JP5731082B2 - An ergothioneine production method and production apparatus using mushroom basidiomycetes using a submerged culture method. - Google Patents
An ergothioneine production method and production apparatus using mushroom basidiomycetes using a submerged culture method. Download PDFInfo
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Description
本発明は、担子菌又は子嚢菌を用いた有用物質の製造方法に関し、特に液体培地中で深部培養した担子菌又は子嚢菌の菌糸体をろ別した後、菌糸体を水分調整して菌糸塊を生成し、菌糸塊を生存環境下に保管して当該菌糸塊に対し低温静止等のストレスを印加することにより、菌糸塊に有用物質を増産させて有用物質を安価に製造する方法及び装置に関する。本発明において、有用物質とはエルゴチオネイン等の抗酸化物質、ペニシリン等の抗生物質、放射線による造血障害抑制物質等を意味し、担子菌とはキノコの菌糸体を意味し、キノコとしてはエノキタケ、タモギタケ、エリンギ、アギタケ、トキイロヒラタケ、霊芝等が挙げられる。子嚢菌の菌糸体とはトリフ、アオカビ等の菌糸体を意味する。 The present invention relates to a method for producing a useful substance using basidiomycetes or ascomycetes, and in particular, after filtering off mycelia of basidiomycetes or ascomycetes that have been submerged in a liquid medium, the mycelium is adjusted to moisture and mycelia And producing a useful substance at low cost by increasing the production of a useful substance in the mycelium by applying a stress such as low-temperature resting to the mycelium and storing the mycelium in a living environment. . In the present invention, a useful substance means an antioxidant substance such as ergothionein, an antibiotic substance such as penicillin, a substance that suppresses hematopoietic damage due to radiation, a basidiomycete means a mycelium of mushrooms, and mushrooms include enokitake mushrooms and tamagotake mushrooms. , Eringi, agitake, tokihirohiratake, ganoderma and the like. The mycelium of ascomycetes means mycelium such as trif and blue mold.
エルゴチオネインの製造方法に関し、特許文献1はキノコの菌糸体を培養して得られた菌糸体よりエルゴチオネインを抽出して精製しエルゴチオネインを製造する方法を開示している。特許文献2は、タモギタケの菌子体をメチオニンを含む培地内で培養し、生成した菌子体からエルゴチオネインを抽出して製造する方法を開示するものであり、ここでは、キノコを、液体培地を用いて培養した後、菌子体をろ別し、凍結乾燥後、粉砕して目的とする有用物質を抽出することにより、有用物質であるエルゴチオネインをろ別した菌糸体を用いて製造する方法を開示している。これらの先行文献では、液体培地を用いてキノコを培養した後、菌子体を取り出し、取り出した菌子体を乾燥してエルゴチオネインを抽出するものである。なお、エルゴチオネインは抗酸化作用や抗老化作用を有することが知られており、化粧品、食品、医薬品、飼料などに利用されている。 Regarding the method for producing ergothioneine, Patent Document 1 discloses a method for producing ergothioneine by extracting and purifying ergothioneine from the mycelium obtained by culturing mycelia of mushrooms. Patent Document 2 discloses a method for cultivating mycelia of Tamogitake in a medium containing methionine and extracting and producing ergothioneine from the produced mycelium, and here, mushrooms are produced using a liquid medium. After culturing, the mycelium is filtered, freeze-dried, pulverized to extract the target useful substance, and a method for producing the mycelium by filtering off the useful substance ergothioneine is disclosed. Yes. In these prior documents, mushrooms are cultured using a liquid medium, and then the mycelium is taken out, and the taken out mycelia are dried to extract ergothioneine. Ergothioneine is known to have an antioxidant effect and an anti-aging effect, and is used in cosmetics, foods, pharmaceuticals, feeds and the like.
このような従来技術においては、液体培養した菌子体をろ別した時点で、培養液を失ったことにより深部培養状態から離脱し、菌糸体による有用物質(エルゴチオネイン)の生産活動は停止する。なお、菌子体をろ別し凍結乾燥した時点では菌子体は死滅する。そのため、有用物質であるエルゴチオネインの菌糸体による生産は液体培養の期間に限定されていた。一方、液体培養には多くのコストが必要となるために目的とする有用物資の製造費用が高価になるという問題点がある。そこで、菌糸体の生産による有用物質を安価に提供する方法が望まれている。 In such a conventional technique, when the mycelia cultured in liquid culture are filtered off, the culture medium is lost, so that it is removed from the deep culture state, and the production activity of useful substances (ergothioneine) by the mycelium is stopped. Note that the mycelium is killed when the mycelium is filtered and lyophilized. Therefore, the production of ergothioneine, a useful substance, by mycelium has been limited to the period of liquid culture. On the other hand, the liquid culture requires a lot of cost, and thus there is a problem that the production cost of the intended useful material becomes expensive. Therefore, a method of providing useful substances at low cost by producing mycelium is desired.
本発明の課題は、上述の問題を解決して安価な菌糸体由来の有用物質を提供することにあり、液体培地中で深部培養した担子菌又は子嚢菌の菌糸体をろ別した後、菌糸体を水分調整して菌糸塊を生成し、菌糸塊を生存環境下に保管して当該菌糸塊に対し低温静止等のストレスを印加することにより有用物質を増産させて有用物質を製造する方法及び製造装置を提供することである。 An object of the present invention is to provide an inexpensive mycelium-derived useful substance by solving the above-mentioned problems, and after filtering the mycelium of basidiomycetes or ascomycetes cultured deeply in a liquid medium, the mycelium A method for producing a useful substance by increasing the production of a useful substance by adjusting moisture in the body to produce a mycelium, storing the mycelium in a living environment, and applying a stress such as low-temperature rest to the mycelium It is to provide a manufacturing apparatus.
本発明において、有用物質はエルゴチオネイン等の抗酸化物質、ペニシリン等の抗生物質、放射線による造血障害抑制物質等であり、担子菌の菌糸体はキノコ菌の菌糸体であり、キノコとしてはエノキタケ、タモギタケ、霊芝、冬虫夏草等が挙げられる。エルゴチオネインの生産に用いるキノコ菌として、エノキタケ菌の他にタモギタケ菌、アギタケ菌、トキイロヒラタケ菌、エリンギ菌等を挙げることができる。子嚢菌の菌糸体はトリフ菌、アオカビ菌等の菌糸体である。 In the present invention, useful substances are antioxidant substances such as ergothioneine, antibiotics such as penicillin, substances that suppress hematopoietic damage due to radiation, mycelia of basidiomycetes are mycelium of mushroom fungi, and mushrooms are enokitake mushroom and tamagotake mushroom. , Reishi, cordyceps and so on. Examples of the mushroom fungus used for the production of ergothionein include Tamogitake fungus, Agitake fungus, Tokihirohiratake fungus, Eringi fungus, etc. in addition to Enokitake fungus. The mycelium of the ascomycete is a mycelium such as Trifungus or Blue mold.
