Method for cultivating Cordyceps

Abstract

To provide a method for cultivating Cordyceps using a culture medium that enables uniform and stable cultivation of Cordyceps. [Solution] The present invention provides a method for culturing Cordyceps, comprising: a culture medium preparation step of preparing a culture medium by adding water; a culture medium storage step of placing the culture medium prepared in the culture medium preparation step into a culture container; a culture medium sterilization step of sterilizing the culture container in which the culture medium was placed in the culture medium storage step under a temperature atmosphere exceeding 100°C; an inoculation step of placing Cordyceps fungi into the culture container after the culture container sterilized in the culture medium sterilization step has been cooled; a mycelial culture step of promoting the formation of mycelium of Cordyceps fungi after the inoculation step; and a mycelial state determination step of determining the state of mycelial formation in the mycelial culture step, wherein the mycelial state determination step is characterized by determining mycelial formation by color determination of at least one of the sides of the culture medium and the bottom of the culture medium.

Description

Technical Field

[0001] The present invention relates to a method for culturing Cordyceps sinensis using an artificial culture medium.

Background Art

[0002] Cordyceps sinensis is often used as a general term for mushrooms parasitic on insects, but it is classified in a genus of Ascomycetes, Hypocreales, and Hypocreaceae. Cordyceps sinensis originally refers to a product in which the ascomycete fungus Ophiocordyceps sinensis parasitizes the larvae of insects such as Lepidoptera, especially bats, and forms sclerotia and fruiting bodies in their bodies. It has been highly regarded as a traditional Chinese medicine since ancient times. In Japan, it is common to call the mushrooms parasitic on insects and spiders Cordyceps sinensis, and it is said that 250 species such as Cicindela dorsalis, Amanita muscaria, and Pentatrichopus japonicus inhabit. According to recent research, it has been reported that Cordyceps sinensis has effects on the central nervous system (sedative effect), immune system, blood vessels (prevention of arteriosclerosis), tonic effect, and blood sugar lowering effect, and is particularly expected to be used as an anticancer agent. In Japan, research on the cultivation of Cordyceps sinensis has been conducted since the late 1970s, and various cultivation methods have been proposed to date (for example, Patent Documents 1 to 27). Among such cultivation methods, as disclosed in Patent Document 28, when the Cordyceps sinensis fungus infects a living insect in the same manner as the infection mechanism in nature, the hyphae can grow sufficiently in the insect's body, and high-quality and stable Cordyceps sinensis can be obtained regardless of the formation of fruiting bodies. However, the method of infecting a living insect with the Cordyceps sinensis fungus requires extremely advanced technology and the need to raise a large number of healthy insects, resulting in a problem of high production costs. On the other hand, as disclosed in each patent document, many methods for culturing Cordyceps sinensis using an artificial culture medium have been proposed. In artificial culture media, insects are sometimes used, but grains are often used. Whether using insects or grains as artificial culture media, it is possible to grow Cordyceps fungi not only to form mycelium but also to form fruiting bodies. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 54-80486 [Patent Document 2] Japanese Patent Application Publication No. 55-26808 [Patent Document 3] Japanese Patent Application Publication No. 59-175825 [Patent Document 4] Japanese Patent Publication No. 107725 / 1983 [Patent Document 5] Japanese Unexamined Patent Publication No. 63-209579 [Patent Document 6] Japanese Patent Application Publication No. 6-233627 [Patent Document 7] Japanese Patent Application Publication No. 8-75 [Patent Document 8] Japanese Patent Application Publication No. 8-172901 [Patent Document 9] Japanese Patent Application Publication No. 9-327232 [Patent Document 10] Japanese Patent Application Publication No. 10-117770 [Patent Document 11] Japanese Patent Application Publication No. 11-103666 [Patent Document 12] Japanese Patent Publication No. 2000-83471 [Patent Document 13] Japanese Patent Publication No. 2001-69850 [Patent Document 14] Special Publication No. 2001-269054 [Patent Document 15] Japanese Patent Publication No. 2003-116342 [Patent Document 16] Japanese Patent Publication No. 2003-250336 [Patent Document 17] Japanese Patent Publication No. 2004-33061

