JP7689875B2 - A method for cultivating Poria columb using corncobs as a substrate - Google Patents

Description

The present invention relates to a method for cultivating Poria columbine (the sclerotium of Pinellia gracilis), and more specifically, to a cultivation method that can produce a high yield of Poria columbine in a short period of time.

Bukryo is the sclerotium of Wolfiporia cocos Ryvarden et Gilbertson (or Poria cocos Wolf), a fungus belonging to the Polyporaceae family. It is used as the herbal medicine "Bukuryo" in many Chinese herbal prescriptions, including Keishibukuryo-gan, Goryo-san, and Bukuryo-in, and has been used since ancient times to treat a variety of illnesses accompanied by chest and side reflux, melancholy, fear, palpitations, pain under the heart, chills and fevers, fullness, burning mouth, dry tongue, and difficulty in urinating. In recent years, in addition to its diuretic effect, it has been recognized to have anti-gastric ulcer, hypoglycemic, and immunostimulating effects.

In the wild, Poria columbine is formed by attaching to the roots of pine trees 10-30 cm underground 3-5 years after they have been felled, but currently such wild plants are hardly collected in Japan, and Poria columbine as a herbal medicine is solely dependent on imports from China. In China, in addition to collecting wild plants, Poria columbine is cultivated artificially using pine logs, but in recent years, areas where felling of forests is restricted have been expanding, raising concerns about whether it will be possible to ensure stable supply and quality in the future. In addition, the cultivation period for log cultivation is long, at about one year, and there is a problem with the heavy workload of burying and digging up the logs.

Therefore, attempts have been made to cultivate Poria columbine using sawdust as a medium. For example, a method has been disclosed in which Poria columbine mycelium is inoculated into a sawdust medium in which a mesh-structured basket is buried, thereby forming Poria columbine inside the basket (Patent Document 1). However, when Poria columbine is cultivated using this method, the sawdust becomes contaminated with the Poria columbine, meaning that it cannot be used as a herbal medicine as is, and the sawdust must be removed, resulting in reduced yields and increased production costs.

In response to this, the applicant has already developed a cultivation method that involves inoculating a mushroom bed made of sawdust with seed fungi of Matsuhodo monad, forming a mycelium mat on the surface of the bed, and then inoculating the seeds of Poria columbine on top of that, allowing the Poria columbine to absorb nutrients through the mycelium of the mycelium mat without coming into direct contact with the sawdust, and preventing the Poria columbine from becoming contaminated by sawdust as it grows. This method has been applied for as a patent (Patent Document 2).

Japanese Patent Application Publication No. 1-111726 Patent No. 5854169

The present invention aims to provide a method for artificially cultivating Poria cocos that can produce high-quality Poria cocos in high yields in a short period of time.

As a result of intensive research conducted by the inventors to solve the above problems, they discovered that by using corncobs as the main substrate for the fungal bed, the sclerotia swell in a shorter period of time and the swelling rate is improved compared to conventional fungal beds using sawdust as the substrate, and thus completed the present invention.

That is, the present invention relates to a method for producing a method for producing a pharmaceutical composition comprising the following steps (1) to (4);
This method for artificially cultivating Poria cocos includes the steps of (1) inoculating a seed fungus of Poria cocos into a fungal bed, (2) cultivating the seed fungus of Poria cocos to form a mycelium mat on the surface of the fungal bed, (3) inoculating a seed Poria cocos onto the mycelium mat formed on the surface of the fungal bed, and (4) cultivating and enlarging the seed Poria cocos on the mycelium mat on the surface of the fungal bed, wherein the fungal bed contains 60 v/v % or more of corn cobs in the substrate.

The cultivation method of the present invention makes it possible to produce high-quality bupleurum in high yields in a short period of time.

1 is a graph showing the mycelium growth rate on each substrate in Test Example 1. 1 is a graph showing the sclerotial enlargement rate for each substrate in Test Example 1.