請求項1に記載の発明は、キノコの担子菌の菌糸体を液体培地中で深部培養し、生成した菌糸体からエルゴチオネインを抽出精製してエルゴチオネインを製造する方法であって、深部培養工程(ステップS1)において培養された菌糸体を深部培養状態から離脱させ、菌糸体と培養タンク内の液体培地とを含む培養液から水分を減じるためのろ過処理を実施し、液体培地が付着した菌糸体を分別するろ別工程(ステップS2)と、前記ろ別した菌糸体の菌糸塊を生成し、当該菌糸塊を気中環境下で生存可能にするために水分含有率を調整する菌糸体水分含有率調整工程(ステップS3)と、前記水分含有率が調整された菌糸塊を生存環境下に保管し、当該菌糸塊に対し静置刺激及び低温環境下における温度刺激または電気刺激印加後の静置刺激及び低温環境下における温度刺激を印加してエルゴチオネインが増量した菌糸塊を生成するストレス印加工程(ステップS4)と、からなる深部培養法を用いたキノコの担子菌によるエルゴチオネイン製造方法であることを特徴とする。 The invention according to claim 1 is a method for producing ergothioneine by deeply culturing mycelia of mushroom basidiomycetes in a liquid medium and extracting and purifying ergothioneine from the produced mycelium, wherein a submerged culture step (step) The mycelium cultured in S1) is removed from the deep culture state, a filtration treatment is performed to reduce water from the culture solution containing the mycelium and the liquid medium in the culture tank, and the mycelium to which the liquid medium is attached is removed. Filtration step (step S2) for sorting, and producing a mycelium of the filtered mycelium, and adjusting the water content to make the mycelium viable in the air environment adjustment process (step S3), and the mycelia mass water content has been adjusted and stored under survival environment, static置刺after thermal stimulation or electrical stimulation applied under static置刺intense and low temperature environment to the hypha masses Wherein the ergothioneine by applying a temperature stimulation is ergothioneine manufacturing method according basidiomycete mushroom using a stress application step of producing increased amounts of mycelia mass (step S4), and submerged culture method consisting and in a low temperature environment And
本発明においては、菌糸体をエノキタケ菌、タモギタケ菌、霊芝菌、冬虫夏草菌から選択されたいずれかの菌糸体とすることができ、本発明においては、菌糸体としてエリンギ菌、アギタケ菌、トキイロヒラタケ菌の菌糸体を利用することができる。また、子嚢菌であるトリフ菌の菌糸体を利用することもできる。 In the present invention, Flammulina fungus mycelium, Pleurotus cornucopiae fungus, Reishibakin, can be any mycelium selected from Cordyceps bacteria, in the present invention, eryngii bacteria, Agitake bacteria as mycelium, collected by The mycelium of the oyster mushroom can be used. Moreover, the mycelium of Trifoccus which is an ascomycete can also be utilized.
本発明においては、菌糸塊に対し与えるストレスを、静置刺激、温度刺激、電気刺激、ガスによる刺激から選択されたいずれかの刺激またはこれら2つ以上を組み合わせた刺激とすることができ、本発明においては、菌糸塊に対し与えるストレスを湿度の変化による刺激、pH環境の変化による刺激、薬品による刺激とすることができる。 In the present invention, the stress applied to the mycelial mass, static置刺stimulation, thermal stimulation, electrical stimulation, can be either stimulated or stimulated with a combination of these two or more selected from stimulation by gas, the In the present invention, the stress applied to the mycelium can be a stimulus by a change in humidity, a stimulus by a change in pH environment, or a stimulus by a drug.
請求項2に記載の発明は、内部に液体培地11を収容してキノコの担子菌12の菌糸体の深部培養を行う培養タンク13と、培養タンクの上部に取り付けられる気密ハッチ14と、培養タンク内の液体培地を攪拌する攪拌機構15と、液体培地に所定量の無菌エアを供給する無菌エア供給機構16と、液体培地のpHを所定の値に調整し維持するpH調整機構17と、液体培地の温度を所定の値に調整し維持する温度調整機構18と、無菌エア供給機構から供給されたエアを培養タンクから外部に排気する排気機構19と、を備え、菌糸体を深部培養する深部培養手段10と、前記深部培養手段により培養された菌糸体を培養タンクから取り出して深部培養状態から離脱させ、菌糸体と培養タンク内の液体培地とを含む培養液CFから水分を減じるためのろ過処理を実施し、当該ろ過処理された培養液をゾル又はゲル状の菌糸体HSとろ別後培養液AFCFとに分別するろ別手段20と、前記ろ別手段によりろ別された菌糸体の菌糸塊HSLを生成し、当該菌糸塊が含有する水分量を菌糸体の生存に最適な水分含有率に調整する菌糸体水分含有率調整手段30と、前記菌糸体水分含有率調整手段により水分含有率が調整された菌糸塊を生存環境下に保管し、当該菌糸塊に対し静置刺激及び低温環境下における温度刺激または電気刺激印加後の静置刺激及び低温環境下における温度刺激を与え、エルゴチオネインの生産を増加したエルゴチオネイン増量菌糸塊IIHSLを生成するストレス印加手段40と、を備えた深部培養法を用いたキノコの担子菌によるエルゴチオネイン製造装置であることを特徴とする。ここでゾル又はゲル状の菌糸体とは液体中に菌糸体が含まれている状態を意味する。
The invention described in claim 2 includes a culture tank 13 for accommodating a liquid medium 11 therein and performing deep culture of mycelia of mushroom basidiomycetes 12, an airtight hatch 14 attached to the upper part of the culture tank, and a culture tank. A stirring mechanism 15 that stirs the liquid medium, a sterile air supply mechanism 16 that supplies a predetermined amount of sterile air to the liquid medium, a pH adjustment mechanism 17 that adjusts and maintains the pH of the liquid medium to a predetermined value, and a liquid. A deep portion for deeply culturing mycelia, comprising a temperature adjustment mechanism 18 for adjusting and maintaining the temperature of the culture medium at a predetermined value and an exhaust mechanism 19 for exhausting air supplied from the sterile air supply mechanism to the outside from the culture tank The mycelium cultured by the culture means 10 and the deep culture means is taken out of the culture tank and removed from the deep culture state, and moisture is removed from the culture solution CF containing the mycelium and the liquid medium in the culture tank. A filtration means 20 for separating the filtered culture liquid into a sol or gel-like mycelium HS and a post-filtration culture liquid AFCF, and the filtration means. A mycelium water content adjusting means 30 for generating a mycelium mass HSL of the mycelium and adjusting the water content contained in the mycelium to an optimum water content for survival of the mycelium, and the mycelium water content adjustment Store the mycelium of which moisture content is adjusted by means in a living environment, and leave the stimulation against static stimulation and temperature stimulation under low-temperature environment or static stimulation after applying electrical stimulation to the mycelium and temperature stimulation under low-temperature environment. the given, the stress applying section 40 for generating a ergothioneine bulking hypha masses IIHSL with increased production of ergothioneine, ergothioneine manufacturing instrumentation by basidiomycete mushroom with deep culture method having a And characterized in that. Here, the sol or gel-like mycelium means a state in which the mycelium is contained in the liquid.
