WO2013161939A1 - Method for culturing fungus - Google Patents

Method for culturing fungus Download PDF

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Publication number
WO2013161939A1
WO2013161939A1 PCT/JP2013/062201 JP2013062201W WO2013161939A1 WO 2013161939 A1 WO2013161939 A1 WO 2013161939A1 JP 2013062201 W JP2013062201 W JP 2013062201W WO 2013161939 A1 WO2013161939 A1 WO 2013161939A1
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Prior art keywords
mycelium
medium
culture
basidiomycete
fruit
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PCT/JP2013/062201
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French (fr)
Japanese (ja)
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池田　順一
大賀　祥治
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共栄社化学株式会社
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Priority to JP2013531808A priority Critical patent/JP5377804B1/en
Publication of WO2013161939A1 publication Critical patent/WO2013161939A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G18/00—Cultivation of mushrooms
    • A01G18/20—Culture media, e.g. compost
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G18/00—Cultivation of mushrooms
    • A01G18/40—Cultivation of spawn

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  • the present invention relates to a fungal culture method for growing basidiomycetes from mycelium to fruit bodies in layers on a sheet using a culture sheet supporting a solid medium or a liquid medium.
  • Various fungi such as venix nokitake (gyudonoshiba), garlic mushroom (Ganoderma lucidum), eucommia serrata (Cycium edulis), and agaricus are used as medicinal ingredients for herbal medicines and as active ingredients for cosmetics and health foods.
  • Natural basidiomycetes are grown on the back of a base material such as a raw tree of a plant that is a nutrient source.
  • Taiwan's endemic species, Benix nokitake grows exclusively on the natural wood of raw gyudon, and is extremely slow growing, coupled with the rough cutting of the base gyudon. It is a rare natural resource that is difficult to collect.
  • Benix nokitake contains almost no medicinal ingredients in its mycelium, and as disclosed in Non-Patent Document 1, triterpenoids exhibiting pharmacological actions such as liver function improving function and antitumor action in the fruit body. Such a medicinal component is contained in many kinds and at a high concentration. Benix nokitake is considered to be artificially produced in liquid culture or solid culture because it has a small collection amount from nature. However, in liquid culture, the culture rate is somewhat faster than natural growth, and a large amount of culture solution must be used. Even if a large-capacity tank is used, the production volume is small for the scale and the production efficiency is poor, and it is not suitable for industrial production.
  • Edible mushrooms such as shiitake mushrooms
  • Patent Document 1 is used for solid culture of fungi such as shiitake mushrooms, and has a plurality of stitch-like pores formed of synthetic fibers as a whole, and has a plurality of thin top portions by press molding and a thin top portion.
  • a molded sheet for fungal culture having a recess is disclosed. Such a molded sheet for culturing fungi is cultivated for transplanting fungi to raw wood, so that solid fungi such as Benicus mushrooms that become membranous fruit bodies from mycelium are not transplanted to the raw wood. It cannot be used for culturing.
  • the present invention has been made in order to solve the above-mentioned problems, and a basidiomycete can be grown from a mycelium to a fruit body in a solid medium or a liquid medium, and can be cultured efficiently and easily with uniform quality. It is an object to provide a method and a culture sheet used therefor.
  • the fungal culture method according to claim 1, which has been made to achieve the above object, is made of metal, synthetic resin, natural resin, and / or paper, from mesh, woven fabric, non-woven fabric, and porous sheet.
  • the mycelium of basidiomycetes is inoculated on the front side by coating or spraying the culture solution, and the mycelium is grown
  • the mycelium growth step for growing the mycelium, and the mycelium grown on the front surface side is attached with a medium for basidiomycetous fruit bodies, And having a fruiting body growth step for growing the mycelium from the mycelium to the fruiting body.
  • the fungal culture method according to claim 2 is the method according to claim 1, wherein the basidiomycete mycelium medium and the basidiomycete fruit body medium are respectively a liquid medium or a solid medium.
  • the fungal culture method according to claim 3 is the method according to claim 2, wherein the basidiomycete mycelium medium comprising the liquid medium is supported on the reinforcing body during the mycelium growth step, During the fruiting body growth step, the medium for basidiomycetous fruiting bodies comprising the liquid medium is supported on the front surface side on the reinforcing body, or the medium for basidiomycetous fruiting bodies comprising the solid medium is the reinforcing body.
  • the basidiomycetous mycelium medium comprising the solid medium is supported on the front side on the reinforcing body, and during the fruiting body growth step, the basidiomycetes comprising the liquid medium
  • a medium for fruiting bodies is carried on the medium for basidiomycetous mycelium or the medium for basidiomycetous fruit bodies consisting of the solid medium is carried on the back side of the reinforcing body, or
  • the basidiomycete mycelium medium comprising the solid medium is supported on the front side on the reinforcing body, and during the fruit body growth step, from the basidiomycete mycelium medium
  • the basidiomycete fruit body culture medium comprising the solid medium is supported on the back side of the reinforcing body from which the reinforcing body has been peeled off together with the mycelium.
  • the fungal culture method according to claim 4 is the method according to claim 3, wherein during the mycelium growth step, the basidiomycete mycelium medium comprising the layered solid medium on the mycelium film, After the reinforcing body is mounted and crimped and integrated, the mycelium culture solution of the basidiomycetous fungus is sprayed to form the mycelium growth culture sheet, and during the fruit body growth step, the front surface side The reinforcing body together with the mycelium grown in step 1 is peeled off from the basidiomycete mycelium medium and the mycelium film, and then the basidiomycetous fruit body medium comprising the layered solid medium on the fruit body film is used. The fruit body growing culture sheet is formed by pressure-bonding the reinforcing body on the back surface side.
  • the method for culturing a fungus according to claim 5 is the method according to any one of claims 1 to 4, wherein the mycelium-growing culture sheet is rolled up to grow the mycelium, and / or The fruit body growing culture sheet is wound up in a roll shape to grow the fruit body.
  • the method for culturing a fungus according to claim 6 is the method according to any one of claims 1 to 5, wherein the basidiomycete is a fungus of the order Hydrangea.
  • the method for culturing fungi according to claim 7 is the method according to claim 6, wherein the fungus of the class Agaricaceae is a chanterelle family, a white mushroom family, a broom bamboo family, a red-branch family, a coral family, a sorghum family, a tobacco scale. It is characterized by being a fungus of the family bambooceae, Idotake, Shirotake, Nikuharitake, Ezoharitake, Ibotake, Ningyotake, Pleurotus, Amanita, or Miyamatonbiidae.
  • the method for culturing fungi according to claim 8 is the method according to any one of claims 1 to 7, wherein the mycelium and / or the fruiting body is subjected to physical stimulation, electrical stimulation, optical stimulation, chemical It is characterized in that at least one treatment selected from a physical stimulus and a biological stimulus is performed.
  • the culture sheet according to claim 9 is selected from a mesh, a woven fabric, a non-woven fabric, and a porous sheet formed of metal, synthetic resin, natural resin, and / or paper, and has a basidiomycetous fungus on its front surface.
  • the reinforcing body for growing the mycelium carries the basidiomycete fruit body culture medium grown from the mycelium to the fruit body.
  • the culture sheet according to claim 10 is the one described in claim 9, wherein the reinforcing body is made of a liquid medium or a solid medium, and the medium for basidiomycetous mycelium in which the mycelium is grown is an unsupported reinforcing body.
  • the basidiomycete fruiting body medium comprising a liquid medium or a solid medium is supported on or supported by the reinforcing body.
  • the culture sheet according to claim 11 is the culture sheet according to any one of claims 9 to 10, and has air permeability due to a mesh-like, lattice-like, gathered-like, irregular wave-like shape, and / or a saw-like gap.
  • a spacer is attached to the reinforcing body.
  • basidiomycetes can be grown from mycelium to fruiting bodies in a short period of time on a solid culture sheet.
  • a solid culture sheet By winding the solid culture sheet and allowing it to stand at a constant temperature, it can be cultured in a large amount in a narrow space. Since a solid culture sheet is used, it can be cultured efficiently and easily with uniform quality.
  • the fungal culture method of the present invention will be described with reference to FIG. This fungal culture method is performed using the mycelium growing solid culture sheet 1 and the fruit body growing solid culture sheet 2 according to the following procedure.
  • the mycelium growth step [I] is performed.
  • a reinforcing body 15 capable of supporting a culture medium is formed and wound up with metal, synthetic resin, natural resin, paper, mesh, woven fabric, nonwoven fabric, and irregularities or holes of a porous sheet.
  • the mycelium film 11 is pulled out from the non-permeable resinous mycelium film roll.
  • the basidiomycete mycelium medium coating solution 13 is applied or sprayed to the mycelium film 11 on the front surface side with a basidiomycete mycelium medium application nozzle 12 to form a mycelium growth agar medium layer which is a solid medium layer.
  • a layered basidiomycete mycelium medium 14 is formed.
  • the reinforcing body 15 and the basidiomycete mycelium medium 14 are integrated.
  • a mycelium culture spray solution 17 of basidiomycetous liquid cultured on the basidiomycete mycelium medium 14 is sprayed onto the basidiomycete mycelium medium 14 from the mycelium culture solution spray nozzle 16, the basidiomycetes 20 become basidiomytes.
  • the medium 14 for fungal mycelium is inoculated. If necessary, a net-like / lattice-like or saw-like spacer (not shown) for preventing adhesion during winding is further provided thereon.
  • the mycelium growing solid culture sheet 1 is obtained.
  • the mycelium growing solid culture sheet 1 is loosely wound up in a roll shape so that the sheets do not adhere to each other, and is left to stand.
  • the basidiomycetes 20 are grown on the basidiomycete mycelium medium 14 and are planarly grown in all directions to form a mycelium layer of the basidiomycetes 20.
  • the fruiting body growth step [II] is performed.
  • the mycelium film 11 is peeled off from the mycelium-growing solid culture sheet 1 together with the basidiomycete mycelium medium 14.
  • the mycelium layer of the basidiomycetes 20 remains on the front side on the reinforcing body 15.
  • the fruiting body film 21 is pulled out from the resinous fruiting body film roll which is impermeable.
  • a basidiomycetous fruit body medium coating liquid 23 is applied or sprayed onto the fruit body film 21 with the basidiomycetous fruit body medium nozzle 22 on the front surface to form a fruit body growing agar medium layer.
  • An entity medium 24 is formed.
  • the reinforcing body 15 When the reinforcing body 15 is pressure-bonded to the basidiomycetous fruit body medium 24 on the fruit body film 21 on the back side, the reinforcing body 15 and the basidiomycete fruit body medium 24 are integrated.
  • the mycelium layer of the basidiomycetes 20 comes into contact with the basidiomycetous fruiting medium 24 and can be grown and grows. Thereby, the fruit body growing solid culture sheet 2 is obtained.
  • the fruit body growing solid culture sheet 2 is loosely wound up in a roll shape so that the sheets do not adhere to each other, and is left to stand. Meanwhile, the basidiomycetes 20 grow and grow from mycelium to fruiting bodies in the basidiomycetous fruiting medium 24, and grow in layers on the sheet in all directions to form a fruiting body layer of the basidiomycetes 20.
  • the mycelium-growing solid culture sheet 1 and the fruit body-growing solid culture sheet 2 can maintain a humidity and aeration state because a gap is generated by a spacer during winding and the adhesion is inhibited.
  • the mycelium layer and fruiting body layer of the basidiomycetes 20 grow while avoiding the mesh-like, lattice-like, or saw-like gaps of the spacers.
