Open access peer-reviewed chapter

Medicinal Mushroom Mycelia: Characteristics, Benefits, and Utility in Soybean Fermentation

Written By

Kohei Suruga, Tsuyoshi Tomita and Kazunari Kadokura

Submitted: 29 November 2021 Reviewed: 07 January 2022 Published: 12 April 2022

DOI: 10.5772/intechopen.102522

From the Edited Volume

Current Topics in Functional Food

Edited by Naofumi Shiomi and Anna Savitskaya

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The medicinal value of mushrooms is long known, but there is increasing awareness of their health benefits and interest in utilizing these in diet as food or nutritional supplement. In this chapter, we discuss the characteristics of 20 wild mushrooms and results from our work on their antioxidant activity, ability to promote nerve growth factor (NGF) synthesis and to convert the glycosylated forms of isoflavones to usable aglycon forms in soybeans fermented with their mycelia. Of the 20 mushroom types, we found that Hericium ramosum (H. ramosum) mycelia had higher antioxidant activity and showed greater capability for increasing the levels of aglycons, such as daidzein, glycitein, and genistein when used for fermentation of soybeans. In general, soybeans fermented with mushrooms increased the levels of aglycons compared to non-fermented ones. Taken together, all these results suggest that mushroom mycelia have a huge potential to be used as food and nutritional supplements for the health benefits they offer and present the prospects for utilizing them in soybean fermentation as natural resources for the large-scale production of aglycons.


  • H. ramosum mycelia
  • antioxidant
  • NGF synthesis
  • soybean fermentation
  • isoflavone

1. Introduction

Mushrooms, their fruiting bodies and mycelia have served as food and food supplements around the world. They are relatively less toxic and are rich in bioactive compounds, such as polysaccharides, proteins, minerals, and other nutrients [1]. Beneficial activities associated with mushroom fruiting bodies and mycelia include antitumor [2], antimutagenic [3], antiviral [4], and antioxidant activities [5]. Some mushrooms alleviate the risk of diseases, such as Parkinson’s and Alzheimer’s disease, and hypertension [6].

Mushroom mycelia contain bioactive compounds as well as mushroom fruiting bodies, which have been investigated for their medicinal value. For example, oral administration of Sparassis crispa mycelia resulted in antitumor responses in tumor-bearing ICR mice [7]. The ability of erinacines, the bioactive compounds of H. erinaceum mycelia, to promote nerve health has been documented [8]. However, published data on the bioactivity of mushroom mycelia are limited compared with those of mushroom fruiting bodies.

In this chapter, we first discuss the antioxidant activity of 20 different species of wild mushroom mycelia [9]. These mushrooms are considered edible in the Tohoku area in northern Japan. Second, we present our findings on the ability of the comb tooth cap medicinal mushroom, H. ramosum mycelia, to promote NGF synthesis [9]. Finally, we discuss our results from using these mushroom mycelia in soybean fermentation [10] and discuss the prospects of utilizing H. ramosum mycelia in soybean fermentation for large-scale production of aglycons.


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 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 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]. 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. Among the mycelia tested, H. ramosum showed maximum antioxidant activity, followed by H. erinaceum, G. frondosa, and L. shimeji.

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).

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 radiation [21]. Generally, these polyphenols are classified as phenolic acids, flavonoids, lignans, and stilbenes [22]. These phenolic compounds possess antioxidant, antiglycemic, 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.

Figure 3.

Direct correlation between DPPH radical scavenging activity and phenolic content [9].


3. NGF synthesis of H. ramosum mycelia

Senile dementia, such as AD, is a severe problem, with no effective therapy [25]. Neurotrophic factors, including NGF, brain-derived neurotrophic factor (BDNF), neurotrophin 3, and glial-derived neurotrophic factor (GDNF), have been implicated in the prevention of neuronal death and promotion of neurite outgrowth [26]. Among them, NGF has been associated with AD [27], with decreased NGF levels in the basal forebrain of AD patients. Intracerebroventricular administration of NGF has been reported to eliminate degeneration and resultant cognitive deficits in rats after brain injury [28]. In rats, poor cognitive effects caused by neuronal degeneration have been shown to be eliminated by intracerebroventricular administration of NGF. However, since NGF cannot cross the blood-brain barrier, utilizing it for therapeutic application will be difficult. Several studies have investigated low-molecular-weight compounds, such as catecholamines [29], benzoquinones [30], hericenones [31], and erinacines [32], for their ability to promote NGF synthesis.