本発明においては請求項5に記載の発明において、菌糸体としてエノキタケ菌、タモギタケ菌、霊芝菌又は冬虫夏草菌の菌糸体を利用することができ、有用物質をエルゴチオネインとすることができる。また、菌糸塊に対し与えるストレスとして、静置刺激、温度刺激、電気刺激、ガスによる刺激から選択されるいずれか、またはこれらの刺激2つ以上を組み合わせた刺激を利用することができる。 In the present invention, the mycelium of Enokitake, Tamogitake, Ganoderma or Cordyceps fungus can be used as the mycelium, and the useful substance can be ergothioneine. In addition, as stress applied to the mycelium, any one selected from static stimulation, temperature stimulation, electrical stimulation, and gas stimulation, or a combination of two or more of these stimulations can be used.
本発明は、深部培養した菌糸体をろ別した後に、菌糸体の水分含有率を調整して菌糸塊を生成する工程と、菌糸塊を生存環境下に保管してストレスを印加する工程を設けたことにより、加えられたストレスに対応するために菌糸塊が有用物質を急速かつ大量に生産することになる。そのため、深部培養した菌糸体を利用して短期間で大量の有用物質を生産でき、有用物質の生産コストを大幅に削減できる。 The present invention provides a process for producing a mycelium by adjusting the moisture content of the mycelium after the submerged mycelium is filtered, and a process for applying stress by storing the mycelium in a living environment. As a result, the mycelium mass rapidly and in large quantities produces useful substances to cope with the applied stress. Therefore, a large amount of useful substances can be produced in a short period of time using mycelia cultured in the deep part, and the production cost of useful substances can be greatly reduced.
また、本発明においてはストレス印加工程を設けたことにより、深部培養工程で有用物質を生産した菌糸体と同じ菌糸体が追加して有用物質を生産するため、深部培養に使用される原料と同じ量の原料から数倍の有用物質を得ることができ、有用物質の原料費を大幅に削減できる。 In the present invention, since the stress application step is provided, the same mycelium as the mycelium that produced the useful substance in the deep culture step is added to produce the useful substance, so that it is the same as the raw material used for the deep culture. Useful materials can be obtained several times from the amount of raw materials, and the raw material costs of useful materials can be greatly reduced.
以下、添付図を参照しながら本発明に係る深部培養法を用いた担子菌又は子嚢菌による有用物質製造方法及び製造装置の実施の形態について説明する。図1は、本発明に係る菌糸体が生産する有用物質の製造手順を示すフロー図である。本発明においては、先ず、液体培地を用いて担子菌又は子嚢菌の菌糸体(以下、「菌糸体」という)を深部培養して菌糸体を増殖する工程(ステップS1)と、培養が完了した菌糸体と培養タンク内の液体培地を含む培養液をろ過して菌糸体を分別するろ別工程(ステップS2)とを実行した後、菌糸体の水分含有率を調整して菌糸塊を生成する菌糸体水分含有率調整工程(ステップS3)と、次いで菌糸塊を生存環境下に保管し、菌糸塊に対し各種ストレスを印加して当該菌糸塊に有用物質の生産を増加させ、有用物質が増量した菌糸塊を生成するストレス印加工程(ステップS4)を実行する。ろ別工程(ステップS2)でろ別された菌糸体は、培養液を失ったことにより深部培養状態から離脱し菌糸体の増殖が止まることにより、菌糸体の増加に伴う有用物質の生産活動は中止されることになる。 Hereinafter, embodiments of a useful substance production method and production apparatus using basidiomycetes or ascomycetes using a deep culture method according to the present invention will be described with reference to the accompanying drawings. FIG. 1 is a flow chart showing a procedure for producing a useful substance produced by a mycelium according to the present invention. In the present invention, first, a process of deeply culturing mycelia of basidiomycetes or ascomycetes (hereinafter referred to as “mycelia”) using a liquid medium to proliferate mycelium (step S1), and the culture is completed. After filtering the culture solution containing the mycelium and the liquid medium in the culture tank and separating the mycelium, the water content of the mycelium is adjusted to produce a mycelium mass Mycelium moisture content adjustment step (step S3), and then storing the mycelium in a living environment, applying various stresses to the mycelium to increase production of useful substances, increasing the useful substances The stress application process (step S4) which produces the mycelium which performed is performed. The mycelium filtered in the filtration step (step S2) is discontinued from the deep culture state due to the loss of the culture solution, and the mycelium stops growing. Will be.
図2は本発明に係る有用物質製造方法を実施するための製造装置の構成例並びに製造工程を説明する図である。本発明に係る有用物質製造装置(以下、「製造装置」という)は、深部培養手段10、ろ別手段20、菌糸体水分含有率調整手段30、ストレス印加手段40により構成されている。 FIG. 2 is a diagram for explaining a configuration example and a manufacturing process of a manufacturing apparatus for carrying out the useful substance manufacturing method according to the present invention. The useful substance manufacturing apparatus (hereinafter referred to as “manufacturing apparatus”) according to the present invention includes a deep culture means 10, a filtering means 20, a mycelium moisture content adjusting means 30, and a stress applying means 40.
