KR101491743B1 - Cultivation method of ascomycota mushroom using nano-gold coloid - Google Patents

Abstract

The present invention relates to a method for cultivating a mushroom fungus using a gold nanocolloid solution, specifically, a method for cultivating mushroom fungus caused by incorporation of atmospheric bacteria, airborne fungi, and harmful bacteria, The method comprising the steps of: (a) cultivating a mixed solution comprising a mixture of a mixture of a solution and a solution in a culture medium; (b) Cultivation method.
Conventional cultivation methods include the death of mushroom species caused by atmospheric bacteria, airborne fungi, and harmful fungi which are inevitably involved in the cultivation process, insufficient formation of fungi due to degradation of viability of seed bacterium, A part of or all of the contamination occurred frequently in a vicious cycle.
However, in the case of the Cordyceps sinensis and the Aspergillus ficus-indica mushroom bacteria described in the embodiment of the present invention, since the gold nanocolloid solution is used to sterilize atmospheric bacteria, airborne bacteria, and harmful bacteria in the immobilized gold nano, The number of fruiting bodies is greatly increased, and the growth period is increased, and it can be confirmed that there is a great effect on the growth of fruit bodies.
In addition, unlike conventional cultivation methods of carrot fungi and porcine fungi that artificially replace the destruction and deficiency of nutrients by utilizing grains, sawdust, and abandoned surfaces, the present invention using a gold nano colloid solution is a method in which a certain amount If only water and nutrients are available, fruiting body can be cultivated by directly cultivating the fungi isolated from the fungus in an animal culture medium such as a fungus such as a fungus and a pupa, and the fungus can be cultivated. Also, Mycelia grows and mycelial mats are formed, and cultivation environment must be established for mushroom cultivation. The present invention can contribute to the cultivation of high-quality mushroom of acanthaceous fungus after expansion, and it can lay the foundation of environment-friendly cultivation, and its technical contribution will be great.
In addition, the gold nanocolloid solution used in the present invention can be used in a wide range of potential applications such as catalysis, medical diagnostic tools, and image sensing in addition to the antimicrobial activity and antioxidation activity of gold. Therefore, It is considered that the components (anti-cancer and anti-inflammatory) are rapidly absorbed by the human body, and each component (anti-cancer and anti-inflammatory) can be absorbed by the human body.

Description

Cultivation method of ascomycota mushroom using nano-gold coloid using gold nanocolloid solution [

The present invention relates to a method for cultivating a mushroom fungus using gold nano colloid solution. More specifically, the present invention relates to a method for cultivating a mushroom of a fungus fungus, comprising the steps of isolating a fungus of a fungus fungus, a step of mass culture with a solid seed or a liquid seed, A step of preparing a growth medium by high temperature sterilization at a high temperature (121 캜 or higher), a step of inoculating the growth medium of the mushroom mycelium into a growth medium, a step of growing into a fruiting body, In the preserving step, a gold nanoparticle, a gold nanocolloid solution, or a mixed (diluted) solution in which water or distilled water is mixed with the nanoparticles is added to the antipyretic fungus Which is characterized in that it is used in the step of mass culture of the mushroom, the preparation stage of the growth medium, and the step of growing and preserving the mushroom fruiting body Pop tall relates to the cultivation method.

Ascomycota is one of the fungus eukaryotes, and it has many kinds of plant pathogens, such as yeast, blue mold, and yeast fungus, as well as a number of plant pathogens, furthermore, mushroom, mushroom, , And mushrooms such as the devil spoon mushroom.

Except for single yeast yeast bacterium, the mycelium is composed of hyphae, and there is diaphragm in mycelium and there is no cross - connection in diaphragm. Propagation is oily and silent, and as a result of metaplastic reproduction, acanthosis is characterized by the formation of acacia and concha spores.

As a representative of the fungus fungus, the mushroom is a broad - leaved insect mushroom, 300 ~ 400 species were found in the world, and about 80 species were found in Korea. As a representative item, the Cordyceps militaris (L.) Link, which is parasitic to the insect pupa of insects, is widely known in Korea.

