Introduction

Over last decades, the interest for cultivation of pharmaceutically significant mushrooms (known as specialty mushrooms) has increased rapidly (Grimm and Wösten 2018). As a result, the share of the common cultivated button mushroom (Agaricus bisporus) declined from 71.6% in 1981 to 15% in 2017, while specialty mushrooms belonging to genera of Lentinula, Pleurotus, Auricularia, and Flammulina account for 70% of the global mushroom production (Royse et al. 2017). The winter mushroom or Enokitake, Flammulina filiformis (previously known as Flammulina velutipes), is the most momentous species of Flammulina which has gained a considerable attention particularly in East Asia (Hughes et al. 1999). The latest phylogenic findings suggest that there are totally 17 species belonging to Flammulina (Wang et al. 2018a), even though no commercial cultivation has been reported for most of them (Redhead et al. 1998; Ripková et al. 2008, 2010; Redhead et al. 2000).

Enokitake has a delightfully crunchy texture and desirable taste with a fragile and sensitive cap of 1–5 cm diameter. Research has also proved that this mushroom possesses substantial nutritional and medicinal properties (Tang et al. 2016). Enokitake has long been successfully cultivated particularly in China and Japan (Royse 2014) as well as Europe and North America (Sharma et al. 2009). China is currently the leading producer of this mushroom with a production of 2.4 million tons annually (Liu et al. 2018). However, commercialization of Enokitake is not yet expanded worldwide and it is still largely behind the other major edible mushrooms in some countries than East Asia (Harith et al. 2014). This issue might be partly explained by the fact that the state of knowledge on composition of locally suitable substrates, agronomic factors, and environmental requirements of Enokitake has not been critically updated since the last available general review on this mushroom (Sharma et al. 2009). By contrast, there is updated technical information on other major edible mushrooms such as Pleurotus spp. (Bellettini et al. 2016). In this context, this mini review aims to consolidate available information on progress made in environmental and agronomic determinants for production of Enokitake. The current limitations of research in both solid-state and submerged cultures are critically evaluated and their contribution to future prospects is also discussed in this review.

It should be noted that Enokitake has been originally described as F. velutipes by many of researchers whose studies were evaluated in this review. However, a recent study proved that all the cultivated strains of Enokitake cited by researchers from East Asia should be treated as a new species namely F. filiformis which is different from the European winter mushroom, F. velutipes (Wang et al. 2018a). Thus, Enokitake or the winter mushroom corresponds to F. filiformis in this review where it is certain that the study comes from East or Southeast Asia.

Composition of lignocellulosic substrates

In solid-state cultivation, substrates directly influence growth, productivity, quality of fruiting bodies, and biological efficiency of specialty mushrooms (Bellettini et al. 2016; Lin et al. 2017) including Flammulina spp. Substrates are mainly lignocellulosic residues containing basic macromolecules such as cellulose, hemicelluloses, and lignin, which serve as main sources to provide nutritious elements and compounds for mushrooms (Elisashvili et al. 2009; Cogorni et al. 2014). Cellulose is a polysaccharide linear homopolymer composed of β-1,4-linked glucosyl units, while hemicelluloses are polysaccharide branched hetropolymers such as xylan, glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan. For example, xylan itself is composed of a 1,4-linked β-d-xylopyranosyl backbone with arabinosyl, O-acetyl, and (4-O-methyl-) glucuronic acid side chains. Contrary to cellulose or hemicellulose, lignin is not a polysaccharide but it is an organic cross-linked polymer composed of three main monolignols, including p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) phenylpropanoid units (Jurak et al. 2015).

From a practical viewpoint, the enzymatic ability of Enokitake to decompose the lignocellulosic polymers is important for successful cultivation, efficient substrate usage, high yield, and production of value-added products (Baldrian 2006). While few studies have addressed events related to enzymatic digestion of lignocellulosic polymers during composting by mushrooms such as A. bisporus (Jurak et al. 2015), the data about how Flammulina spp. degrade cellulose, hemicellulose, and lignin is scarce. It was only recently found that F. filiformis cultivated on ramie stalk produced enzymes such as cellulase, hemicellulose, and ligninolytic, which degraded lignocellulosic polymers at various rates and showed a positive relationship with the mushroom yield (Xie et al. 2017). Similar to other mushrooms (Isikhuemhen and Mikiashvilli 2009), further studies involving biochemical analyses of substrates (before and after fruiting) are thus needed to determine what is happening to the substrate in order to gain a whole picture of bioconversion efficiency of agro wastes by Flammulina spp.

