a e e A. arvensis, A. bitorquis A. brasil- 1.3 Cafeic 1.4 1.7 1.5 1.1 1.0 1.0 1.0 1.0 2.2 0.1 0.3 strain Kanmycel 3-1, , iensis, A. campestris and A. silvaticus had TP at the level >600 mg GAE/100 g DW, while other strains of white ± 0.3 ± 0.5 ± 0.1 ± 0.2 ± 0.2 ± 0.2 ± 0.7 ± 0.2 ± 0.1 ± 0.3 bc ± 0.1 ± 0.1 d a ab bc cd bc cd ef bc f f A. bisporus (besides Kanmycel 3-1) had a lower content 6.7 7.9 9.2 8.4 7.8 7.3 8.0 7.3 6.0 7.9 5.5 5.6 Gallic Phenolic acids 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, 36–38]. 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 species (mg/100 g DW) Content of phenolic acids in fruiting bodies of Agaricus present study is consistent with the investigation of Gan et al. [37], which indicated that A. brasiliensis was richer A. M7400 arvensis , Mycelia A. F599 bisporus (white), Italspawn 3 Table test in the Tukey’s to at p ≤ 0.05 according same columns mean diferences letters diferent by followed Values detected nd not Samples C9 A. Hollander Spawn bisporus (brown), 767 A. bisporus (white), Sylvan 2600 A. bisporus (white) Amycel 3-1 A. bisporus (white), Kanmycel K2 A. bisporus (white), Kanmycel A15 A. bisporus (white), Sylvan A. bitorquis A. brasiliensis A. campestris A. silvaticus in phenolics than A. bisporus. Öztürk et al. [40] studying

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Table 4 Total phenolic content, antioxidant activity and ergosterol content of Agaricus species

Sample no TP (mg GAE/100 g DW) EC50 (mg/mL) DPPH (RSA %) Ergosterol (mg/100 g DW)

A. arvensis, Mycelia M7400 711.0bc ± 22.2 0.8g ± 0.0 90.7ab ± 2.0 2.68fg ± 0.8 A. bisporus (brown), Hollander Spawn C9 756.3b ± 22.2 1.4ef ± 0.0 89.3abc ± 1.5 26.4c ± 1.5 A. bisporus (white), Sylvan 767 344.3f ± 34.6 1.9d ± 0.2 70.3d ± 2.5 9.50e ± 1.2 A. bisporus (white) Amycel 2600 542.7e ± 24.3 2.7bc ± 0.1 68.0de ± 2.6 21.7d ± 1.2 A. bisporus (white), Kanmycel 3-1 616.0de ± 29.8 2.4c ± 0.1 71.3d ± 2.1 36.1b ± 3.0 A. bisporus (white), Italspawn F599 230.7g ± 23.7 3.0ab ± 0.1 66.3de ± 3.1 4.0fg ± 0.7 A. bisporus (white), Kanmycel K2 346.7f ± 27.3 2.6c ± 0.2 65.0de ± 1.0 18.4d ± 1.0 A. bisporus (white), Sylvan A15 132.7 h ± 14.2 3.2a ± 0.2 62.7e ± 2.5 1.1g ± 0.2 A. bitorquis 654.7cd ± 25.5 1.2f ± 0.1 91.3a ± 2.1 35.3b ± 0.5 A. brasiliensis 1154.7a ± 65.7 0.8g ± 0.0 90.0abc ± 2.5 6.0ef ± 0.3 A. campestris 767.3b ± 19.3 1.6e ± 0.2 84.3bc ± 3.1 42.4a ± 0.4 A. silvaticus 638.3cd ± 38.1 1.6e ± 0.1 83.7c ± 2.1 45.8a ± 2.1

Values followed by diferent letters in the same columns mean diferences at p ≤ 0.05 according to Tukey’s test