The main constituents of Cordyceps are 18.2% crude protein, 15.8% crude fat (13% saturated fatty acid, 82% unsaturated fatty acid), crude fiber 11.2%, carbohydrate 51.0%, ash 2.8% and other minor constituents such as cordisepin and millitarin. Cordicepin is a nucleic acid that is an isomer of quinic acid. It is involved in the genetic information of cells and activates the degraded immune function to prevent normal cells from being transformed into cancer cells. Its content is about 7% Militarin is a new physiologically active substance with antitumor activity. It contains about 6.1 mg per 100 g of Cordyceps sinensis, and it is known that Cordyceps is effective for anti-inflammation, anti-cancer, blood circulation, and improvement of the constitution.

As is the case with most fungus fungus mushrooms, Cordyceps is also found mainly in lowland forests, high valleys, and areas where streams meet, but its size and natural habitat is very small. Recently, The collection of caterpillar fungus is in a more difficult situation and continuous research is ongoing to mass-produce it.

A conventional method of artificial cultivation of Cordyceps mushroom is as follows: a) Separating the germs by germinating spores from cotyledonary fruiting bodies which are readily sold or purchased from pre-sale institutions such as the Korean caterpillar fungus (KEFC), b) A step of pre-culturing the recipient bacterium, a step of pre-culturing the plate-cultured inoculum (primary culture), a step of culturing a solid culture of a pre-cultured liquid seed bacterium or a solid medium such as a cereal bacterium, and c) D) cultivation of mycelium on the growth medium and inoculation of the mycelium on the growth medium of the growth medium and formation of the spermatozoa from the mycelium, And the step of cultivating Cordyceps mushroom until it grows into fruiting bodies.

The method of cultivation of other fungus fungus mushrooms is different from the specific materials used and cultivation environment in the separation step of fungi, seed culture, preparation of seed culture medium and seeding stage, and fruiting body growth stage. A method of cultivating a caterpillar fungus.

In the case of the method of cultivating the mushroom fungus represented by the conventional Cordyceps, the atmospheric and harmful microorganisms and the harmful microbes which can be inevitably incorporated into each step are implanted in the seed culture medium, or the growth medium is sprouted in the growth medium, Contamination, yield of fruiting body per unit area is reduced, and it is difficult to keep the raw state in the preservation process after growing, and the malicious cycle is repeated in the whole cultivation.

In order to overcome this vicious cycle, conventional cultivation methods use a liquid culture medium for high temperature and high pressure sterilization of cultivation equipment and culture medium, and easy aseptic automatic inoculation, and even if the seeds have grown into fruiting bodies, And so on. However, since this effort can not fundamentally solve atmospheric bacteria, harmful bacteria, and airborne bacteria, alternatives are urgent.

The present invention aims at solving the problems of conventional cultivation in the process of artificially cultivating and then mass-producing the mycelial fungus Mycobacterium tuberculosis in order to improve its quality, productivity and preservability.

As means for solving the above-mentioned problems,

The method for growing mushroom fungus according to the present invention comprises

a) a step of isolating the mushroom bacterium of the collected fungus fungus or the step of distributing the bacterium b) the step of mass-culturing the bacterium as a solid seed or liquid seed bacterium, c) a step of inoculating the seed bacterium, And d) inoculating the growth medium to the growth medium and cultivating the mushroom of the acanthosis fungus,

In step b), one or more of gold nanoparticles, gold nanocolloid solution, or a mixed solution of gold nanocolloid and water or distilled water may be added to the seed culture medium or seed culture to sterilize the germs,

In step c), at least one of gold nanoparticles, gold nanocolloid solution or mixed solution of gold nanocolloid and water or distilled water is added to the growth medium to sterilize the germs,

In step (d), the fungus body is sterilized by spraying at least one of a gold nanoparticle, a gold nanocolloid solution, or a mixed solution of gold nanocolloid and water or distilled water to the fruiting body immediately before transferring the growth medium to the general culture chamber.