In addition to basic lignocellulosic residues, a substrate may be properly enriched with various supplements (Table 1) to provide an appropriate formulation for mushroom cultivation. However, Flammulina spp. have shown the ability to grow on basic lignocellulosic substrates such as sawdust even without any supplementation (Rezaeian and Pourianfar 2017). Table 1 illustrates best formulations/combinations of basic lignocellulosic residues enriched with supplements reported to give highest yield or biological efficiency of Enokitake. In some studies reported in Table 1, the information on biological efficiency (weight of fresh basidiocarps / weight of dry substrate × 100) has been given, while the others lack such information. Biological efficiency is one of the key determinants of cultivation of specialty mushrooms in a substrate. In addition, some of the substrate formulae indicated in Table 1 have been selected based on comparative studies, indicating the ability of Enokitake to grow on a wide range of basic substrates and supplementations, depending on the availability for the region.

Table 1 Lignocellulosic substrate formulations for cultivation of Enokitake
Full size table

Wild and cultivated strains of Enokitake

Wild mushrooms have long been used as human food and appreciated for their texture and flavors as well as medicinal properties (Ferreira et al. 2010). Accordingly, production of non-Agaricus mushrooms, including Enokitake, in the East and Southeast Asia has been common for centuries and people of these regions have been fully familiar with these mushrooms and their medicinal and nutritional properties. Enokitake is one of the specialty mushrooms that can grow wild in many regions in the world (Hughes et al. 1999; Ge et al. 2015; Rezaeian and Pourianfar 2017). Wild Enokitake has light or dark brown caps with short stems, while the commercial strains have narrow white caps with long bases (Ge et al. 2015). In addition, the wild strains possess greater genetic diversity than the cultivars do (Liu et al. 2016).

A large body of research has reported adaptation of wild mushrooms indigenous to the original countries (Rezaeian et al. 2015; Tan et al. 2015; Sulistiany et al. 2016; Luangharn et al. 2017). In addition, biological activities of various wild mushrooms species have been compared with their cultivate counterparts, including antioxidant activities (Tajalli et al. 2015; Rezaeian et al. 2015), antimicrobial (Soltanian et al. 2016), and anticancer activities (Rezaeian and Pourianfar 2018). However, very limited reports are available on domestication or nutritional analysis of wild Enokitake (Table 1). This issue might be attributed to the high number of wild mushroom species growing in different habitats, the high variability in chemical composition within individual species, and the difficulty in artificial production of fruiting bodies (Kalač 2013). The use of domesticated wild mushrooms ensures a continuous supply of the mushroom, reproducibility and reliability of research, and the conservation of rare wild species (Lindequist et al. 2005; Tan et al. 2015). A recent study reported successful cultivation of a well-identified wild strain of Enokitake in locally available substrates, indicating there were also interaction effects between substrate formulations and mushroom strains (i.e., wild versus cultivar) (Rezaeian and Pourianfar 2017). These findings together with the fact that Enokitake does not necessarily need supplementation and may grow on low-cost basic substrates (such as sawdust, wheat straw, paddy straw, and ramie stalk) might have economic implications for developing countries. Thus, it is essential to seek for ideal formulation of locally available lignocellulosic substrates to cultivate wild-growing strains of this mushroom.

Nitrogen content of substrate/media and its interaction with carbon

A substrate containing high levels of nitrogen content may increase the mycelium growth rate of Flammulina spp. as compared with a low-level nitrogen substrate (Harith et al. 2014). While carbon is provided by basic lignocellulosic residues, nitrogen content of a substrate is mostly determined by supplementations added to it, including wheat bran, sugarcane bagasse, and rice bran. However, as stated previously, mycelia of Enokitake is able to grow to some extend in a condition without nitrogen supplementation.

Different sources of nitrogen might have different effects on mycelia of Enokitake (Hassan et al. 2012b; Harith et al. 2014). The growth of mycelia is greater with organic nitrogen sources such as yeast extract compared with inorganic ones (e.g., urea). This difference lies in the fact that yeast extract is composed of nutrient compounds such as carbohydrates, vitamins, and amino acids (Hassan et al. 2012b). Similarly, it was shown that amino acids and ammonium compounds effectively encourage mycelium growth and fruiting body induction in Enokitake (Sharma et al. 2009).

Nitrogen in substrate may also interact with carbon as the ratio of C/N. Important processes such as lignocellulosic degradation are related to the C/N ratio in substrate (Xie et al. 2017). Research has shown that F. filiformis can degrade substrates with low ratio of C/N much more effective than those with high ratio of C/N (Xie et al. 2017; Harith et al. 2014).

Unlike mycelium, fruiting body production and basidiocarp size in Enokitake might be negatively influenced by nitrogen supplementation. While formation of basidiocarp decreases under high levels of nitrogen (Harith et al. 2014), nitrogen starvation induces fruiting body formation in mushrooms (Sakamoto 2018). It has been found that a C/N ratio of 30 might be the best for fruiting body formation (Xie et al. 2017). Thus, accurate analysis of carbon and nitrogen contents in substrate or media and proper utilization of a C/N ratio would lead to enhancement of mycelial growth rate, yield, and biological efficiency. Further investigations would be also warranted to determine the role C/N ratio might play in protein content or production of metabolites of Enokitake in submerged cultures or solid-state cultivation.

