Cultivation method of Poria cocos using corncob as a substrate

Abstract

To provide a method for artificially cultivating Poria Sclerotium. which can produce high-quality Poria Sclerotium in a short cultivation period with a higher yield.SOLUTION: There is provided, a method for artificially cultivating Poria Sclerotium, the method comprising the following steps (1) to (4): (1) inoculating an inoculum of Wolfiporia extensa into a mushroom bed; (2) culturing the inoculum of Wolfiporia extensa to form a mycelial mat on the surface of the mushroom bed; (3) inoculating the seed Poria Sclerotium onto the mycelial mat formed on the surface of the mushroom bed; and (4) cultivating and enlarging the seed Poria Sclerotium on the mycelial mat on the surface of the mushroom bed. The mushroom bed contains 60 v / v% or more of corncob in the base material.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a cultivation method of Bukuryo (sclerotium of Wolfiporia cocos), and more particularly to a cultivation method capable of producing a high yield of Bukuryo in a short period of time.

Background Art

[0002] Bukuryo is the sclerotium of a fungus belonging to the genus Wolfiporia cocos Ryvarden et Gilbertson (or Poria cocos Wolf) of the family Polyporaceae. As a crude drug, "Poria cocos", it is formulated in many traditional Chinese medicine prescriptions such as Keishi-Bukuryo-Gan, Gokuryo-San, and Bukuryo-In, and has been used since ancient times for various diseases accompanied by reverse qi in the chest and hypochondrium, melancholy, fright, palpitations, binding pain in the epigastrium, fever and chills, vexation and fullness, dry mouth, dry tongue, and dysuria. In recent years, in addition to its diuretic effect, it has been found to have anti-ulcer, blood sugar-lowering, and immunostimulating effects.

[0003] In its natural state, Bukuryo is formed by adhering to the roots 10 to 30 cm underground of pine trees that have been felled for 3 to 5 years. However, in Japan today, almost no wild products are collected, and Bukuryo as a crude drug depends entirely on imports from China. In China, in addition to collecting wild products, artificial cultivation using pine logs is carried out. However, in recent years, the areas where forest logging is restricted have been expanding, and there are concerns about whether the supply volume and quality stability can be ensured in the future. In addition, in log cultivation, the cultivation period is as long as about one year, and there is a problem that the work load such as burying and excavating logs is large.

[0004] Therefore, attempts have been made to cultivate Poria cocos using sawdust as a medium. For example, a method has been disclosed in which mycelium of Tricholoma matsutake is inoculated into a sawdust medium in which a basket having a mesh structure is buried, and Poria cocos is formed inside the basket (Patent Document 1). However, when cultivating Poria cocos by this method, sawdust is mixed into the Poria cocos, so it cannot be used as a crude drug as it is, and it is necessary to remove the sawdust. As a result, there are problems such as a decrease in yield and an increase in production cost.

[0005] In contrast, the applicant of the present application has already inoculated the inoculum of Tricholoma matsutake into a sawdust-based mushroom bed to form a mycelium mat on the surface of the mushroom bed, and inoculated the Poria cocos as a seed thereon, so that the Poria cocos does not come into direct contact with the sawdust, and can absorb nutrients through the mycelium of the mycelium mat, and has developed a cultivation method capable of preventing the mixing of sawdust into the Poria cocos during hypertrophy, and has filed a patent application (Patent Document 2).

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0007] An object of the present invention is to provide a method for artificially cultivating Poria cocos capable of producing high-quality Poria cocos with high yield in a shorter period.

Means for Solving the Problems

[0008] As a result of intensive studies to solve the above problems, the present inventors have found that by using corn cobs as the main base material constituting the mushroom bed, compared with the conventional mushroom bed using sawdust as the base material, the sclerotia grow hypertrophied in a short period of time and the hypertrophy rate is also improved, thus completing the present invention.

[0009] That is, the present invention comprises the following steps (1) to (4); (1) A step of inoculating the seed fungus of Matsutake into the mushroom bed (2) A step of culturing the seed fungus of Matsutake to form a mycelial mat on the surface of the mushroom bed (3) A step of inoculating the seed of Shiraia bambusicola on the mycelial mat formed on the surface of the mushroom bed (4) A step of culturing and hypertrophying the seed of Shiraia bambusicola on the mycelial mat on the surface of the mushroom bed An artificial cultivation method of Shiraia bambusicola containing the above steps, characterized in that the mushroom bed contains 60 v / v% or more of corn cobs in the base material.

