JP6037676B2 - Cordyceps culture method and culture apparatus used therefor - Google Patents

Description

  The present invention relates to a method for cultivating cordyceps and a culture apparatus used therefor.

Cordyceps spp. (Cordyceps spp. And Paecilomyces spp.) Are insect parasitic fungi.
Cordyceps and its products are nutrients that are claimed to be beneficial in preventing and / or delaying diabetes, cardiovascular disease, cancer and metabolic diseases, but lacking good scientific evidence. Widely used (Non-Patent Document 1).
In vivo effect on decrease in insulin resistance and increase in insulin secretion by water extract of Cordyceps militaris (Non-patent document 2), anti-tumor activity by Plutenoglucan plutoglucan complex (Non-patent document 3) ), The antihyperlipidemic effect (Non-patent Document 4) of hot water extraction of Tibetan Cordyceps sinensis is worthy of attention.
Non-Patent Document 5 shows that the Tibetan Cordyceps extract may improve the learning and memory ability of mice administered with D-galactose.
Non-Patent Document 6 shows that a hot water extract of Paecilomyces tenuipes cultured in an insect body specifically using silkworms as a host may prevent brain function decline in aging mice.
However, Cordyceps (Cordiseptus and Pekyromyces) are already rare resources and are rarely collected in nature. In addition, the destruction of nature by collecting Tibetan cordyceps is a social problem. Therefore, artificial cultivation of cordyceps is attracting attention.
In Japan, in Tsuwano Town (former Hihara Town), Shimane Prefecture, a method for cultivating Cordyceps sinensis (Sanatake, Sasatake and Sasatake) in insects with silkworms as a specific host has been established (Patent Document 1). In the same area, silkworms bred on mulberry leaves are used for cordyceps by the Tsuwano culture method, and research is also being conducted in parallel to find new bioactive functions related to cordyceps by the Tsuwano culture method. Patent Document 7).
Cordyceps by the Tsuwano culture method is characterized by inoculating and cultivating silkworms of live silkworms. Since the fruiting body is formed after the fungal nuclei are reliably produced in the silkworm, not only the smell is low, but also a high bioactivity function is expected.
Inoculation should be performed on silkworms that have emerged from the upper silkworm (usually about 16 days from the upper silkworm day). However, from the upper eyelid to the seventh day, the body surface of the eyelid is soft and may be damaged during the inoculation process. Furthermore, even if it is 7 to 16 days ago, the pupae that have grown will emerge without infection. Therefore, the inoculation period is very limited.
In Tsuwano Town, Shimane Prefecture, due to the decline in sericulture farmers in the area due to aging, 200,000 cocoons were brought from other areas in 2010. The production of was hit hard. In subsequent studies, it was found that the cause was the growth of the cocoon.
By the way, anesthesia using carbon dioxide gas is used not only for animals (Patent Document 2) but also for fish (Patent Documents 3 and 4).
Carbon dioxide anesthesia is also used for insects at research sites (Non-patent Document 8).

Japanese Patent No. 3593429 Japanese Patent No. 2620121 JP 2007-186476 A JP 2008-54559 A

Paterson, R.A. R. M.M. (2008) Cordyceps-A traditional Chinese medicine and another funeral therapeutic biofactory? Phytochemistry, 69, 1469-1495 Choi, S .; B. Park, C.I. H. Choi, M .; K. Jun, D.C. W. and Park, S.A. (2004) Improvement of Insulin Resistance and Insulin section by water extracts of Cordyceps militaris, Pellinus linteus and Pecilometices. Biosci. Biotechnol. Biochem. 68, 2257-2264 Nakamura, T .; Matsugo, S .; Uzuka, Y. et al. Matsuo, S .; and Kawagishi, H .; (2004) Fractionation and anti-tumor activity of the myceria of liquid-cultured pellinus linteus. Biosci. Biotechnol. Biochem. 68, 868-872 Koh, J.H. -H. Kim, J .; -M. Chang, U.K. -J. and Suh, H .; -J. (2003) Hypocholesteric effect of hot-water extract from myceria of Cordyceps sinensis. Biol. Pharm. Bull. , 26, 84-87 Ji, D.D. -B. Ye, J .; , Li, C.I. -L. Wang, Y .; -H. Zhao, J. et al. and Cai, S.A. -Q. (2009) Antiaging effect of Cordyceps sinensis extract. Physother. Res. , 23, 116-122 Tsushima, M .; Yamamoto K. Goryo, M .; Suzuki, F .; and Suzuki, K (2010) Hot-water extract of Paecilomyces teneupes from the siliworm pupae improves D-galactose-induced brainaging. Journal of Insect Biotechnology and Seriology. 79-2, 45-51 Suzuki Koichi (2005) Insect Technology Overview-Research and Development Trends-In: Insect Technology Research and Industrial Use, pp. 3-12, CMC Publishing Tokyo Boorman, J.M. (1975) Semi-automatic device for inoculation of small incidents with viruses. Lab Pract. , 24-2, 90