Inoculum size and spawn rate

Increasing the spawn rate up to 25% of the substrate (dry weight) accelerates mycelial growth of Enokitake (Leifa et al. 2001; Murthy and Manonmani 2008). However, no significant difference has been found between the amounts of spawn rates ranging from 10 to 25% in terms of mushroom yield or protein and fiber contents. From an economics point of view, a spawn rate of 10% would be recommended as appropriate, while a rate less than 10% might facilitate contaminations and reduce biological efficiency and yield production (Murthy and Manonmani 2008; Leifa et al. 2001).

The effect of inoculum size in submerged cultures was also investigated. If a seed culture liquid medium containing vegetative mycelia is considered as the inoculum for a 1000-mL fermentation medium, an inoculum size up to 8–9% of fermentation medium (V/V) may increment dry cell weight and mycelial biomass of F. filiformis (Hassan et al. 2012b). Thus, it is important to utilize an appropriate amount of actively growing mycelia for both solid-state cultivation and production of mycelial biomass and metabolites in liquid fermentation.

The effect of pH

Morphological and developmental changes of the winter mushroom are largely affected by pH in media and substrate (Harith et al. 2014). In media, the degree of pH may influences cell membrane function, cell morphology and structure, nutrient absorbance, and biomass production (Osman et al. 2014). A range of pH values from 4 to 8 has been shown to support mycelium growth in many of mushroom species (Kim et al. 2002b), including Flammulina spp. (Osman et al. 2014; Hassan et al. 2012b; Kozhemyakina et al. 2010). However, the highest biomass of mycelia of Flammulina spp. may be achieved at pH 5.0–6.5 (Kozhemyakina et al. 2010; Fidler et al. 2015b). In addition, pH might have a moderate effect on mineral adsorption in submerged cultures. For instance, rate of selenite uptake was reduced when pH increased from 5.5 to 7.5 (Wang et al. 2016).

The strain of Enokitake may also interact with pH value. A study showed that the biomass yield of strain Fv. 19 reached the maximum level (6.37 g/l) by increasing pH value from 4 to 6, while the greatest biomass yield for strain Fv. 01 strain was obtained at pH values ranging 6.5–7.5 (Hassan et al. 2012b). Besides, different stages of fructification are affected by pH of substrate (Khan and Chandra 2017) so that changes in pH of substrate at the end of vegetative growth stage could stimulate fruiting body formation (Harith et al. 2014). A relatively neutral situation (pH 6–7) is the best for fruiting body formation in Flammulina spp. (Harith et al. 2014; Khan and Chandra 2017), while mushrooms such as Lentinula edodes (shiitake) produce fruiting body at pH values as low as 4 (Ohga 1999). Moreover, changes in pH from a neutral to acidic may induce an apical stipe wall extension in Enokitake (Fang et al. 2014). Overall, due to the direct influence of pH on the fungus nutrient metabolism, pH in substrate or media affects a wide range of the growth characteristics, including biological efficiency, yield, flushing of mushrooms, time of pinning, number of pileus, metabolic processes, and the ability to adsorb certain nutrients.

Supplementation of substrate

Minerals

The uptake capacity of mushrooms for minerals explains the presence of nutritional minerals in their fruiting bodies (Lee et al. 2009). In addition to the nutritional values and biological roles of minerals in mushroom fruiting bodies in terms of human consumption, they also serve as micronutrients and enzyme cofactors for functions of mushrooms, which eventually enhance both mycelial growth and yield (Rodriguez Estrada and Royse 2007). It has been known that the composition and nature of substrates determine profile of minerals in substrates (Koutrotsios et al. 2018), making it necessary to supplement substrates or liquid fermentation media of Flammulina spp. with proper amounts of minerals.

Research has shown that there are differences among minerals in terms of their uptake by Enokitake. While potassium was particularly abundant in both substrates and fruiting bodies, calcium was very low in the fruiting bodies despite a high level of calcium in the substrate (Lee et al. 2009; Hashemian et al. unpublished data). These findings indicated that the winter mushroom does not have specific channels for calcium uptake or bioavailable form of calcium in the cultivation substrates is rare (Lee et al. 2009). Differences in the uptake capacity of minerals may also yield information regarding the type of minerals that could be added to substrate or fermentation medium as supplements in order to maximize productivity of Enokitake.

At the present, limited data is available regarding how addition of minerals to cultivation substrate may influence yield and biological efficiency of Enokitake. According to Dhiman (2009), the mycelium growth happened only in the basic salt medium with trace elements such as magnesium, iron, zinc, and manganese. In contrast, it was shown that increasing levels of magnesium and phosphate could improve the growth of mycelium of Enokitake in submerged cultures (Osman et al. 2014). Some other trace minerals including zinc (2 mg/L), manganese (2 mg/L), iron (1 mg/L), and molybdate (10 mg/L) have also been used in submerged medium in order to obtain the best mycelial growth (Sharma et al. 2005).