Effect of the Invention

[0010] According to the cultivation method of the present invention, it is possible to produce high-quality Shiraia bambusicola with a high yield in a short period of time.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

Mode for Carrying Out the Invention

[0012] In the cultivation method of the present invention, first, inoculate the seed fungus of Matsuhodo into the fungus bed (step (1)), and use corn cob as the main base material constituting the fungus bed. The corn cob is obtained by pulverizing the core part excluding the seeds of corn, and is also called corn cob meal. Corn cobs produced in China, Indonesia, etc. are commercially available from Shimizu Flour Mill Co., Ltd., Taiko Co., Ltd., Orient General Co., Ltd., Takamisawa Co., Ltd., etc. The content of corn cob in the fungus bed is preferably 60 v / v% or more, more preferably 75 v / v% or more, and even more preferably 90 v / v% or more from the viewpoints of improving the sclerotium hypertrophy rate of Bukuryo and shortening the cultivation period.

[0013] As the base material of the fungus bed, sawdust can be used as a secondary base material in addition to the corn cob. Examples of the sawdust include Japanese red pine, Korean pine, Hokkaido pine, black pine, Chinese red pine, Japanese white pine, Yunnan pine, sugi, Chinese cinnamon (scientific name: Cinnamomum cassia Blume), etc. Among these, Japanese red pine, Korean pine, black pine, Chinese red pine, Yunnan pine, and Hokkaido pine are suitable, and Japanese red pine or Korean pine 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 fungus bed.

[0014] Mix the above-mentioned corn cob and sawdust used as necessary to prepare a base material, and put this into a container with an appropriate capacity such as a pot, a cultivation bag, a cultivation bin, etc. for culturing.

[0015] The spawn bed may be composed only of the base material such as the above-mentioned corncob, or nutrient components may be further added. Such nutrient components include ammonium salts. Examples of ammonium salts include ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium carbonate, ammonium phosphate, etc. Among these, when ammonium sulfate, ammonium nitrate, or ammonium chloride is added, the growth of the mycelium is promoted, a mycelium mat is rapidly formed on the surface of the spawn bed, and the biting of the base material into the mycelium is reduced, which is preferable. Also, if fruiting bodies are formed in the middle to lower layers of the spawn bed while the mycelium mat is being formed, even if the spawn fruiting bodies are inoculated and cultured later, the nutrients are often absorbed by the fruiting bodies formed in the middle to lower layers of the spawn bed, and the spawn fruiting bodies cannot grow sufficiently. However, when ammonium sulfate or ammonium nitrate is added, the formation of fruiting bodies in the middle to lower layers of such a spawn bed is suppressed, which is suitable. On the other hand, the fruiting bodies formed in the upper layer of the spawn bed hardly grow, so they do not compete with the inoculated spawn fruiting bodies and inhibit their growth. Here, the upper layer of the spawn bed generally means from the surface of the spawn bed to about 1 / 5 to 1 / 3 of the height of the spawn bed. The addition amount of ammonium salt in the spawn 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 base material. Within this range, the growth rate of the mycelium is fast, a mycelium mat is rapidly formed, the biting of the base material is reduced, and the formation of fruiting bodies in the middle to lower layers of the spawn bed can also be suppressed. On the other hand, if more than 1.0% by mass is added, the growth of the mycelium may be suppressed.

[0016] By adding potassium salts in addition to ammonium nitrate to the mushroom bed, the growth of mycelia can be promoted, and the formation of mold in the middle to lower layers of the mushroom bed can be suppressed. Examples of potassium salts include potassium phosphate, potassium carbonate, potassium chloride, etc. Among these, potassium phosphate is suitable because it is excellent in promoting mycelial growth and the like. The addition amount of potassium salt in the mushroom bed is preferably 0.01 to 4.0% by mass, more preferably 0.01 to 0.6% by mass, based on the dry mass of the base material. If the addition amount of potassium salt is lower than 0.1% by mass, the promotion of mycelial growth may be insufficient, and if it is higher than 4.0% by mass, the growth of mycelia may be suppressed.

[0017] By further adding calcium salts to the mushroom bed, the growth of mycelia is promoted and the thickness of the mycelial mat formed increases. Examples of calcium salts include calcium chloride, calcium carbonate, calcium sulfate, calcium phosphate, etc. Among these, calcium chloride is preferably used because it is excellent in promoting mycelial growth and the like. The addition amount of calcium salt in the mushroom bed is preferably 0.001 to 1.0% by mass, more preferably 0.01 to 0.2% by mass, based on the dry mass of the base material, from the viewpoint of the mycelial growth effect.