深部培養手段10において、菌糸体を培養する培養タンク13は内部に液体培地11を収容して菌糸体12の深部培養を行う容器である。気密ハッチ14は培養タンク13の上部に取り付けられ、原料の投入やタンク内の清掃用として用いられる。攪拌機構15は培養タンクに接続され培養タンク内の液体培地を攪拌する。無菌エア供給機構16は培養タンクに接続され液体培地に所定量の無菌エアを供給する。pH調整機構17は培養タンクに接続され液体培地のpH値を所定の値に調整して維持する。温度調整機構18は培養タンクに接続され液体培地の温度を所定の値に調整して維持する。排気機構19は無菌エア供給機構から供給されたエアを培養タンクから外部に排気する。 In the deep culture means 10, a culture tank 13 for culturing mycelium is a container for accommodating a liquid medium 11 therein and performing deep culture of mycelium 12. The airtight hatch 14 is attached to the upper part of the culture tank 13 and used for charging raw materials and cleaning the tank. The stirring mechanism 15 is connected to the culture tank and stirs the liquid medium in the culture tank. The sterile air supply mechanism 16 is connected to the culture tank and supplies a predetermined amount of sterile air to the liquid medium. The pH adjusting mechanism 17 is connected to the culture tank and adjusts and maintains the pH value of the liquid medium to a predetermined value. The temperature adjustment mechanism 18 is connected to the culture tank and adjusts and maintains the temperature of the liquid medium to a predetermined value. The exhaust mechanism 19 exhausts the air supplied from the sterile air supply mechanism to the outside from the culture tank.
培養液CFは、深部培養手段10から送出されろ別手段20に供給されるものであり、培養タンク内部で深部培養された菌糸体並びに液体培地を含む液体である。ろ別手段20は、深部培養手段10の培養タンク13から培養液CFの供給を受け、培養液を菌糸体HSと、ろ別後培養液AFCFに無菌的に分離して菌糸体を深部培養から離脱させる。菌糸体HSはろ別手段20により培養液CFから分離して取り出されたゾル又はゲル状の菌糸体であり、ろ別後培養液AFCFは培養液CFから菌糸体HSを取り出した残りの培養液である。 The culture solution CF is delivered from the deep culture means 10 and supplied to the filter separation means 20, and is a liquid containing mycelium and a liquid medium that have been deeply cultured inside the culture tank. The filtering means 20 is supplied with the culture solution CF from the culture tank 13 of the deep culture means 10, and the culture solution is aseptically separated into the mycelium HS and the filtered culture solution AFCF to remove the mycelium from the deep culture. Let go. The mycelium HS is a sol or gel mycelium separated and removed from the culture solution CF by the filtering means 20, and the post-filtering culture solution AFCF is the remaining culture solution obtained by removing the mycelium HS from the culture solution CF. is there.
菌糸体水分含有率調整手段30は、ろ別手段20から菌糸体HSの供給を受け、当該菌糸体HSが含有する水分量を菌糸体の生存に最適な含有率に無菌的に調整し、菌糸塊HSLを生成する。菌糸塊HSLは菌糸体HSが含有する水分量が菌糸体の生存に最適な状態に調整され、深部培養から離脱した状態であって気中で生存している菌糸の塊である。ストレス印加手段40は、菌糸体水分含有率調整手段30から菌糸塊HSLの供給を受け、当該菌糸塊HSLに対し各種ストレスSTを与える。菌糸塊は、ストレス印加手段40によりストレスを受けることにより、目的とする有用物質の生産活動を活発化して有用物質を大幅に増加生産する。このようにストレス印加手段40は有用物質が増加された有用物質増量菌糸塊IIHSLを生成する。 The mycelium moisture content adjusting means 30 receives the mycelium HS supplied from the filtering means 20 and aseptically adjusts the water content contained in the mycelium HS to the optimum content for survival of the mycelium. Generate a mass HSL. The mycelium lump HSL is a mycelium lump that has been adjusted to an optimal state for the survival of the mycelium and the mycelium HS has been detached from the submerged culture and is alive in the air. The stress applying unit 40 receives the mycelium lump HSL from the mycelium moisture content adjusting unit 30 and applies various stresses ST to the mycelium lump HSL. When the mycelium is subjected to stress by the stress applying means 40, the production activity of the intended useful substance is activated and the useful substance is greatly increased. Thus, the stress applying means 40 generates the useful substance-increasing mycelium IIHSL in which the useful substance is increased.
次に、図2を参照しながら本発明に係る有用物質の製造手順について詳細に説明する。先ず、気密ハッチ14を開いて液体培地11を培養タンク13内に導入する。温度制御機構18がタンク内を121℃周辺の高温に維持して内部の液体培地を滅菌する。滅菌が終了すると温度制御機構18は培養タンクを冷却して内部の液体培地を菌糸の接種に適した温度、5℃〜35℃程度に調整して当該温度を維持する。 Next, the production procedure of the useful substance according to the present invention will be described in detail with reference to FIG. First, the airtight hatch 14 is opened, and the liquid medium 11 is introduced into the culture tank 13. The temperature control mechanism 18 maintains the inside of the tank at a high temperature around 121 ° C. to sterilize the liquid medium inside. When the sterilization is completed, the temperature control mechanism 18 cools the culture tank and adjusts the internal liquid medium to a temperature suitable for inoculation of mycelia to about 5 ° C. to 35 ° C. to maintain the temperature.
そして、気密ハッチ14から担子菌又は子嚢菌を培養タンク内の液体培地11に接種する。液体培地攪拌機構15は培養タンク内の液体培地を攪拌する。無菌エア供給機構16は液体培地に気泡の形で空気を導入し深部培養を行う。培養タンク13に導入された空気は排気機構19により外部に排出される。pH調整機構17は深部培養期間中の液体培地のpH値をpH3〜pH9程度に調整して当該pH値を維持する。これにより深部培養工程(ステップS1)が実行されることになる。深部培養が終了すると、液体培地内で担子菌又は子嚢菌の菌糸体が伸長して培養液CFが得られる。 Then, basidiomycetes or ascomycetes are inoculated from the airtight hatch 14 into the liquid medium 11 in the culture tank. The liquid medium stirring mechanism 15 stirs the liquid medium in the culture tank. The sterile air supply mechanism 16 introduces air into the liquid medium in the form of bubbles and performs deep culture. The air introduced into the culture tank 13 is discharged to the outside by the exhaust mechanism 19. The pH adjusting mechanism 17 adjusts the pH value of the liquid medium during the submerged culture period to about pH 3 to pH 9, and maintains the pH value. Thereby, the deep culture process (step S1) is executed. When the deep culture is completed, the mycelium of basidiomycete or ascomycete is elongated in the liquid medium to obtain the culture solution CF.