H. erinaceum is a common fungus found in the East Asian diet. Hericenones [33] have been isolated from the fruiting bodies of H. erinaceum and erinacines [34] have been identified in H. erinaceum mycelia. H. erinaceum may be valuable in the treatment and prevention of dementia [35, 36]. However, to our knowledge, no reports have shown the induction of NGF synthesis by H. ramosum mycelia. In this section, we show the results of our assessment on the ability of H. erinaceum and H. ramosum mycelia to induce NGF synthesis.

3.1 Methods

NGF synthesis was measured as described by Hazekawa et al. [37]. Male ddY mice (25–30 g weight) obtained from Kiwa Laboratory Animals Co., Ltd. (Wakayama, Japan), were housed under a 12-h light/dark cycle at room temperature and 55 ± 5% humidity. The lyophilized mycelia from H. erinaceum and H. ramosum were suspended in purified water and the samples were orally administrated to mice once a day for 14 days. NGF levels were analyzed in the cortex, striatum, and hippocampus 24 h after the last administration. Results are expressed as mean ± standard error of mean (SEM). The standard dose (300 mg/kg body weight) was determined based on Hazekawa et al. [37]. More detailed methodology can be found in Suruga et al. [9].

3.2 Stimulation of NGF synthesis by H. ramosum and H. erinaceum mycelia

The effects of 14-days of oral administration of 300 mg/kg H. erinaceum mycelia and H. ramosum mycelia on NGF levels in intact mouse brains are shown in Figure 4. H. ramosum mycelia were more potent than H. erinaceum mycelia in terms of NGF stimulation in the hippocampus of intact mice. Processing of H. ramosum mycelia over time significantly increased NGF levels in the hippocampus.

Figure 4.

Activation of NGF synthesis with wild H. erinaceum and H. ramosum mycelia [9]. NGF levels in various parts of the brain were measured after 14 days of repeated oral administration of H. erinaceum and H. ramosum mycelia (300 mg/kg). 1, Cortex; 2, striatum; and 3, hippocampus. Data are expressed as the mean ± SEM. *p < 0.05, compared with vehicle (Student’s test).

Different regions of the mouse brain responded differently to application of varying concentrations of H. ramosum mycelia on NGF synthesis [9]. The NGF levels in hippocampus increased with increased concentration of H. ramosum mycelia, while such dose-dependent response was not seen in cortex or striatum (Figure 5).

Figure 5.

Effect of varying concentrations of wild H. ramosum mycelia on NGF synthesis in different parts of mouse brain [9]. 1, Cortex; 2, striatum; and 3, hippocampus. Data are expressed as the mean ± SEM. *p < 0.05 and **p < 0.01, compared with vehicle (Student’s test).


4. Soybean fermentation using mushroom mycelia

The legume soybean is highly proteinaceous (36% protein in dried beans), rich in major nutrients essential for human nutrition and can potentially be a good replacement for animal-derived proteins [38, 39, 40, 41]. It can be used both in fermented and non-fermented forms [42]. While soybeans are rich in flavonoid groups such as genistein, daidzein, and glycitein isoflavones that have tremendous health benefits [43], they are not easily absorbed and incorporated in their natural glycosylated forms unless hydrolyzed by the microflora of the intestine through their beta-glucosidase production [44]. Isoflavones have health benefits against several diseases and hormone-related issues [45, 46, 47, 48]. The easily absorbable form of flavones is the aglycon form, which is abundant in fermented sources of soybean, such as tempeh, miso, and natto [49].