The carbon dioxide anesthesia of Patent Document 2 prevents live birds from rampaging when slaughtering.
In addition, carbon dioxide anesthesia for live fish proposed in Patent Document 3 and Patent Document 4 is used to prevent damage and physical exhaustion of the fish body by preventing rampage in the same manner as anesthesia with drugs. It is used as an alternative to drugs because of edible safety and residual toxicity issues.
In the case of Non-Patent Document 8, carbon dioxide is used to stop the movement of insects.
Thus, carbon dioxide anesthesia is generally used to stop movement.
According to the Tsuwano-style cordyceps culture, insects have a biological defense mechanism against fungi, and Cordyceps fungus has a breakthrough mechanism against the insect's biological defense mechanism. Anesthesia acts on both insects and fungi. The infection rate cannot be improved.
For example, in experimental insects, chilled anesthesia is sometimes used together with carbon dioxide anesthesia. However, refrigerated anesthesia stops the fungal activity together with the insects, and the infection rate of the bacteria cannot be increased.
By the way, the cocoon that has grown has emerged without being infected by bacteria, but the occurrence of emergence is not only a problem of yield but also a problem of environmental deterioration due to urine, so that the cocoon has grown Realization of infection is strongly desired.

  Therefore, an object of the present invention is to realize mass stable production of cordyceps by suppressing emergence even in the case of growing cocoons.

The method for cultivating Cordyceps sinensis of the present invention according to claim 1 is a method for cultivating Cordyceps sinensis using an insect as a host, using a cocoon moth as the insect, and inoculating the Cordyceps fungus on the moth , to the pupae before emergence, by the action of 43% or more of the concentration of carbon dioxide gas, characterized in anesthesia and to Rukoto the pupae are.
The culture device of the present invention described in claim 2, a culture apparatus for use in a method of culturing Cordyceps claim 1, and anesthesia chamber to put the insects after the inoculation, the carbon dioxide gas to the anesthesia chamber And at least two anesthesia containers as the anesthesia chamber, the anesthesia container on the upstream side and the anesthesia container on the downstream side are connected by a connecting pipe, and the gas generation source The carbon dioxide gas is introduced into the anesthesia container on the upstream side, the carbon dioxide gas is guided to the downstream anesthesia container by the connecting pipe, and at least the upper part of the anesthesia container on the downstream side is led out of air A hole is provided.

  According to the present invention, after the fungus of Cordyceps is inoculated into insects, the biological activity of the insects is suppressed by causing carbon dioxide to act on the insects, so that the bacteria are easily infected.

The figure which shows the experimental method by the carbon dioxide anesthesia by one Example of this invention The figure which shows the experimental method by the cold storage anesthesia by the Example The figure which shows the experimental result by the carbon dioxide anesthesia by the same Example The figure which shows the experimental result by the cold storage anesthesia by the same Example The figure which shows the comparison of the emergence rate of the control group and the refrigerated anesthesia group by the Example The figure which shows the influence on the emergence of the eyelid by the carbon dioxide anesthesia by the same Example The figure which shows the influence on the infection by the carbon dioxide anesthesia days by the same Example Configuration diagram of carbon dioxide anesthesia device used in the same example Configuration diagram of carbon dioxide anesthesia apparatus according to another embodiment of the present invention The block diagram of the carbon dioxide anesthesia apparatus used for the further another Example of this invention The figure which shows the experimental result about the effectiveness by the carbon dioxide anesthesia by the Example

Hereinafter, a method for cultivating Cordyceps sinensis according to an embodiment of the present invention will be described.
Cordyceps according to the present invention is not limited to Tibetan cordyceps, which is referred to as Cordyceps in China, but is an insect parasite that clings to insects such as the Ascomycetes, Bacchikinidae, stink bug, butterfly, coleoptera, bee, grasshopper, Host insects such as dragonflies and flies are also diverse.
However, the method for cultivating cordyceps in the present invention is intended for inoculating cordyceps fungus on living insects.
In addition to using ascospores or conidia formed on the fruit bodies of Cordyceps sinensis, mycelium can also be used for inoculation. In addition to direct inoculation of insects, inoculation may be performed on the body surface or on food.

