In this study, we optimized culture conditions and methods for producing ergothioneine yields by the basidiomycete, Schizophyllum commune (NBRC 30749). Mycelium cultivation was carried out by shaking at 25℃ for 42 days in bonito soup stock. In addition, a malt culture medium made with bonito soup stock that is commonly used for mycelial culture was used as a control. In addition to a multi-bead shocker that is conventionally used to disrupt mycelia, we incorporated a pestle and a mortar step, which was particularly effective in increasing ergothioneine yields. The supernatant obtained by centrifugation was used as a cell-free extract, and the ergothioneine content was measured by HPLC. The optimal conditions for ergothioneine production comprised rotational shaking of the basidiomycete culture for 42 days in a culture medium with a glucose concentration of 3% and an amino acid and a yeast extract added to bonito soup stock. Further improvements in ergothioneine yields are expected through modification of culture conditions, additives and culture period.

The authors previously reported in research using the fermentation capabilities of basidiomycetes that the split gill mushroom (Schizophyllum commune) grew rapidly on various food materials and could produce ergothioneine.

The production pathway of ergothioneine has been reported to be produced by basidiomycetes with cysteine and histidine as its backbone. It was believed that investigating the base substrate and the components to be added with a focus on the ergothioneine production pathway could increase the amount of ergothioneine produced. Bonito stock, which is rich in histidine, was used in the base substrate. With the ergothioneine production pathway revealed, it is suggested that PLP (pyridoxal phosphate) acts as a coenzyme promoting ergothioneine production. Therefore, cultivation methods for producing high amounts of ergothioneine were considered, focusing on yeast extract containing PLP.

In this research, split gill mushroom Schizophyllum commune was used. The S. commune strain NBRC30749 was provided by the Biotechnology Center of the National Institute of Technology and Evaluation. A total of 19.5 g of Nissui potato dextrose agar medium was dissolved in 500 mL of tap water, autoclaved for sterilisation, and poured into petri dishes to solidify. The S. commune spawn was then inoculated on the agar medium and cultivated statically.

2. Substrate preparation and cultivation method
The bonito stock substrate was prepared by adding 400g of bonito (Yamaki Co., Ltd.) to 4L of boiling tap water, simmering for 30 minutes, then cooling before filtering with a cloth. The filtered liquid was the bonito stock substrate, with 100mL added to 200mL flasks.

The malt substrate was prepared by dissolving 2g of malt extract in 100mL of tap water. A 25% glucose solution was created and the final glucose concentration in the bonito stock and malt substrates was adjusted to 1%, 2%, or 3%. Solutions of cysteine (500 mM), histidine (500 mM) and methionine (250 mM) were prepared, and each amino acid was added and adjusted so the final concentration was 5mM of each per 5 mL of bonito stock substrate and malt substrate. One gram of yeast extract was added to achieve a final concentration of 1%. All substrates were sterilised by autoclave after the glucose and yeast extracts were added. The amino acids were sterilised using a 0.2 μm membrane filter in a clean bench and added to each substrate that had been autoclaved (at 121°C for 20 minutes). There were three types of substrate conditions using bonito stock and malt as the base substrates: those with glucose added, those with glucose and amino acids added, and those with glucose, amino acids, and yeast extract added. The glucose concentrations were set at 1%, 2% and 3%. The S. commune mycelium grown on potato dextrose agar medium was cut into 5mm agar squares with 5 pieces added to each liquid substrate and then cultivated by rotary shaking at 25°C for 42 days.

4. Ergothioneine measurements
Ergothioneine was measured using high-performance liquid chromatography (HPLC). The column used was CAPCELLPAKC18MG II (4.6 mm ID × 250 mm, Shiseido), the pump was DP-8020 (Tosoh), and the detector was UV-8020 (Tosoh). A sample volume of 50 μL was injected. Eluent A was a 10mM citric acid buffer (pH 6.2) and a 75mM sodium perchlorate solution, and eluent B was 50% acetonitrile (v/v). The gradient conditions for solution B was 5% at sample injection, with the gradient starting simultaneously with the analysis. After 15 minutes, solution B was 8.8% then 100%. After 20.1 minutes, it returned to 5%. The column temperature was set to 40°C and the detection wavelength to 260 nm.