培養液CFをろ別手段20に供給する。ろ別工程(ステップS2)において、ろ別手段20は無菌環境、例えば外気閉鎖環境等の下でフィルター濾過、遠心濾過、真空濾過又はサイクロン濾過を用いて、培養液CFを菌糸体とろ別後培養液とにろ別分離し、ゾル又はゲル状の菌糸体HSを生成する。ろ別された菌糸体HSは、培養液を失ったことにより深部培養状態から離脱し菌糸体の増殖活動を中止する。 The culture solution CF is supplied to the filtration means 20. In the filtration process (step S2), the filtration means 20 uses a filter filtration, a centrifugal filtration, a vacuum filtration or a cyclone filtration in an aseptic environment, for example, an outside air closed environment, and the culture solution CF is separated from the mycelium and cultured. It is separated from the liquid by filtration to produce sol or gel mycelium HS. The mycelium HS that has been filtered out is disengaged from the deep culture state due to the loss of the culture solution and stops the mycelial growth activity.
ろ別手段20は菌糸体HSを菌糸体水分含有率調整手段30に供給する。菌糸体水分含有率調整工程(ステップS3)において、菌糸体水分含有率調整手段30は、無菌環境の下で圧搾、乾燥、遠心脱水又は自重による水分のドリップにより、菌糸体HSの水分含有率を菌糸が空気中で生存するための最適な値、75%〜99%程度に調整して菌糸塊HSLを生成する。菌糸塊HSLは、生存に最適な水分含有率を有し、生存に最適な温度5℃〜35℃、湿度50%〜100%の空気環境下に置かれるため死滅せずに目的とする有用物質の生産活動を継続している。 The filtering unit 20 supplies the mycelium HS to the mycelium moisture content adjusting unit 30. In the mycelium moisture content adjusting step (step S3), the mycelium moisture content adjusting means 30 adjusts the moisture content of the mycelium HS by drip of moisture by pressing, drying, centrifugal dehydration or dead weight under an aseptic environment. The mycelium HSL is generated by adjusting the hyphae to an optimum value for survival in the air, about 75% to 99%. The mycelium lump HSL has a moisture content that is optimal for survival, and it is placed in an air environment with a temperature of 5 ° C to 35 ° C and a humidity of 50% to 100%, which is optimal for survival. Has continued production activities.
菌糸体水分含有率調整手段30は生存している菌糸塊HSLをストレス印加手段40に供給する。ストレス印加工程(ステップS4)において、ストレス印加手段40は菌糸塊HSLに対し以下のような各種のストレス(刺激)STを加える。菌糸塊HSLを6℃前後の低温又は30℃前後の高温の環境を維持して、あるいは低温と高温を変化させる環境下に、2〜20日間程度静置保管する(静置刺激、温度刺激)。菌糸塊HSLに対し直流電流による火花放電を2〜10秒間程度印加する等の電気刺激を与える。菌糸塊HSLに対し、空気環境に炭酸ガスやオゾンガスを所定濃度で一定時間加えてガスによる刺激を与える。また、菌糸塊HSLを60%前後の低湿又は90%前後の高湿の環境を維持して、あるいは低湿と高湿を変化させる環境下に、2〜20日間程度静置保管する(湿度刺激)。菌糸塊HSLをpH5前後又はpH8前後の環境を維持して、あるいはpH値を変化させる環境下に、2〜20日間程度静置保管する(pH環境の変化による刺激)。菌糸塊HSLに対し滅菌水で所定濃度に希釈した硫酸や苛性ソーダ溶液を噴霧して、2〜20日間程度静置保管する(薬品による刺激)。なお、本発明においては、上記ストレスから選ばれるいずれか一つのストレスを採用することができるが、これらのストレス2つ以上を組み合わせたものを採用することができる。菌糸塊HSLはストレスSTを受けて、菌糸体がストレスの印加による環境変化に対応するために有用物質の生産活動を活発化する。これにより有用物質が増加された有用物質増量菌糸塊IIHSLが生成される。 The mycelium moisture content adjusting means 30 supplies the surviving mycelial mass HSL to the stress applying means 40. In the stress applying step (step S4), the stress applying means 40 applies various stresses (stimulations) ST as described below to the mycelium lump HSL. The mycelium lump HSL is stored for about 2 to 20 days in a low temperature around 6 ° C or a high temperature around 30 ° C, or in an environment where the low and high temperatures are changed (stationary stimulation, temperature stimulation). . An electrical stimulus such as applying a spark discharge by direct current to the mycelium lump HSL for about 2 to 10 seconds is given. The mycelium HSL is stimulated by gas by adding carbon dioxide gas or ozone gas to the air environment at a predetermined concentration for a certain period of time. In addition, the mycelium lump HSL is kept stationary for about 2 to 20 days in a low humidity of about 60% or a high humidity of about 90%, or in an environment where low and high humidity are changed (humidity stimulation). . The mycelium HSL is stored for about 2 to 20 days in an environment where the pH value is maintained around 5 or around 8 or the pH value is changed (stimulation due to a change in pH environment). The mycelium HSL is sprayed with sulfuric acid or caustic soda solution diluted to a predetermined concentration with sterilized water, and left to stand for about 2 to 20 days (stimulation with chemicals). In the present invention, any one stress selected from the above stresses can be employed, but a combination of two or more of these stresses can be employed. The mycelium HSL receives the stress ST and activates the production activity of useful substances in order for the mycelium to cope with the environmental change due to the application of stress. As a result, useful substance-increasing mycelium IIHSL with increased useful substance is produced.
