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2. Characteristics of wild mushroom mycelia 1 Collection of mushrooms and separation of mycelia
We investigated the characteristics of 20 species of mushrooms: #1, A. brasiliensis (Agaricaceae); #2, Mycoleptodonoides aitchisonii (Climacodontaceae); #3, Ganoderma applanatum and #4, G. lusidum (Ganodermataceae); #5, H. erinaceum and #6, Hericium ramosum (Hericiaceae); #7, Inonotus obliquus (Hymenochaetaceae); #8, Lentinus edodes (Pleurotaceae); #9, Dendropolyporus umbellatus; #10, Grifola frondosa;
#11, Laetiporus sulphureus; #12, Polyporellus badius and #13, Polyporus tuberaster (Polyporaceae); #14, Sparassis crispa (Sparassidaceae); #15, Pholiota aurivella and
#16, Pholiota nameko (Strophariaceae); #17, Hypsizygus marmoreus, #18, Lepista nuda;
#19, Lyophyllum shimeji and #20, Panellus serotinus (Tricholomataceae).
Nineteen of these (#2–20) wild mushroom fruiting bodies were collected from the Akita and Iwate prefectures in the Tohoku area in northern Japan. A. brasil- iensis (#1) mycelia were provided by Dr. Makoto Yoneyama, I.M.C. Institution (Yamanashi Prefecture, Japan). Pieces of mushroom fruiting bodies collected from natural sites were plated in a 90-mm Petri dish containing potato dextrose agar (PDA) medium and incubated at 25°C for 2 days until the mycelia germi- nated. Mycelia were allowed to germinate and then cultured for 14 days at 25°C, after which period, they were maintained at 3°C on PDA medium. Mushroom mycelia were grown in submerged culture following the methods of A. brasiliensis mycelia cultivation, as described previously [11]. The culture was incubated at 25°C for 14 days with gentle shaking and the mycelia were lyophilized by freeze- drying after cultivation.
2.2 Ethanol extract preparation from mushroom mycelia
Mushroom mycelia extraction with ethanol was performed following methods described in previous reports [12, 13] with a few modifications. Lyophilized mush- room mycelia (0.1 g) were extracted with 80% ethanol (10 mL) at 25°C for 24 h and the resulting solutions were concentrated and lyophilized to a powder.
2.3 Antioxidant activity of wild mushroom mycelia
Free radicals exert tissue damage through reactive oxygen species (ROS)- induced oxidative stress, which can be counterbalanced by antioxidants [14, 15].
ROS, such as superoxide anion radicals, hydroxyl radicals, and hydrogen peroxide (H2O2), induce aging and cell damage [16, 17], and have been implicated in sev- eral diseases [18]. Recent epidemiological data indicate the association between inactivation of ROS and the disease-prevention benefits resulting from consum- ing food containing antioxidants, such as fruits, vegetables, and certain cereals [19]. As a result, there is an increasing trend worldwide in the incorporation of antioxidant compounds and foods into regular diet. We measured the antioxidant activity of the 20 wild mushrooms listed above by DPPH radical scavenging activity assay.
2.3.1 Methods
Measurement of 2,2-diphenyl-1-picryhydrazyl (DPPH) radical scavenging activity of mushroom mycelia was performed as previously described [20]. Ethanol extracts of mushroom mycelia (0.3 mL) were mixed with 0.6 mL of 100 mM MES buffer (pH 6.0)/10% ethanol solution, and 0.3 mL of 400 μM DPPH in ethanol.
The absorbance of the reaction mixture was quantified at 520 nm after the reaction was set to complete for 20 minutes at RT. The DPPH radical scavenging activity of mushroom mycelia was calculated from assay lines of Trolox (0, 5, 10, 15, 20, and 25 μM) and expressed as μmol Trolox/g dry powder.
2.3.2 DPPH free radical scavenging activity of mushroom mycelia
Eighty-percent ethanol extracts of mushroom mycelia were used for antioxidant activity measurements using DPPH radical scavenging activity (Figure 1). Among the 20 mushroom mycelia analyzed, L. shimeji (#19), G. frondosa (#10), H. erinaceum (#5), and H. ramosum (#6) showed more robust DPPH radical scavenging activities.
Figure 1.
DPPH radical scavenging activity of mycelial extracts from the 20 wild mushrooms [9]. 1, A. brasiliensis; 2, M. aitchisonii; 3, G. applanata; 4, G. lucidum; 5, H. erinaceum; 6, H. ramosum; 7, I. obliquus; 8, L. edodes;
9, D. umbellatus; 10, G. frondosa; 11, L. sulphureus; 12, P. badius; 13, P. tuberaster; 14, S. crispa; 15, P.
aurivella; 16, P. nameko; 17, H. marmoreus; 18, L. nuda; 19, L. shimeji; and 20, P. serotinus. The DPPH radical scavenging activity of mushroom mycelia was calculated and expressed as the Trolox equivalent. Data represent the mean ± SD (n = 5).
Among the mycelia tested, H. ramosum showed maximum antioxidant activity, fol- lowed by H. erinaceum, G. frondosa, and L. shimeji.
2.4 Total phenolic content of the wild mushroom mycelia
Phenolic compounds are secondary metabolites of plants produced as defensive responses to threatening environments, including pathogen attack and UV radia- tion [21]. Generally, these polyphenols are classified as phenolic acids, flavonoids, lignans, and stilbenes [22]. These phenolic compounds possess antioxidant, antigly- cemic, anticarcinogenic, and anti-inflammatory properties and can protect against bacterial and viral infections [23]. We analyzed the total phenolic content of the mushroom mycelia.
2.4.1 Methods
Folin & Ciocalteu method [24] with catechin as a standard was used for analysis. Ethanol extracts of mushroom mycelia (1 mL) were mixed with 0.5 mL of Folin & Ciocalteu solution and 5 mL of 0.4 M sodium carbonate solution. The absorbance of the reaction mixture was quantified at 660 nm after the reaction was set to complete for 30 minutes at 30°C. Methods are described in detail in Suruga et al. [9].
2.4.2 Measurement of total phenolic content
The phenolic contents of the samples were expressed as mg of catechin equivalent/g dry powder in Figure 2. H. ramosum (#6) showed the highest amount of phenol contents, followed by H. erinaceum (#5), G. frondosa (#10), A. brasiliensis (#1), L. shimeji (#19), E. applanata (#3), G. lucidum (#4), and H. marmoreus (#17).
Figure 2.
Total phenolic content of wild mushrooms mycelia extracts [9]. 1, A. brasiliensis; 2, M. aitchisonii; 3, G.
applanata; 4, G. lucidum; 5, H. erinaceum; 6, H. ramosum; 7, I. obliquus; 8, L. edodes; 9, D. umbellatus; 10, G. frondosa; 11, L. sulphureus; 12, P. badius; 13, P. tuberaster; 14, S. crispa; 15, P. aurivella; 16, P. nameko; 17, H. marmoreus; 18, L. nuda; 19, L. shimeji; and 20, P. serotinus. Data represent the mean ± SD (n = 5).
2.5 Phenolic compounds enable the DPPH radical scavenging capacity of mushroom mycelia
DPPH radical scavenging activity showed a significant correlation (R2 = 0.7929) with the total phenolic content in the wild mushroom mycelia extracts (Figure 3).
The Hericiaceae group, including H. erinaceum (#5) and H. ramosum (#6), which had a higher total phenolic content, showed stronger antioxidant potential. All these results suggest that the DPPH radical scavenging capacity of these extracts is driven by the phenolic compounds.