  • the mycelium-growing solid culture sheet 1 is prepared, wound and allowed to stand, and the basidiomycetes 20 are grown and propagated. 20 mycelium layers are formed (see FIG. 1).
  • a fruit body growth step [II] is performed.
  • the basidiomycetous fungus body medium coating solution 23 is sprayed onto the basidiomycete mycelium medium 14 on the front side of the mycelium-growing solid culture sheet 1 with the basidiomycetous fruit body medium nozzle 22 to spray the fruiting body growing liquid medium.
  • the basidiomycetous fruiting body medium 24 to be layered is formed. Thereby, the fruit body growing solid culture sheet 2 is obtained.
  • the fruit body growing solid culture sheet 2 is loosely wound up in a roll shape so that the sheets do not adhere to each other, and is left to stand. Meanwhile, the basidiomycetes 20 grow from the mycelium to the fruiting bodies and grow in the basidiomycetous fruiting medium 24 and multiply in a planar manner to form a fruiting body layer of the basidiomycetes 20.
  • the fungal culture method is suitable for culturing basidiomycetes.
  • Basidiomycetes can be expressed according to the classification of Mushrooms, Newly-developed Sankei Field Books 7 (June 10, 2006, first edition, first edition, published by Yamato Valley Co., Ltd.). It is preferable that the order is Lepidoptera-H.
  • Subtilis-Hymenaceae For example, chanterelles, sphagnum mushrooms, blossoms mushrooms, blossoms mushrooms, coral mushrooms, sorghum mushrooms, cigarette mushrooms, iridaceae, mushroom mushrooms, mushroom mushrooms, mushroom mushrooms, mushroom mushrooms, mushroom mushrooms, Family fungi of the family, Koyakutake department, Manentake department, Miyamatonbimai family.
  • chanterelles Cantharellus cibarius
  • white-spotted bamboo Clavaria vermicularis
  • broom bamboo Ramaria ⁇ botrytis
  • red-bellied bamboo Sparassis crispa
  • coral harimatake Hericium ramosun
  • fusahari cyno bat
  • Beech haritake Mycoleptodonoides aitcbisonii
  • Maitake mushroom Grifola ⁇ frondosa
  • Giant mushroom Trametes gibbosa
  • Agaric mushroom Phanerochaete chrysorbiza
  • Olive mushroom Lopharia cinerascens
  • Pepper mushroom patite
  • Ningyotake Albatrellus confluens
  • Aurouji Albatrellus caeruleoporus
  • Numeraitake Albatrellus yasudai
  • Kotake Kurokawa
  • the medium for basidiomycete mycelium and the medium for basidiomycete fruit bodies may be a solid medium such as an agar medium or a liquid medium.
  • the solid medium is, for example, a hydrogel-forming polymer such as polyalginate and agar in 1 L of medium alone or mixed, 3 to 20 g thereof, 3 to 5 g of potato extract, glucose, dextrose, fructose, sucrose , Cellobiose, or trehalose alone, or 10 to 30 g of a saccharide such as a mixture of any of them.
  • a hydrogel-forming polymer such as polyalginate and agar in 1 L of medium alone or mixed, 3 to 20 g thereof, 3 to 5 g of potato extract, glucose, dextrose, fructose, sucrose , Cellobiose, or trehalose alone, or 10 to 30 g of a saccharide such as a mixture of any of them.
  • the liquid medium is, for example, 10 to 30 g each of yeast extract and malt extract in 1 L of medium, dextrose, dextrose, fructose, sucrose, cellobiose, trehalose alone, or a mixture of any of them.
  • the fruit body When the fruit body is grown, it is formed on the solid body-cultivating solid culture sheet 2, or during the formation of the fruit body, or after the formation of one month, physical stimulation, electrical stimulation, optical stimulation, biological stimulation, chemistry You may perform the process of at least any one of an irritation
  • physical stimulation, electrical stimulation, and optical stimulation irradiation with pulse laser at 20 to 100 mW at intervals of 10 nanoseconds to 100 milliseconds, preferably several tens of nanoseconds to several tens of milliseconds, 20 to Examples include plasma treatment with a pulsed plasma generator at 90 kV, preferably 30 to 70 kV and 100 ⁇ A to 1 mA at intervals of 100 to 500 milliseconds for 30 to 60 seconds, and light irradiation treatment with a wavelength of 500 to 800 nm.
  • physical stimulation and electrical stimulation may not be applied directly to the fruit body of the fruit body growing solid culture sheet 2. For example, as shown in FIG.
  • a pulse voltage 26a is generated by the pulse voltage electrode 25a toward the fruit body growing solid culture sheet 2
  • / or the pulse voltage 26b is generated by the pulse voltage electrode 25b of the fruit body growing solid culture sheet 2. It may be generated for air sterilization without directing it upwards.
  • plasma treatment with a pulsed plasma generator at 50 to 90 kV, preferably 70 to 90 kV, 100 ⁇ A to 1 mA and 100 to 500 milliseconds for 1 to 10 seconds is continued once per minute.
  • the reinforcing body 15 is a cell culture mesh, for example, having an opening of 50 to 1000 microns and a thickness of 30 to 300 microns.
  • the material is a metal (for example, stainless steel), a synthetic resin (for example, nylon, Polyester, polyethylene, polypropylene, fluorine fiber, polyvinylidene chloride, polyvinyl alcohol, ethylene-vinyl acetate copolymer, polyurethane, polycellulose, etc.) mesh cloth and porous sheet, woven fabric of natural fibers (eg silk, cotton) , Paper, and non-woven fabric.
  • the width is, for example, 200 to 1100 mm.
  • the mycelium film 11 and the fruit body film 21 are support films, and are formed of a heat-resistant resin exemplified by polyester, polypropylene, and polyvinylidene chloride.
  • the thickness is, for example, 10 to 200 microns, and the width is, for example, 200 to 1100 mm.
  • pretreatment such as pulse voltage treatment, pulse laser irradiation treatment, plasma treatment, and light irradiation treatment may be performed as necessary for sterilization and germination promotion of basidiomycetes.
  • the spacer is a mesh or lattice, for example, a gathered shape that is creased at intervals of 1 to 3 cm, and a concavo-convex wave having continuous irregularities of sine wave shape, cone shape, bell shape, or truncated cone shape at intervals of 0.1 to 3 cm. It may be air permeable due to its shape and / or slat-like gap. This unevenness is to allow the mycelium-growing solid culture sheets 1 or the fruiting body-growing solid culture sheets 2 to be in line contact / point contact with each other to give air permeability.
  • the mycelium-growing solid culture sheet 1 and the fruit body-growing solid culture sheet 2 may be used while being stretched, or may be used by being folded or wound, or may be used after being cut to an appropriate size. When a continuous long sheet is rolled up and used, it contributes to an improvement in productivity in a narrow space.
  • Ion exchanged water was added to make the total volume 1 L, transferred to a flask, and autoclaved at 121 ° C. for 15 minutes to prepare an agar medium (hereinafter referred to as PDA medium).
  • PDA medium an agar medium
  • the pH was adjusted to 5 to 7 with a phosphate buffer.
  • the obtained medium was cooled in a sterile draft by adding 15 to 20 mL of PDA medium to a petri dish having a diameter of 9 cm in the case of (i) solid culture.
  • the flask containing the PDB medium was inoculated and cultured after cooling to room temperature. All the cultures were performed at a constant temperature of 25 ° C. in an incubator.
  • Example 1 Culture on a planar solid culture sheet
  • a polyethylene terephthalate (PET) film which is a mycelium film 11 having a thickness of 100 microns and a thickness of 20 ⁇ 25 cm, was laid on a bat having a size of 25 ⁇ 30 cm.
  • PDA medium which is a basidiomycete mycelium medium 14
  • 15 ⁇ 20 cm A medium was prepared by laying a PET mesh, which is a reinforcing member 15 of the above. The medium was cooled to room temperature.
  • the vat was cultured at 25 ° C. under aseptic conditions for 30 days.
  • the resulting mycelium-attached mesh was transferred together with the basidiomycetes 20 onto the medium described in Example 3 (3-2) to obtain a fruit body growing solid culture sheet 2. This was cultured for 60 days.
  • Example 2 Culture in a roll-shaped solid culture sheet
  • a PET film, a PET film and a rolled PET film having a width of 300 mm are used, and a 1 cm square mesh spacer made of porous vinyl chloride resin having a diameter of 1 to 2 mm is wound between layers in each step.
  • the mycelium growing solid culture sheet 1 and the fruiting body growing solid culture sheet 2 were prepared and subjected to plasma treatment with a pulse plasma generator for 30 seconds at 400 ⁇ A at 30 kV and at intervals of 100 milliseconds. After 24 days from the start of the culture, it was applied once a day for 2 days. After 60 days, the culture on the mesh was observed for spores and clamps under a microscope. When extracted and analyzed by HPLC, ergosterols, which are fruit body-containing components, were also detected. This confirmed that a child entity was generated.
  • the method for culturing fungi using the solid culture sheet of the present invention is a method of artificially cultivating basidiomycetes that grow and produce a film-like fruit body on a smooth or rough surface and used as a crude drug in medicines or health foods It is used to be used as an active ingredient.
  • 1 is a solid culture sheet for growing mycelium
  • 2 is a solid culture sheet for growing fruit bodies
  • 11 is a film for mycelium
  • 12 is a medium application nozzle for basidiomycete mycelium
  • 13 is a medium application solution for basidiomycetous mycelium
  • 14 is a basidiomycete Medium for fungal mycelium
  • 15 reinforcement 16 mycelium culture spray nozzle
  • 17 mycelium culture spray nozzle
  • 20 basidiomycetes 21 fruit body film
  • 22 basidiomycete culture medium nozzle 23 is a basidiomycete fruit body medium coating solution
  • 24 is a basidiomycete fruit body medium
  • 25a and 25b are pulse voltage electrodes
  • 26a and 26b are pulse voltages.

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Abstract

Provided is a method for culturing a fungus, which enables a mycelium of a basidiomycete to be grown into a fruiting body in a solid culture medium or a liquid culture medium and therefore can culture the basidiomycete with uniform quality, with high efficiency and in a simple manner. The method for culturing a fungus comprises: a mycelium proliferation step [I] for forming a reinforcing body (15) selected from a mesh, a woven fabric, a non-woven fabric and a porous sheet using a metal, a synthetic resin, a natural resin and/or paper, applying a basidiomycetous mycelium culture medium (14) onto the reinforcing body (15), then inoculating a mycelium of a basidiomycete (20) onto the front surface side of the reinforcing body (15) by coating or spraying a culture liquid (17) of the mycelium onto the front surface side to form a sheet (1) for mycelium-growing/culturing purposes, and then growing the mycelium; and a fruiting body proliferation step [II] for applying a basidiomycetous fruit body culture medium (24) to the mycelium that has been grown on the front surface side to form a sheet (2) for fruit body-growing/culturing purposes and then allowing the mycelium to be grown into a fruiting body.

Description

菌類培養方法Fungal culture method
　本発明は、固体培地又は液体培地を担持する培養シートを用いて、担子菌類を菌糸体から子実体へシート上で層状に生育する菌類培養方法に関するものである。 The present invention relates to a fungal culture method for growing basidiomycetes from mycelium to fruit bodies in layers on a sheet using a culture sheet supporting a solid medium or a liquid medium.
