**2. Characteristics of wild mushroom mycelia**

#### **2.1 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. brasiliensis* (#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 germinated. 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 freezedrying 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 mushroom 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].

## *Medicinal Mushroom Mycelia: Characteristics, Benefits, and Utility in Soybean Fermentation DOI: http://dx.doi.org/10.5772/intechopen.102522*

ROS, such as superoxide anion radicals, hydroxyl radicals, and hydrogen peroxide (H2O2), induce aging and cell damage [16, 17], and have been implicated in several diseases [18]. Recent epidemiological data indicate the association between inactivation of ROS and the disease-prevention benefits resulting from consuming 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, followed by *H. erinaceum*, *G. frondosa*, and *L. shimeji*.