Mushroom mycelia can be used as a source of beta glucosidase to convert isoflavone glycosides to their aglycon form. For example, G. lusidum, belonging to the basidiomycetes group, has been shown to increase serum concentration of the aglycon form of isoflavones in soybeans [50].

Studies from our laboratory investigated the health effects of fermentation using mushrooms, such as G. lucidum, H. erinaceum, and H. ramosum [10]. We measured DPPH scavenging activity, total phenolic content, antioxidant activity, alpha glucosidase inhibition, and isoflavone concentration, few major health parameters of paramount importance, in soybeans fermented with different mushroom types and compared them with non-fermented soybeans.

Soybean fermentation was carried out as described in Suruga et al. [10]. We found that G. lucidum was more effective in quickly fermenting soybeans compared to the other two mushroom types (Figure 6).

Figure 6.

G. lucidum was faster in fermenting soybeans compared to other types (a) Control (non-fermented soybeans); (b) G. lucidum; (c) H. erinaceum; (d) H. ramosum [10].

4.1 Antioxidant activity of fermented soybean

4.1.1 Methods

The DPPH radical scavenging activity and total phenolic content of fermented soybeans were analyzed using the methods described in Subsections 2.3.1 and 2.4.1. Oxygen radical absorbance capacity (ORAC) was determined using the OxiSelect™ ORAC Activity Assay Kit (Cell Biolabs Inc., San Diego, CA, USA) [51]. The assay was performed as described in Suruga et al. [10]. Briefly, fluorescence activity of the reaction mixture with antioxidant and fluorescein solution was measured after adding the free radical initiator. Increasing Trolox concentrations were used for the standard curve, and extracts were quantified and expressed as μmol Trolox equivalents/g of dry fermented soybean powder.

4.1.2 Total phenolic content and antioxidant activity of fermented soybean by mushroom mycelia

Total phenolic content was higher in all the fermented extracts compared to non-fermented control soybeans. Both DPPH radical scavenging activity and antioxidant activity were higher in fermented soybeans than in non-fermented ones (Table 1).

ControlG. lucidumH. erinaceumH. ramosum
Total phenolic content (mg/g dry powder)1.547 ± 0.0682.304 ± 0.0352.074 ± 0.0662.160 ± 0.014
DPPH radical scavenging activity (μmol Trolox/g dry powder)1.847 ± 0.0734.246 ± 0.0102.246 ± 0.0612.367 ± 0.173
ORAC (μmol Trolox/g dry powder)49.763 ± 2.85660.090 ± 1.50666.147 ± 1.89872.897 ± 2.113

Table 1.

Total phenolic content and antioxidant activity of soybeans fermented by mushroom mycelia [10].

4.2 Alpha-glucosidase inhibitory activity of soybeans fermented with mushroom mycelia

4.2.1 Methods

Yeast alpha-glucosidase inhibitory activity was measured using methods reported before [52] with modifications as described in Suruga et al. [10]. Briefly, yeast alpha-glucosidase was incubated with fermented soybean extract solutions and then p-nitrophenyl α-D-glucopyranoside (pNP-glucoside) was added and absorbance was determined at 400 nm. For the mammalian reaction, alpha glucosidase from rat intestinal acetone powder [53] was incubated with fermented soybean extract and the amount of glucose released was measured. We also used maltose as the substrate and calculated the % inhibition rate of alpha glucosidase [54].

4.2.2 Fermented soybean showed higher inhibition of alpha-glucosidase activity compared to non-fermented ones

Comparison of control (non-fermented soybeans) to soybeans fermented with mushroom mycelia showed that significant alpha-glucosidase inhibition could be achieved in the fermented soybeans using both pNP-glucoside and maltose (Figure 7A and B). Yeast alpha-glucosidase inhibition was the highest with H. ramosum compared to G. lucidum and H. erinaceum, while the mammalian alpha-glucosidase inhibition was significantly higher with G. lucidum fermentation (Figure 7A-C).

Figure 7.