目的とする有用物質は有用物質増量菌糸塊IIHSLから抽出し、得られた抽出液を遠心分離、結晶化、限外ろ過、エバポレーター、活性炭処理等の公知の方法により精製することができる。なお、培養液CF中に例えばペニシリン等のような有用物質が含有される場合は、ろ別後培養液AFCF及び有用物質増量菌糸塊IIHSLの抽出液から有用物質を公知の方法により精製することができる。本発明によれば、深部培養において生産されて得られる有用物質に加えて、ストレスの印加により有用物質が大幅に増加して生産された有用物質を得ることができる。 The intended useful substance can be extracted from the useful substance-enriched mycelium IIHSL, and the resulting extract can be purified by known methods such as centrifugation, crystallization, ultrafiltration, evaporator, activated carbon treatment and the like. When a useful substance such as penicillin is contained in the culture broth CF, the useful substance can be purified from the extract of the culture broth AFCF after filtration and the hyphae IIHSL after increasing the useful substance by a known method. it can. According to the present invention, in addition to the useful substance produced and obtained in the submerged culture, the useful substance produced by greatly increasing the useful substance by applying stress can be obtained.
上述したように深部培養し、菌糸体を増殖する工程(ステップS1)において菌糸体により生産された有用物質に加えて、培養液から菌糸体をろ別(ステップS2)した後、さらに菌糸体の水分含有率を調整して菌糸塊を生成する工程(ステップS3)並びに菌糸塊を生存環境下に保管してストレスを印加する工程(ステップS4)を実行することにより、菌糸体は、培養液の中での良好な深部培養環境から気中環境に置かれることによって深部培養状態から離脱し、周囲に栄養分が乏しい厳しい環境の下で加えられるストレスに対応するために有用物質を急速に大量に生産すると考えられる。 In addition to the useful substance produced by the mycelium in the step of culturing deeply and growing the mycelium as described above (step S1), the mycelium is filtered from the culture solution (step S2), By executing the step of adjusting the moisture content to generate a mycelial mass (step S3) and the step of storing the mycelial mass in a living environment and applying stress (step S4), the mycelium is obtained from the culture solution. Rapidly produce a large amount of useful substances to cope with the stress applied under severe conditions where surrounding nutrients are scarce by leaving the deep culture state from the favorable deep culture environment in the atmosphere I think that.
本発明に係る製造装置を使用して、キノコ菌糸としてエノキタケ菌糸を用いて以下のようにエルゴチオネインの製造を実施した。一次培養として、エノキタケ菌糸をポテトデキストロース寒天(PDA)培地で平板培養して元種菌を得た。二次培養として、一次培養で得られた元種菌から平板地ごとキノコ菌そうを、コルクボーラーで直径5mmに抜き取り、三角フラスコ内で滅菌、放冷された200ml(ミリリットル)のMYPG培地(麦芽抽出物5.76g/l(リットル)、酵母抽出物3.84g/l、グルコース3.84g/l、豚膵消化カゼインペプトン3.84g/l)等に2片接種し、1週間振とう培養して液体種菌を得た。 Using the production apparatus according to the present invention, ergothioneine was produced as follows using enokitake mycelia as mushroom mycelia. As primary culture, enokitake mushroom mycelia were plated on potato dextrose agar (PDA) medium to obtain the original seed fungus. As a secondary culture, the mushroom fungus with a flat plate was removed from the original inoculum obtained in the primary culture to a diameter of 5 mm with a cork borer, sterilized and cooled in an Erlenmeyer flask, and 200 ml (milliliter) of MYPG medium (malt extraction) 2.76 g / l (liter), yeast extract 3.84 g / l, glucose 3.84 g / l, porcine pancreatic digested casein peptone 3.84 g / l), etc. Liquid inoculum was obtained.
水に30g/lの大豆粉を加えて30分間抽出した抽出液に、砂糖20g/lと、硫酸マグネシウム七水和物0.5g/lと、燐酸2水素カリウム0.5g/lと、シリコン消泡剤0.5g/lを加えた培養液を、気密ハッチから培養タンクに充填した。培養タンク内部の培養液を121℃の温度で20分程度滅菌した後に放冷し培養液の温度を20℃程度に維持した。 30 g / l soy flour added to water and extracted for 30 minutes, sugar 20 g / l, magnesium sulfate heptahydrate 0.5 g / l, potassium dihydrogen phosphate 0.5 g / l, silicon The culture solution to which 0.5 g / l of antifoam was added was filled into the culture tank from an airtight hatch. The culture solution inside the culture tank was sterilized at a temperature of 121 ° C. for about 20 minutes and then allowed to cool to maintain the temperature of the culture solution at about 20 ° C.
そして、気密ハッチから前記二次培養で得られた液体種菌を培養タンク内の培養液に接種し、液体培地に対し無菌エア供給機構から空気を気泡の形で導入し2週間程度深部培養を行った。培養タンクに導入した空気は排気し、深部培養が終了すると液体培地内でエノキタケ菌糸が伸長した培養液となった。ここで深部培養工程(ステップS1)が終了する。 Then, the liquid inoculum obtained from the secondary culture from the airtight hatch is inoculated into the culture solution in the culture tank, and air is introduced into the liquid medium from the aseptic air supply mechanism in the form of bubbles and deep culture is performed for about two weeks. It was. The air introduced into the culture tank was evacuated, and when the submerged culture was completed, a culture solution in which enokitake mushrooms were elongated in the liquid medium was obtained. Here, the deep culture process (step S1) ends.
深部培養で得た培養液を、メッシュ状のステンレスフィルターを用いて、エノキタケ菌糸体と培養液とに気密環境下で無菌的にろ別し、ゾル状あるいはゲル状のエノキタケ菌糸体を得た(ろ別工程ステップS2)。ここで菌糸体は培養液を失ったことにより深部培養状態から離脱することになる。続いて、上記ゾル状あるいはゲル状のエノキタケ菌糸体を、無菌環境の下で、ステンレスメッシュの上に置き、菌糸体の上に適度な重りを載せ、圧搾による水分のドリップにより、菌糸体の水分含有率を80〜98%に調整して菌糸体の塊(菌糸塊)を得た(菌糸体水分含有率調整工程ステップS3)。ここで、無菌的に生成した菌糸塊は、生存に最適な上記水分含有率を有し、生存に最適な温度並びに湿度の環境下に保管したので生存が確認された。 The culture solution obtained in the submerged culture was aseptically filtered in an airtight environment into enokitake mycelium and culture solution using a mesh-like stainless steel filter to obtain sol-like or gel-like enokitake mycelium ( Filtration process step S2). Here, the mycelium is detached from the deep culture state due to the loss of the culture solution. Subsequently, the sol-form or gel-form enokitake mycelium is placed on a stainless steel mesh in a sterile environment, an appropriate weight is placed on the mycelium, and the moisture of the mycelium is dripped by drip of the water by pressing. The content rate was adjusted to 80 to 98% to obtain a mycelium mass (mycelium mass) (mycelium moisture content adjustment step S3). Here, the mycelial mass produced aseptically had the above-mentioned water content optimal for survival, and was stored in an environment of temperature and humidity optimal for survival, so that survival was confirmed.