Furthermore, the method of cultivating acarbose mushroom according to the present invention

Gold nano-colloid solution is a gold nanoparticle prepared by reducing HAuCl _{4 with a} phytochemical, or a gold nanoparticle coated with a polymer to ensure pH stability in vivo,

When the content of the gold nanoparticles in the gold nanocolloid solution is 6.0 g / L as a standard, the mixing ratio of water or distilled water: standard gold nanocolloid solution is 1: 0.0002 to 0.002 (specific example: 20 L : Gold nanocolloid solution 4 to 40 ml).

According to the present invention,

Since gold nanocolloid solution can selectively sterilize germs while maintaining the vitality of the yeast and seed bacterium,

First, it is possible to shorten the cultivation period of the fungus fungus mushroom compared to the conventional fungus fungus mushroom cultivation method,

Second, mushrooms of excellent quality (length, weight) can be provided in a large amount compared with the conventional method of cultivating fungus fungus mushroom,

Third, since the growth period is longer than that of the conventional fungus fungus mushroom cultivation method, it is possible to extend the preservation period of normal temperature (20 ° C) and slow cooling (10 ° C or less, at least 5 ° C)

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Advantageous effect.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a flow chart illustrating a cultivation method of a Chinese caterpillar fungus cultivated using a gold nanocolloid solution according to a representative embodiment of the present invention. FIG.
FIGS. 2A to 2C are photographs showing pre-cultivation (primary culture) of seedlings cultured on a flat plate through a sterile (clean chamber) according to the method of cultivating a Chinese cabbage harvesting with gold nanocolloid solution according to a representative embodiment of the present invention to be.
FIG. 3A and FIG. 3B are photographs of the inoculum solution and the composition of the inoculum of the Chinese cabbage seedlings cultured in a large amount according to the method of cultivating the Chinese cabbage using the gold nanocolloid solution according to the exemplary embodiment of the present invention.
FIG. 4 is an initial photograph of Cordyceps fruiting body formed on a growth medium containing a gold nanocolloid solution according to a method of cultivating a Chinese cabbage using a gold nanocolloid solution according to a representative embodiment of the present invention.
5a and 5b are photographs showing a cucumber herbaceous fruiting body cultured for 5 to 6 weeks according to a method of cultivating cucurbitacea using a gold nanocolloid solution according to a representative embodiment of the present invention, This is a comparative photograph of fruiting bodies of caterpillar fungus grown for 6 weeks.

Advantages and features of the present invention and methods for achieving the same will be apparent from the following description of a preferred embodiment of the method for cultivating a Chinese cabbage, which will be described in detail with reference to the accompanying drawings. However, as stated above, the present invention is not limited to the representative embodiments of Cordycephalus described in the specification, but can be applied and embodied in other forms or items of Carrot Fungus. Rather, the exemplary embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

As used herein, the term "Cordyceps chinensis" refers to Cordyceps mushroom (fruiting body) of the carrot fungus. To avoid confusion, the term " The mushrooms belonging to the fungi are used as the term "fungus fungus mushroom" and they are called "germs" for the atmospheric bacteria, airborne fungi, fungus fungi, Legionella fungi, .

The Cordyceps, described as a representative embodiment of the present invention, is applied to the Cordyceps militaris using the brown rice medium to which the core technology of the present invention is applied. In addition, Cordiceps sphecocephala, Cordiceps sinensis, , Paecilomyces sin clarii, Paecilomyces tenuipes, Paecilomyces japonica, and the like, and the broad range includes carrot fungus edible mushrooms.