In the cultivation method of the present invention, the seed fungus of Poria cobia is first inoculated into the fungal bed (step (1)), and corn cobs are used as the main substrate for the fungal bed. Corn cobs are made by grinding the core of corn, excluding the kernel, and are also called corn cob meal. Corn cobs from China, Indonesia, etc. are commercially available from Shimizu Flour Mill Co., Ltd., Taiko Co., Ltd., Orient Generator Co., Ltd., Takamizawa Co., Ltd., etc. From the viewpoint of improving the sclerotial enlargement rate of Poria cobia and shortening the cultivation period, the content of corn cobs in the fungal bed is preferably 60 v/v% or more, more preferably 75 v/v% or more, and even more preferably 90 v/v% or more.

In addition to corncobs, sawdust can be used as a secondary substrate for the mushroom bed. Examples of sawdust include red pine, larch, Abies sachalinensis, black pine, pine serrata, Japanese stone pine, Unnanmatsu, Japanese cedar, and Chinese cinnamon (Cinnamomum cassia Blume). Among these, red pine, larch, black pine, pine serrata, Unnanmatsu, and Abies sachalinensis are preferred, and red pine or larch is particularly preferred.
The average particle size of the sawdust is preferably 0.5 to 20 mm. If it is larger than 20 mm, moisture may not be sufficiently retained in the culture bed.

The above corncobs and sawdust, which is used as needed, are mixed to prepare a substrate, which is then placed in a container of appropriate capacity, such as a pot, cultivation bag, or cultivation bottle, for cultivation.

The fungal bed may be composed of only the substrate such as the corn cob, but nutrients may also be added. Examples of such nutrients include ammonium salts. Examples of ammonium salts include ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium carbonate, and ammonium phosphate. Among these, the addition of ammonium sulfate, ammonium nitrate, and ammonium chloride is preferable because it promotes the growth of the mycelium, quickly forms a mycelium mat on the surface of the fungal bed, and reduces the substrate being bitten by the mycelium. Also, if a porgy is formed in the middle to lower layers of the fungal bed during the formation of the mycelium mat, even if a seed porgy is then inoculated and cultured, the nutrients are often absorbed by the porgy formed in the middle to lower layers of the fungal bed, and the seed porgy does not grow sufficiently. However, the addition of ammonium sulfate or ammonium nitrate is preferable because it suppresses the formation of porgy in the middle to lower layers of such a fungal bed. On the other hand, the porgy formed in the upper layer of the fungal bed hardly grows, so it does not compete with the inoculated seed porgy to inhibit its growth. Here, the upper layer of the fungal bed generally means from the surface of the fungal bed to about one-fifth to one-third of the height of the fungal bed. The amount of ammonium salt added to the fungal bed is preferably 0.01 to 1.0% by mass, more preferably 0.1 to 0.8% by mass, based on the dry mass of the substrate. Within this range, the mycelium grows quickly, a mycelium mat is formed quickly, and the substrate is less bitten, and the formation of porphyra in the middle to lower layers of the fungal bed can be suppressed. On the other hand, if more than 1.0% by mass is added, mycelium growth may be suppressed.

By adding potassium salt to the fungal bed in addition to ammonium nitrate, the growth of mycelium is promoted and the formation of porphyra in the middle to lower layers of the fungal bed can be suppressed. Examples of potassium salts include potassium phosphate, potassium carbonate, and potassium chloride, and among these, potassium phosphate is preferred because of its excellent effect of promoting mycelium growth. The amount of potassium salt added to the fungal bed is preferably 0.01 to 4.0 mass% based on the dry mass of the substrate, and more preferably 0.01 to 0.6 mass%. If the amount of potassium salt added is less than 0.1 mass%, mycelium growth may not be sufficiently promoted, and if it is more than 4.0 mass%, mycelium growth may be suppressed.

Addition of a calcium salt to the fungal bed promotes mycelial growth and increases the thickness of the mycelial mat that is formed. Examples of calcium salts include calcium chloride, calcium carbonate, calcium sulfate, and calcium phosphate. Among these, calcium chloride is preferably used because of its excellent mycelial growth promotion effect. The amount of calcium salt added to the fungal bed is preferably 0.001 to 1.0% by mass based on the dry mass of the substrate, and from the viewpoint of mycelial growth effect, 0.01 to 0.2% by mass is preferable.