Patent Document 18

Patent Document 19

Patent Document 20

Patent Document 21

Patent Document 22

Patent Document 23

Patent Document 24

Patent Document 25

Patent Document 26

Patent Document 27

Patent Document 28

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, in the method of culturing Cordyceps sinensis using an artificial culture medium, the hyphae are not uniformly formed like in the body of a living insect and are formed only in a part of the medium. Thus, when the formation of hyphae is insufficient, the components of the artificial culture medium remain as they are, not only reducing the productivity of Cordyceps sinensis, but also, for example, components specific to the medium remaining in the extracted extract component, or when using insects in the artificial culture medium, the odor specific to insects remaining.

[0005] Therefore, an object of the present invention is to provide a method for culturing Cordyceps sinensis that can perform the culturing of Cordyceps sinensis using a medium uniformly and stably.

Means for Solving the Problems

[0006] The method for culturing Cordyceps sinensis according to claim 1 of the present invention includes a medium adjustment step of adjusting a medium by containing moisture, a medium accommodation step of accommodating the medium adjusted in the medium adjustment step in a culture container, a medium sterilization step of sterilizing the culture container containing the medium in a temperature atmosphere exceeding 100°C, an inoculation step of putting the fungus of Cordyceps sinensis into the culture container after cooling the culture container sterilized in the medium sterilization step, a mycelium culture step of promoting the formation of the mycelium of the Cordyceps sinensis fungus after the inoculation step, and a mycelium state determination step of determining the formation state of the mycelium in the mycelium culture step. In the mycelium state determination step, the determination of mycelium formation is performed by color determination of at least one of the side surface of the medium and the bottom surface of the medium. The present invention according to claim 2 is characterized in that, in the method for culturing Cordyceps sinensis according to claim 1, in the mycelium state determination step, voids are formed in the medium determined to have insufficient formation of the mycelium, and after forming the voids, the mycelium culture step is continued. The present invention according to claim 3 is characterized in that, in the method for culturing Cordyceps sinensis according to claim 2, the voids in the medium are formed between the side surface of the medium and the side surface of the culture container or between the bottom surface of the medium and the bottom surface of the culture container by moving or dividing the medium inside the culture container.

Effect of the Invention

[0007] According to the method for culturing Cordyceps sinensis of the present invention, the state of mycelium formation can be accurately determined by performing the determination of mycelium formation by color determination. By performing this color determination using at least one of the side surface of the medium and the bottom surface of the medium, the culture of Cordyceps sinensis using the medium can be carried out uniformly and stably without reducing productivity.

Brief Description of the Drawings

[0008] [Figure 1] FIG. 1 is a flowchart showing each step of the method for culturing Cordyceps sinensis according to an embodiment of the present invention. [Figure 2] (a) is a photograph of the culture medium from above after the mycelial culture process, and (b) is a photograph of the culture medium from above after the fruiting body formation process. [Figure 3] Photograph of the bottom of the culture medium in the culture vessel. [Figure 4] Photographs of the bottom surfaces of 10 culture vessels and a diagram showing these images in binarized form. [Figure 5] A diagram showing the effect of transferring the culture medium to a different culture vessel on mycelial formation. [Modes for carrying out the invention]

[0009] A method for cultivating Cordyceps according to the first embodiment of the present invention comprises a culture medium preparation step of preparing a culture medium by adding water, and a culture medium storage step of storing the culture medium prepared in the culture medium preparation step in a culture vessel, The method comprises a culture medium sterilization step in which the culture vessel containing the culture medium is sterilized in an atmosphere at a temperature exceeding 100°C, an inoculation step in which Cordyceps fungi are placed into the culture vessel after the culture vessel has been cooled after the culture medium sterilization step, a mycelial culture step in which the mycelium of Cordyceps fungi is promoted after the inoculation step, and a mycelial state determination step in which the state of mycelial formation in the mycelial culture step is determined. In the mycelial state determination step, mycelial formation is determined by color determination of at least one of the sides and bottom of the culture medium. According to this embodiment, the state of mycelial formation can be accurately determined by color determination. In particular, by performing this color determination using at least one of the sides and bottom of the culture medium, the cultivation of Cordyceps using the culture medium can be carried out uniformly and stably without reducing productivity.

