**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 (*R*<sup>2</sup> = 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 (*R*<sup>2</sup> = 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 (*R*<sup>2</sup> = 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 *Esch***e***richia coli* [60]. *L. connatum* fruiting bodies (Oshiroishimeji) have been shown to contain new ceramides [61]. The antitumor activities of (1 → 3)-ß-Dglucan 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

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

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* [81–83].

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 pNPglucoside 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 3.2.1.21) 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 betaglucosidase 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

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

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, 92–95], 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.