[0018] By further adding vitamins to the mushroom bed, the growth of mycelia can be promoted. Examples of vitamins include vitamin B1 (thiamine), vitamin C (ascorbic acid), vitamin B7 (biotin), vitamin B3 (nicotinic acid), vitamin B6 (pyridoxine), vitamin B9 (folic acid), vitamin B2 (riboflavin), vitamin B5 (pantothenic acid), etc. Among these, vitamin B1 is preferably used because it is excellent in promoting mycelial growth. The addition amount of vitamins in the mushroom 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 base material.

[0019] By further adding carbohydrates to the spawn bed, the growth of hyphae is promoted. As the carbohydrates, monosaccharides such as glucose, fructose, arabinose, xylose, disaccharides such as sucrose, maltose, trehalose, galactose, lactose, polysaccharides such as cellulose, starch, and sugar alcohols such as glycerol, mannitol, sorbitol can be used. Among these, glucose, maltose, and sucrose are suitable because they have a high effect of promoting the growth of hyphae, and glucose is particularly preferred. The addition amount of carbohydrates relative to the dry mass of the base material is preferably 0.1 to 15.0% by mass, and more preferably 1.0 to 5.0% by mass.

[0020] The spawn bed prepared by mixing the above base material and the nutrient components added as required is preferably adjusted to have a moisture content of 40% by mass or more and less than 60% by mass, and more preferably 42 to 45% by mass. If it is less than 40% by mass, the spawn bed is likely to dry, and the growth of hyphae may be inhibited by drying. If it is 60% by mass or more, mycelial masses are formed in the middle to lower layers of the spawn bed, and when inoculating and culturing the inoculum mycelial masses thereafter, the hypertrophy of the inoculum mycelial masses may be inhibited.

[0021] The spawn bed prepared as described above is inoculated with the spawn of Matsutake. When inoculating the spawn of Matsutake, for example, the spawn pre-cultured in a spawn bed medium using the above corn cobs or sawdust as the base material and adding nutrient components such as wheat bran and glucose may be added in an amount of about 5 to 10 g per 1 kg of the spawn bed. As the hyphae of Matsutake inoculated in the pre-culture, those collected from the wild or those obtained by assignment from preservation institutions such as ATCC (American Type Culture Collection) and NIAS GeneBank (National Institute of Agrobiological Sciences GeneBank) can be used.

[0022] Next, the inoculated Matsutake spawn is cultured to form a mycelial mat on the surface of the spawn bed (step (2)). The culture temperature is preferably 20 to 32°C, and more preferably 22 to 30°C. Within this range, it is preferable because the growth of hyphae is accelerated. Also, the humidity is preferably 10 to 90%, and more preferably 60 to 80%.

[0023] By culturing the matsutake spawn under the above conditions, the mycelium spreads throughout the mushroom bed, and then at least a part of the mycelium is exposed on the surface of the mushroom bed to form a mycelium mat. By growing the mycelium on the surface of the mushroom bed in this way and forming a mycelium mat, since the substrate is less likely to be bitten into during the growth process of the mycelium, it is possible to prevent the substrate from mixing into the mushroom when the mycelium fuses with the mushroom spawn. The thickness of this mycelium mat is preferably about 0.5 to 2 cm, more preferably 1 cm or more. By forming a mycelium mat with such a thickness, when inoculating and enlarging the mushroom spawn on it, the contact between the mushroom spawn and the substrate is blocked to prevent the mixing of the substrate, and at the same time, sufficient nutrients are supplied from the substrate to the mushroom spawn through the mycelium mat, so the enlargement is promoted. In addition, although the mycelium in the culture process is vegetative mycelium, when the vegetative mycelia adhere to each other and the internal moisture escapes and the tissue becomes hard and dense, the mushroom sclerotium is formed, and at that time, it is considered that the vegetative mycelium morphologically changes into sclerotium mycelium. The mycelium mat is in a state where the vegetative mycelia are in close contact, but in a part of it, it may morphologically change into sclerotium mycelium and form the mushroom.

[0024] On the mycelium mat formed in this way, inoculate the mushroom spawn (step (3)). From the viewpoint of the efficiency of mushroom enlargement, for example, when the mass of the mushroom bed is 2.5 kg, the mass per piece is preferably 15 g to 40 g, more preferably 20 g to 30 g. Also, the mushroom spawn can be either immature or mature, or it can be a divided large one. The skin of the mushroom spawn may be left as it is or removed. Such a mushroom spawn may be placed in the central part on the mycelium mat formed on the surface of the mushroom bed. The mushroom spawn may be inoculated with two or more on one medium, but from the viewpoint of the enlargement efficiency, it is preferably inoculated with only one.