A study showed fruiting body production and basidiocarp formation of Enokitake were affected by the micronutrients and mushroom yields two to seven times more than the control. Moreover, copper sulfate and calcium sulfate stimulated primordial appearance in a shorter time and number of mushrooms enhanced with application of ferrous iron (Dhiman 2009).

Elicitors

Supplementation of substrate of Enokitake with various types of elicitors including chitosan, triacontanol, and 2,4-dichlorophenoxyacetic acid (2,4-D) has been shown to influence growth characteristics such as time to appearance of fruiting bodies, the number of mushroom, and yield (Tuheng et al. 2017). Hormones such as alkylal 30 + gibberellin (Sharma et al. 2009) and gibberellic acid (5 ppm) were found as the best growth regulators followed by succinic acid 2,2-dimethyl hydrazide and morphactin (5 ppm) (Rangad 1981). Another study showed that indole-3-butyric acid (20 mg/L) is the best growth hormone for enhancing mycelium growth. Besides, β-indole acetic acid combined with 6-benzylaminopurine (at 0.5 mg/L concentration each) increased mycelium growth of Enokitake cultured in media (Harith et al. 2014). Among vitamins, thiamine or thiamine hypochlorite showed the most impact on the mycelial growth of Enokitake in media (Dhiman 2009). The exogenous supply of thiamine at 0.025 ppm followed by 0.025 ppm of biotin significantly enhanced mycelial growth of this mushroom. On the contrary, a decline in the growth was recorded with a mixture of pentothenic acid, biotin, folic acid, and riboflavin (Dhiman 2009).

Effects of environmental factors

Heat treatment of substrate

The yield of Enokitake can be different among sterilized, pasteurized, or unpasteurized substrates. While a high yield was achieved with sterilized and pasteurized substrates, the yield obtained with unpasteurized substrate was very low due to high rate of contamination (Hassan et al. 2012a). Earlier studies have also evaluated different combinations of time and temperature, including 100 °C for 4 h, 100 °C for 3 h, 121 °C for 1 h, and 121 °C for 1 h. Over the first and second flushes, the most contamination and lowest yield were observed in substrates sterilized at 121 °C for 1 h, while the least contamination and highest yield were achieved with 100 °C for 4 h (Sharma et al. 2009). However, more recent studies indicate that the best method of sterilization is 121 °C, 15 psi, for 2–3 h (Xie et al. 2017; Rezaeian and Pourianfar 2017; Harith et al. 2014).

Temperature of the culture house and media

The colonization of Enokitake mycelia may occur in temperatures between 3 and 34 °C (Harith et al. 2014), while the optimal temperature was reported to be 18–25 °C (Xie et al. 2017; Rezaeian and Pourianfar 2017; Harith et al. 2014; Liu et al. 2016). At very low temperatures (3–4 °C), the mycelial growth rate would be limited but still it could survive. But, temperatures over 34 °C may stop the vegetative growth and promote contaminants (Sharma et al. 2009; Harith et al. 2014).

Fructification in Flammulina spp. is often induced after significantly altering environmental conditions, including temperature (Sharma et al. 2009). Accordingly, the temperature should be reduced to 5–8 °C in order to induce formation of primordia. The best temperature for the maintenance and growth of fruiting bodies ranges from 15 to 18 °C (Xie et al. 2017; Harith et al. 2014; Ohga 1999; Miyazaki et al. 2011).

In submerged cultures, temperature may also affect biomass production of Enokitake so that the highest values were obtained at 25 °C (Hassan et al. 2012b; Osman et al. 2014). The highest mycelial biomass amounts of Enokitake strains Fv. 19 and Fv. 01 at 25 °C for 15 days were 5.56 and 5.19 g/l, respectively (Hassan et al. 2012b).

Moisture and humidity

The optimal level of substrate moisture for mycelial growth of Flammulina spp. is associated with the formulation of substrate (Leifa et al. 2001). A range of 50–80% moisture for wooden and non-wooden substrates has been recommended for Enokitake (Xie et al. 2017; Harith et al. 2014; Sangkaew and Koh 2017; Park et al. 2014). To reach this level of moisture, it is normally required to soak raw substrates in water for 14–16 h prior to sterilization. Wooden substrates need lower moisture compared to non-wooden substrates in order to increase substrate porosity and improve oxygen exchange (Leifa et al. 2001; Kües and Liu 2000). A 45% decrease in moisture of the substrate was shown to have negative influence on growth characteristics such as mycelial growth in Enokitake (Leifa et al. 2001).

There are contradictions regarding the desirable relative humidity of the culture house for primordial development of Flammulina spp. While some studies have reported 60–70% as the best relative humidity for stimulating primordial formation (Rezaeian and Pourianfar 2017; Harith et al. 2014), other studies demonstrated that a humidity of 90–95% is the optimal condition for fructification (Sangkaew and Koh 2017; Wang et al. 2016). This contradiction is most likely due to the fact that different compositions of substrates have been used. Following primordial appearance, a high relative humidity (90–95%) is required to maintain conditions for fruiting body maturation in this mushroom (Xie et al. 2017; Khan and Chandra 2017; Miyazaki et al. 2011; Park et al. 2014).