The present invention is characterized in that carbon dioxide anesthesia is used for insects after inoculation to the insects. Carbon dioxide anesthesia here is characterized by the action of carbon dioxide gas, the insects in the assassination state, the sleep state, It means falling into a paralyzed state, and some kind of suppression of life activity has occurred.
As will be described later, in the case of a sputum, there is a case where it completely awakens and emerges after anesthesia, but for example, even if it awakens, it may not emerge after that.
That is, the state of anesthesia in the present invention refers to a state in which some vital activity is suppressed with respect to the host insect, and compared to a case where carbon dioxide gas is not allowed to act, To do.

The method of culturing Cordyceps according to the first embodiment of the present invention, the fungus Cordyceps sinensis, after inoculated into pupae, against pupae before emergence, the pupa by the action of 43% or more of the concentration of carbon dioxide anesthesia is a state and you shall. According to the present embodiment, the action of carbon dioxide suppresses the life activity of insects, and bacteria are likely to be infected. In addition, in the case of a bamboo shoot, which is a moth, it is possible to infect the cocoon that has progressed particularly in the pupae that has been grown. In addition, the appearance activity of the moth moth stops, and even the moth just before its emergence can be reliably infected.

According to a second embodiment of the present invention, there is provided an anesthesia room in which an insect after inoculation is placed and a gas generation for introducing carbon dioxide gas into the anesthesia room in the culture apparatus used for the method for cultivating cordyceps by the first embodiment. An anesthesia chamber using at least two anesthesia containers, and connecting an upstream anesthesia container and a downstream anesthesia container with a connecting pipe, and carbon dioxide from a gas generation source is used for anesthesia on the upstream side. Carbon dioxide gas is introduced into the container and the downstream anesthesia container is guided by a connecting pipe, and an air outlet hole is provided at least in the upper part of the downstream anesthesia container . According to the present embodiment, the air in the anesthesia chamber can be led out to form a carbon dioxide gas atmosphere, and the influence on insects due to an increase in pressure can be eliminated. Further, even in an anesthesia room made of a material having a low gas barrier property, carbon dioxide gas can be made to act reliably.

Silkworms (Shunrei x Shogetsu) were grown on artificial feed up to 4 years and on mulberry leaves after 4 years.
Cordyceps militaris (NBRC No.1000074) was purchased from (National Institute of Technology and Evaluation Technology).

First, an experimental method using carbon dioxide anesthesia will be described.
FIG. 1 shows an experimental method using carbon dioxide anesthesia.
The cocoon was taken out of the cocoon on the eighth day after the upper eve. In one experiment, 200 wrinkles with eye spots developed on the same day were used.
Forty days were inoculated into 40 pupae every day for 5 days (from “1 day after” to “5 days after” in FIG. 2) after the onset of eye spots. Twenty of the 40 were placed in a carbon dioxide anesthesia apparatus, and the remaining 20 were used as a control group. The inoculation was performed using a syringe in the sputum.
The sputum in the carbon dioxide anesthesia apparatus was taken out of the carbon dioxide anesthesia apparatus two days after the inoculation, and infection was confirmed on the 10th to 25th days after the onset of the eye point. Thereafter, the test was performed under the same conditions as in the control group. Infection was judged by hardening of sputum, and those that did not harden even after 25 days from inoculation were judged to be spoiled.
Six experiments were performed by the method shown in FIG.