The three major components of the mushroom cultivation of Cordyceps mushroom including Cordyceps are largely divided into seed, medium, and growth environment. The proper amount of oxygen is required for each step, and the mushroom fungus, Especially at the end of growth. This means that the proper inflow of oxygen and the proper discharge of carbon dioxide are essential. In particular, in the case of caterpillar fungus, such oxygen inflow and carbon dioxide emission are indispensable processes in the cultivation process and method, Contamination due to the incorporation of aerobic bacteria and atmospheric bacteria in the process soon causes a deterioration in the quality, productivity and preservation of fruiting body, thereby causing a great obstacle to mass production of high quality cordyceps and mushrooms.

Examples of the germs that inhibit the stable cultivation and growth of the conventional Cordyceps and Mycorrhizal fungi include airborne bacteria such as green fungus, white fungus, and yeast fungus, insect parasites, yeast enrichment, and Legionella bacteria And the like. Leptin (Leguminosae) and Legionella are bacteria that grow in the soil (or polluted water) and air, and inhibit the growth and growth of the mushroom and the growable mushroom fungus.

In particular, in the present specification, the pupae of Cordyceps mushroom described as a representative of the fungus fungus mushroom is inevitably involved in the decay of the protein component, and thus it is inevitably caused by the formation of the fungus, which inhibits the growth of the fruit body after germination of the fungus It destroys moisture and nutrients and causes rapid drying of fruiting body. In addition, Legionella is generally known to live in the soil and reproduce easily from water. However, it is called from 24 ℃ beyond the proper temperature (20 ℃ ± 2) , It is analyzed that the mixing and cultivation in the process of collecting the germ cells and the characteristics of the culture media (containing moisture) are evoked. In addition, if the fermentation of the brown rice medium is insufficient or when the water content is too high, the growth of Legionella and the bacteria becomes more active at the same time when the seedlings of the Chinese Cordyceps spp.

The reason why it is difficult to sterilize or sterilize the germs in the cultivation of the fungus fungus mushroom is due to the fact that these germs belong to the germ fungus mainly.

The inventors of the present invention reduced the limit of sterilization and disinfection of germs by the cultivation method of conventional fungus fungus mushroom and solved this problem. At the same time, the inventors of the present invention found that the fungus of the fungus fungus, While researching sterilization and sterilization methods effectively, it has attracted attention to a wide range of potentially usable properties of gold nanoparticles, including excellent sterilization performance, catalysis, medical diagnostic tools, image sensing, and the like. It has been found that only germs can be selectively sterilized or sterilized by diluting the particles to a certain concentration.

Any gold nanoparticle or gold nanocolloid solution may be used for the cultivation of the mushroom fungus mushroom using the gold nanocolloid solution of the present invention. However, gold nanoparticles or gold nanocolloid solution may be used. However, gold nanoparticles or gold nanoparticles prepared by reducing phthalocyanine (HAuCl ₄ ) Or a gold nanocolloid solution in which gold nanoparticles coated with a polymer are dispersed so as to ensure pH stability in vivo is preferable from the viewpoint of food safety. Examples of the phytochemicals used as a reducing agent include gallic acid, protocatechinic acid, and isoflavone, which can be extracted from natural plants such as soybean, and examples of the polymer used as the coating agent include polyethylene glycol and the like.

The gold nanoparticle or gold nanocolloid solution used as the bactericide in the method of cultivating the mushroom mycelium using the gold nanocolloid solution of the present invention may be diluted with water or distilled water.

The size of the gold nanoparticles used in the cultivation method of the fungus fungus mushroom of the present invention is approximately 1 to 120 nm. The gold nanocolloid solution used in the method of cultivating the mushroom of the present invention is characterized in that electrically neutral gold nanoparticles are stable Which is a colloidal solution of a purple color which is not precipitated.

The diluted solution containing the gold nanoparticles was prepared by mixing water or distilled water and a standard gold nanocolloid solution (based on the gold nanoparticle content of 6.0 g / l) in a volume ratio of 1: 0.0002 to 0.002, It was found that immobilized gold nanoparticles selectively sterilized all kinds of germs and sterilized the remaining germs even during the high temperature and high pressure sterilization process of the cultivation equipment.