Oxygen and carbon dioxide

Similar to other mushrooms, the winter mushroom also requires oxygen to decompose the substrate during vegetative growth of mycelia, resulting in production of large amounts of carbon dioxide. Besides, carbon dioxide at concentrations less than 0.3–0.5% is required for vegetative growth of mycelia and stimulating primordia development of Flammulina spp. (Long 1966; Lee et al. 2009; Harith et al. 2014; Bellettini et al. 2016). During fructification, however, it is important to reduce carbon dioxide levels and increase the concentration of oxygen (Bellettini et al. 2016). Concentrations more than 0.1–0.2% of carbon dioxide might increase stipe elongation, decrease pileus diameter, and suppress basidiospore development. On the contrary, short and thick stipes along with opened caps appear in Enokitake under low concentrations of carbon dioxide (Bellettini et al. 2016; Kurtzman and Martínez-Carrera 2013). Practically, a number of holes are normally made on the cultivation plastic sheets to allow for gas exchange. The size of holes is important, as larger holes leads to less mycelia growth, small mushrooms, and yield reduction (Bellettini et al. 2016).

Light requirement

Considerable progress has been made with regard to the role light may play in different developmental stages of Enokitake. While Flammulina spp. mycelia may grow in dark conditions (Xie et al. 2017; Rezaeian and Pourianfar 2017; Leifa et al. 2001), light is crucial for the morphogenesis, fruiting body induction, and spore viability (Kurtzman and Martínez-Carrera 2013; Sakamoto et al. 2002; Sakamoto et al. 2004; Fuller et al. 2015). Fruiting bodies of Enokitake grown in the dark produce long and narrow stipes with undeveloped pileus on the top (Xie et al. 2017; Harith et al. 2014; Sakamoto et al. 2004; Miyazaki et al. 2011). Under light exposure, stipe elongation stops and it becomes thicker (Sakamoto et al. 2004). However, stipe thickening is suppressed and elongation of stipe will be re-started 8 days after light illumination. The color of Enokitake fruiting bodies is also affected by light. The pinhead fruiting bodies formed in the dark conditions are whiter than those that grow under light exposure.

Light intensity, light wavelength, and time of irradiation are important factors that influence mushroom development and yields during fructification (Nakano et al. 2010). A light intensity up to 100 lx increases the diameter of the pileus and instantly stimulates pileus differentiation in Enokitake fruiting body (Sakamoto et al. 2004; Sakamoto et al. 2007). Other studies have shown that the optimal intensity of light and exposure time for the fruiting body development is 70–150 lx and 8–12 h/day, respectively (Xie et al. 2017; Harith et al. 2014; Miyazaki et al. 2011). Among light wavelengths, blue light is specially reported to associate with photomorphogenesis (Harith et al. 2014; Idnurm et al. 2010). Red and blue coherent light of 632.8 and 488 nm, respectively, stimulated the germination of basidiospores and growth of monokaryons (Poyedinok et al. 2015). It was also shown that production of Enokitake increased after exposure of vegetative mycelial growth to either blue LED or white fluorescent lamps, while UV illumination had no significant effect on the yield (Miyazaki et al. 2011).

Effect of agronomic and environmental conditions on pharmaceutically important bioactive compounds

Recent updates on Enokitake bioactive compounds with inhibition activities

There has been a growing interest in medicinal mushrooms as a source of biologically active metabolites which provide remarkable potential applications for medicinal purposes (Rathee et al. 2012). Enokitake, as a medicinal mushroom, has long been recognized to harbor a broad spectrum of biologically active components whose inhibitory effects against a number of diseases or disorders have been proven (Yeh et al. 2014; Tang et al. 2016). In this mushroom, both cellular components and secondary metabolites have been shown to contribute to a variety of inhibitory activities. Among cellular components, bioactive proteins such as fungal immunomodulatory proteins and ribosome inactivating proteins (flammin, velin, velutin, and flammulin) have significantly shown immunomodulatory, anticancer, and anti-inflammatory properties. In addition, glycoprotein enzymes such as proflamin and asparaginase have been contributed to anticancer effects (Tang et al. 2016). Polysaccharides of Enokitake were also the subject of a recent study where a novel polysaccharide FVPB2 purified from fruiting bodies of Flammulina spp. affected the activation of B cells and releasing of IgG and IgM, in addition to previous proven effects on T cells (Wang et al. 2018b). Another study showed that active polysaccharides such as dietary fiber from Enokitake could accelerate the decomposition of cholesterol and reduce LDL level in the blood (Yeh et al. 2014), while a recent study reported strong antibacterial and antioxidant properties for polysaccharide-iron (III) complexes (FVP-Fe and FVP2-Fe) obtained from Enokitake (Dong et al. 2017). In addition to proteins and polysaccharides, lipids such as sterols (particularly ergosterol) extracted from Enokitake were also demonstrated to possess antiproliferative activities against several cancer cell lines (Tang et al. 2016).