　ベニクスノキタケ(牛樟芝)、マンネンタケ(霊芝)、トウチュウカソウ(冬虫夏草)、アガリクスなどの各種菌類が、生薬医薬品の薬効成分として、また化粧品や健康食品の有効成分として、用いられている。 Various fungi such as venix nokitake (gyudonoshiba), garlic mushroom (Ganoderma lucidum), eucommia serrata (Cycium edulis), and agaricus are used as medicinal ingredients for herbal medicines and as active ingredients for cosmetics and health foods.
　天然の担子菌類は、栄養源である植物の原木などの基物に背着し、菌糸体から、子実層托が平滑又は粗面で膜状の子実体を作って、育成する。それら天然の担子菌類の中でも特に需要の大きな台湾固有種のベニクスノキタケは、天然樹木の牛樟原木で専ら増殖するもので、生育が極めて遅く、基物となる牛樟の乱伐と相俟って、採取し難い希少な天然資源である。 Natural basidiomycetes are grown on the back of a base material such as a raw tree of a plant that is a nutrient source. Among these natural basidiomycetes, Taiwan's endemic species, Benix nokitake, grows exclusively on the natural wood of raw gyudon, and is extremely slow growing, coupled with the rough cutting of the base gyudon. It is a rare natural resource that is difficult to collect.
　ベニクスノキタケは、その菌糸体に薬効成分は殆ど含まれておらず、非特許文献１に開示されているように、子実体に肝機能改善機能や抗腫瘍作用などの薬理作用を示すトリテルペノイドのような薬効成分が、多数種、高濃度で含まれている。ベニクスノキタケは、天然からの採取量が少ないため、液体培養や固体培養で人工的に生産することが検討されている。しかし液体培養は、培養速度が天然での育成よりも幾分速い程度であり、多量の培養液を用いなければならない。たとえ大容量タンクを用いてもそのスケールの割に生産量が少なくて生産効率が悪く、工業的製造に向かない。一方、従来の固体培養は、寒天培地容器を多数並べ、無菌室で培養しなければならず面倒なうえ、寒天培地容器毎の生育速度・薬効成分含量の格差が大きく、容積が限られた無菌室に寒天培地容器を然程並べられないので工業的製造に向かない。 Benix nokitake contains almost no medicinal ingredients in its mycelium, and as disclosed in Non-Patent Document 1, triterpenoids exhibiting pharmacological actions such as liver function improving function and antitumor action in the fruit body. Such a medicinal component is contained in many kinds and at a high concentration. Benix nokitake is considered to be artificially produced in liquid culture or solid culture because it has a small collection amount from nature. However, in liquid culture, the culture rate is somewhat faster than natural growth, and a large amount of culture solution must be used. Even if a large-capacity tank is used, the production volume is small for the scale and the production efficiency is poor, and it is not suitable for industrial production. On the other hand, conventional solid culture requires a lot of agar medium containers to be cultivated in an aseptic room and is troublesome. In addition, there is a large difference in the growth rate and medicinal component content among the agar medium containers, and the volume is limited. It is not suitable for industrial production because the agar containers cannot be arranged so much in the room.
　椎茸などの食用きのこは、ベニクスノキタケよりも遥かに栽培し易く、菌類を培地で培養し原木に移植して栽培される。特許文献１に、椎茸などの菌類を固体培養するために用いられるもので、合成繊維で形成され編目状の細孔を全体的に有し、プレス成形で上端部分が厚く底部分が薄い複数の凹部を有する菌類培養用成形シートが、開示されている。このような菌類培養用成形シートは、菌類を原木に移植するために培養するものであるから、菌糸体から膜状の子実体になるベニクスノキタケなどの担子菌類を原木に移植せずに固体培養するのに用いることができない。 Edible mushrooms, such as shiitake mushrooms, are much easier to cultivate than Benicus mushrooms, and are cultivated by culturing fungi in a medium and transplanting them into raw wood. Patent Document 1 is used for solid culture of fungi such as shiitake mushrooms, and has a plurality of stitch-like pores formed of synthetic fibers as a whole, and has a plurality of thin top portions by press molding and a thin top portion. A molded sheet for fungal culture having a recess is disclosed. Such a molded sheet for culturing fungi is cultivated for transplanting fungi to raw wood, so that solid fungi such as Benicus mushrooms that become membranous fruit bodies from mycelium are not transplanted to the raw wood. It cannot be used for culturing.
特許第３１３９８３７号公報Japanese Patent No. 3139837
　本発明は前記の課題を解決するためになされたもので、担子菌類を、固体培地又は液体培地で菌糸体から子実体に、育成して、均一な品質で効率良く、簡便に培養できる菌類培養方法、及びそれに用いられる培養シートを提供することを目的とする。 The present invention has been made in order to solve the above-mentioned problems, and a basidiomycete can be grown from a mycelium to a fruit body in a solid medium or a liquid medium, and can be cultured efficiently and easily with uniform quality. It is an object to provide a method and a culture sheet used therefor.
　前記の目的を達成するためになされた請求の範囲の請求項１に記載の菌類培養方法は、金属、合成樹脂、天然樹脂、及び／又は紙で、メッシュ、織布、不織布、多孔質シートから選ばれる補強体を形成し、担子菌類菌糸体用培地を付してから、それのおもて面側に担子菌類の菌糸体をそれの培養液の塗工又は噴霧により接種し、菌糸体育成培養シートを形成した後、前記菌糸体を育成する菌糸体増殖工程と、前記おもて面側で育成した前記菌糸体に、担子菌類子実体用培地を付して、子実体育成培養シートを形成した後、前記菌糸体から子実体へ育成する子実体増殖工程とを、有することを特徴とする。 The fungal culture method according to claim 1, which has been made to achieve the above object, is made of metal, synthetic resin, natural resin, and / or paper, from mesh, woven fabric, non-woven fabric, and porous sheet. After forming the selected reinforcing body and attaching a medium for basidiomycete mycelium, the mycelium of basidiomycetes is inoculated on the front side by coating or spraying the culture solution, and the mycelium is grown After forming the culture sheet, the mycelium growth step for growing the mycelium, and the mycelium grown on the front surface side is attached with a medium for basidiomycetous fruit bodies, And having a fruiting body growth step for growing the mycelium from the mycelium to the fruiting body.
　請求項２に記載の菌類培養方法は、請求項１に記載されたもので、前記担子菌類菌糸体用培地と前記担子菌類子実体用培地とがそれぞれ、液体培地又は固体培地であることを特徴とする。 The fungal culture method according to claim 2 is the method according to claim 1, wherein the basidiomycete mycelium medium and the basidiomycete fruit body medium are respectively a liquid medium or a solid medium. And
　請求項３に記載の菌類培養方法は、請求項２に記載されたもので、前記菌糸体増殖工程中、前記液体培地からなる前記担子菌類菌糸体用培地を、前記補強体に担持し、前記子実体増殖工程中、前記液体培地からなる前記担子菌類子実体用培地を前記補強体に前記おもて面側で担持し、若しくは前記固体培地からなる前記担子菌類子実体用培地を前記補強体にうら面側で担持し、又は、
　前記菌糸体増殖工程中、前記固体培地からなる前記担子菌類菌糸体用培地を、前記補強体に前記おもて面側で担持し、前記子実体増殖工程中、前記液体培地からなる前記担子菌類子実体用培地を前記担子菌類菌糸体用培地に担持し若しくは前記固体培地からなる前記担子菌類子実体用培地を前記補強体にうら面側で担持し、又は、
　前記菌糸体増殖工程中、前記固体培地からなる前記担子菌類菌糸体用培地を、前記補強体に前記おもて面側で担持し、前記子実体増殖工程中、前記担子菌類菌糸体用培地から前記菌糸体と共に前記補強体を剥離した前記補強体にうら面側で、前記固体培地からなる前記担子菌類子実体用培地を、担持することを特徴とする。 The fungal culture method according to claim 3 is the method according to claim 2, wherein the basidiomycete mycelium medium comprising the liquid medium is supported on the reinforcing body during the mycelium growth step, During the fruiting body growth step, the medium for basidiomycetous fruiting bodies comprising the liquid medium is supported on the front surface side on the reinforcing body, or the medium for basidiomycetous fruiting bodies comprising the solid medium is the reinforcing body. Supported on the back side, or
During the mycelium growth step, the basidiomycetous mycelium medium comprising the solid medium is supported on the front side on the reinforcing body, and during the fruiting body growth step, the basidiomycetes comprising the liquid medium A medium for fruiting bodies is carried on the medium for basidiomycetous mycelium or the medium for basidiomycetous fruit bodies consisting of the solid medium is carried on the back side of the reinforcing body, or
During the mycelium growth step, the basidiomycete mycelium medium comprising the solid medium is supported on the front side on the reinforcing body, and during the fruit body growth step, from the basidiomycete mycelium medium The basidiomycete fruit body culture medium comprising the solid medium is supported on the back side of the reinforcing body from which the reinforcing body has been peeled off together with the mycelium.
　請求項４に記載の菌類培養方法は、請求項３に記載されたもので、前記菌糸体増殖工程中、菌糸体用フィルム上の層状の前記固体培地からなる前記担子菌類菌糸体用培地に、前記補強体を搭載して圧着し一体化してから、前記担子菌類の菌糸体の培養液を噴霧し、前記菌糸体育成培養シートを形成し、前記子実体増殖工程中、前記おもて面側で育成した前記菌糸体と共に前記補強体を、前記担子菌類菌糸体用培地及び菌糸体用フィルムから剥離した後、子実体用フィルム上の層状の前記固体培地からなる担子菌類子実体用培地を前記補強体にそのうら面側で圧着させて、前記子実体育成培養シートを形成することを特徴とする。 The fungal culture method according to claim 4 is the method according to claim 3, wherein during the mycelium growth step, the basidiomycete mycelium medium comprising the layered solid medium on the mycelium film, After the reinforcing body is mounted and crimped and integrated, the mycelium culture solution of the basidiomycetous fungus is sprayed to form the mycelium growth culture sheet, and during the fruit body growth step, the front surface side The reinforcing body together with the mycelium grown in step 1 is peeled off from the basidiomycete mycelium medium and the mycelium film, and then the basidiomycetous fruit body medium comprising the layered solid medium on the fruit body film is used. The fruit body growing culture sheet is formed by pressure-bonding the reinforcing body on the back surface side.
　請求項５に記載の菌類培養方法は、請求項１～４の何れかに記載されたもので、前記菌糸体育成培養シートを、ロール状に巻き取って前記菌糸体を育成し、及び／又は前記子実体育成培養シートを、ロール状に巻き取って前記子実体を育成することを特徴とする。 The method for culturing a fungus according to claim 5 is the method according to any one of claims 1 to 4, wherein the mycelium-growing culture sheet is rolled up to grow the mycelium, and / or The fruit body growing culture sheet is wound up in a roll shape to grow the fruit body.
　請求項６に記載の菌類培養方法は、請求項１～５の何れかに記載されたもので、前記担子菌類が、ヒダナシタケ目の菌類であることを特徴とする。 The method for culturing a fungus according to claim 6 is the method according to any one of claims 1 to 5, wherein the basidiomycete is a fungus of the order Hydrangea.
　請求項７に記載の菌類培養方法は、請求項６に記載されたもので、前記ハラタケ綱の菌類が、アンズタケ科、シロソウメンタケ科、ホウキタケ科、ハナビラタケ科、サンゴハリタケ科、サルノコシカケ科、タバコウロコタケ科、イドタケ科、ウロコタケ科、カンゾウタケ科、ニクハリタケ科、エゾハリタケ科、イボタケ科、ニンギョウタケモドキ科、コウヤクタケ科、マンネンタケ科、又はミヤマトンビマイ科の菌類であることを特徴とする。 The method for culturing fungi according to claim 7 is the method according to claim 6, wherein the fungus of the class Agaricaceae is a chanterelle family, a white mushroom family, a broom bamboo family, a red-branch family, a coral family, a sorghum family, a tobacco scale. It is characterized by being a fungus of the family Bambooceae, Idotake, Shirotake, Nikuharitake, Ezoharitake, Ibotake, Ningyotake, Pleurotus, Amanita, or Miyamatonbiidae.