When the mixing ratio of water or distilled water and the standard gold nanocolloid solution is less than 1: 0.0002, the bactericidal effect on the germs is insignificant in the preparation of the liquid culture broth and preparation of the growth medium, And it was repeated that the same kind of calligraphy as the conventional cultivation method was repeated. When the ratio exceeded 1: 0.002, it was confirmed that not only germ cells, but also the seeds of Cordyceps sinensis and Acanthopanax senticosus mushrooms were all killed.

In order to clarify the above contents in more detail, the conventional method for cultivating Cordyceps is described below in comparison with the method for growing Cordyceps, using the gold nanocolloid solution according to the present invention, and the invention The contents and techniques will be explained and presented in addition.

S-0. Collection of Cordyceps

The strain used in the method for cultivating the insecticide using the gold nanocolloid solution of the present invention may be obtained by germinating the spore derived from the collected Cordyceps sinensis by collecting the germ. However, in consideration of the economic cost of the product and the identity of the cultivated product, Won-kyung and Seongbun of Seongnam-moth, Cordyceps, recommended by Korea Institute of Science and Technology can be used.

S-1. Isolation of Cordyceps

Cordyceps and acanthosis fungus are germinated and formed mycelium when they are taken out and transplanted into the medium to isolate the Chinese cabbage. Among them, the agar medium used for the isolation and culture of the Cordycephalomyces sp. Are PDA (potato dextrose agar) medium and SDAY (sabouraud dextrose agar with yeast extract) medium. Among them, PDA medium is representative. Conventionally, to prevent contamination by germs, pieces of fruiting bodies are immersed in sodium hypochlorite or sterilized using a high-temperature and high-pressure sterilizer.

In contrast, in the method for cultivating the insecticide using the gold nanocolloid solution of the present invention, the gold nanocolloid solution (water or distilled water 1: standard gold nano-colloidal solution 0.0002 to 0.002) is used for the equipment (plate, test tube, , And the method is characterized in that the pieces of fruiting bodies are immersed in the above mixed solution or the above mixed solution is added to the agar medium to effectively isolate the germs in the air permeated during the transplantation process with the separation of the germ and the agar medium .

S-2. Cultivation of Cordyceps spp.

S-2-1. Flat culture

Plate culture is performed by transplanting into a plate incubator such as a petri dish for expansion of the yeast cells. The medium was placed in a large Erlenmeyer flask and dissolved in distilled water until it became transparent. The inlet of the Erlenmeyer flask was closed with a sommerson cover, covered with aluminum foil, sterilized at 121 DEG C and 15 psi for about 20 minutes, Place in an incubator. At this time, the inlet of the test tube is flame-sterilized and the mycelium is removed with the platinum-iodate medium and inoculated in the center of the plate-type incubator. Place the inoculated plate incubator in a thermostat (about 25 ℃, humidity 60 ~ 70%) so that mycelium grows quickly and vitally. Such an operation is generally performed in a clean room since it must be performed in a state of being blocked from other germs.

However, in the conventional case, the inlet of the test tube was sterilized by flame sterilization and the medium was sterilized by high temperature and high pressure in a clean room to prevent contamination by germs. However, the present plate culture is not a sterilization process which is 100% Therefore, there is a limit to the complete sterilization which is necessary for the optimum environment of the plate culture.

Therefore, in the method of cultivating the insect hatchery using the gold nanocolloid solution of the present invention, various equipment (flask, test tube, petri dish, platinum beet, etc.) and medium are mixed with gold nanocolloid solution (Water or distilled water 1: standard gold nanocolloid solution 0.0002 to 0.002) and then mixed with gold nanocolloid solution (water or distilled water 1: standard gold nanocolloid solution 0.0002 to 0.002) The microorganisms penetrated by the microorganisms are automatically sterilized during the plate culture. As a result, the same concept and plate culture method were applied to the fungus fungus mushroom in the same way, and the result showed the same sterilizing power.

S-2-2. Liquid culture

Since it is advantageous to prepare and use liquid germs in order to prevent the emergence of germs in the conventional cultivation method and speedy activation of mycelium, liquid culture is also used in the present invention.