[0025] Cultivate and enlarge the inoculated spawn mushroom in the manner described above (step (4)). The spawn mushroom can be placed on the mycelium mat formed on the surface of the mushroom bed and cultivated as it is, but if sand is layered on the mycelium mat and the spawn mushroom is cultivated with part or all of it buried therein, the shape of the enlarged mushroom will become spherical, resulting in less loss during peeling and improved yield, which is preferable. If part of the spawn mushroom is cultivated in an exposed state, the surface of the spawn mushroom may dry out and crack, etc., so it is preferably cultivated in a state of being completely buried in the sand. As the sand, river sand, sea sand, mountain sand, etc. can be used. 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 spherical mushrooms can be obtained by cultivating with the spawn mushroom completely buried therein.

[0026] For the cultivation to enlarge the spawn mushroom, a temperature of 20 to 32°C is preferable, and 22 to 30°C is more preferable. Also, the humidity of the cultivation environment is not preferably in a too dry state, and it is preferably 60% or more. Further, during cultivation, a higher carbon dioxide concentration is preferable, and for this reason, it is preferably cultivated in a container with high airtightness. Under such conditions, for example, by cultivating for 30 to 90 days, preferably 40 to 60 days, the mushroom can be enlarged to 3 times or more, preferably 6 times or more, more preferably 7 times or more the mass of the inoculated spawn mushroom.

Example

[0027] Hereinafter, the invention will be described based on examples, etc. It should be noted that the present invention is not limited by examples, etc.

[0028] Test Example 1 Influence of the type of substrate on the mycelium growth rate and sclerotium formation: As the base material for the mushroom bed, corn cob and sawdust of Cryptomeria japonica, Pinus densiflora or Larix kaempferi were used. Each base material wetted to a moisture content of 50% by mass was filled into mushroom bed bags at 250 g each. The mushroom bed was sterilized at 120°C for 60 minutes, cooled, and then about 1.5 g of inoculum of Matsuhodo pre-cultured under the following conditions was inoculated into the mushroom bed. After inoculation, it was cultured at 23.5°C for 14 days. During the culture, from 2 days after inoculation (5 days after inoculation for Cryptomeria japonica), the distance from the inoculation point of the inoculum to the tip of the mycelium was measured every day to evaluate the mycelial growth rate. After the culture was completed, about 13 - 15 g of inoculum of Shubukuryo was inoculated onto the mycelial mat formed on the surface of the mushroom bed. Sand sterilized at 120°C for 60 minutes was layered thereon to cover the sclerotia. While appropriately adding sand, stirring, adding water, etc., it was harvested 2 months after inoculation of the inoculum, and the weight of the sclerotia was measured. The results of the mycelial growth rate are shown in Figure 1, and the results of the sclerotia yield are shown in Figure 2, respectively. (Pre-culture conditions) A mushroom bed medium in which corn cob and rice bran were mixed at a volume ratio of 3:1 as the base material was adjusted to a moisture content of about 50%, and the mycelium of Matsuhodo as the mother strain was inoculated therein and cultured for 30 days in an environment of 23.5°C and 60% humidity.

[0029] From Figures 1 and 2, when corn cob was used as the base material, although the mycelial growth rate was equivalent to that of other base materials, the sclerotia clearly swelled, and a significantly higher yield was obtained.