Next, an experimental method using refrigerated anesthesia will be described.
FIG. 2 shows an experimental method using refrigerated anesthesia.
The cocoon was taken out of the cocoon on the eighth day after the upper eve. In one experiment, 200 wrinkles with eye spots developed on the same day were used.
Forty days were inoculated into 40 pupae every day for 5 days (from “1 day after” to “5 days after” in FIG. 2) after the onset of eye spots. Twenty of the 40 were placed in a refrigerated anesthesia apparatus (refrigerated at 5 ° C.), and the remaining 20 were used as a control group. The inoculation was performed using a syringe in the sputum.
The sputum in the refrigerated anesthesia apparatus was taken out of the refrigerated anesthesia apparatus two days after the inoculation, and the infection was confirmed on the 10th to 25th days after the onset of the eye point. Thereafter, the test was performed under the same conditions as in the control group. Infection was judged by hardening of sputum, and those that did not harden even after 25 days from inoculation were judged to be spoiled.
Three experiments were performed by the method shown in FIG.
In addition, the date of emergence of cocoons that emerged in three experiments was confirmed.

  ANOVA with Dunnett test was used for the infection effect by carbon dioxide anesthesia, Student t test was used for the infection effect by chilled anesthesia, and Kaplan-Meier method (failure rate plot) was used for the emergence rate by chilled anesthesia. When P <0.05, it is considered that there is a statistically significant difference.

In FIG. 3, the experimental result by carbon dioxide anesthesia is shown.
As the wrinkle grew, the number of infections decreased as shown in the control group (P <0.001 on the 4th day versus P <0.0001 on the 5th day). Most of this decrease was due to emergence. The total of the 6 groups in the control group was 600, the emergence was 286, and the corruption was 7.
On the other hand, in the carbon dioxide anesthesia group, most of the plants that were inoculated after the growth of the sputum were infected (P = 0.817, 5 days on the 4th day compared to the 1st day). Eye P = 0.2441). The total of 6 groups in the carbon dioxide anesthesia group was 600, 10 emergence and 15 rot.
From the above results, in the carbon dioxide anesthesia group, the infection rate did not decrease even when the growth of the eyelids progressed after days from the onset of eye point.

In FIG. 4, the experimental result by refrigeration anesthesia is shown.
As the pupae grew, the number of infections decreased as it appeared in the control. Most of this decrease was due to emergence. The total of the three groups in the control group was 300, the emergence was 158, and the corruption was 9.
In the refrigerated anesthesia group, the number of infections decreased as in the control group. Most of this decrease was due to emergence. The total of the three groups in the control group was 300, 142 emerged and 21 spoiled.
When comparing the refrigerated anesthesia group and the target group, the Student t test showed no statistically significant difference between the two from the first day to the fifth day.

FIG. 5 shows the rate of emergence of 158 animals that emerged in the control group and 142 animals that emerged in the refrigerated anesthesia group. It can be seen that the emergence was clearly delayed in the refrigerated anesthesia group (average 8.845, standard error 0.118) compared to the control group (average 7.025, standard error 0.108) (P <0.0001). ).
From the above results, in the refrigerated anesthesia group, the occurrence of emergence was delayed compared to the control group, but the infection rate was not improved.

FIG. 6 is a diagram showing the influence of cocoon gas anesthesia on the emergence of wrinkles, where the vertical axis indicates the number of emergence and the horizontal axis indicates the number of days of carbon dioxide anesthesia.
In this case, the emergence was confirmed when the number of days of carbon dioxide anesthesia was changed to 1, 2, and 3. Ten cocoons, each not inoculated, were used.
On the 1st, 9 animals emerged, but on the 3rd, most did not emerge. In order to suppress the emergence with certainty, it is preferable to leave it in a carbon dioxide gas environment for a long period of time. However, it is preferable to keep the anesthesia period to 2 days or less because it is possible to suppress the spoilage by suppressing damage to the sputum as much as possible. In this experiment, the carbon dioxide gas concentration was measured with a high concentration gas detector (XP-3140 Rex Corporation) and set to 85% or more. When the carbon dioxide concentration was lowered, the number of emergence increased even if the anesthetic period was prolonged.
In this experiment, the carbon dioxide gas concentration was set to 85% or more, but it was confirmed that the activity of the sputum stopped and anesthesia was reached at 43% or more.

FIG. 7 is a diagram showing the effects on infection when the number of days of carbon dioxide anesthesia is 1 and 2. The vertical axis represents the number of infections, and the horizontal axis represents the date of inoculation (the course from the occurrence of eye spots). Number of days).
In this experiment, 20 wrinkles were used, and wrinkles in which eye points developed on the 11th day from the upper eyelid were used.
When the number of days of carbon dioxide anesthesia was 1, the number of infections was improved compared to the control group, but was inferior compared to 2 days.
Therefore, the anesthetic period is preferably 2 days.