[0010] A second embodiment of the present invention is a method for culturing Cordyceps according to the first embodiment, wherein in the mycelial state determination step, voids are formed in the culture medium where mycelial formation is determined to be insufficient, and the mycelial cultivation step is continued after the voids are formed. According to this embodiment, by forming voids in the culture medium, mycelial formation is promoted, and the mycelial formation rate of Cordyceps using the culture medium can be increased.

[0011] A third embodiment of the present invention is a method for cultivating Cordyceps according to the first embodiment, wherein voids in the culture medium are formed between the side of the culture medium and the side of the culture container, or between the bottom of the culture medium and the bottom of the culture container, by moving or dividing the culture medium inside the culture container. According to this embodiment, by moving or dividing the culture medium inside the culture container to form voids in the culture medium, mycelial formation is promoted, and the mycelial formation rate of Cordyceps using the culture medium can be increased. [Examples]

[0012] Figure 1 is a flowchart showing the steps of a method for cultivating Cordyceps according to one embodiment of the present invention. The Cordyceps used in the present invention is cultivated using a culture medium mainly composed of silkworm pupae or a culture medium mainly composed of grains. In the culture medium preparation step (S1), water is added to prepare culture media mainly composed of silkworm pupae or grains. The silkworm pupae used as the main component of the culture medium can be live silkworm pupae extracted from cut cocoons, or dried silkworm pupae that have been dried at the cocoon stage. Furthermore, live silkworm pupae or dried silkworm pupae after silk reeling can also be used. Post-reeling pupae contain 60% crude protein, 9% total nitrogen, as well as ash, glycogen, and other nutrients necessary for the development of Cordyceps. When using dried silkworm pupae as a culture medium, include 80 ml to 120 ml of water per 100 g of dried silkworm pupae. When using dried grains (such as soybeans or adzuki beans) as a culture medium, it is preferable to include 120 ml to 560 ml of water per 100 g of dried grain. When using dried grains, it is preferable to add a nutrient solution such as glucose, peptone, or silkworm pupa powder, or pupal liquid extracted by boiling silkworm pupae, as disclosed in the patent documents listed as prior art. Dried silkworm pupae or dried grains are crushed in a mixer, sieved to remove foreign matter and irregular shapes, water is added and mixed, and left to stand for about 20 minutes to prepare a wetland culture medium.

[0013] In the culture medium placement step (S2), the culture medium prepared in the culture medium preparation step (S1) is placed into the culture vessel. The culture vessel should have a lid, be airtight enough to maintain humidity inside the vessel, and preferably have a heat resistance of 120°C or higher, preferably around 140°C. It is preferable that the vessel be made of polypropylene resin or glass and have a certain degree of transparency. It is preferable that the length and width of the vessel are greater than the depth. For example, a vessel with length and width dimensions of 10cm to 40cm and a depth of 5cm to 20cm can be used. The culture medium should fill the container to a depth of 20% to 80%, preferably 40% to 60%.

[0014] In the culture medium sterilization step (S3), the culture vessels containing the culture medium in the culture medium containment step (S2) are sterilized under a temperature atmosphere exceeding 100°C. Sterilize the sealed culture vessels in an autoclaver at 121°C for 60 minutes. In addition, uncrushed pupae can be used as a culture medium. Even with uncrushed pupae, water should be added and sterilized using an autoclaver at 121°C for 15 minutes, followed by natural cooling.