A conventional method of culturing liquid seeds is as follows.

Unlike the culture medium for plate culture, there is a difference in that agar, which is a coagulant, is not added to the liquid medium used for liquid culture. The medium components suitable for liquid culture include carbon sources and nitrogen sources suitable for the growth of Chinese cabbage, and include vitamins, trace elements (inorganic salts) and the like.

(W / v) of a carbon source, 0.1 to 1.0% (w / v) of a nitrogen source, 0 to 0.15% (w / v) of KH2PO4, 0 to 0.15% of MgSO4 .7H2O in a pre- (w / v), and the remaining distilled water was prepared. Next, the inoculated culture of the Cordyceps mushroom fungus cultured in the pre-culture was inoculated, and then the inoculated preculture was inoculated at 80 to 200 rpm under the condition of 22 to 30 ° C Lt; / RTI > incubation for 6-8 days. Then, when mycelium grows about 70% in the flask, the flask is inoculated with liquid culture medium.

(W / v) of a carbon source, 0.1 to 1.0% (w / v) of a nitrogen source, 0 to 0.15% (w / v) of KH2PO4, 0 to 0.15% (w / v) of MgSO4. 7H2O, v), and the remaining distilled water is inoculated aseptically into the preculture inoculation source, and the liquid culture liquid: the precultured inoculum is inoculated at a ratio of 20 to 200: 1. The liquid culture inoculated as described above is cultured in air at a temperature of 22 to 28 ° C in a sterilized air of about 0.1 to 1.0 vvm for 5 to 12 days to carry out a large-scale culture of the seeds.

As the carbon source, one or more of sugar, maltose, fructose, glucose, sucrose, malt extract, and syrup can be selectively used. As the nitrogen source, one or two of soybean powder or peptone can be used together.

However, the conventional culture process of the Cordyceps sinensis liquid described above can not completely control the germs introduced in the air during the cultivation of the air with the sterilized air, and the problem of the carbon source itself used during the above process and the limit of the sterilization have.

First of all, it is said that the air in the air is sterilized during the aeration culture. However, the conventional sterilization system can not completely sterilize the germs that are infiltrated in real time, and the defective rate (contamination rate) of the culture stage is increased by calling out the germs during the aeration culture period.

In addition, one or more of sugar, maltose, fructose, glucose, sucrose, malt extract and syrup which are used as conventional carbon sources are selected and used. These products are already processed, and even if they are sterilized during processing, When high temperature and high pressure are applied to the constituents, it is impossible to say that the individual components have not originally blocked mechanisms that can react with equipment and atmospheric bacteria.

On the other hand, in the case of the present invention, since a liquid culture liquid is prepared by mixing gold nanocolloid solution with water or distilled water (water or distilled water 1: standard gold nanocloid solution 0.0002-0.0005) The immobilized gold nanoparticles were effectively blocked and sterilized even in the secondary germs generated by the unsuitable microorganisms and the sterilization process at the high temperature and high pressure and the failure to maintain the proper temperature and humidity. In addition, even if bacteria in the air which have not yet been sterilized during the sterilization process of the aeration culture are implanted in the culture medium during the culturing process, the gold nanoparticles already immobilized in the culture medium are automatically sterilized by a transmission electron microscope (TEM) And confirmed by scanning electron microscopy (FE-SEM).

In addition, if the method of cultivating the fungus fungus mushroom including the Cordyceps sinensis has the problem that the equipment and the seedlings are exposed to the air at the stage of progressing from the plate culture to the liquid culture, the pollution rate by the germs is the highest, In order to prevent intrusion of the germs and to prevent the call, the equipment was immersed or washed in a gold nanocolloid mixed solution to prevent contamination.