そして、菌糸塊を6℃前後の低温環境下に20日間静置保管することにより、菌糸塊に対し静置刺激および温度刺激(酸化ストレス)を与えた(ストレス印加工程ステップS4)。ここで、菌糸塊は酸化ストレス環境下に置かれたことにより、内部で抗酸化物質であるエルゴチオネインを急速に大量生産することになると考えられ、エルゴチオネインが多く含む菌糸塊(エルゴチオネイン増量菌糸塊)を得ることができた。そして、菌糸塊の静置保管を開始した日(初発)、1日目、3日目、6日目、9日目、12日目、20日目それぞれの日に、菌糸塊を乾燥後に粉砕したものから有機溶媒を使用してエルゴチオネインを抽出し、得られた抽出液を一般的な方法で精製した精製物のエルゴチオネイン含有量を計測した。ここに菌糸塊の静置保管経過日と当該日の精製物のエルゴチオネイン含有量との関係を示したHPLC分析結果を表1に示す。 Then, the mycelial mass was stored in a low temperature environment of about 6 ° C. for 20 days, so that the mycelial mass was subjected to static stimulation and temperature stimulation (oxidation stress) (stress application step S4). Here, it is considered that the mycelial mass is rapidly put into mass production of the antioxidant ergothioneine by being placed in an oxidative stress environment, and the mycelial mass containing a large amount of ergothioneine (ergothioneine-enriched mycelial mass) I was able to get it. Then, the hyphae lump is dried and pulverized on the first day, the first day, the third day, the sixth day, the ninth day, the twelfth day, and the twentieth day. Ergothioneine was extracted from the resulting product using an organic solvent, and the ergothioneine content of a purified product obtained by purifying the resulting extract by a general method was measured. Table 1 shows the HPLC analysis results showing the relationship between the stationary storage elapsed date of the mycelium and the ergothioneine content of the purified product on that day.
表1に示すように、エノキタケ菌糸塊への静置刺激および温度刺激(低温静置ストレス)を加えることによるエルゴチオネインの含有量は12日目で最大値(1.70)となり、培養液からろ別したエノキタケ菌糸体の生産量にほぼ相当する、静置保管を開始した日(初発)の含有量(0.28)の約6.07倍となっていることが分かる。 As shown in Table 1, the content of ergothioneine by adding static stimulation and temperature stimulation (low-temperature static stress) to the enokitake mushroom mycelium reached the maximum value (1.70) on the 12th day. It can be seen that the content is about 6.07 times the content (0.28) of the day (first time) when the stationary storage is started, which is almost equivalent to the production amount of another enokitake mycelium.
実施例1と同様にして得たエノキタケ菌糸塊に対してDC100KVの火花放電を3秒間印加(電圧印加ストレス)した後に、当該菌糸塊を6℃前後の低温環境下に20日間静置保管した。そして、実施例1と同様に、静置保管を開始した日(初発)、1日目、3日目、6日目、9日目、12日目、20日目それぞれの日に、エルゴチオネインを抽出精製した精製物のエルゴチオネイン含有量を計測した。ここに電圧印加後の菌糸塊の静置保管経過日と当該日の精製物のエルゴチオネイン含有量との関係を示したHPLC分析結果を表2に示す。 After applying a spark discharge of DC 100 KV for 3 seconds to the enokitake mushroom mycelium obtained in the same manner as in Example 1 (voltage application stress), the mycelial mass was stored in a low temperature environment of about 6 ° C. for 20 days. And, as in Example 1, on the day of starting stationary storage (first departure), the first day, the third day, the sixth day, the ninth day, the twelfth day, and the twentieth day, ergothioneine The ergothioneine content of the extracted and purified product was measured. Table 2 shows the HPLC analysis results showing the relationship between the stationary storage elapsed day of the mycelial mass after voltage application and the ergothioneine content of the purified product on that day.
表2に示すように、電圧印加後のエノキタケ菌糸塊に低温静置ストレスを加えることによるエルゴチオネインの含有量は9日目で最大値(1.92)となり、静置保管を開始した日(初発)の含有量(0.28)の約6.85倍となっていることが分かる。なお、静置保管を開始した日(初発)のエルゴチオネインの含有量は、培養液からろ別したエノキタケ菌糸体の含有量(従来技術に係る含有量)にほぼ相当する。この事実は、低温静置に電圧印加を加えたストレスを与えた実施例2は、低温静置ストレスのみを与えた実施例1と比較して初発から3日間早くエルゴチオネイン含有量の最大値に到達し、またその含有量も0.22増加していることが分かる。 As shown in Table 2, the content of ergothioneine by applying low-temperature static stress to the enokitake mushroom mycelium after voltage application reached the maximum value (1.92) on the 9th day, and the day when the static storage was started (first time ) Content of about 6.85 times (0.28). In addition, the content of ergothioneine on the day when the stationary storage is started (first time) substantially corresponds to the content of enokitake mycelium separated from the culture solution (content according to the prior art). This fact is that Example 2 which applied stress by applying voltage to low temperature standing reached the maximum value of ergothioneine content three days earlier than the first example which applied only low temperature standing stress. It can also be seen that the content is increased by 0.22.
上述の実施例から明らかなように、ろ別工程を実行した後、菌糸体水分含有率調整工程および各種ストレス印加工程を実行することにより、菌糸体は、深部培養工程において生産したエルゴチオネインに加えて抗酸化物質であるエルゴチオネインを急速に大量生産する。また、電圧印加後の菌糸塊に低温静置ストレスを加えることによりエルゴチオネインの増加速度を増すことができると共に含有量を増加させることができる。なお、エルゴチオネインの生産に用いるキノコ菌として、エノキタケ菌の他にタモギタケ菌、霊芝菌、冬虫夏草菌、アギタケ菌、トキイロヒラタケ菌、エリンギ菌等を用いても良い。また子嚢菌としてトリフ菌等を用いても良い。 As is clear from the above examples, the mycelium is added to the ergothionein produced in the submerged culture step by performing the mycelium moisture content adjustment step and various stress application steps after performing the filtration step. Rapidly mass-produces ergothioneine, an antioxidant. Moreover, by adding low temperature static stress to the mycelium after voltage application, the increase rate of ergothioneine can be increased and the content can be increased. As mushroom fungi used for the production of ergothioneine, in addition to Enokitake fungi, Tamogitake fungi, Ganoderma fungi, Cordyceps fungi, Agitake fungi, Tokihirohiratake fungi, Eringi fungi and the like may be used. Moreover, you may use a Trifungus etc. as an ascomycete.