Next, the configuration of the carbon dioxide anesthesia apparatus used in the above embodiment will be described below.
FIG. 8 is a configuration diagram of the carbon dioxide anesthesia apparatus used in this example.
The carbon dioxide anesthesia device connects a carbon dioxide generation container (gas generation source) 1 and an anesthesia container (anesthetic room) 3 that exposes the inoculated sputum 2 in a carbon dioxide atmosphere with a vinyl tube (connecting pipe) 4. Then, the dry ice 5 was put in the carbon dioxide generating container 1, and the carbon dioxide generated from the dry ice 5 was continuously supplied to the anesthetic container 3.
Dry ice (Continental Corporation) used 1.5 kg × 2 (3 kg set) / day.

An 11 L container (250 mm × 300 mm × 150 mm) was used as the carbon dioxide generating container 1, and a 350 L container (440 mm × 740 mm × 350 mm) was used as the anesthetic container 3.
Carbon dioxide gas was generated in the carbon dioxide gas generation container 1, carbon dioxide gas was led out from the lower part of the carbon dioxide gas generation container 1, and carbon dioxide gas was supplied from the lower part of the anesthetic container 3. In order to reliably supply carbon dioxide gas, the carbon dioxide generating container 1 and the anesthetic container 3 are provided with a predetermined height difference (80 cm), and in order to avoid the influence of cooling by the dry ice 5, the vinyl tube 4 is sufficient. The carbon dioxide gas was heated to room temperature and supplied to the anesthesia container 3.
The anesthesia container 3 is an upper lid type, and a small hole (air outlet hole) 3b is provided in the upper lid 3a. By making the anesthesia container 3 an upper lid type, the influence of the outflow of carbon dioxide gas to the outside when the basket 2 is put in and out is reduced. Further, by providing a small hole 3b in the upper lid 3a, nitrogen and oxygen can be easily removed from the anesthesia container 3, thereby preventing the internal pressure of the anesthesia container 3 from rising and eliminating the influence of the pressure.

Next, the configuration of a carbon dioxide anesthesia apparatus according to another embodiment of the present invention will be described below.
FIG. 9 is a configuration diagram of the carbon dioxide anesthesia apparatus used in this example.
In this embodiment, a carbon dioxide cylinder 7 is used instead of the carbon dioxide generating container 1. As in the present embodiment, a carbon dioxide gas cylinder 7 can be used as a carbon dioxide gas generation source, and a vinyl tube 4 is connected to the carbon dioxide gas cylinder 7 via a regulator 6.
Other configurations are the same as those of the previous embodiment, and thus the same reference numerals are given and description thereof is omitted.

Next, the configuration of a carbon dioxide anesthesia apparatus according to still another embodiment of the present invention will be described below.
FIG. 10 is a configuration diagram of the carbon dioxide anesthesia apparatus used in this example.
In the present embodiment, a carbon dioxide gas cylinder 7 is used, and the regulator 6 is branched into a plurality of paths, and the anesthesia container 3 is provided in series in each path. A small hole (air outlet hole) 3b is formed in the upper lid 3a of the anesthesia container 3 on the most downstream side. Each anesthesia container 3 is connected by a connecting pipe 4 that connects the upper position and a connecting pipe 4 that connects the lower position. A pair of connecting pipes 4 that connect the upper positions are provided near both ends of the anesthetic container 3, and a pair of connecting pipes 4 that connect the lower positions are also provided near both ends of the anesthetic container 3.
The concentration measuring device 8 is provided in the anesthesia container 3 on the most downstream side.

By providing a plurality of anesthesia containers 3 as in this embodiment, each anesthesia container 3 can be made independent, and the decrease in carbon dioxide gas concentration when the sputum is put in and out is limited only to the anesthesia container 3 to be opened. The influence on other anesthesia containers 3 can be reduced. In addition, by providing the connecting tube 4 at a position above the anesthetic container 3, it is possible to prevent oxygen from remaining in the upper space of the anesthetic container 3. Further, by providing the connecting pipe 4 also at a position below the anesthetic container 3, the supply speed of carbon dioxide gas to the anesthetic container 3 located on the downstream side can be increased.
Further, by forming the small hole 3b in the most downstream anesthetic container 3, the flow of carbon dioxide gas from the upstream to the downstream can be promoted. Further, since the small hole 3b is formed in the upper lid 3a, oxygen can be discharged in a state where carbon dioxide gas having a high specific gravity is retained in the anesthetic container 3.