Among secondary metabolites of Enokitake, sesquiterpenes, norsesquiterpenes (flammulinolides), and phenolic compounds (such as protocatechuic acid, p-coumaric, and ellagic acid) have been linked to anticancer, antioxidant, anti-atherosclerotic, and anti-thrombosis effects. In addition, enokipodins and flamvelutpenoids (belonging to sesquiterpenoids) were proven to inhibit the growth of pathogenic microorganisms. Lovastatin and γ-aminobutyric acid (GABA) in the fruiting body of Enokitake inhibited biosynthesis of cholesterol and hypertension, thereby reducing the risk of coronary heart disease (Tang et al. 2016). A recent study also confirmed the presence of several antibacterial and antioxidant phenolic compounds in the water extract of Enokitake, including chromogenic acid, methyl-5-O-caffeoylquinate, Kukoamine A, Kushenol K, Methyl Kushenol C, Glabrol, Sanggenon J, Corylin, and Moracenin C (Shah et al. 2018). In addition, six flavonoid substances from Enokitake were identified as arbutin, epicatechin, phillyrin, apigenin, kaempferol, and formononetin, all of which demonstrated antioxidant capabilities, effects against H2O2-induced neurotoxicity in PC12 cells, and the potential for neuroprotection (Hu et al. 2016).

Similar to other mushroom species, composition and concentration of biologically active components of the winter mushroom can be highly influenced by agronomic and environmental conditions such as substrate composition, fructification conditions, and stage of development (Smith 2014). Similarly, medium components (e.g., carbon, nitrogen, and inorganic acids) and the fermentation parameters (e.g., temperature, pH, aeration, shear stress, and inoculation density) may affect production of bioactive compounds in submerged cultures (Lin et al. 1997). Therefore, a number of studies have been conducted to optimize solid-state cultivation and submerged culture conditions in order to maximize production or bioactivity of metabolites from Flammulina spp. some of which have also investigated inhibitory effects of these metabolites.

Influential factors in solid-state cultivation

Substrate composition and condition

Formulation of a substrate and its supplementation may influence the content of metabolites in Enokitake. For example, nitrogen in raw ingredients of the cultivation substrate was shown to be a critical nutrient for the synthesis of proteins, nucleic acids bases, and polysaccharides (Hoa and Wang 2015). The investigation of substrate formulation has shown that Enokitake cultivated in cooked rice-based substrate produced several secondary metabolites, including six new cuparene sesquiterpenes, enokipodins E-J (1–6), two new sterpurane sesquiterpenes, sterpurols A (10) and B (11), and four known sesquiterpenes, 2,5-cuparadiene-1,4-dione (7), enokipodins B (8) and D (9), and sterpuric acid (12). Further bioactivity screening assay indicated that compounds 5–9 displayed weak antibacterial activity against Bacillus subtilis, while compounds 2, 3, and 5 showed weak antifungal activity against Aspergillus fumigatus. Compounds 6–9 showed moderate cytotoxicity against the human tumor cell lines (HepG2, MCF-7, SGC7901, and A549) and antioxidant activity in DPPH scavenging assay (Wang et al. 2012). In addition, moisture of substrates was shown to affect the production of polysaccharides in Enokitake grown in soybean curd residue (Shi et al. 2012). Similarly, protein content of fruiting bodies of Enokitake was shown to decline when the substrate moisture decreased to 45% (Leifa et al. 2001). Supplementation of substrates of Enokitake with various types of elicitors including chitosan, triacontanol, and 2,4-D have also been shown to influence production of protein and polysaccharides (Tuheng et al. 2017).

Developmental stages of mushrooms

It has been known that the stage of growth of mushrooms affects their bioactive substances, including protein content in fruiting bodies which directly associates with human nutrition and a variety of important biological activities (Lau et al. 2012). Thus, it is important to know which developmental stage is the best in terms of protein content and quality (amino acid composition). So far, very little is known about how developmental stages of Enokitake affect the protein content and quality in fruiting bodies of this mushroom. We recently studied changes in the protein profile of Flammulina spp. cultivated in 40% wheat straw + 40% sawdust + 18% wheat bran + 1% lime + 1% gypsum wheat bran + 1% lime + 1% gypsum. At 3–5 days after appearance of primordia, fruiting bodies of Enokitake produced high amounts of protein with a various range of high and low molecular weights (Hashemian et al. unpublished data). Similar results were also reported with Volvariella volvacea where the highest protein content and quality were noted in stage III−IV, while the lowest was observed in stage I of fruiting body development (Eguchi et al. 2015).