　請求項８に記載の菌類培養方法は、請求項１～７の何れかに記載されたもので、前記菌糸体及び／又は前記子実体に、物理的刺激、電気的刺激、光学的刺激、化学的刺激、及び生物的刺激から選ばれる少なくとも何れかの処理を施すことを特徴とする。 The method for culturing fungi according to claim 8 is the method according to any one of claims 1 to 7, wherein the mycelium and / or the fruiting body is subjected to physical stimulation, electrical stimulation, optical stimulation, chemical It is characterized in that at least one treatment selected from a physical stimulus and a biological stimulus is performed.
　請求項９に記載の培養シートは、金属、合成樹脂、天然樹脂、及び／又は紙で形成された、メッシュ、織布、不織布、多孔質シートから選ばれ、そのおもて面で担子菌類の菌糸体を育成している補強体が、前記菌糸体から子実体へ育成している担子菌類子実体用培地を担持していることを特徴とする。 The culture sheet according to claim 9 is selected from a mesh, a woven fabric, a non-woven fabric, and a porous sheet formed of metal, synthetic resin, natural resin, and / or paper, and has a basidiomycetous fungus on its front surface. The reinforcing body for growing the mycelium carries the basidiomycete fruit body culture medium grown from the mycelium to the fruit body.
　請求項１０に記載の培養シートは、請求項９に記載されたもので、前記補強体は、液体培地又は固体培地からなり、前記菌糸体を育成した担子菌類菌糸体用培地が未担持補強体に担持され又は担持されてから剥離されており、液体培地又は固体培地からなる前記担子菌類子実体用培地が、前記補強体に担持されていることを特徴とする。 The culture sheet according to claim 10 is the one described in claim 9, wherein the reinforcing body is made of a liquid medium or a solid medium, and the medium for basidiomycetous mycelium in which the mycelium is grown is an unsupported reinforcing body. The basidiomycete fruiting body medium comprising a liquid medium or a solid medium is supported on or supported by the reinforcing body.
　請求項１１に記載の培養シートは、請求項９～１０の何れかに記載されたもので、網目状、格子状、ギャザー状、凹凸波形状、及び／又はすのこ状の間隙によって通気性を有するスペーサが、前記補強体に付されていることを特徴とする。 The culture sheet according to claim 11 is the culture sheet according to any one of claims 9 to 10, and has air permeability due to a mesh-like, lattice-like, gathered-like, irregular wave-like shape, and / or a saw-like gap. A spacer is attached to the reinforcing body.
　本発明の培養シートを用いた菌類培養方法によれば、担子菌類を、固体培養シートで菌糸体から子実体に、短期間で育成することができる。固体培養シートを巻き取り定温で静置することにより、狭いスペースで、大量に培養できる。固体培養シートを用いるから、均一な品質で効率良く、簡便に培養できる。 According to the fungal culture method using the culture sheet of the present invention, basidiomycetes can be grown from mycelium to fruiting bodies in a short period of time on a solid culture sheet. By winding the solid culture sheet and allowing it to stand at a constant temperature, it can be cultured in a large amount in a narrow space. Since a solid culture sheet is used, it can be cultured efficiently and easily with uniform quality.
本発明を適用する菌類培養方法を示す概要図である。It is a schematic diagram which shows the fungi culture | cultivation method to which this invention is applied. 本発明を適用する別な菌類培養方法を示す概要図である。It is a schematic diagram which shows another fungi culture | cultivation method to which this invention is applied.
　以下、本発明を実施するための形態を詳細に説明するが、本発明の範囲はこれらの形態に限定されるものではない。 Hereinafter, modes for carrying out the present invention will be described in detail, but the scope of the present invention is not limited to these modes.
　本発明の菌類培養方法は、図１を参照しながら説明する。この菌類培養方法は、菌糸体育成固体培養シート１と子実体育成固体培養シート２とを用いて、以下の手順で行われる。 The fungal culture method of the present invention will be described with reference to FIG. This fungal culture method is performed using the mycelium growing solid culture sheet 1 and the fruit body growing solid culture sheet 2 according to the following procedure.
　先ず、菌糸体増殖工程［Ｉ］が行われる。金属、合成樹脂、天然樹脂、紙で、メッシュ、織布、不織布、多孔質シートの凹凸又は孔によって培地の担持が可能な補強体１５を、形成し、巻き取っておく。非浸透性で樹脂製の菌糸体用フィルムロールから菌糸体用フィルム１１を引き出す。担子菌類菌糸体用培地塗布ノズル１２で担子菌類菌糸体用培地塗布液１３を、菌糸体用フィルム１１へおもて面側で、塗布し又は吹き付け、固体培地層である菌糸体育成寒天培地層になる層状の担子菌類菌糸体用培地１４を形成する。ロール状の補強体１５を、引き出して、担子菌類菌糸体用培地１４へ圧着して付すと、補強体１５と担子菌類菌糸体用培地１４とが、一体化する。補強体１５のおもて面で担子菌類菌糸体用培地１４に、菌糸体培養液噴霧ノズル１６から、液体培養された担子菌類の菌糸体培養噴霧液１７を噴霧すると、担子菌類２０が、担子菌類菌糸体用培地１４に、接種される。必要に応じて、さらにその上に、巻取り時の密着防止用の網状・格子状又はすのこ状のスペーサ（不図示）を付す。これにより、菌糸体育成固体培養シート１が、得られる。菌糸体育成固体培養シート１を、シート同士が密着し合わないように緩くロール状に巻き取り、静置する。その間、担子菌類２０は、担子菌類菌糸体用培地１４で、育成し、四方八方へ平面的に増殖し、担子菌類２０の菌糸体層を形成する。 First, the mycelium growth step [I] is performed. A reinforcing body 15 capable of supporting a culture medium is formed and wound up with metal, synthetic resin, natural resin, paper, mesh, woven fabric, nonwoven fabric, and irregularities or holes of a porous sheet. The mycelium film 11 is pulled out from the non-permeable resinous mycelium film roll. The basidiomycete mycelium medium coating solution 13 is applied or sprayed to the mycelium film 11 on the front surface side with a basidiomycete mycelium medium application nozzle 12 to form a mycelium growth agar medium layer which is a solid medium layer. A layered basidiomycete mycelium medium 14 is formed. When the roll-shaped reinforcing body 15 is pulled out and pressed onto the basidiomycete mycelium medium 14, the reinforcing body 15 and the basidiomycete mycelium medium 14 are integrated. When a mycelium culture spray solution 17 of basidiomycetous liquid cultured on the basidiomycete mycelium medium 14 is sprayed onto the basidiomycete mycelium medium 14 from the mycelium culture solution spray nozzle 16, the basidiomycetes 20 become basidiomytes. The medium 14 for fungal mycelium is inoculated. If necessary, a net-like / lattice-like or saw-like spacer (not shown) for preventing adhesion during winding is further provided thereon. Thereby, the mycelium growing solid culture sheet 1 is obtained. The mycelium growing solid culture sheet 1 is loosely wound up in a roll shape so that the sheets do not adhere to each other, and is left to stand. Meanwhile, the basidiomycetes 20 are grown on the basidiomycete mycelium medium 14 and are planarly grown in all directions to form a mycelium layer of the basidiomycetes 20.
　次いで、子実体増殖工程［II］が行われる。菌糸体育成固体培養シート１から、担子菌類菌糸体用培地１４ごと菌糸体用フィルム１１を剥ぎ取る。すると補強体１５上におもて面側で、担子菌類２０の菌糸体層が残存する。非浸透性で樹脂製の子実体用フィルムロールから子実体用フィルム２１を引き出す。担子菌類子実体用培地ノズル２２で担子菌類子実体用培地塗布液２３を、子実体用フィルム２１へおもて面で、塗布し又は吹き付け、子実体育成寒天培地層になる層状の担子菌類子実体用培地２４を形成する。補強体１５を、そのうら面側で、子実体用フィルム２１上の担子菌類子実体用培地２４へ圧着して付すと、補強体１５と担子菌類子実体用培地２４とが一体化する。担子菌類２０の菌糸体層は、担子菌類子実体用培地２４と接触し、育成可能となり、増殖する。これにより、子実体育成固体培養シート２が得られる。子実体育成固体培養シート２を、シート同士が密着し合わないように緩くロール状に巻き取り、静置する。その間、担子菌類２０は、担子菌類子実体用培地２４で、菌糸体から子実体へ成長して育成し、四方八方へシート上で層状に増殖し、担子菌類２０の子実体層を形成する。 Next, the fruiting body growth step [II] is performed. The mycelium film 11 is peeled off from the mycelium-growing solid culture sheet 1 together with the basidiomycete mycelium medium 14. Then, the mycelium layer of the basidiomycetes 20 remains on the front side on the reinforcing body 15. The fruiting body film 21 is pulled out from the resinous fruiting body film roll which is impermeable. A basidiomycetous fruit body medium coating liquid 23 is applied or sprayed onto the fruit body film 21 with the basidiomycetous fruit body medium nozzle 22 on the front surface to form a fruit body growing agar medium layer. An entity medium 24 is formed. When the reinforcing body 15 is pressure-bonded to the basidiomycetous fruit body medium 24 on the fruit body film 21 on the back side, the reinforcing body 15 and the basidiomycete fruit body medium 24 are integrated. The mycelium layer of the basidiomycetes 20 comes into contact with the basidiomycetous fruiting medium 24 and can be grown and grows. Thereby, the fruit body growing solid culture sheet 2 is obtained. The fruit body growing solid culture sheet 2 is loosely wound up in a roll shape so that the sheets do not adhere to each other, and is left to stand. Meanwhile, the basidiomycetes 20 grow and grow from mycelium to fruiting bodies in the basidiomycetous fruiting medium 24, and grow in layers on the sheet in all directions to form a fruiting body layer of the basidiomycetes 20.
　菌糸体育成固体培養シート１や子実体育成固体培養シート２は巻取り時にスペーサによって間隙が生じて密着が阻害され、湿度及び通気状態を維持できる。担子菌類２０の菌糸体層や子実体層は、スペーサの網目状、格子状、又はすのこ状間隙を避けて、増殖する。 The mycelium-growing solid culture sheet 1 and the fruit body-growing solid culture sheet 2 can maintain a humidity and aeration state because a gap is generated by a spacer during winding and the adhesion is inhibited. The mycelium layer and fruiting body layer of the basidiomycetes 20 grow while avoiding the mesh-like, lattice-like, or saw-like gaps of the spacers.
　菌糸体増殖工程［Ｉ］及び子実体増殖工程［II］中、何れも担子菌類菌糸体用培地及び担子菌類子実体用培地として、固体培地を用いた例を示したが、何れか一方を液体培地とし他方を固体培地としてもよく、何れも液体培地としてもよい。例えば、別な菌類培養方法として、図２を参照しながら説明すると、以下の手順で行われる。 In the mycelium growth step [I] and the fruiting body growth step [II], both showed examples in which a solid medium was used as a basidiomycete mycelium medium and basidiomycete fruiting body medium. The medium may be a solid medium, and the other may be a liquid medium. For example, another fungal culture method will be described with reference to FIG. 2 according to the following procedure.