S-3. Preparing the Growth Medium for Cordyceps

It is known that the growth medium used for artificial cultivation of Chinese caterpillar fungus is advantageous in the formation of fruiting body by using a cereal medium made by mixing noodle powder with corn grain such as brown rice or comfort rice. At present, the commonly used curd culture media has a gap formed between the curved grains, which facilitates the activation of mycelial growth and maintains moisture and ventilation, thereby contributing to the formation of fruiting bodies. Also, There are advantages.

The main reason and advantage of utilizing the conventional corn grain medium is that when the medium component is inevitably steamed at a high temperature and a high pressure for the sterilization of the germ at the time of preparing the medium, the medium components such as the sawdust It is not able to be sterilized by high temperature and high pressure, and there is a limit in that the main medium and other additives are destroyed and lost due to high temperature and high pressure even when sterilized. On the other hand, the corn grain medium has an advantage that it can contain moisture required for germination as a fruiting body as well as nutrients of the corn grain itself even under high temperature and high pressure, due to the surface area and elasticity of the corn grain itself.

On the other hand, the advantage of such a curd medium acts as a disadvantage. First, the conventional corn grain medium was sterilized at a high temperature and a high pressure in order to prevent the growth of germs generated from the carbon source, the nitrogen source and the grains themselves. However, this process alone could not sterilize the germs 100% If the content exceeds 5% of the proper amount, the growth of the hypha of the medium is promoted in the entire medium, and the productivity of the fruit body is directly decreased.

However, in the preparation step of the growth medium of the method for cultivating the insect hatchery using the gold nanocolloid solution of the present invention, a mixture of water and distilled water 1: 0.0002-0.002 of a standard gold nanocolloid solution is mixed with the grain used for the growth medium, The gold nanoparticles immobilized thereon are effectively inhibited or sterilized even if the germs in the medium adhere to the medium and the moisture remains in the cereal medium for more than the proper amount. It is possible to completely prevent a call or corruption of the medium.

These results show that the use of gold nanocolloid solution in the whole growth medium of Cordyceps militaris and Fungus fungus mushroom as well as the limit of the conventional grape media and its problems are confirmed, The inventors used a gold nanocolloid solution to make a growth medium of Cordyceps mellifera using a dodec rather than a grain, inoculate the seeds and observe the progress of the growth.

The growth medium using doduck was purchased from farmhouse, and the impurities on the surface were completely removed by using a knife, finely pulverized and mixed with only gold nanocolloid solution, and the medium was used as a medium. After the inoculation of mycelium Germination and germination into fruiting bodies were confirmed by transmission electron microscopy and field emission scanning electron microscopy. In addition, nutrients (such as vitamins and trace elements) added to increase the active ingredient artificially in the existing culture were not added to the growth medium.

This process and medium composition suggests that conventional methods of cultivating cucurbits and cucurbit fungus mushrooms using a grain medium artificially injected carbon sources and nitrogen sources for the proliferation of the coryneform bacteria, However, in case of using gold nanocolloid solution, when the seedlings are inoculated on the growth medium of natural plants such as Doduck, the mycelium is only allowed to grow at the maximum temperature of 20 ± 1 ° C, And mushroom seedlings were able to grow naturally by absorbing (absorbing) carbon source and nitrogen source, and the germs were automatically sterilized in the process, so that the conventional problems of the growth medium could be completely solved.

In addition, not only the vegetable medium such as dodec which does not cause the loss of natural nutrients, but also the animal (insect) growth medium which can not be used due to the corruption of the protein component, is directly inoculated with the culture solution containing the gold nanocolloid solution, Which is a new method of cultivation.

S-4. Inoculation

As mentioned above, in culturing the Chinese cabbage, it is necessary to take careful measures to prevent contamination by the germs, and when the cultivated liquid is inoculated into the culture bottle, care must be taken to prevent the inclusion of germs. First, aseptic automatic inoculation equipment is used to ensure rapid inoculation without being exposed to the outside air. When transferring the inoculum (liquid seedlings) cultured in the Erlenmeyer flask to a sterilized inoculation apparatus, The contamination of germs should be minimized by touching as far away from the entrance as possible. Connect the connection pipe of the inoculant containing the inoculation source and the connection pipe of the culture bottle, and blow the air into the air inlet of the inoculant equipped with the filter to inject the inoculum into the culture bottle. Since this process is the most likely contamination during the inoculation, flame sterilization or alcohol sterilization is required between the connection tubes.