Inhibition of alpha-glucosidase activity soybeans fermented by mushroom mycelia. (A) Yeast alpha-glucosidase inhibition using pNP-glucoside as substrate, (B) mammalian alpha-glucosidase inhibition using maltose as substrate, and (C) mammalian alpha-glucosidase inhibition using sucrose as substrate. Results are expressed as mean ± SD (n = 3). N.D.: not detectable. 1: p < 0.01 vs. control, 2: p < 0.01 vs. G. lucidum, 3: p < 0.01 vs. H. erinaceum, 4: p < 0.01 vs. H. ramosum, and 5: p < 0.05 vs. H. ramosum [10].

4.3 Comparison of isoflavone concentrations in soybeans fermented with mycelia versus non-fermented soybeans

4.3.1 Methods: high-performance liquid chromatography (HPLC) analysis

We followed the methods described in Kudou et al. [55] for measuring isoflavone concentrations in fermented and non-fermented soybeans. An LC-6A system (Shimadzu, Kyoto, Japan) equipped with a PEGASIL-ODS (4.6 mm i.d. × 250 mm) HPLC column (Senshu Scientific, Tokyo, Japan) was used for analysis. HPLC parameters used for the measurement of different isoflavones, such as genistein, daidzein, glycitein, daidzin, and glycitin, both in the glycosylated and in aglycone forms, are detailed in Suruga et al. [10].

4.3.2 Methods: liquid chromatography/mass spectrometry (LC/MS) analysis

An ACQUITY UPLC apparatus (Waters MS Technologies, Manchester, UK) equipped with a reversed-phase Acquity UPLC CHS C18 column with a particle size of 2.1 mm × 100 mm × 1.7 μm (Waters MS Technologies) was used for LC/MS analysis. Parameters of analysis are documented in detail in Suruga et al. [10].

4.3.3 Fermentation with mushrooms decreased the concentrations of glycosylated forms of isoflavones and increased the concentrations of aglycon forms

LC/MS profile was shown in Figure 8. The concentration of glycosylated forms of isoflavones, such as daidzin, glycitin, and genistin was about 95.6% in non-fermented soybeans, while it was reduced to 52.5, 15.8, and 17.6% in soybeans fermented by the G. lucidum, H. erinaceum, and H. ramosum mycelia, respectively. The aglycone forms of these isoflavones, on the other hand, increased from 4.4% in the non-fermented controls to 47.5, 84.2, and 82.4% in soybeans fermented by G. lucidum, H. erinaceum, and H. ramosum mycelia, respectively. LC/MS profile shown in Figure 8 corroborate these results. Based on the retention time and MS data, molecular formula and identity of compounds corresponding to 11 of the 12 peaks have been predicted: peak #1, daidzin; peak #2, glycitin; peak #3, 8-hydroxydaidzein; peak #4, genistin; peak #5, 6″-O-malonyldaidzin; peak #7, 6″-O-acetyldaidzin; peak #8, 6″-O-malonylgenistin; peak #9, daidzein; peak #10, glycitein; peak #11, 6″-O-acetylgenistin; and peak #12, genistein, respectively.

Figure 8.

LC/MS profile of soybean fermented using mushroom mycelia: a, control (non-fermented soybeans); b, G. lucidum; c, H. erinaceum; and d, H. ramosum [10].


5. Discussion

5.1 Characteristics of H. ramosum mycelia and other mushroom mycelia

While the beneficial effects of mushrooms in human health and nutrition have long been known and their pharmacological use has been studied in several types of mushrooms, including Pleurotus, Ganoderma, Cordyceps, Lentinus, Grifola, and Hericium [56], there are plenty of rare species of mushrooms that have not been investigated yet in terms of their biological functions, such as antioxidant activity, induction of NGF synthesis, etc. For example, there is only a single report that investigated the kappa opioid receptor binding activity of erinacine E on H. ramosum, indicating the rarity of this mushroom. Our analysis has provided a vast amount of data on the potential value of this mushroom. Given their vast health benefits and medicinal value, finding new mushrooms and analyzing their biological and pharmacological properties is of tremendous importance toward utilizing them in the development of new drugs and food supplements. We have investigated 20 mushroom types for their health benefits.