　先ず、前記と同様にして菌糸体増殖工程［Ｉ］を行うことによって、菌糸体育成固体培養シート１を調製し、巻き取って静置して、担子菌類２０を育成して増殖させ、担子菌類２０の菌糸体層を形成する（図１参照）。次いで、図２に示すように、子実体増殖工程［II］が行われる。菌糸体育成固体培養シート１のおもて面側の担子菌類菌糸体用培地１４に、担子菌類子実体用培地ノズル２２で担子菌類子実体用培地塗布液２３を、吹き付け、子実体育成液体培地層になる担子菌類子実体用培地２４を形成する。これにより、子実体育成固体培養シート２が得られる。子実体育成固体培養シート２を、シート同士が密着し合わないように緩くロール状に巻き取り、静置する。その間、担子菌類２０は、担子菌類子実体用培地２４で、菌糸体から子実体へ成長して育成し、平面的に増殖し、担子菌類２０の子実体層を形成する。 First, by performing the mycelium growth step [I] in the same manner as described above, the mycelium-growing solid culture sheet 1 is prepared, wound and allowed to stand, and the basidiomycetes 20 are grown and propagated. 20 mycelium layers are formed (see FIG. 1). Next, as shown in FIG. 2, a fruit body growth step [II] is performed. The basidiomycetous fungus body medium coating solution 23 is sprayed onto the basidiomycete mycelium medium 14 on the front side of the mycelium-growing solid culture sheet 1 with the basidiomycetous fruit body medium nozzle 22 to spray the fruiting body growing liquid medium. The basidiomycetous fruiting body medium 24 to be layered is formed. Thereby, the fruit body growing solid culture sheet 2 is obtained. The fruit body growing solid culture sheet 2 is loosely wound up in a roll shape so that the sheets do not adhere to each other, and is left to stand. Meanwhile, the basidiomycetes 20 grow from the mycelium to the fruiting bodies and grow in the basidiomycetous fruiting medium 24 and multiply in a planar manner to form a fruiting body layer of the basidiomycetes 20.
　菌類培養方法は、担子菌類を培養するのに適している。担子菌類は、きのこ　新装版山渓フィールドブックス７（２００６年６月１０日、初版第１刷、山と渓谷社発行）の分類に拠って表わすと、菌界－担子菌亜門－真正担子菌綱－帽菌亜綱－ヒダナシタケ目であることが好ましい。ヒダナシタケ目は、例えばアンズタケ科、シロソウメンタケ科、ホウキタケ科、ハナビラタケ科、サンゴハリタケ科、サルノコシカケ科、タバコウロコタケ科、イドタケ科、ウロコタケ科、カンゾウタケ科、ニクハリタケ科、エゾハリタケ科、イボタケ科、ニンギョウタケモドキ科、コウヤクタケ科、マンネンタケ科、ミヤマトンビマイ科の菌類が挙げられる。より具体的には、アンズタケ（Cantharellus cibarius）、シロソウメンタケ（Clavaria vermicularis）、ホウキタケ(Ramaria botrytis)、ハナビラタケ（Sparassis crispa）、サンゴハリタケ(Hericium ramosun)、フサハリタケ(Creoloplus cirrbatus)、カノシタ（Hydnum repandum）、ブナハリタケ（Mycoleptodonoides aitcbisonii）、マイタケ（Grifola frondosa）、オオチリメンタケ（Trametes gibbosa）、ヒイロハリタケ（Phanerochaete chrysorbiza）、オリーブウロコタケ（Lopharia cinerascens）、カンゾウタケ（Fistulina hepatica）、イボラシャタケ（Tomentella crinalis）、カラスタケ（Polyzellus multiplex）、ニンギョウタケ（Albatrellus confluens）、アオロウジ（Albatrellus caeruleoporus）、ヌメリアイタケ（Albatrellus yasudai）、コウタケ（Sarcodon aspratus）、クロカワ（Boletopsis leucomelas）、センニンタケ（Albatrellus pascaprae）、チョレイマイタケ（Polyporus umbellatus）、マスタケ（Laetiporus sulphureus）、ヒイロタケ（Pycnoporus coccineus）、ヒメシロアミタケ(Antrodia albida)、ミヤマシロアミタケ（Antrodia beteromorpha）、ベニクスノキタケ（Antrodia camphorata）、Antrodia salmonea（和名なし）、クロサルノコシカケ（Melanoporia castanea）、アナタケ（Schizopora flavipora）、ツガノマンネンタケ（Ganoderma mastporum）、カバノアナタケ（Inonotus oblicuus）、カワタケ（Peniophora quercina）等が挙げられる。 The fungal culture method is suitable for culturing basidiomycetes. Basidiomycetes can be expressed according to the classification of Mushrooms, Newly-developed Sankei Field Books 7 (June 10, 2006, first edition, first edition, published by Yamato Valley Co., Ltd.). It is preferable that the order is Lepidoptera-H. Subtilis-Hymenaceae. For example, chanterelles, sphagnum mushrooms, blossoms mushrooms, blossoms mushrooms, coral mushrooms, sorghum mushrooms, cigarette mushrooms, iridaceae, mushroom mushrooms, mushroom mushrooms, mushroom mushrooms, mushroom mushrooms, Family fungi of the family, Koyakutake department, Manentake department, Miyamatonbimai family. More specifically, chanterelles (Cantharellus cibarius), white-spotted bamboo (Clavaria vermicularis), broom bamboo (Ramaria ハ botrytis), red-bellied bamboo (Sparassis crispa), coral harimatake (Hericium ramosun), fusahari cyno (bat) Beech haritake (Mycoleptodonoides aitcbisonii), Maitake mushroom (Grifola 、 frondosa), Giant mushroom (Trametes gibbosa), Agaric mushroom (Phanerochaete chrysorbiza), Olive mushroom (Lopharia cinerascens), Pepper mushroom (patite) ), Ningyotake (Albatrellus confluens), Aurouji (Albatrellus caeruleoporus), Numeraitake (Albatrellus yasudai), Kotake (Sarcodon aspratus), Kurokawa (Boletopsis leucomelas), Sennintake (Albatrellus pas) caprae), choreimaitake (Polyporus umbellatus), mustard (Laetiporus sulphureus), oyster mushroom (Pycnoporus coccineus), Japanese oyster mushroom (Antrodia albida), yamashiroamitake (Antrodia beteromorpha) No Japanese name), Melanoporia castanea, Anatake (Schizopora flavipora), Tsunonomanentake (Ganoderma mastporum), Bamboo shoot (Inonotuslicoblicuus), Kawatake (Peniophora quercina), and the like.
　担子菌類菌糸体用培地や担子菌類子実体用培地は、寒天培地のような固体培地であっても液体培地であってもよい。 The medium for basidiomycete mycelium and the medium for basidiomycete fruit bodies may be a solid medium such as an agar medium or a liquid medium.
　固体培地は、例えば、培地１Ｌ中に、ポリアルギン酸塩、寒天等のハイドロゲル形成ポリマーを単独、或いは混合し、その３～２０ｇ、ポテト抽出物の３～５ｇ、グルコース、デキストロース、フルクトース、シュークロース、セロビオース、又はトレハロースの単独、又はそれらのうちの何れかの混合物のような糖類の１０ｇ～３０ｇが含まれている。必要に応じて、ｐＨ調整剤、窒素源やミネラル源となる無機塩類、おがくず、ハト麦、オーツ麦や小麦、大麦、麹を始めとした動植物体やそれらの抽出物、二次代謝物などを適宜加えてもよい。 The solid medium is, for example, a hydrogel-forming polymer such as polyalginate and agar in 1 L of medium alone or mixed, 3 to 20 g thereof, 3 to 5 g of potato extract, glucose, dextrose, fructose, sucrose , Cellobiose, or trehalose alone, or 10 to 30 g of a saccharide such as a mixture of any of them. As necessary, pH regulators, inorganic salts that serve as nitrogen and mineral sources, sawdust, pigeons, oats and wheat, barley, straw, and other animals and plants, their extracts, secondary metabolites, etc. You may add suitably.
　液体培地は、例えば、培地１Ｌ中に、酵母エキスと麦芽エキスとの各１０～３０ｇ、デキストロース、デキストロース、フルクトース、シュークロース、セロビオース、トレハロースの単独、又はそれらのうちの何れかの混合物のような糖類の１０ｇ～３０ｇが含まれている。必要に応じてｐＨ調整、窒素源やミネラル源となる無機塩類、動植物抽出物やおがくず、ハト麦、オーツ麦、小麦、大麦、麹を始めとした動植物体やそれらの抽出物、二次代謝物などを適宜加えてもよい。 The liquid medium is, for example, 10 to 30 g each of yeast extract and malt extract in 1 L of medium, dextrose, dextrose, fructose, sucrose, cellobiose, trehalose alone, or a mixture of any of them. Contains 10-30 g of sugar. Adjust pH as necessary, inorganic salts as nitrogen and mineral sources, animal and plant extracts and sawdust, animal and plant bodies such as wheat, oats, wheat, barley and straw, their extracts, secondary metabolites Etc. may be added as appropriate.
　子実体育成の際に子実体育成固体培養シート２に、それの形成時、又は形成から１箇月経過時までの間に、形成物理的刺激、電気的刺激、光学的刺激、生物的刺激、化学的刺激の少なくとも何れかの処理を、施してもよい。物理的刺激、電気的刺激、光学的刺激として、２０～１００ｍＷにて、１０ナノ秒～１００ミリ秒、好ましくは数十ナノ秒～数十ミリ秒の間隔でのパルスレーザーの照射処理、２０～９０ｋＶ好ましくは３０～７０ｋＶで１００μＡ～１ｍＡにて１００～５００ミリ秒間隔で３０～６０秒間のパルスプラズマ発生装置によるプラズマ処理、及び波長が５００～８００ｎｍの光照射処理が、挙げられる。物理的刺激、電気的刺激、光学的刺激の中でも、物理的刺激や電気的刺激は、直接、子実体育成固体培養シート２の子実体へ向けて施さなくてもよい。例えば、図２に示すように、パルス電圧電極２５ａでパルス電圧２６ａを子実体育成固体培養シート２に向け生じさせ、及び／又はパルス電圧電極２５ｂでパルス電圧２６ｂを子実体育成固体培養シート２の上方でそれへ向けずに大気殺菌のために生じさせてもよい。一例として５０～９０ｋＶ好ましくは７０～９０ｋＶで１００μＡ～１ｍＡにて１００～５００ミリ秒間隔で１～１０秒間のパルスプラズマ発生装置によるプラズマ処理を毎分１回照射し続ける。生物的刺激、化学的刺激として、乳酸菌、酵母、糀菌のような子実体生育阻害しない異種の菌類の熱処理物、又は子実体育成すべき同種の菌類の別ロットの生菌類や熱処理物を共存させたり、テルペン類やテルペン類を産生している樟や牛樟のおがくずを混入させたりする処理が、挙げられる。形成物理的刺激、電気的刺激、光学的刺激、生物的刺激、化学的刺激のような処理により、子実体の生成量・生成速度・生成密度が、大幅に向上し、例えば、パルス電圧処理やパルスレーザー照射処理で、生成量が、約２０～５０％増量する。 When the fruit body is grown, it is formed on the solid body-cultivating solid culture sheet 2, or during the formation of the fruit body, or after the formation of one month, physical stimulation, electrical stimulation, optical stimulation, biological stimulation, chemistry You may perform the process of at least any one of an irritation | stimulation. As physical stimulation, electrical stimulation, and optical stimulation, irradiation with pulse laser at 20 to 100 mW at intervals of 10 nanoseconds to 100 milliseconds, preferably several tens of nanoseconds to several tens of milliseconds, 20 to Examples include plasma treatment with a pulsed plasma generator at 90 kV, preferably 30 to 70 kV and 100 μA to 1 mA at intervals of 100 to 500 milliseconds for 30 to 60 seconds, and light irradiation treatment with a wavelength of 500 to 800 nm. Among physical stimulation, electrical stimulation, and optical stimulation, physical stimulation and electrical stimulation may not be applied directly to the fruit body of the fruit body growing solid culture sheet 2. For example, as shown in FIG. 2, a pulse voltage 26a is generated by the pulse voltage electrode 25a toward the fruit body growing solid culture sheet 2, and / or the pulse voltage 26b is generated by the pulse voltage electrode 25b of the fruit body growing solid culture sheet 2. It may be generated for air sterilization without directing it upwards. As an example, plasma treatment with a pulsed plasma generator at 50 to 90 kV, preferably 70 to 90 kV, 100 μA to 1 mA and 100 to 500 milliseconds for 1 to 10 seconds is continued once per minute. As biological and chemical stimuli, coexisting heat-treated products of heterogeneous fungi such as lactic acid bacteria, yeast, and koji molds that do not inhibit fruit body growth, or live bacteria and heat-treated products of different lots of the same type of fungi to grow fruit bodies And treatment of mixing terpenes and sawdust from beef cake producing terpenes and terpenes. Processes such as formation physical stimulation, electrical stimulation, optical stimulation, biological stimulation, and chemical stimulation significantly improve the production amount, generation rate, and generation density of fruiting bodies. With the pulsed laser irradiation treatment, the production amount is increased by about 20 to 50%.