In the above embodiment, the case where the sputum is put into the carbon dioxide anesthesia apparatus immediately after the inoculation (within several hours) has been explained. Experiments were conducted on the effectiveness of gas anesthesia.
The results are shown in FIG.
On the 9th day after inoculation, the pupae that were alive in an unemerged state were placed in a carbon dioxide device and carbon dioxide anesthetized. The period of carbon dioxide anesthesia was 2 days, 3.5 days, and 5 days.
With 2 days of anesthesia, 16 animals emerged, but 9 animals were infected. The reason why the sum of the number of emergence and the number of infections was small compared to the total number was because of decay without emergence.
Although not shown in FIG. 11, as a comparative example, on the 9th day from the inoculation date, 25 pupae living in an unemerged state were left without carbon dioxide anesthesia. Has emerged.
Therefore, it has been clarified that carbon dioxide anesthesia works effectively even before days have passed since the inoculation.

In the above embodiment, the case where carbon dioxide gas is continuously supplied has been described. After the inside of the anesthesia container 3 reaches a predetermined concentration of carbon dioxide gas, the introduction from the vinyl tube 4 is stopped, and a small hole (air derivation) is performed. The hole 3b may be closed and the anesthetic container 3 may be sealed. However, when the airtight container 3 does not have a gas barrier function, the carbon dioxide gas concentration decreases with the passage of time, so it is preferable to intermittently supply the carbon dioxide gas.
Further, instead of the anesthesia container 3, carbon dioxide gas may be sealed using a plastic bag or a nylon bag. However, in this case as well, it is necessary to pay attention to a decrease in carbon dioxide gas concentration if the gas barrier function is not provided. is there.

In order to cultivate cordyceps by receiving cocoon moths from sericulture farmers who remain in the country, it is necessary to consider the growth of moths during the transportation period. Although the growth can be suppressed by refrigerated transport, it is not possible to obtain a sufficient cooling effect when transporting a large amount of strawberries, and if it is subdivided into small portions to obtain a cooling effect, enormous transport costs are incurred. Resulting in.
In addition, in the cultivation of Cordyceps sinensis in living moths, emergence must be dealt with by contamination with manure, and deterioration of the working environment due to odor is also very painful for workers.
Therefore, it is very effective in the culture of Cordyceps medicinal plants by living moths to suppress the emergence of wings that have grown by the present invention.
With this carbon dioxide anesthesia, it may be possible to stably cultivate the kind of cordyceps that was difficult until now, and a new cordyceps that could not be cultured until now by applying to insects other than moths. Can be expected.
In this example, Sanagitake was used as a fungus of Cordyceps sinensis, but it has already been confirmed that the mechanism of infection of moths using Hananagitake and Konanagitake which is different from the shape of fruit bodies from Sanagitake is almost the same, The present invention can be applied to other Cordyceps fungus fungi, such as Hanasa Nagatake and Konasa Nagatake.

  The present invention is suitable for a method for cultivating cordyceps using live insects.

1 Carbon dioxide gas generation container (gas generation source)
2 ３ 3 Anesthesia container (anesthesia room)
3a Top lid 3b Small hole (Air outlet hole)
4 Vinyl tube (connecting tube)
5 Dry ice 6 Regulator 7 Carbon dioxide gas cylinder

Claims (2)

A method for cultivating Cordyceps sinensis using an insect as a host,
Using insect moths as the insects,
After inoculating the fungus of Cordyceps sinensis on the cocoon , the cocoon soy grass is anesthetized by acting carbon dioxide at a concentration of 43% or more on the cocoon before emergence. Method. A culture apparatus for use in the method for cultivating Cordyceps as claimed in claim 1 ,
An anesthesia room for placing the insect after inoculation;
A gas generation source for introducing the carbon dioxide gas into the anesthesia room,
As the anesthesia room, at least two anesthesia containers are used,
The upstream anesthetic container and the downstream anesthetic container are connected by a connecting pipe,
Introducing the carbon dioxide gas from the gas generation source into the anesthetic container upstream;
The carbon dioxide gas is guided by the connecting pipe to the anesthetic container on the downstream side,
An culturing apparatus comprising an air outlet hole at least in an upper part of the anesthesia container on the downstream side .