DPPH radical scavenging activity is a good indicator of antioxidant properties. Our study indicated that several mushrooms (L. shimeji #19, G. frondosa #10, H. erinaceum #5, and H. ramosum #6) were potent scavengers of DPPH (Figure 1). We also found a direct correlation (R2 = 0.7929) between total phenolic content and DPPH radical scavenging activity (Figure 3). A direct relationship between total phenolic content and DPPH scavenging activity has been demonstrated in several studies. For example, a direct correlation (R2 = 0.9788) between total phenolic content and total antioxidant activity has been shown in 11 kinds of fruits by Sun et al. [57]. A direct relationship (R2 = 0.8181) between total phenolics volume and DPPH radical scavenging activity has also been reported in the fruiting bodies of 14 different kinds of commercially available mushrooms by Abdullah et al. [58]. A direct relationship was reported between the high antioxidant activity observed in rice fermented by Monascus mycelia and its high total phenolic compound levels [59]. Our results corroborate the findings from these reports.

From our analyses, we have found that L. shimeji (#19) and G. frondosa (#10) had potent DPPH radical-scavenging activities. Several studies have investigated the applications of these and other mushrooms in various diseases and for other purposes. Pyranose oxidase, a flavoprotein from L. shimeji (Honshimeji in Japanese), has been studied and its heterologous expression is reported to be under the control of the T7 promoter in Escherichia coli [60]. L. connatum fruiting bodies (Oshiroishimeji) have been shown to contain new ceramides [61]. The antitumor activities of (1 → 3)-ß-D-glucan and (1 → 6)-ß-D-glucan from L. decastes (Hatakeshimeji) hot water extract against Sarcoma 180 have also been described [62].

Several compounds responsible for DPPH and antioxidant activity have been isolated from mushrooms and studied in detail. However, little information has been published regarding the DPPH scavenging activity of active compounds from mycelia of L. shimeji and G. frondosa. DPPH active compounds ergothioneine, N-hydroxy-N′,N′-dimethylurea, connatin, and ß-hydroxyergothioneine have been isolated from L. connatum fruiting bodies [63]. Yeh et al. described the antioxidant compounds β-tocopherol and flavonoids in ethanol extracts of G. frondosa fruiting bodies, which are edible mushrooms in Japan [64]. Zhang et al. isolated three analogues of ergosterol from G. frondosa mycelia as compounds with antioxidant activity [65]. Reis et al. investigated the effects of five kinds of mushroom mycelia (A. bisporus-white, A. bisporus-brown, P. ostreatus, P. eryngii, and L. edodes) on antioxidant activity. The authors reported that the antioxidant compounds of these mushroom mycelia were gallic acid, protocatechuic acid, p-hydroxybenzoic acid, and p-coumaric acid [66]. Considering these observations, the potent DPPH scavenging activity of L. shimeji, G. frondosa, and other mushrooms could be attributed to the polyphenols ergothioneine, N-hydroxy-N′,N′-dimethylurea, connatin, ß-hydroxyergothioneine ergosterol, α-tocopherol, flavonoids, gallic acid, protocatechuic acid, p-hydroxybenzoic acid, and p-coumaric acid.

The present findings indicate that the DPPH radical scavenging activity of the Hericiaceae group, including H. erinaceum (#5) and H. ramosum (#6), was stronger than those of other mushroom mycelia. The antioxidant activity of some phenolic compounds has been reported in the H. erinaceum and its mycelial extracts [67]. A strong antioxidant activity has also been shown in vitro in polysaccharides derived from an ethanol extract of H. erinaceum grown on tofu [68]. Thus, there has been minimal effect on H. ramosum mycelia which contain phenolic compounds and polysaccharides with strong antioxidant activity.