TP in A. bitorquis estimated the content at 13.06–37.94 µg/ 43]. The present investigation confrmed the strong nega- mg extract (1306–3794 mg/100 g extract) depending on tive correlation between ­EC50 value and TP (r = −0.82). the solvent used for extraction. Woldegiorgis et al. [33] Additionally, a very strong positive correlation was found showed that TP in A. campestris was 14.6 mg/g extract between antioxidant activity expressed as % of inhibition (1460 mg/100 g extract). Phenolic compounds are one of of DPPH radical with TP (r = 0.81). This shows the pos- the main groups of antioxidants in mushrooms [41, 42]. sibly antioxidative function of phenolic compounds acting Therefore, the antioxidant activity of the obtained extract as scavengers of free radicals. However, the ­EC50 value was measured. was only weakly correlated with cafeic acid (r = −0.34) Antioxidant activity was estimated according to a com- and moderately correlated with syringic acid (r = −0.452) monly used method for testing the ability to scavenge and protocatechuic acid (r = 0.44). Moreover, TP was DPPH radicals. The results are presented in Table 4. The correlated only with syringic acid (r = 0.62). Therefore, ­EC50 values ranged from 0.8 to 3.2 mg/mL depending it could be assumed that antioxidant properties may also on the species of mushrooms. The lower values of EC­ 50 result from the presence of other substances in the etha- indicate better scavenging ability. The cultivated species nolic extract and their synergic efect with phenolics, A. brasiliensis and A. arvensis were distinguished by the particularly since the Folin–Ciocalteu assay can be over- lowest value of 0.8 mg/mL, respectively. Among difer- estimated by ascorbic acid, sugars and some amino acids ent A. bisporus strains, the greatest scavenging ability was such as: tryptophan or tyrosine [32]. The signifcant cor- recorded for brown Agaricus, which was comparable to relation between scavenging activity and TP has also been the wild growing form. All white strains of A. bisporus confrmed by other studies on diferent species of mush- had signifcantly higher EC­ 50 values than other tested Aga- rooms [5, 34, 44]. The authors point out that the antioxi- ricus species. The ­EC50 for A. brasiliensis has confrmed dant properties of phenolics could be associated with the that it possesses great ability to scavenge DPPH radicals, ability to donate a hydrogen to scavenging DPPH˙ [5, 36]. ranging from 0.084 to <3 mg/mL [17, 37, 39, 43]. The The pattern of the sterol profle shows that ergosterol ­EC50 value of A. bisporus was between 2.77 and 4.57 mg/ is the main compound synthesized by wild growing and mL, depending on the solvent [37]. The percentage of cultivated mushrooms [45, 46]. Because of its antimicro- inhibition of DPPH˙ (%RSA) at a concentration of 12 mg/ bial, antitumor and anticomplementary activities, ergos- mL was between 62.7 and 91.3%. The order of inhibition terol and its peroxidation derivatives are currently under was as follows: A. bitorquis > A. arvensis > A. brasil- frequent investigation [47–49]. To our knowledge, most iensis > brown A. bisporus > A. campestris > A. silvati- research on ergosterol in Agaricus species concerns white cus > white A. bisporus. Phenolic compounds are known and brown A. bisporus [47, 50, 51]. There are no reports to be highly efective antioxidants and previous research of prior investigations on ergosterol in other than Agari- has indicated the correlation between antioxidant proper- cus bisporus species such as: A. arvensis A. bitorquis, A. ties and the content of phenolics in mushrooms [34, 37, brasiliensis, A. campestris and A. silvaticus. The level of

1 3 Eur Food Res Technol ergosterol in the studied Agaricus species ranged from appropriate credit to the original author(s) and the source, provide a 1.1 to 45.8 mg/100 g DM (Table 4). link to the Creative Commons license, and indicate if changes were made. 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 men- References tioned above. The present investigation revealed signifcant variety in the content of ergosterol, not only among diferent 1. Walleyn R, Rammeloo J (1994) The poisonous and useful fungi species of Agaricus. Huge variation in the content of ergos- of Africa South of the Sahara. Scripta Botanica Belgica 10. In: terol was observed among diferent strains of A. bisporus. Heinemann P (ed) National Botanic Garden of Belgium Ergosterol content in white A. bisporus ranged from 1.1 to 2. 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