The growth of mycelium can be promoted by further adding vitamins to the fungal bed. Examples of vitamins include vitamin B1 (thiamine), vitamin C (ascorbic acid), vitamin B7 (biotin), vitamin B3 (nicotinic acid), vitamin B6 (pyridoxine), vitamin B9 (iodine), vitamin B2 (lactoflavin), and vitamin B5 (pantothenic acid). Of these, vitamin B1 is preferably used because of its excellent effect of promoting mycelium growth. The amount of vitamins added to the fungal bed is preferably 0.001 to 0.01% by mass, more preferably 0.002 to 0.005% by mass, based on the dry mass of the substrate.

The growth of mycelia can be promoted by further adding carbohydrates to the fungal bed. Examples of carbohydrates that can be used include monosaccharides such as glucose, fructose, arabinose, and xylose; disaccharides such as sucrose, maltose, trehalose, galactose, and lactose; polysaccharides such as cellulose and starch; and sugar alcohols such as glycerol, mannitol, and sorbitol. Among these, glucose, maltose, and sucrose are preferred because of their high effect of promoting the growth of mycelia, and glucose is particularly preferred. The amount of carbohydrate added relative to the dry mass of the substrate is preferably 0.1 to 15.0% by mass, and more preferably 1.0 to 5.0% by mass.

The moisture content of the fungal bed prepared by mixing the above-mentioned base material and nutrients added as necessary is preferably adjusted to 40% by mass or more and less than 60% by mass, more preferably 42 to 45% by mass. If it is less than 40% by mass, the fungal bed is prone to drying, which may inhibit mycelial growth, and if it is 60% by mass or more, porphyra will form in the middle to lower layers of the fungal bed, which may inhibit the growth of the seed porphyra when it is inoculated and cultured.

The mushroom bed prepared as described above is inoculated with the seed fungus of Matsuhodo. When inoculating the seed fungus of Matsuhodo, for example, about 5 to 10 g of the seed fungus pre-cultured in a mushroom bed medium containing the above-mentioned corn cob or sawdust as a base material and added with nutrients such as wheat bran and glucose may be added per 1 kg of mushroom bed. As the mycelium of Matsuhodo to be inoculated in the pre-culture, it is possible to use collected wild mycelium or that provided by a preservation institution such as the ATCC (American Type Culture Collection) or the National Institute of Agricultural Science and Technology (NIAS GeneBank).

Next, the inoculated Matsuhodo seed fungus is cultured to form a mycelium mat on the surface of the fungal bed (step (2)). The culture temperature is preferably 20 to 32°C, more preferably 22 to 30°C. This range is preferable because it allows mycelium growth to proceed quickly. The humidity is also preferably 10 to 90%, more preferably 60 to 80%.

By culturing the seed fungus of Matsuhodo under the above conditions, the mycelium spreads over the entire fungal bed, and then at least a portion of the mycelium is exposed on the surface of the fungal bed to form a mycelium mat. By growing the mycelium on the surface of the fungal bed in this way and forming a mycelium mat, the substrate is less likely to be bitten during the mycelium growth process, which prevents the substrate from being mixed into the seed porgy when the mycelium fuses with it. The thickness of this mycelium mat is preferably, for example, about 0.5 to 2 cm, and more preferably 1 cm or more. By forming a mycelium mat of such thickness, when the seed porgy is inoculated on it and allowed to swell, contact between the seed porgy and the substrate is blocked to prevent the substrate from being mixed in, and sufficient nutrients are supplied from the substrate to the seed porgy via the mycelium mat, promoting its swell-up. In addition, the hyphae during the cultivation process are vegetative hyphae, but when the vegetative hyphae come into close contact with each other, lose internal moisture, and the tissue becomes hard and dense, a sclerotium is formed, and it is thought that at this time the vegetative hyphae change form into sclerotinia hyphae. A hyphal mat is a state in which vegetative hyphae are in close contact with each other, but some of them may change form into sclerotinia hyphae and form sclerotium.

The mycelium mat thus formed is inoculated with seed staghorns (step (3)). From the viewpoint of the efficiency of staghorn enlargement, when the mass of the fungal bed is 2.5 kg, the mass of each seed staghorn is preferably 15 g to 40 g, and more preferably 20 g to 30 g. The seed staghorns may be either immature or mature, or may be large staghorns divided into pieces. The skin of the seed staghorns may be left as is, or may be removed. Such a seed staghorn may be placed in the center of the mycelium mat formed on the surface of the fungal bed. Two or more seed staghorns may be inoculated on one medium, but from the viewpoint of the efficiency of enlargement, it is preferable to inoculate only one seed staghorn.