[0015] After the culture vessels have been sterilized in the culture medium sterilization step (S3), they are cooled to approximately room temperature in a clean bench before being inoculated (S4). In the inoculation process (S4), the Cordyceps fungus suspension is sprinkled evenly into the culture container. After sprinkling the Cordyceps fungus into the culture container, the container is closed with a lid. The Cordyceps fungi used as materials in this invention may be either Cordyceps types that produce ascospores or Isaria types that produce naked conidia. For example, Cordyceps militaris, Cordyceps pupa, Cordyceps japonica, and Cordyceps verrucosa can be used. The fungus used in the inoculation process (S4) can be ascospores or conidia formed on the fruiting body of Cordyceps sinensis, or secondary spores obtained by culture, and mycelium can also be used.

[0016] After the inoculation process (S4), a mycelial culture process (S5) is performed to promote the formation of Cordyceps mycelium in the culture medium. The mycelial culture process (S5) lasts approximately 60 days, although this varies depending on the fungal species and the temperature inside the culture chamber. Mycelial culture is carried out in a culture chamber at a temperature of around 20°C and a humidity of around 60%. UV sterilization or ozone sterilization may be performed as appropriate. In the mycelial culture process (S5), the humidity inside the culture container is checked by the presence or absence of water droplets adhering to the inner surface of the container. If it is determined that the humidity inside the container is not good, that is, that adequate humidity is not being maintained (NO in S6), moisture is supplied to the culture container by spraying sterile water into the container (S7).

[0017] Furthermore, in the mycelial culture process (S5), mycelial formation is assessed after approximately 30 days (S8). In the mycelial state determination step (S8), the state of mycelial formation in the mycelial culture step (S5) is determined by color determination of at least one of the sides of the culture medium and the bottom surface of the culture medium. In the mycelial state determination step (S8), if the culture medium is determined to have insufficient mycelial formation (NO in S8), voids are formed (S9), and after void formation, the mycelial culture step (S5) is continued for another 20 to 30 days. In the void formation step (S9), voids in the culture medium are formed between the side of the culture medium and the side of the culture container, or between the bottom of the culture medium and the bottom of the culture container, by moving or dividing the culture medium inside the culture vessel. In methods of moving the culture medium inside the culture vessel, for example, by turning it upside down or vibrating the culture vessel, a gap is formed between the bottom of the culture vessel and the culture medium. In the method of dividing the culture medium inside the culture vessel, the lid is opened and the medium is divided into multiple portions using a knife or similar tool. Note that the medium does not necessarily need to be completely separated; simply making cuts is sufficient. This void formation process (S9) allows hyphal formation to continue.

[0018] In the mycelial state determination step (S8), if the mycelial body is determined to be in good condition (YES in S8) and fruiting body formation is not necessary (NO in S10), the mycelial culture step (S5) is continued for another 20 to 30 days, after which the culture medium is divided (S11) and dried (S12). If fruiting body formation is required (YES in S10), the fruiting body formation process (S13) is carried out, followed by the division of the culture medium (S11) and drying (S12). In the fruiting body formation process (S13), after the mycelium has formed well (YES in S8), cultivation is carried out for a further 60 days. The fruiting body formation process (S13) is carried out in a culture room at a temperature of around 20°C or slightly higher than that of mycelial culture, with a humidity of around 60%. The fruiting body formation process (S13) ends when the fruiting body begins to turn white and spores begin to emerge. In this embodiment, the determination of the mycelial state (S8) is described as being performed at an intermediate stage of the mycelial culture process (S5). However, it is preferable to perform the determination at the end of the mycelial culture process (S5). In particular, for those in the void formation process (S9), the mycelial culture process (S5) should be continued for another 20 to 30 days after void formation, and then the determination of the mycelial state (S8) should be performed again. Therefore, all those that underwent drying (S12) were determined to have good mycelial formation.

[0019] Figure 2(a) is a photograph of the culture medium from above after the mycelial culture process, and Figure 2(b) is a photograph of the culture medium from above after the fruiting body formation process.

[0020] Figure 3 is a photograph of the bottom of the culture medium in the culture vessel. The whitish areas are where mycelium is formed, and it can be seen that the rate of mycelial formation differs depending on the culture vessel.