　補強体１５は、菌体培養用メッシュであり、例えば、目開きが５０～１０００ミクロンで厚さが３０～３００ミクロンであって、素材が金属（例えば、ステンレス）、合成樹脂（例えば、ナイロン、ポリエステル、ポリエチレン、ポリプロピレン、フッ素繊維、ポリ塩化ビニリデン、ポリビニルアルコール、エチレン－酢酸ビニル共重合体、ポリウレタン、ポリセルロースなど）のメッシュクロスや多孔質シート、天然繊維（例えば、絹、綿）の織布、紙、不織布が挙げられる。その幅は、例えば２００～１１００ｍｍである。 The reinforcing body 15 is a cell culture mesh, for example, having an opening of 50 to 1000 microns and a thickness of 30 to 300 microns. The material is a metal (for example, stainless steel), a synthetic resin (for example, nylon, Polyester, polyethylene, polypropylene, fluorine fiber, polyvinylidene chloride, polyvinyl alcohol, ethylene-vinyl acetate copolymer, polyurethane, polycellulose, etc.) mesh cloth and porous sheet, woven fabric of natural fibers (eg silk, cotton) , Paper, and non-woven fabric. The width is, for example, 200 to 1100 mm.
　菌糸体用フィルム１１や子実体用フィルム２１は、支持フィルムであり、ポリエステル、ポリプロピレン、ポリ塩化ビニリデンで例示される耐熱性の樹脂で形成されている。その厚さは、例えば１０～２００ミクロンであり、その幅は、例えば２００～１１００ｍｍである。 The mycelium film 11 and the fruit body film 21 are support films, and are formed of a heat-resistant resin exemplified by polyester, polypropylene, and polyvinylidene chloride. The thickness is, for example, 10 to 200 microns, and the width is, for example, 200 to 1100 mm.
　菌類培養方法の際、必要に応じて、殺菌、担子菌類の発芽促進のため、パルス電圧処理、パルスレーザー照射処理、プラズマ処理、光照射処理のような前処理を、行ってもよい。 In the fungus culture method, pretreatment such as pulse voltage treatment, pulse laser irradiation treatment, plasma treatment, and light irradiation treatment may be performed as necessary for sterilization and germination promotion of basidiomycetes.
　スペーサは、網目状、格子状、例えば１～３ｃｍ間隔にひだ折りしたギャザー状、０．１～３ｃｍ間隔で四方へ正弦波状・コーン状・釣鐘状・円錐台状の連続した凹凸を有する凹凸波形状、及び／又はすのこ状の間隙によって通気性を有していてもよい。この凹凸は、菌糸体育成固体培養シート１同士や子実体育成固体培養シート２同士を線接触・点接触させて、通気性を持たせるものである。 The spacer is a mesh or lattice, for example, a gathered shape that is creased at intervals of 1 to 3 cm, and a concavo-convex wave having continuous irregularities of sine wave shape, cone shape, bell shape, or truncated cone shape at intervals of 0.1 to 3 cm. It may be air permeable due to its shape and / or slat-like gap. This unevenness is to allow the mycelium-growing solid culture sheets 1 or the fruiting body-growing solid culture sheets 2 to be in line contact / point contact with each other to give air permeability.
　菌糸体育成固体培養シート１や子実体育成固体培養シート２は、延ばしたまま用いても、折り曲げ又は巻き取って用いてもよく、適当な大きさで裁断して用いてもよい。連続した長尺のシートをロール状に巻き取って使用すると、狭いスペースでの生産性の向上に資する。 The mycelium-growing solid culture sheet 1 and the fruit body-growing solid culture sheet 2 may be used while being stretched, or may be used by being folded or wound, or may be used after being cut to an appropriate size. When a continuous long sheet is rolled up and used, it contributes to an improvement in productivity in a narrow space.
　本発明を適用する培養シートを用いた菌類培養方法により、ベニクスノキタケを培養した例を示す。なお、特に商品名・メーカーを記した試薬以外は、市販の試薬特級を用いた。 An example of cultivating Benicaria mushroom by a fungal culture method using a culture sheet to which the present invention is applied is shown. In addition, a reagent special grade which is commercially available was used except for the reagent in which the trade name and manufacturer were written.
（調製例）
（１）培地の調製
　ジャガイモ２００ｇの皮を剥き、１ｃｍ角に切り、１Ｌの沸騰水中で２０分間茹で、ガーゼで濾したもの、グルコース２０ｇにイオン交換水を加え全量を１Ｌとし、１２１℃、２０分間オートクレーブ処理してポテトデキストロース培地（以下、ＰＤＢ培地という）を調製した。この時リン酸緩衝液にてｐＨを５～７に調製した。
　また、ジャガイモ２００ｇの皮を剥き、１ｃｍ角に切り、１Ｌの沸騰水中で２０分間茹で、ガーゼで濾したもの、グルコース２０ｇに寒天（日水製薬株式会社製）１５～２０ｇを加えて攪拌し、イオン交換水を加え全量を１Ｌとしフラスコに移し、１２１℃で１５分間、オートクレーブ処理し、寒天培地（以下、ＰＤＡ培地という）を調製した。この時リン酸緩衝液にてｐＨを５～７に調製した。
　なお、得られた培地は無菌ドラフト内で（ｉ）固体培養の場合、直径９ｃｍのペトリ皿に、ＰＤＡ培地を１５～２０ｍＬを加え冷却した。（ｉｉ）液体培養の場合、ＰＤＢ培地の入ったフラスコを常温に冷却したのち接種及び培養した。培養は、全てインキュベーター内で、２５℃の定温で行った。 (Preparation example)
(1) Preparation of medium Peel 200 g of potato, cut into 1 cm square, boiled in 1 L boiling water for 20 minutes, filtered with gauze, added ion exchange water to 20 g of glucose to a total volume of 1 L, 121 ° C., 20 A potato dextrose medium (hereinafter referred to as PDB medium) was prepared by autoclaving for minutes. At this time, the pH was adjusted to 5 to 7 with a phosphate buffer.
Also, peel off 200 g of potato, cut into 1 cm square, boiled in 1 L boiling water for 20 minutes, filtered with gauze, added 15 to 20 g of agar (Nissui Pharmaceutical Co., Ltd.) to 20 g of glucose, and stirred. Ion exchanged water was added to make the total volume 1 L, transferred to a flask, and autoclaved at 121 ° C. for 15 minutes to prepare an agar medium (hereinafter referred to as PDA medium). At this time, the pH was adjusted to 5 to 7 with a phosphate buffer.
The obtained medium was cooled in a sterile draft by adding 15 to 20 mL of PDA medium to a petri dish having a diameter of 9 cm in the case of (i) solid culture. (Ii) In the case of liquid culture, the flask containing the PDB medium was inoculated and cultured after cooling to room temperature. All the cultures were performed at a constant temperature of 25 ° C. in an incubator.
（２）ベニクスノキタケの野生種の固体継体培養
　ガスバーナーの火炎により消毒したピンセットで、ベニクスノキタケの野生種の裏側の組織の２ｍｍ×３ｍｍ片を剥がし、ＰＤＡ培地に接種した。１０日後、直径約２．５ｃｍのコロニーとなった菌糸体を、５ｍｍ四方の角片として切り出し、新しいＰＤＡ培地に接種した。その後、約２１日ごとに、継体培養を実施し、本発明の菌類培養方法に供した。
　なお、継体培養中、第１世代から第１０世代まで、育成速度に変化が認められなかった。ＤＮＡアナライザーを用いて第２世代菌糸体について、ＤＮＡ分析を行った結果、塩基配列が９６％（２６６／２７６）の相同性を示し、この菌糸体が、ベニクスノキタケであると同定された。 (2) Solid Passage Culture of Wild Species of Benicus mushroom A 2 mm x 3 mm piece of the tissue behind the wild Species of Benicus mushroom was peeled off with tweezers sterilized by a flame of a gas burner, and inoculated into PDA medium. Ten days later, the mycelium that became a colony having a diameter of about 2.5 cm was cut out as a 5 mm square piece and inoculated into a new PDA medium. Thereafter, the passage culture was carried out approximately every 21 days and used for the fungal culture method of the present invention.
In addition, during the subculture, no change was observed in the growth rate from the first generation to the tenth generation. As a result of DNA analysis of the second generation mycelium using a DNA analyzer, the base sequence showed a homology of 96% (266/276), and this mycelium was identified as Benicus nokitake.
（３）固体培地の最適化
（３－１）培地の選定
　ＰＤＡ培地中、糖類であるグルコースと、それに代えてデキストロース、フルクトース、又はスクロースとをそれぞれ炭素源として０．５％、１．０％、１．５％、２．０％、及び３．０％添加した被検試料を、別々に調製し、継体培養したベニクスノキタケ株を３０日間培養した後、菌糸体のコロニーの直径を計測した。それぞれのコロニー直径は、グルコースとデキストロース又はフルクトースとを含む培地で１．０％以上の場合略同じく５０ｍｍΦ程度であり、スクロースを含む培地で濃度非依存的に２５ｍｍΦ程度であった。このことから、グルコース、デキストロース、フルクトース、とりわけ安価なグルコースが、ベニクスノキタケ菌糸体の培養に適していた。カンファーを５００ｐｐｍ混ぜ合わせたものはともに６０ｍｍΦとなり、これらは生育促進に適していた。 (3) Optimization of solid medium (3-1) Selection of medium In PDA medium, glucose, which is a saccharide, and dextrose, fructose, or sucrose instead of 0.5% and 1.0%, respectively, as a carbon source , 1.5%, 2.0%, and 3.0% test samples were prepared separately, and after passage culture, the diameter of the mycelium colony was measured. did. Each colony diameter was about 50 mmΦ in the case of 1.0% or more in a medium containing glucose and dextrose or fructose, and about 25 mmΦ in a concentration-independent medium in a medium containing sucrose. For this reason, glucose, dextrose, fructose, and particularly inexpensive glucose were suitable for cultivation of Benix noctum mycelium. The mixture of camphor with 500 ppm both had a diameter of 60 mm, which was suitable for promoting growth.