Environmental conditions

Content and quality of bioactive compounds of mushrooms might be affected by environmental conditions (Li et al. 2017). Light was shown to increase accumulation of phenolic pigments in the fruiting body of the winter mushroom so that they became brown 2 days after light exposure (Sakamoto et al. 2007). Besides, light exposure caused an increase in the activity of phenol oxidases (Harith et al. 2014). Another research group reported the effect of light-emitting diodes on the cultivation and the amounts of bioactive components in the winter mushroom. The crude fat content, crude fiber content, polysaccharide content, and ergosterol content were found at concentrations of 2.9 g/100 g, 7.9 g/100 g, 3.9 g/100 g, and 1.4 mg/g, respectively. The winter mushroom cultivated under different lighting conditions showed different profiles for proximate composition, nutritional compounds, and principal fatty acids (Tsai et al. 2017). The inductive impact of light on expression of pileus-specific hydrophobin-like protein has also been evidenced. This protein is a cell wall-associated protein with serine and threonine-rich domains in F. filiformis pileus (Sakamoto et al. 2007). Moreover, some proteins express specifically during fruiting body development of F. filiformis after light irradiation (Sakamoto 2010).

Inoculum size was also found to be an influential factor for the production of bioactive compounds. The highest polysaccharide production in solid-state fermentation of Enokitake was earned with an inoculum size of 9.69% (Shi et al. 2012). Further investigations are warranted to evaluate the effect of other environmental conditions such as humidity on production and content of bioactive metabolites in Enokitake.

Influential factors in submerged cultures

Nutrient content

Production of polysaccharides, terpenoids, triterpenoids, diterpenoids, sesquiterpenoids, and ergosterol produced in submerged liquid fermentation heavily depends on growth parameters, growth timing, and their nutritional requirements (Kim et al. 2002b; Gregori et al. 2007; Lin et al. 2008). The influence of nutritional and environmental factors on the production of Enokipodins A, B, C, and D (α-cuparene-type sesquiterpenoids that are antimicrobial metabolites) in the stationary stage of Flammulina sp. mycelia was investigated. The findings revealed that the greater antimicrobial metabolite production occurred in complete Pontecorvo’s culture medium (De Melo et al. 2009). Another study revealed that Enokipodins C and D had antimicrobial activity against Cladosporium herbarum, B. subtilis, and Staphylococcus aureus (Ishikawa et al. 2001).

Nutritional requirements for the production of angiotensin-converting enzyme (which has inhibitory activities) from Enokitake were studied. Sucrose, ammonium acetate, and glutamic acid were chosen for the maximum production of this inhibitory substance. The optimal medium composition included sucrose (20 g/L), ammonium acetate (5 g/L), glutamic acid (2 g/L), KH2PO4 (3 g/L), MgSO4·7H2O (0.8 g/L), and yeast extract (0.5 g/L). Under the optimal culture conditions, the inhibitory effect of angiotensin-converting enzyme was more than 80% (Kim et al. 2002a).

The biosynthetic potential of Enokitake along with some other mushroom species for production of intracellular (endo-polysaccharides and lipids) and extracellular (exo-polysaccharides) compounds was examined in liquid media with glucose as substrate. Polysaccharides and lipids synthesized at the early growth stages were subjected to degradation as the fermentation proceeded. Mycelial lipids of all strains were highly unsaturated, dominated by linoleic acid, whereas glucose was the main building block of endo-polysaccharides (Diamantopoulou et al. 2014).

Another report of optimization of submerged culture conditions of Flammulina spp. indicated that Sorghum bicolor biomass medium increased specific bioactive compounds (Fidler et al. 2015b). In addition, a high production of anti-complementary exo-polysaccharides (with immunomodulating and antitumor effects) in the submerged culture of Flammulina spp. was obtained in the medium with galactose (15 g/L), sodium nitrate (5 g/L), glutamic acid (3 g/L), KH2PO4 (2.5 g/L), and MgSO4·7H2O (0.6 g/L) (Shin et al. 2007).

Alcoholic extracts obtained from Enokitake mycelium biomass cultured on wine yeast liquid medium had higher antibacterial and antioxidant activities as compared to those obtained in sorghum deposit medium. These activities were contributed to phenolic compounds in the extracts (Fidler et al. 2015a). Similar findings were obtained with other mushroom species such as Polyporus tricholoma, where the type of media affected antibacterial activity of sesquiterpenes in the mycelium extracts (Vieira et al. 2008).

Time

Time of collection has been shown to influence content and activity of metabolites in submerged cultures of other mushrooms (Shittu et al. 2005). Specifically for Enokitake, it was shown that the anti-complementary activity of exo-polysaccharides increased sharply after 4 days of cultivation. The content of exo-polysaccharides was at the highest level at 5–6 days of cultivation in submerged culture of Enokitake (Shin et al. 2007).

pH

Production of crude polysaccharide in Enokitake was shown to reach 20.8 mg/g dry weight in pH 6. By contrast, increasing pH up to 8 might cause a significant decrease in polysaccharide content as well as mycelium growth rate (Hassan et al. 2012b). In addition, optimal production of anti-complementary exo-polysaccharides in the submerged culture of Flammulina spp. was achieved at pH = 3.5–5.5 and 25 °C (Shin et al. 2007). Similar to Enokitake, a decrease in pH of the medium caused an increase in extracellular and intracellular polysaccharide production in the Ganoderma species (Fang and Zhong 2002) and antibacterial activity of sesquiterpenes in P. tricholoma (Vieira et al. 2008).