[0021] Figure 4 shows photographs of the bottom surfaces of 10 culture vessels and binarized versions of these images. The black areas after binarization represent areas where hyphal formation has not yet occurred. Odd-numbered vessels (1, 3, 5, 7, 9) have a hyphal coverage rate of less than 50% and are culture media judged to have insufficient hyphal formation in the hyphal state determination step (S8). The even-numbered culture media shown in Figure 4 (2, 4, 6, 8, 10) have a mycelial coverage rate of 95% or more and are judged to have good mycelial formation in the mycelial state determination step (S8). Although Figure 4 shows the bottom surface of the culture vessel, the same determination can be made on the sides of the culture vessel, and the determination results for the bottom surface and the sides of the culture vessel are almost identical. In this way, the state of mycelial formation can be accurately determined by using color determination. In particular, by performing this color determination using at least one of the sides and bottom surfaces of the culture medium, it is possible to cultivate Cordyceps using the medium uniformly and stably without reducing productivity.

[0022] Figure 5 shows the effect of transferring the culture medium to a different culture vessel on mycelial formation. Figure 5 shows a comparative experiment between a control group (where culture medium after 30 days of mycelial culture was continuously cultured in the same culture vessel) and a treatment group (where culture medium after 30 days of mycelial culture was transferred to a different culture vessel and continuously cultured). On day 50 of cultivation, 15 cultures were randomly selected as either the control group (not transferred) or the treatment group (transferred). Before drying, the cultures were cut with a knife, the cut surfaces were photographed, and the proportion of mycelium was determined from the area of ​​the cut surface and the area of ​​mycelium. The mycelial proportion data was confirmed to be homovariate using an F-test, followed by a Student's t-test. The results showed a significant difference between the experimental groups (df=28, t=3.430, p=0.0019). This indicates that mycelial formation is promoted by transferring the culture medium. Furthermore, on day 30 of cultivation, the culture vessels were transferred, and on day 50 of cultivation, 15 cultures were randomly selected from those in which fruiting bodies had developed and 15 cultures in which fruiting bodies had not developed. The proportion of mycelium in the cross-section of the culture vessels was compared, but no significant difference was observed. Figure 5 shows the effect of transferring cultures to different containers. However, it was found that hyphal formation is promoted by moving or dividing the culture medium within the culture container to create voids in the medium, without transferring the cultures to different containers. [Industrial applicability]

[0023] According to the present invention's method for culturing Cordyceps, it is possible to cultivate Cordyceps using a culture medium in a uniform and stable manner.

Claims

1. A culture medium preparation step involves preparing the culture medium by adding moisture, A culture medium preparation step involves placing the culture medium prepared in the culture medium preparation step into a culture vessel, A culture medium sterilization step is performed in which the culture vessel containing the culture medium in the culture medium containing step is sterilized in an atmosphere at a temperature exceeding 100°C. After cooling the culture vessel that has been sterilized in the culture medium sterilization step, the inoculation step involves placing the Cordyceps fungus into the culture vessel. Following the inoculation step, a mycelial culture step is performed to promote the formation of the mycelium of the Cordyceps fungus, A mycelial state determination step for determining the formation state of the mycelium in the mycelial culture step, It has, In the mycelial state determination step, mycelial formation is determined by color determination of at least one of the sides of the culture medium and the bottom surface of the culture medium. A method for cultivating Cordyceps fungi characterized by the following features.

2. In the mycelial state determination step, if the mycelial body is determined to be insufficiently formed, voids are formed in the culture medium, and the mycelial culture step is continued after the voids are formed. The method for cultivating Cordyceps sinensis according to feature 1.

3. The voids in the culture medium are formed between the side surface of the culture medium and the side surface of the culture container, or between the bottom surface of the culture medium and the bottom surface of the culture container, by moving or dividing the culture medium inside the culture container. The method for cultivating Cordyceps according to feature 2.

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