Results and discussion
1. Mycelial morphology in each substrate
Figure 1 shows the morphology of the mycelium cultivated in each substrate condition. Characteristic differences in the growth and appearances of the mycelium were observed in the different combinations of glucose, amino acids and yeast extract dissolved in the bonito stock substrate and malt substrate. In the bonito stock substrates with glucose only (Figure 1A) and glucose and amino acids (Figure 1B), 3-5 large mycelia and several smaller mycelia grew robustly, with short hyphae extending from their spherical surfaces. In contrast, in the substrate with glucose, amino acids, and yeast extract (Figure 1C), all mycelia were of similar size with smaller mycelia forming in the substrate with only glucose and substrate with glucose and amino acids, extending shorter hyphae. No morphological differences in mycelium due to differences in glucose concentration were observed in each of the different substrates.

Meanwhile, the mycelia in the malt substrates were morphologically different from those cultivated in the bonito stock substrates. Mycelium with a bumpy surface grew robustly in the glucose-only substrate (Figure 1D). The mycelia in the glucose and amino acid substrate (Figure 1E) showed limited growth, some with short hyphae and some with no bumps. In the substrate with glucose, amino acids and yeast extract (Figure 1F), mycelia grew robustly but with short hyphae.

Figure 2 shows the mycelial yield when cultivated in the substrates with different additives combinations. Of the bonito stock substrates, the one with with 3% glucose and amino acids (Figure 2A) had the highest mycelial yield with 8.89g. In the glucose-only substrate and the substrate with glucose and amino acids, the wet weight of the mycelium increased as the glucose concentrations increased, but increased glucose concentration in the substrate with glucose, amino acids and yeast extract resulted in little change in mycelial yield. Meanwhile, of the malt substrates, the highest mycelial yield of 12.31g was in the one with glucose, amino acids, and yeast extract (Figure 2B). A comparison of the mycelial yield between the bonito stock and malt substrates indicated that the malt substrates supported more mycelial growth when glucose and yeast extract were added. When mycelium was cultivated in the malt substrates, the yield was lower in the glucose and amino acid substrate than in the glucose-only substrate, suggesting that the addition of a certain amount of amino acids suppresses mycelial growth.

3. Ergothioneine production
Figure 3 shows the ergothioneine production per 1g of mycelium grown in substrates with different additives. The highest amount of ergothioneine at 1.6 mg/g was produced in the bonito stock substrate with 3% glucose, amino acids and yeast extract (Figure 3A). Despite variations in glucose concentration, a high amount of ergothioneine was produced when yeast extract was added, suggesting that adding yeast extract promotes ergothioneine production. Conversely, of the malt substrates, the one with 1% glucose, amino acids and yeast extract (Figure 3B) produced the highest ergothioneine amount of 0.25 mg/g. However, unlike the bonito stock substrate, the addition of yeast extract to the malt substrates with glucose concentrations of 2% and 3% did not increase ergothioneine yield more than that in the glucose-only substrate. Further, a comparison of the ergothioneine production between the highest producing substrates with glucose, amino acids and yeast extract in both bonito stock and malt substrates suggested that the production of ergothioneine was more effective in the bonito stock substrate, indicating that certain substances inherent in bonito stock may enhance ergothioneine production when yeast extract is added. Additionally, the reason for the high ergothioneine production in the substrates with yeast extract may be related to the biosynthetic pathway of ergothioneine. It has been reported that PLP is involved as a coenzyme in the production of ergothioneine from its precursor, starting with the binding of cysteine to histidine. Therefore, it is thought that PLP in the added yeast extract promoted the conversion of the precursor to ergothioneine. Moreover, bonito stock contains histidine (72.2 μmol/mL) and cysteine (0.53 μmol/mL), implying that bonito stock substrate, which is richer in the histidine and cysteine needed for ergothioneine biosynthesis than malt substrate, led to an increase in ergothioneine production. Methionine was added alongside cysteine and histidine, which are necessary for ergothioneine production in this study, as it was included in a patented method for producing ergothioneine.