The seed porgy inoculated as described above is cultured and allowed to swell (step (4)). The seed porgy may be placed on the mycelium mat formed on the surface of the fungal bed and cultured as is, but it is preferable to layer sand on the mycelium mat and culture the seed porgy with part or all of it buried in it, as this will result in the swelled porgy becoming spherical in shape, reducing loss during peeling and improving yield. If the seed porgy is cultured with part exposed, the surface of the seed porgy may dry out and crack, so it is preferable to culture the seed porgy completely buried in sand. River sand, sea sand, mountain sand, etc. can be used as sand. For example, river sand with an average particle size of about 0.5 to 2.0 mm is layered on the mycelium mat to a thickness of about 10 to 30 cm, preferably about 20 to 40 cm, and the seed porgy is cultured completely buried, resulting in spherical porgy.

The temperature for culturing the seed buds to enlarge is preferably 20 to 32°C, more preferably 22 to 30°C. The humidity of the culture environment should not be too dry, and is preferably 60% or higher. A high carbon dioxide concentration is also preferred during culture, and for this reason, it is preferable to culture in a highly sealed container. By culturing under these conditions for, for example, 30 to 90 days, preferably 40 to 60 days, the buds will enlarge, for example to 3 times or more, preferably 6 times or more, more preferably 7 times or more the mass of the inoculated seed buds.

The invention will be described below with reference to examples. Note that the present invention is not limited to these examples.

Test Example 1
Effect of substrate type on mycelial growth rate and sclerotium formation:
Corn cobs and sawdust from Japanese cedar, red pine or larch were used as substrates for the fungal bed. Each substrate was filled in a fungal bed bag in an amount of 250 g, with water added to the substrate to a moisture content of 50% by mass. The fungal bed was sterilized at 120°C for 60 minutes, allowed to cool, and then about 1.5 g of the seed fungus of Matsuhodo pre-cultured under the following conditions was inoculated into the fungal bed. After inoculation, the fungal bed was cultured at 23.5°C for 14 days. During the culture, starting from 2 days after inoculation (5 days after inoculation for Japanese cedar), the distance from the time of inoculation of the seed fungus to the position of the tip of the hyphae was measured every day to evaluate the mycelial growth rate. After the culture was completed, about 13 to 15 g of the seed porgy was inoculated on the mycelial mat formed on the surface of the fungal bed. Sand sterilized at 120°C for 60 minutes was layered on top of it to cover the sclerotium. Sand was appropriately added, agitated, water was added, etc., and the mixture was harvested 2 months after inoculation of the seed fungus, and the weight of the sclerotium was measured. The results of mycelial growth rate are shown in FIG. 1, and the results of sclerotium yield are shown in FIG.
(Preculture conditions)
The substrate was a fungal bed medium made by mixing corn cobs and rice bran in a volume ratio of 3:1, and the moisture content was adjusted to approximately 50%. The parent strain, mycelium of Matsukoji, was inoculated into the medium and cultured for 30 days at a temperature of 23.5°C and humidity of 60%.

As can be seen from Figures 1 and 2, when corncobs were used as the substrate, the mycelium growth rate was equivalent to that of other substrates, but the sclerotia were clearly enlarged and a significantly higher yield was obtained.

Test Example 2
Effect of the mixture ratio of corn cobs and red pine sawdust on the cultivation period and sclerotinia yield (1):
As the substrate for the fungal bed, corn cobs and red pine were mixed at a content ratio (volume ratio; corn cobs: red pine) of 100:0, 75:25, 50:50, and 0:100, respectively. A fungal bed was prepared by adding 0.2% by mass of ammonium sulfate ((NH4)2SO4) to 2.0 kg of sawdust adjusted to a moisture content of 50% by mass, and further adding 0.03% by mass of potassium phosphate (K2HPO4, KH2PO4), 0.03% by mass of calcium chloride (CaCl2.H2O) / kg, 0.002% by mass of vitamin B1, and 1% by mass of white sugar. Each substrate was filled in a fungal bed bag at 1 kg each. The fungal bed was sterilized at 103 ° C for 180 minutes and allowed to cool, and then about 1.5 to 2 g of the seed fungus of Matsuhodo pre-cultured under the conditions of Test Example 1 was inoculated into the fungal bed. After inoculation, the mixture was cultured for 14 days at 22°C and humidity of 60-70%. After the culture was completed, about 30 g of seed Poria columbine was inoculated onto the mycelium mat formed on the surface of the fungal bed. The sclerotia were covered on top of the inoculated mixture with sand that had been sterilized at 120°C for 60 minutes. Cultivation was continued while adding sand, stirring, adding water, etc. as appropriate, and the point at which the weight of the sclerotia reached a maximum or plateau was defined as the end of cultivation. The number of days from Poria columbine seed inoculation to the end of cultivation in the substrates with each content ratio was calculated as the cultivation period. The swelling rate of the sclerotia in the substrates with each content ratio ((Poria columbine harvest weight/seed Poria columbine weight) x 100) was also calculated. The results are shown in Table 1.