NGF plays a crucial role in nerve growth and neuronal cell function, and protection of neurons. NGF has been implicated in various diseases, including in Alzheimer’s disease, the most common type of dementia that affects language, memory, processing of visual cues, judgment, and mood [69]. Reduced levels of NGF or increased accumulation of β-amyloid peptide and tau protein have been suggested as causes of AD [70]. Given its importance, there has been a demand for finding natural inducers of NGF synthesis. Natural compounds such as hericenones and erinacines isolated from H. erinaceum have been shown to induce NGF synthesis [33, 71]. We have shown that H. ramosum mycelia induced stronger NGF synthesis activity compared to H. erinaceum mycelia in the hippocampus of intact mice, and that processing of H. ramosum mycelia over time elevated the levels of NGF levels (Figure 4). We also found a dose-dependent response of NGF with increasing concentrations of H. ramosum mycelia in the hippocampi of intact mice (Figure 5). However, we have not determined the active compounds in the mycelia responsible for NGF synthesis. There are mounting evidence suggesting that erinacine species could be responsible, with the isolation of erinacine E from H. ramosum [72], and the observation that active substances other than hericenones stimulated NGF synthesis through c-Jun N-terminal kinase activation in H. erinaceum [73]. This evidence indicates that erinacine species could be involved in the induction of NGF synthesis in H. ramosum mycelia. There may be involvement from other unknown compounds as well, as our data comes from mycelia and not the fruiting bodies.

5.2 Soybean fermentation of mushroom mycelia

Mushrooms are effective in combating issues caused by obesity, diabetes, and other health issues [74]. The medicinal value of mushrooms has been known for thousands of years [75, 76] and they have been incorporated in nutrition supplements [74] and in the production of fermented foods, such as soybean-based foods, bread and cheese, and in alcoholic beverages [77]. However, detailed analysis of soybeans fermented by mushroom mycelia has not been conducted, insofar as their oxidative properties or alpha-glucosidase inhibitory activity are concerned. Our study analyzed all these properties and the LC/MS profiles of the bioactive products to glean more insights into the medicinal value of fermented soybeans.

We found that soybeans fermented with mushroom mycelia had stronger DPPH radical scavenging activity and ORAC than the non-fermented control ones. We also found that H. ramosum mycelia were more potent in DPPH radical scavenging and oxygen radical absorbance compared to all the other 19 mushroom groups we had tested (Figure 1) [9]. While this result was consistent in our subsequent study, we also found that DPPH radical scavenging activity and total phenolic content of G. lucidum mycelia-fermented soybeans was higher than soybeans fermented with H. erinaceum and H. ramosum mycelia [10].

The compound 8-hydroxydaidzein (peak #3 in Figure 8b) and one unidentified compound (peak #6) were identified by LC/MS analysis in soybeans fermented using G. lucidum mycelia. While we are investigating the identity of this unknown compound, we believe that this could possibly be 6-hydroxydaidzein or 3-hydroxydaidzein based on mass spectrometry analysis results. 6-Hydroxydaidzein has been isolated from soybean koji fermented with Aspergillus oryzae [78] and was found to be more potent in terms of antioxidative properties compared to daidzein [79], suggesting that phenolic compounds such as hydroxydaidzeins could influence the antioxidant effects of soybeans fermented with G. lucidum mycelia. Since oxidative stress is linked to several diseases [80], mushroom mycelia showing antioxidant activity is of much relevance toward producing antioxidant foods and nutritional supplements. We have shown high antioxidant activity in G. lucidum mycelia-fermented soybeans [10], as well as in fermented soy residue (“okara”) with Rhizopus oligosporus [81, 82, 83].