4. Relationship between mycelial growth and ergothioneine
When cultivated in the malt substrate, S. commune had robust mycelial growth but low ergothioneine production. Conversely, the bonito stock substrate had higher amounts of histidine and cysteine, the backbone of ergothioneine biosynthesis, which is presumed to promote ergothioneine production more than the malt substrate. Furthermore, in the bonito stock substrates with glucose-only or glucose and amino acids, increased glucose concentrations did not result in greater ergothioneine amounts despite increased mycelial yield. Meanwhile, in the substrate with glucose, amino acids and yeast extract, higher glucose concentrations did not increase mycelial yield, but did increase ergothioneine production. This study suggested that the morphology of mycelium is also associated with ergothioneine production. Additionally, it is inferred that glucose and amino acids promote mycelium formation while yeast extract extract promotes ergothioneine production. Adding yeast extract may shift the metabolism from mycelial growth to ergothioneine production, underscoring the need for further investigation into substrates and cultivation period.

Fukuda E., Aoyanagi Y., Yamagishi K., Gasa S. and Chiji H. (2012). Tamogitake kara tanri shita erugochionein no kanetsu oyobi kaku pH ni okeru kōsan kasei e no eikyō (Research on the effects of heat and pH on the antioxidative activity of ergothioneine from Pleurotus cornucopiae). Bulletin of Fuji Women`s University 49 (II), 51-55.

Gasa S., Yamagishi K. and Toyama T. (2010). Tamogitake yurai erugochionein no teiryō to seiseihō no kakuritsu (An analysis and a purification of ergothioneine from Pleurotus cornucopiae var. citrinopileatus). Journal of Center for Medical Education, Sapporo Medical University 1, 67-70.

Keirinbo K. and Dogasaki T. (2007). Shokuyōtakechū no erugochionein ni kansuru kiso kenkyū (Foundational research on ergothioneine in edible mushrooms). Journal of Azabu University 15-16, 65-66.

Maekawa T., Kanja S., Ishimori Y., Ebara S and Watanabe T. (2007). Katsuobushi oyobi konbu no chūshutsueki ni okeru aminosan sosei no sanchi ni yoru hikaku kentō (A comparative study on amino acid composition in bouillons extracted from bonitos and seaweeds). Trace Nutrients Research 24, 191-197.

Matsui T., Kagemori T., Fukuda S. and Osugi M (2009). Characteristics of wine produced by mushroom fermentation using Schizophyiiom commune NBRC4929. Mushroom Science and Bio-technology 17, 107-111.

Matsui T. and Osugi M. (2006). Kinoko o mochiita miso no seizō (The use of mushrooms in miso production). Journal of the Brewing Society of Japan 101, 833-838.

Melville, D. B., Horner, W. H. and Otken, C. C. (1955). Studies on the origin of ergothioneine in animals. J Biol Chem 213, 61-68.

Patent Report (2012). Erugochionein no seizō hōhō (Method for producing ergothioneine). Patent held by Shimizu Sachiko.

Sameshima Y., Sawa N., Tabata M. and Matsui T. (2017). Katsuodashi o riyō shita erugochionein no seisan (Production of ergothioneine using bonito soup stock). Japanese Society of Mushroom Science and Biotechnology 25(3), 100-103.

Seebeck, F. P. (2010). In Vitro Reconstitution of Mycobacterial Ergothioneine Biosynthesis, J Am Chem Soc 132, 6632-6633.

Tabata M., Hayashi T. and Matsui T. (2009). Tanshikin yurai gyōnyū kōso no kassei sokutei e no koaguromētā no tekiyō (Coagulometer assay for determination of milk clotting activity in mushroom enzymes). Japanese Society of Mushroom Science and Biotechnology 17(4), 151-154.

This English translation was done for the Japanese Exotic Mushroom Journal, and any errors in terminology, transcription, or in the presentation of the data are the responsibility of the Japanese Exotic Mushroom Journal.

About the Translator: Naomi Roulston has over a decade of experience as a Japanese to English translator and specialises in food, mushrooms, nutrition and lifestyle medicine, and travel and tourism. She has a postgraduate degree in translation, is NAATI-certified, and holds a Kinoko Meister certificate from the Japan Kinoko (mushroom) Meister Association.