Minerals

An increase in levels of magnesium and phosphate up to 3 g/L in submerged cultures of Enokitake resulted in enhancing endo-polysaccharide content (Osman et al. 2014). Phosphate is also shown to influence the production yield of endo-polysaccharides in Enokitake media, where high concentrations (more than 3 g/L) negatively affected polysaccharide accumulation.

Temperature

Production of exo-polysaccharides and endo-polysaccharides by Enokitake cultivated in liquid culture was also shown to be changed by temperature (Osman et al. 2014). An increase in temperature from 25 to 37 °C on the 15th day of Enokitake mycelia culture in malt extract and peptone broth optimized antimicrobial metabolite production (De Melo et al. 2009).

C/N ratio

In submerged cultivation of Flammulina spp., the C/N ratio also affects fatty acid composition and the relationship between saturated and unsaturated fatty acid. The best ratio of C/N was found to be 15 for both total free fatty acid synthesis and the optimal production of unsaturated fatty acid (Bespalova et al. 2002). In addition, mycelium growth and water-soluble endo-polysaccharides were affected by C/N ratio in the medium so that the maximum mycelium growth and endo-polysaccharide yield from F. filiformis were found in a C/N ratio of 10 (Osman et al. 2014).

Gas content

Aeration is known to positively influence mycelial growth and bioactive compound production in liquid media of mushrooms (Diamantopoulou et al. 2014). It was shown that when oxygen supply was limited, biosynthesis of pigments and citrinin was occurred as primary metabolites in Monascus spp. By contrast, citrinin was produced as a secondary metabolite and pigment formation decreased dramatically at high amounts of oxygen (Lin et al. 2008). With regard to Enokitake, anti-complementary activity of exo-polysaccharides increased under aeration rate of 2 vvm, agitation speed of 150 rev/min, and working volume 3 L (Shin et al. 2007).

Elicitors

In addition to solid-state cultivation, elicitors may be also added into liquid culture for metabolite production. In any case, one should consider variables such as temperature, treatment time, time of action, time intervals for harvesting, and concentration of elicitor in order to measure amount of metabolites. Further HPLC analyses could also provide information on identification and quantitative determination of metabolites (Attaran Dowom et al. 2017)

Extraction condition

The research has shown that the optimal production of polysaccharides of Enokitake was achieved at the following conditions: a ratio of 50:1 for water to material, initial pH of 6.0, extracting temperature of 85 °C, and extracting time of 6 h. Antioxidative and anticancer properties of these polysaccharides have also been proved (Zhao et al. 2013). Another study revealed that water and methanol extracts of fresh and lyophilized fruiting bodies of Flammulina spp. had stronger antitumor effects than those from mycelia and broth (Younis et al. 2014).

Conclusion and future prospects

Commercialization or domestication of Enokitake is largely determined by both agronomic and environmental factors. However, it appears that the substrate composition has the most immediate impact on all the growth characteristic of Enokitake in solid-state cultivation. Although remarkable achievements have been made in the field of substrate composition of Flammulina spp., formulation of an economic and locally available substrate to reduce production costs should be sought prior to mushroom cultivation (Yang et al. 2016). The basic lignocellulosic substrates may include sawdust, wheat straw, paddy straw, and ramie stalk, while supplementations such as wheat bran, rice bran, and sugarcane bagasse could improve biological efficiency.

Further studies are needed to gain an in-depth understanding of biochemical changes and the fate of lignocellulosic and non-lignocellulosic macromolecules before and after fruiting of Enokitake. Such studies would be useful to evaluate bioconversion of agro wastes by the mushroom, which could have implications for efficient formulation of substrate and the use of spent substrates for animal feeding, recovery of enzymes, and other industrial uses (Phan and Sabaratnam 2012). Despite wide industrial applications having been reported for spent substrates from Pleurotus spp., L. edodes, and A. bisporus (Phan and Sabaratnam 2012), very limited studies have addressed applications of spent substrates of Enokitake, including recovering xylanase (Ko et al. 2005) and organic fertilizers (Rugolo et al. 2016).

Considerable progress has also been made regarding myco-chemistry and biological activities of various compounds isolated from Enokitake, proving this mushroom as a potential in pharmaceutical drug development (Tang et al. 2016). In addition, various studies have evaluated environmental and agronomic factors affecting the content and yield of bioactive compounds of Enokitake in both solid-state cultivation and submerged cultures. Particularly, submerged cultures of Enokitake could be promising tools to efficiently obtain mycelial biomass and pharmaceutically valuable metabolites in a short period of time. Yet, further research is warranted to gain deeper insight into how environmental and agronomic factors may change capability and efficacy of bioactive metabolites of Enokitake.