本発明によれば、飼料、化粧品、薬品等に利用できるエルゴチオネイン等の抗酸化物質、抗生物質等をキノコ菌糸体により安価に製造できるので、本発明はキノコ業界のみならず、水産業、畜産業、医療業等において広く貢献でき、医薬品、化粧品、健康食品等広い用途がある。 According to the present invention, antioxidants such as ergothioneine that can be used in feeds, cosmetics, medicines, antibiotics, etc. can be produced at low cost by the mushroom mycelium. It can contribute widely in the medical industry and has a wide range of uses such as pharmaceuticals, cosmetics, and health foods.
10 深部培養手段
11 液体培地
12 担子菌又は子嚢菌
13 培養タンク
14 気密ハッチ
15 撹拌機構
16 無菌エア供給機構
17 pH調整機構
18 温度調整機構
19 排気機構
20 ろ別手段
30 菌糸体水分率調整手段
40 ストレス印加手段
CF 培養液
HS 菌糸体
AFCF ろ別後培養液
HSL 菌糸塊
ST ストレス
IIHSL 有用物質増量菌糸塊
DESCRIPTION OF SYMBOLS 10 Deep culture | cultivation means 11 Liquid culture medium 12 Basidiomycetes or Ascomycetes 13 Culture tank 14 Airtight hatch 15 Stirring mechanism 16 Aseptic air supply mechanism 17 pH adjustment mechanism 18 Temperature adjustment mechanism 19 Exhaust mechanism 20 Filtration means 30 Mycelium moisture content adjustment means 40 Stress application means CF culture medium HS mycelium AFCF culture medium after filtration HSL mycelial mass ST stress II HSL useful substance increase mycelial mass
Claims (2)
深部培養工程において培養された菌糸体を深部培養状態から離脱させ、菌糸体と培養タンク内の液体培地とを含む培養液から水分を減じるためのろ過処理を実施し、液体培地が付着した菌糸体を分別するろ別工程と、
前記ろ別した菌糸体の菌糸塊を生成し、当該菌糸塊を気中環境下で生存可能にするために水分含有率を調整する菌糸体水分含有率調整工程と、
前記水分含有率が調整された菌糸塊を生存環境下に保管し、当該菌糸塊に対し静置刺激及び低温環境下における温度刺激または電気刺激印加後の静置刺激及び低温環境下における温度刺激を印加してエルゴチオネインが増量した菌糸塊を生成するストレス印加工程と、
からなることを特徴とする深部培養法を用いたキノコの担子菌によるエルゴチオネイン製造方法。 A method for producing ergothioneine by deeply culturing mycelia of mushroom basidiomycetes in a liquid medium, extracting and purifying ergothioneine from the produced mycelium,
The mycelium cultivated in the deep culture step is removed from the deep culture state, filtered to reduce the moisture from the culture solution containing the mycelium and the liquid medium in the culture tank, and the mycelium to which the liquid medium is attached A filtration process for separating
Producing a mycelium of the filtered mycelium, and adjusting the water content to adjust the water content in order to make the mycelium viable in an air environment; and
Store the mycelium of which the moisture content is adjusted in a living environment, and subject the mycelium to a static stimulus in a static environment and a low temperature environment or a static stimulus after applying an electrical stimulus and a thermal stimulus in a low temperature environment. Applying a stress to produce a mycelium of increased ergothioneine by application,
The manufacturing method of the ergothioneine by the mushroom basidiomycetes using the deep culture method characterized by comprising .
前記深部培養手段により培養された菌糸体を培養タンクから取り出して深部培養状態から離脱させ、菌糸体と培養タンク内の液体培地とを含む培養液から水分を減じるためのろ過処理を実施し、当該ろ過処理された培養液をゾル又はゲル状の菌糸体とろ別後培養液とに分別するろ別手段と、
前記ろ別手段によりろ別された菌糸体の菌糸塊を生成し、当該菌糸塊が含有する水分量を菌糸体の生存に最適な水分含有率に調整する菌糸体水分含有率調整手段と、
前記菌糸体水分含有率調整手段により水分含有率が調整された菌糸塊を生存環境下に保管し、当該菌糸塊に対し静置刺激及び低温環境下における温度刺激または電気刺激印加後の静置刺激及び低温環境下における温度刺激を与え、エルゴチオネインの生産を増加したエルゴチオネイン増量菌糸塊を生成するストレス印加手段と、
を備えたことを特徴とする深部培養法を用いたキノコの担子菌によるエルゴチオネイン製造装置。 A culture tank that contains a liquid medium inside and performs deep culture of mycelium of mushroom basidiomycetes , an airtight hatch attached to the top of the culture tank, a stirring mechanism that stirs the liquid medium in the culture tank, and a liquid medium A sterile air supply mechanism for supplying a predetermined amount of sterile air to the liquid, a pH adjusting mechanism for adjusting and maintaining the pH of the liquid medium to a predetermined value, and a temperature adjusting mechanism for adjusting and maintaining the temperature of the liquid medium to a predetermined value An exhaust mechanism for exhausting the air supplied from the sterile air supply mechanism to the outside from the culture tank, and a deep culture means for deeply culturing mycelium,
The mycelium cultured by the deep culture means is removed from the culture tank and removed from the deep culture state, and a filtration treatment is performed to reduce water from the culture solution containing the mycelium and the liquid medium in the culture tank. A filtration means for separating the filtered culture solution into a sol or gel mycelium and a culture solution after filtration;
Generating a mycelium of the mycelium filtered by the filtering means, and adjusting the moisture content contained in the mycelium to an optimal moisture content for survival of the mycelium; and
The mycelium whose water content is adjusted by the mycelia water content adjusting means is stored in a living environment, and the mycelium is subjected to static stimulation and static stimulation after application of temperature stimulus or electrical stimulation in a low temperature environment. and giving thermal stimulation in a low temperature environment, and stress applying means for generating a ergothioneine bulking mycelial mass increased production of ergothioneine,
An apparatus for producing ergothioneine using mushroom basidiomycetes using a submerged culture method.
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