（３－２）子実体育成の培地の選定
　培地１Ｌ中に、酵母エキス、麦芽エキス、各１０ｇ、デキストロース２０ｇ、寒天１５ｇ、ハト麦破砕物１００ｇから成る培地に、継体培養したベニクスノキタケ株を、接種してから３ヶ月間培養した後、菌体の一部を顕微鏡で観察したところ、子実体形成を示すクランプ及び胞子の存在が、確認できた。また、得られた菌体を乾燥後、メタノールで抽出し高速液体クロマトグラフ法（ＨＰＬＣ）分析を行ったところ、子実体含有成分であるエルゴステロール類も検出された。これらのことから、菌糸体育成には、ＰＤＡ培地が適しており、子実体育成には、上記培地が適していることが分かった。 (3-2) Selection of fruiting body growth medium In 1 L of medium, Benix nokitake strain that has been subcultured in a medium consisting of 10 g of yeast extract, malt extract, 20 g of dextrose, 15 g of agar, and 100 g of crushed pigeons. After culturing for 3 months after inoculation, a part of the cells were observed with a microscope, and the presence of clamps and spores indicating fruiting body formation could be confirmed. Moreover, when the obtained microbial cells were dried and extracted with methanol and subjected to high performance liquid chromatography (HPLC) analysis, ergosterols as fruit body-containing components were also detected. From these results, it was found that PDA medium is suitable for mycelium growth, and that the medium is suitable for fruiting body growth.
（４）液体培地の最適化
（４－１）液体培養可能性検討
　３００ｍＬのエレンマイヤーフラスコに、ＰＤＢ培地の２００ｍＬを加え、滅菌後、ベニクスノキタケを担持した５ｍｍΦのＰＤＡ培地を５個添加し、２５℃に保った恒温室で毎分１５０回転で振蘯下、７日間液体培養し、菌糸体を得た。 (4) Optimization of liquid medium (4-1) Examination of liquid culture feasibility Add 200 mL of PDB medium to a 300 mL Erlenmeyer flask, and after sterilization, add five 5 mmΦ PDA medium carrying Benix nokitake. Then, liquid culture was performed in a thermostatic chamber maintained at 25 ° C. with shaking at 150 rpm for 7 days to obtain mycelia.
（実施例１：平面状固体培養シートでの培養）
　２５×３０ｃｍの大きさのバットに、２０×２５ｃｍの厚さ１００ミクロンの菌糸体用フィルム１１であるポリエチレンテレフタレート（ＰＥＴ）フィルムを敷いた。その上に凡そ１５×２０ｃｍとなるように、予め１２１℃で３０分間オートクレーブ処理し、６０℃以下に冷却した担子菌類菌糸体用培地１４であるＰＤＡ培地の約１５０ｇを流した後、１５×２０ｃｍの補強体１５であるＰＥＴメッシュを敷き、培地を調製した。この培地を室温に冷却した。予めＰＤＢ培地で液体培養し、ホモジナイザーで均質化したベニクスノキタケ培養液を、この培地に均一に塗付し、菌糸体育成固体培養シート１を得た。このバットを無菌の下２５℃で３０日間培養した。
　得られた菌糸体付着メッシュを担子菌類２０ごと、上記実施例３の（３－２）に記載の培地上に移殖し、子実体育成固体培養シート２を得た。これを６０日間培養した。この時３０ｋＶで４００μＡにて１００ミリ秒間隔で３０秒間のパルスプラズマ発生装置によるプラズマ処理を培養開始から２４時間ごとに一日１回ずつ２日間施し、得られたメッシュ上の培養物は、顕微鏡下で、胞子及びクランプが観察され、また、得られた菌体を乾燥後、メタノールで抽出しＨＰＬＣ分析を行ったところ子実体含有成分であるエルゴステロール類も検出された。このことにより、子実体が生成されていると確認できた。 (Example 1: Culture on a planar solid culture sheet)
A polyethylene terephthalate (PET) film, which is a mycelium film 11 having a thickness of 100 microns and a thickness of 20 × 25 cm, was laid on a bat having a size of 25 × 30 cm. After flowing about 150 g of PDA medium, which is a basidiomycete mycelium medium 14, which was autoclaved at 121 ° C. for 30 minutes in advance and cooled to 60 ° C. or less so as to be approximately 15 × 20 cm, 15 × 20 cm A medium was prepared by laying a PET mesh, which is a reinforcing member 15 of the above. The medium was cooled to room temperature. A liquid culture of Benix nokitake, which had been previously cultured in a PDB medium and homogenized with a homogenizer, was evenly applied to this medium to obtain a mycelium-growing solid culture sheet 1. The vat was cultured at 25 ° C. under aseptic conditions for 30 days.
The resulting mycelium-attached mesh was transferred together with the basidiomycetes 20 onto the medium described in Example 3 (3-2) to obtain a fruit body growing solid culture sheet 2. This was cultured for 60 days. At this time, plasma treatment with a pulsed plasma generator at 30 kV and 400 μA at intervals of 100 milliseconds for 30 seconds was performed once every day for 24 days from the start of the culture for 2 days. Below, spores and clamps were observed, and the obtained microbial cells were dried, extracted with methanol, and subjected to HPLC analysis. As a result, ergosterols, which are fruit body-containing components, were also detected. This confirmed that a child entity was generated.
（実施例２：ロール状固体培養シートでの培養）
　実施例１において、ＰＥＴフィルム、ＰＥＴメッシュとして幅３００ｍｍのロール状ＰＥＴフィルムとし、各工程において直径１～２ｍｍの多孔質塩化ビニル樹脂で出来た１ｃｍ角の網目状スペーサを層間にはさみながら巻き取ること以外は実施例１と同様にして、菌糸体育成固体培養シート１、子実体育成固体培養シート２を作製し、３０ｋＶで４００μＡにて１００ミリ秒間隔で３０秒間のパルスプラズマ発生装置によるプラズマ処理を培養開始から２４時間ごとに一日１ずつ回２日間施し、６０日後、メッシュ上の培養物は、顕微鏡下で、胞子及びクランプが観察され、また、得られた菌体を乾燥後、メタノールで抽出しＨＰＬＣ分析を行ったところ子実体含有成分であるエルゴステロール類も検出された。このことにより、子実体が生成されていると確認できた。 (Example 2: Culture in a roll-shaped solid culture sheet)
In Example 1, a PET film, a PET film and a rolled PET film having a width of 300 mm are used, and a 1 cm square mesh spacer made of porous vinyl chloride resin having a diameter of 1 to 2 mm is wound between layers in each step. In the same manner as in Example 1, the mycelium growing solid culture sheet 1 and the fruiting body growing solid culture sheet 2 were prepared and subjected to plasma treatment with a pulse plasma generator for 30 seconds at 400 μA at 30 kV and at intervals of 100 milliseconds. After 24 days from the start of the culture, it was applied once a day for 2 days. After 60 days, the culture on the mesh was observed for spores and clamps under a microscope. When extracted and analyzed by HPLC, ergosterols, which are fruit body-containing components, were also detected. This confirmed that a child entity was generated.
　本発明の固体培養シートを用いた菌類培養方法は、平滑又は粗面で膜状の子実体を作って育成する担子菌類を、人工的に培養して、生薬として医薬品に用いられたり、健康食品の有効成分として用いられたりするのに、用いられる。 The method for culturing fungi using the solid culture sheet of the present invention is a method of artificially cultivating basidiomycetes that grow and produce a film-like fruit body on a smooth or rough surface and used as a crude drug in medicines or health foods It is used to be used as an active ingredient.
　１は菌糸体育成固体培養シート、２は子実体育成固体培養シート、１１は菌糸体用フィルム、１２は担子菌類菌糸体用培地塗布ノズル、１３は担子菌類菌糸体用培地塗布液、１４は担子菌類菌糸体用培地、１５は補強体、１６は菌糸体培養液噴霧ノズル、１７は菌糸体培養液噴霧ノズル、２０は担子菌類、２１は子実体用フィルム、２２は担子菌類子実体用培地ノズル、２３は担子菌類子実体用培地塗布液、２４は担子菌類子実体用培地、２５ａ・２５ｂはパルス電圧電極、２６ａ・２６ｂはパルス電圧である。 1 is a solid culture sheet for growing mycelium, 2 is a solid culture sheet for growing fruit bodies, 11 is a film for mycelium, 12 is a medium application nozzle for basidiomycete mycelium, 13 is a medium application solution for basidiomycetous mycelium, and 14 is a basidiomycete Medium for fungal mycelium, 15 reinforcement, 16 mycelium culture spray nozzle, 17 mycelium culture spray nozzle, 20 basidiomycetes, 21 fruit body film, 22 basidiomycete culture medium nozzle , 23 is a basidiomycete fruit body medium coating solution, 24 is a basidiomycete fruit body medium, 25a and 25b are pulse voltage electrodes, and 26a and 26b are pulse voltages.

Claims (11)

  1. 　金属、合成樹脂、天然樹脂、及び／又は紙で、メッシュ、織布、不織布、多孔質シートから選ばれる補強体を形成し、担子菌類菌糸体用培地を付してから、それのおもて面側に担子菌類の菌糸体をそれの培養液の塗工又は噴霧により接種し、菌糸体育成固体培養シートを形成した後、前記菌糸体を育成する菌糸体増殖工程と、
    　前記おもて面側で育成した前記菌糸体に、担子菌類子実体用培地を付して、子実体育成培養シートを形成した後、前記菌糸体から子実体へ育成する子実体増殖工程とを、
    有することを特徴とする菌類培養方法。     Form a reinforcement selected from mesh, woven fabric, non-woven fabric, and porous sheet with metal, synthetic resin, natural resin, and / or paper, attach a medium for basidiomycete mycelium, After inoculating the mycelium of basidiomycetes on the surface side by coating or spraying the culture solution thereof to form a mycelium-growing solid culture sheet, a mycelium growth step for growing the mycelium,
    The mycelium grown on the front side is attached with a basidiomycetous fruit body medium to form a fruit body growth culture sheet, and then a fruit body growth step for growing from the mycelium to the fruit body ,
    A method for culturing fungi, comprising:
  2. 　前記担子菌類菌糸体用培地と前記担子菌類子実体用培地とがそれぞれ、液体培地又は固体培地であることを特徴とする請求項１に記載の菌類培養方法。 2. The fungal culture method according to claim 1, wherein the basidiomycete mycelium medium and the basidiomycete fruit body medium are respectively a liquid medium or a solid medium.