Alpha-glucosidases are the primary enzymes responsible for hydrolyzing carbohydrates into glucose. Inhibition of alpha-glucosidase activity, therefore, is a strategy for controlling increase in blood glucose levels in diabetic conditions. We have shown that soybeans fermented with mushroom mycelia have significantly higher alpha-glucosidase activity than the non-fermented control groups. When pNP-glucoside was used as a substrate, the yeast alpha-glucosidase activity was inhibited in soybeans fermented with H. erinaceum, H. ramosum, and G. lucidum mycelia, with fermentation using the Hericiaceae members showing higher inhibition than with G. lucidum mycelia. Similar inhibition of alpha-glucosidase using pNP-glucoside has been achieved by the commercial soy isoflavone genistein by Lee et al. [84], suggesting that genistein might play a role. Despite pNP-glucoside’s wide usage in testing anti-diabetic agents, maltose and sucrose are biologically more relevant as substrates than pNP-glucoside for mammalian systems. Therefore, we used these two substrates for testing inhibition of alpha-glucosidase activity in soybeans fermented using H. erinaceum, H. ramosum, and G. lucidum mycelia. We found that all three were able to inhibit alpha-glucosidase activity with varying degrees, with G. lucidum mycelia exhibiting higher inhibition with both maltose and sucrose as substrates compared to the other mushroom species. We suspect that in addition to genistein, hydroxydaidzein in soybeans fermented using G. lucidum mycelia could facilitate this inhibition. The precise identification of the active compounds in fermented soybeans using mushroom mycelia is yet to be completed, but fermented soybeans have potential use as nutritional supplements for treating diabetes.

The beta-glucosidase enzyme (EC produced by microbes facilitates the breakdown of glycosylated isoflavones to their aglycon form, which is more easily absorbable [85]. Several microbes, including Aspergillus niger [86], A. oryzae [87], Penicillium brasilianum [88], and Phanerochaete chrysosporium [89], are being tapped for this fermentation purpose. We found that the levels of aglycons (daidzein, glycitein, and genistein), were higher in soybeans fermented with mycelia compared to non-fermented soybeans. While one previous report has shown the conversion of isoflavone glucosides to aglycons using G. lucidum mycelia to ferment soybeans [50], not many studies have investigated soybean fermentation using H. erinaceum and H. ramosum mycelia. We have shown that fermentation using these mycelia increased the amount of the aglycon form compared to non-fermented ones. The amount of aglycons was higher with H. erinaceum and H. ramosum mycelia compared to that when G. lucidum mycelia were used, possibly because the former produces more beta-glucosidase enzyme than the latter.

Our mass spectrometry analysis data revealed that the aglycon form of isoflavones obtained in soybeans fermented with G. lucidum mycelia contained 8-hydroxydaidzein and an unidentified compound, which we assumed to be 6-hydroxydaidzein or 3-hydroxydaidzein based on m/z data and molecular formula derived from LC/MS analysis. 8-Hydroxydaidzein was first isolated from Streptomyces sp. fermentation broth [90] and has also been obtained from A. oryzae and recombinant Pichia pastoris, in addition to 6-hydroxydaidzein and 3-hydroxydaidzein [91]. This compound has been shown to have anti-proliferative, tyrosinase inhibition, aldose reductase inhibition, anti-inflammatory, and antioxidant activities [79, 92, 93, 94, 95], indicating that soybeans fermented with G. lucidum mycelia might also have these properties. Since the mechanism of conversion of hydroxydaidzein to daidzin is known [96], and given its valuable properties, synthetic hydroxydaidzein is produced at the commercial level, but the process has its own limitations, such as the formation of undesirable by-products, lengthy reaction steps and low yield [97]. Large-scale production of hydroxydaidzein using natural resources such as A. oryzae are being investigated [96, 98]. Our results have added several suitable candidates for this purpose. In particular, soybeans fermented using G. lucidum mycelia have enormous potential to be used as food, nutritional supplement and as a source for commercial production of hydroxydaidzein.



We are grateful to the Chairman and CEO Masahito Hoashi, Kibun Foods Inc., for his support of this study.


Conflict of interest

The authors declare that there are no conflicts of interest.


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Written By

Kohei Suruga, Tsuyoshi Tomita and Kazunari Kadokura

Submitted: 29 November 2021 Reviewed: 07 January 2022 Published: 12 April 2022