[0030] Test Example 2 Influence of the mixing ratio of corn cob and Pinus densiflora sawdust on the cultivation period and sclerotia yield (1): As substrates for the spawn beds, corn cobs and Japanese red pine sawdust were mixed and used at volume ratios (corn cob:Japanese red pine sawdust) of 100:0, 75:25, 50:50, and 0:100, respectively. For the spawn beds, 2.0 kg of sawdust with the moisture content of the spawn bed adjusted to 50% by mass was used, and ammonium sulfate ((NH4)2SO4) was added at 0.2% by mass. Additionally, a spawn bed was prepared by adding 0.03% by mass of potassium phosphate (K2HPO4, KH2PO4), 0.03% by mass of calcium chloride (CaCl2·H2O) per kg, 0.002% by mass of vitamin B1, and 1% by mass of granulated sugar. Each substrate was filled into spawn bed bags at 1 kg each. The spawn beds were sterilized at 103°C for 180 minutes, and after cooling, approximately 1.5 - 2 g of the inoculum of Tricholoma matsutake pre-cultured under the conditions of Test Example 1 was inoculated into the spawn beds. After inoculation, the cultures were incubated at 22°C and a humidity of 60 - 70% for 14 days. After the cultivation was completed, approximately 30 g of the inoculum of Sclerotium cepivorum was inoculated onto the mycelium mat formed on the surface of the spawn bed. The sclerotia were covered with sand sterilized at 120°C for 60 minutes. Cultivation was continued while appropriately adding sand, stirring, and adding water, etc., and the end of cultivation was defined as the time when the weight of the sclerotia reached the maximum or a plateau. The number of days from the inoculation of the Sclerotium cepivorum inoculum to the end of cultivation for each substrate with each mixing ratio was determined as the cultivation period. Also, the hypertrophy rate of the sclerotia ((weight of harvested Sclerotium cepivorum / weight of inoculated Sclerotium cepivorum) × 100) for each substrate with each mixing ratio was calculated. The results are shown in Table 1.

[0031]

Table 1

[0032] As shown in Table 1, it was revealed that the higher the proportion of corn cobs, the higher the hypertrophy rate.

[0033] Test Example 3 Influence of the mixing ratio of corn cobs and Japanese red pine sawdust on the cultivation period and sclerotium yield (2): As substrates for the spawn beds, cultivation was carried out in the same manner as in Test Example 2 except that the volume ratios (corn cob:Japanese red pine sawdust) of corn cobs and Japanese red pine sawdust were 50:50, 60:40, 90:10, and 100:0, and the hypertrophy rate of the sclerotia was determined. The results are shown in Table 2.

[0034]

Table 2

Industrial Applicability

[0035] According to the cultivation method of Buchu of the present invention, it is possible to produce high-quality Buchu in a short period of time with a high yield without mixing the base material, which is useful for the stable supply of Buchu as a crude drug.

Claims

1. The following steps (1) to (4); (1) A step of inoculating the seed fungus of Matsuhodo into the mushroom bed (2) A step of culturing the seed fungus of Matsuhodo to form a mycelium mat on the surface of the mushroom bed (3) A step of inoculating the seed of Bukuryo on the mycelium mat formed on the surface of the mushroom bed (4) A step of culturing and growing the seed of Bukuryo on the mycelium mat on the surface of the mushroom bed An artificial cultivation method of Bukuryo containing the above steps, characterized in that the mushroom bed contains 60 v / v% or more of corn cobs in the substrate.

2. The artificial cultivation method of Bukuryo according to Claim 1, wherein the mushroom 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 artificial cultivation method of Bukuryo according to Claim 2, wherein the addition amount of the ammonium salt is 0.01 to 1.0% by mass based on the dry mass of the substrate.

4. The artificial cultivation method of Bukuryo according to any one of Claims 1 to 3, wherein the mushroom bed further contains a potassium salt selected from the group consisting of potassium phosphate, potassium carbonate and potassium chloride.

5. The artificial cultivation method of Bukuryo according to any one of Claims 1 to 4, wherein the mushroom bed further contains a calcium salt selected from the group consisting of calcium chloride, calcium carbonate, calcium sulfate and calcium phosphate.

6. The artificial cultivation method of Bukuryo according to any one of Claims 1 to 5, wherein the mushroom bed further contains vitamins 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 (folic acid), vitamin B2 (riboflavin) and vitamin B5 (pantothenic acid).

7. The artificial cultivation method of Bukuryo according to any one of Claims 1 to 6, wherein the mushroom bed further contains saccharides selected from the group consisting of glucose, fructose, arabinose, xylose, sucrose, maltose, trehalose, galactose, lactose, cellulose, starch, glycerol, mannitol and sorbitol.

8. The following steps (1) to (4); (1) A step of inoculating the seed fungus of Matsuhodo into the mushroom bed (2) A step of culturing the seed fungus of Matsuhodo to form a mycelium mat on the surface of the mushroom bed (3) A step of inoculating the spawn of *Pleurotus eryngii* on the mycelium mat formed on the surface of the mushroom bed (4) A step of culturing and growing the spawn of *Pleurotus eryngii* on the mycelium mat on the surface of the mushroom bed In a method for artificially cultivating *Pleurotus eryngii* containing the above, a method for increasing the yield of *Pleurotus eryngii*, characterized in that 60 v / v% or more of corn cob is added to the base material of the mushroom bed.

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