  3. 　前記菌糸体増殖工程中、前記液体培地からなる前記担子菌類菌糸体用培地を、前記補強体に担持し、前記子実体増殖工程中、前記液体培地からなる前記担子菌類子実体用培地を前記補強体に前記おもて面側で担持し、若しくは前記固体培地からなる前記担子菌類子実体用培地を前記補強体にうら面側で担持し、又は、
    　前記菌糸体増殖工程中、前記固体培地からなる前記担子菌類菌糸体用培地を、前記補強体に前記おもて面側で担持し、前記子実体増殖工程中、前記液体培地からなる前記担子菌類子実体用培地を前記担子菌類菌糸体用培地に担持し若しくは前記固体培地からなる前記担子菌類子実体用培地を前記補強体にうら面側で担持し、又は、
    　前記菌糸体増殖工程中、前記固体培地からなる前記担子菌類菌糸体用培地を、前記補強体に前記おもて面側で担持し、前記子実体増殖工程中、前記担子菌類菌糸体用培地から前記菌糸体と共に前記補強体を剥離した前記補強体にうら面側で固体培地からなる前記担子菌類子実体用培地を、担持することを特徴とする請求項２に記載の菌類培養方法。     During the mycelium growth step, the basidiomycetous mycelium medium composed of the liquid medium is supported on the reinforcing body, and during the fruiting body propagation step, the basidiomycetous fruit body culture medium composed of the liquid medium is reinforced. The body is carried on the front side, or the basidiomycete fruiting body medium comprising the solid medium is carried on the back side on the reinforcing body, or
    During the mycelium growth step, the basidiomycetous mycelium medium comprising the solid medium is supported on the front side on the reinforcing body, and during the fruiting body growth step, the basidiomycetes comprising the liquid medium A medium for fruiting bodies is carried on the medium for basidiomycetous mycelium or the medium for basidiomycetous fruit bodies consisting of the solid medium is carried on the back side of the reinforcing body, or
    During the mycelium growth step, the basidiomycete mycelium medium comprising the solid medium is supported on the front side on the reinforcing body, and during the fruit body growth step, from the basidiomycete mycelium medium 3. The fungal culture method according to claim 2, wherein the basidiomycete fruiting body medium comprising a solid medium is supported on the back side of the reinforcing body from which the reinforcing body has been peeled off together with the mycelium.
  4. 　前記菌糸体増殖工程中、菌糸体用フィルム上の層状の前記固体培地からなる前記担子菌類菌糸体用培地に、前記補強体を搭載して圧着し一体化してから、前記担子菌類の菌糸体の培養液を噴霧し、前記菌糸体育成培養シートを形成し、
    　前記子実体増殖工程中、前記おもて面側で育成した前記菌糸体と共に前記補強体を、前記担子菌類菌糸体用培地及び菌糸体用フィルムから剥離した後、子実体用フィルム上の層状の前記固体培地からなる担子菌類子実体用培地を前記補強体にそのうら面側で圧着させて、前記子実体育成培養シートを形成することを特徴とする請求項３に記載の菌類培養方法。     During the mycelium growth step, the basidiomycetous mycelium medium comprising the layered solid medium on the mycelium film is mounted by pressing and integrating the reinforcement, and then the mycelium of the basidiomycetes is integrated. Spray the culture solution to form the mycelium growth culture sheet,
    During the fruiting body growth step, after peeling the reinforcing body together with the mycelium grown on the front surface side from the basidiomycete mycelium medium and mycelium film, The fungus cultivation method according to claim 3, wherein the basidiomycete fruit body culture medium comprising the solid medium is pressure-bonded to the reinforcing body on the back surface side to form the fruit body growth culture sheet.
  5. 　前記菌糸体育成培養シートを、ロール状に巻き取って前記菌糸体を育成し、及び／又は前記子実体育成培養シートを、ロール状に巻き取って前記子実体を育成することを特徴とする請求項１～４の何れかに記載の菌類培養方法。 The mycelium growing culture sheet is wound up in a roll shape to grow the mycelium, and / or the fruit body growing culture sheet is wound up in a roll shape to grow the fruit body. Item 5. The fungal culture method according to any one of Items 1 to 4.
  6. 　前記担子菌類が、ヒダナシタケ目の菌類であることを特徴とする請求項１～５の何れかに記載の菌類培養方法。 The method for culturing fungi according to any one of claims 1 to 5, wherein the basidiomycetous fungi are fungi of the order Cleoptera.
  7. 　前記ハラタケ綱の菌類が、アンズタケ科、シロソウメンタケ科、ホウキタケ科、ハナビラタケ科、サンゴハリタケ科、サルノコシカケ科、タバコウロコタケ科、イドタケ科、ウロコタケ科、カンゾウタケ科、ニクハリタケ科、エゾハリタケ科、イボタケ科、ニンギョウタケモドキ科、コウヤクタケ科、マンネンタケ科、又はミヤマトンビマイ科の菌類であることを特徴とする請求項６に記載の菌類培養方法。 The fungus of the Agaricaceae is chanterelles, white mushrooms, brooms, coral, family coral, cigarettes, cigarettes, iridaceae, agaricaceae, agaricaceae, agaricaceae, agaricaceae, 7. The method for culturing fungi according to claim 6, wherein the fungus is a member of the family Ningyotake, Amanita, Amanentake, or Miyamatonbiidae.
  8. 　前記菌糸体及び／又は前記子実体に、物理的刺激、電気的刺激、光学的刺激、化学的刺激、及び生物的刺激から選ばれる少なくとも何れかの処理を施すことを特徴とする請求項１～７の何れかに記載の菌類培養方法。 The mycelium and / or the fruiting body is subjected to at least one treatment selected from physical stimulation, electrical stimulation, optical stimulation, chemical stimulation, and biological stimulation. The fungal culture method according to any one of 7.
  9. 　金属、合成樹脂、天然樹脂、及び／又は紙で形成された、メッシュ、織布、不織布、多孔質シートから選ばれ、そのおもて面で担子菌類の菌糸体を育成している補強体が、前記菌糸体から子実体へ育成している担子菌類子実体用培地を担持していることを特徴とする培養シート。 A reinforcement body made of metal, synthetic resin, natural resin, and / or paper, selected from mesh, woven fabric, non-woven fabric, and porous sheet, and growing mycelium of basidiomycetes on its front surface A culture sheet, which carries a medium for basidiomycete fruit bodies grown from the mycelium to the fruit bodies.
  10. 　前記補強体は、液体培地又は固体培地からなり、前記菌糸体を育成した担子菌類菌糸体用培地が未担持補強体に担持され又は担持されてから剥離されており、液体培地又は固体培地からなる前記担子菌類子実体用培地が、前記補強体に担持されていることを特徴とする請求項９に記載の培養シート。 The reinforcing body is composed of a liquid medium or a solid medium, and the basidiomycetous mycelium medium in which the mycelium is grown is supported on or unloaded from the unsupported reinforcing body, and is composed of a liquid medium or a solid medium. The culture sheet according to claim 9, wherein the medium for basidiomycete fruit bodies is supported on the reinforcing body.
  11. 　網目状、格子状、ギャザー状、凹凸波形状、及び／又はすのこ状の間隙によって通気性を有するスペーサが、前記補強体に付されていることを特徴とする請求項９～１０の何れかに記載の培養シート。 11. The spacer having air permeability by a mesh-like, lattice-like, gather-like, uneven wave-like shape, and / or slat-like gap is attached to the reinforcing body. The culture sheet as described.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104277983A (en) * 2014-09-17 2015-01-14 浙江中医药大学 Culture medium for fungal culture and preparation method of culture medium
JP2019170161A (en) * 2018-03-26 2019-10-10 国立大学法人名古屋大学 Production method of ganoderma lucidum, antioxidant, medicine, food product and skin external preparation
WO2020237201A1 (en) * 2019-05-23 2020-11-26 Bolt Threads, Inc. A composite material, and methods for production thereof
JP2021023173A (en) * 2019-08-01 2021-02-22 株式会社島津製作所 Cell culture apparatus and cell dissemination method
JP2023510573A (en) * 2020-01-15 2023-03-14 チャン，ジュイ－ナン Cultivation method of Antrodia cinnamomea and porous carrier for cultivating Antrodia cinnamomea

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103548563B (en) * 2013-10-23 2016-08-17 关岭百龙汇生物科技有限公司 A kind of fragmentation processing method for germination bacterium liquid strain mycelium pellets
CN104920073A (en) * 2015-06-30 2015-09-23 兴安县铭晖食用菌种植有限公司 Cultivating method of tricholoma giganteum
CN108531402A (en) * 2017-03-03 2018-09-14 天明制药股份有限公司 Liquid culture medium of inonotus obliquus and preparation method thereof
DE102022112282A1 (en) 2022-04-28 2023-11-02 Alexander Robin Kerpe Method and device for growing mushrooms

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03195422A (en) * 1990-06-01 1991-08-27 Konpetsukusu:Kk Culture promoting sheet of mushroom spawn in bed log with small diameter
JP2004065165A (en) * 2002-08-08 2004-03-04 Iwate Prefecture Method for producing inoculating sheet for mycorrhizal mushroom

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03195422A (en) * 1990-06-01 1991-08-27 Konpetsukusu:Kk Culture promoting sheet of mushroom spawn in bed log with small diameter
JP2004065165A (en) * 2002-08-08 2004-03-04 Iwate Prefecture Method for producing inoculating sheet for mycorrhizal mushroom

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FUMIHIKO YOSHIMURA: "Artificial formation of Tricholoma matsutake mycorrhiza. Success in inoculation using matsutake-sheet. Expectation for fruit body generation", KAGAKU TO SEIBUTSU, vol. 43, no. 5, 1 May 2005 (2005-05-01), pages 285 - 287 *
MASAKI NARIMATSU: "Akamatsu Bayashi no Tamenteki Katsuyo - Matsutake no Zosan o Mezashite", MOKUZAI HOZON, vol. 32, no. 4, 25 July 2006 (2006-07-25), pages 172 - 176 *
MASAKI NARIMATSU: "Kinshi Tantai o Mochiita Ekitai Baiyo ni Okeru Baiyo Joken ga Matsutake Kinshi no Zoshoku ni Ataeru Eikyo", BULLETIN OF THE IWATE PREFECTURAL FORESTRY TECHNOLOGY CENTER, no. 14, March 2006 (2006-03-01), pages 31 - 36 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104277983A (en) * 2014-09-17 2015-01-14 浙江中医药大学 Culture medium for fungal culture and preparation method of culture medium
CN104277983B (en) * 2014-09-17 2017-09-29 浙江中医药大学 It is a kind of for culture medium of fungal culture and preparation method thereof
JP2019170161A (en) * 2018-03-26 2019-10-10 国立大学法人名古屋大学 Production method of ganoderma lucidum, antioxidant, medicine, food product and skin external preparation
JP2022163106A (en) * 2018-03-26 2022-10-25 国立大学法人東海国立大学機構 Methods for producing ganoderma lucidum, antioxidant, pharmaceutical, food and external preparation for skin
JP7170255B2 (en) 2018-03-26 2022-11-14 国立大学法人東海国立大学機構 Method for producing Ganoderma lucidum, antioxidant, pharmaceutical, food, and topical skin preparation
JP7362014B2 (en) 2018-03-26 2023-10-17 国立大学法人東海国立大学機構 Production method of Cinnamon mushrooms, antioxidants, medicines, foods, and skin external preparations
WO2020237201A1 (en) * 2019-05-23 2020-11-26 Bolt Threads, Inc. A composite material, and methods for production thereof
US11015059B2 (en) 2019-05-23 2021-05-25 Bolt Threads, Inc. Composite material, and methods for production thereof
US11891514B2 (en) 2019-05-23 2024-02-06 Bolt Threads, Inc. Composite material, and methods for production thereof
JP2021023173A (en) * 2019-08-01 2021-02-22 株式会社島津製作所 Cell culture apparatus and cell dissemination method
JP2023510573A (en) * 2020-01-15 2023-03-14 チャン，ジュイ－ナン Cultivation method of Antrodia cinnamomea and porous carrier for cultivating Antrodia cinnamomea
JP7430356B2 (en) 2020-01-15 2024-02-13 チャン，ジュイ－ナン Cultivation method of Antrodia camphorata and porous carrier for cultivation of Antrodia camphorata

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