As shown in Table 1, it was found that the higher the proportion of corn cobs, the higher the enlargement rate.

Test Example 3
Effect of the mixture ratio of corn cobs and red pine sawdust on the cultivation period and sclerotium yield (2):
The cultivation was carried out in the same manner as in Test Example 2, except that the substrate used for the fungal bed was a mixture of corn cob and red pine in a ratio (volume ratio; corn cob:red pine) of 50:50, 60:40, 90:10, or 100:0, and the swelling rate of the sclerotium was determined. The results are shown in Table 2.

The cultivation method for Poria columbine of the present invention makes it possible to produce high-quality Poria columbine in high yields in a short period of time without contamination with the substrate, and is useful for ensuring a stable supply of the herbal medicine Poria columbine.

Claims (8)

The following steps (1) to (4):
(1) a step of inoculating a seed fungus of Matsuhodo japonica into a fungal bed; (2) a step of cultivating the seed fungus of Matsuhodo japonica to form a mycelium mat on the surface of the fungal bed; (3) a step of inoculating a seed Poria columbine onto the mycelium mat formed on the surface of the fungal bed; and (4) a step of cultivating and enlarging the seed Poria columbine on the mycelium mat on the surface of the fungal bed, wherein the fungal bed contains 60 v/v % or more of corn cobs in the substrate. 2. The method for artificially cultivating Poria cocos according to claim 1, wherein the fungal bed further contains an ammonium salt selected from the group consisting of ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium carbonate and ammonium phosphate. 3. The method for artificially cultivating Poria cocos according to claim 2 , wherein the amount of ammonium salt added is 0.01 to 1.0% by mass based on the dry mass of the substrate. The method for artificially cultivating Poria cocos according to any one of claims 1 to 3, further comprising adding a potassium salt selected from the group consisting of potassium phosphate, potassium carbonate, and potassium chloride to the fungal bed. The method for artificially cultivating Poria cocos according to any one of claims 1 to 4, further comprising adding a calcium salt selected from the group consisting of calcium chloride, calcium carbonate, calcium sulfate and calcium phosphate to the fungal bed. The method for artificially cultivating Poria cocos according to any one of claims 1 to 5, further comprising in the fungal bed a vitamin selected from the group consisting of vitamin B1 (thiamine), vitamin C (ascorbic acid), vitamin B7 (biotin), vitamin B3 (nicotinic acid), vitamin B6 (pyridoxine), vitamin B9 (iodine), vitamin B2 (lactoflavin) and vitamin B5 (pantothenic acid). The method for artificially cultivating Poria cocos according to any one of claims 1 to 6, further comprising adding to the fungal bed a carbohydrate selected from the group consisting of glucose, fructose, arabinose, xylose, sucrose, maltose, trehalose, galactose, lactose, cellulose, starch, glycerol, mannitol and sorbitol. The following steps (1) to (4):
A method for artificially cultivating Poria cocos, comprising the steps of (1) inoculating a seed fungus of Poria cocos into a fungal bed, (2) cultivating the seed fungus of Poria cocos to form a mycelium mat on the surface of the fungal bed, (3) inoculating a seed Poria cocos onto the mycelium mat formed on the surface of the fungal bed, and (4) cultivating and enlarging the seed Poria cocos on the mycelium mat on the surface of the fungal bed, characterized in that corn cobs are added to the substrate of the fungal bed at 60 v/v% or more.