However, in the conventional case, even if flame sterilization or alcohol sterilization is performed between the connection tubes, there is a limitation that all kinds of germs distributed in the air can not be completely blocked.

Therefore, in the present invention, in the inoculation process as in the plate culture process, the various kinds of equipment (such as the inoculation apparatus) are immersed or washed in a mixed solution of water or distilled water and a standard gold nanocolloid solution to sterilize the remaining germs.

S-5. Growing of Cordyceps

The inoculum (liquid seedlings) is inoculated into the growth medium of the Cordyceps sinensis cultivated in the culture bottle and the mycelium is activated throughout the growth medium. When the spermatozoa are formed from the mycelium, the cordyceps are grown until the seedlings are grown .

It is advantageous for the initial activation of mycelia to proceed in sterile culture room with temperature 20 ~ 24 ℃, humidity 70 ~ 80%, fluorescent lamp for vegetable incubation (500 ~ 1,000 Lux) It takes about 10 to 15 days. Initially, the mycelium that has become active develops a milky light, but gradually develops yellowish light. It should be noted that at 24 ° C, mycelial growth is vigorous, but growth of fruiting body is inhibited by the activation of airborne bacteria, so the temperature of the culture room is lowered to 20 ° C and the humidity is adjusted to about 80 to 90% . It grows on the surface of the head of the thyroid gland until it grows into mature fruit body. It takes 50 ~ 60 days to grow.

In order to prevent contamination by germs, the growth of Cordyceps is usually carried out in a sterilized culture room until at least the time when fruiting bodies are formed. Although the fruiting body can be grown in the aseptic culture chamber after the fruiting body is formed, the size of the aseptic culture chamber is increased and the maintenance cost is increased to increase the economic burden. Also, since the maximum respiration rate of the fruit body grows, it needs to be transferred to the general culture room When fruiting bodies mature, it is common that they are transferred to a general culture room. However, the problem is that the rate of contamination of fruiting bodies by germs in the process of growing fruiting bodies in a general culture room reaches about 10 ~ 25% It is a point.

In the method for cultivating the insect hallow weaving according to the present invention, the mixed solution obtained by mixing the distilled water 1: the standard gold nano-colloidal solution 0.0002-0.002 immediately before transferring the growth medium in which the fruiting body is formed to the general culture chamber is added to the fruiting body once . By spraying gold nanocolloid solution at the end of the growth period when the respiration rate of the fruiting body becomes maximum, it is possible to prevent pollution caused by germs in the atmosphere penetrating in the growing process of fruiting bodies, thereby preventing the nutrients supplied from the growth medium from being lost to the germs (Length, etc.) of the fruiting body and the preservation period were also increased. The above procedure was applied to other fungus fungus mushrooms in the same manner, and the same results were obtained in the cultivation step and preparation of the growth medium.

Based on the economics of the use of the gold nanocolloid solution and the results of the experiment in the cultivation of the mushroom mycelium using the gold nanocolloid solution described so far, the proper mixing ratio of the gold nanocolloid solution is, in the volume ratio, Is preferably 0.0002 to 0.002 when used for disinfection of various equipments, and when it is mixed with distilled water in the liquid culture preparation step or growth medium preparation step, 0.0002-0. 0.0005 was the most suitable, and when the ratio of the gold nanocolloid solution was more than 0.002, both the seed bacteria and the germs of the mushroom fungus were killed.

Experimental examples of Cordyceps cultivated by the method of cultivating Cordyceps mugwort using the gold nanocolloid solution of the present invention and Cordyceps cultivated by the conventional cultivation method are listed as follows and the use of gold nanocolloid solution Productivity, economic efficiency, and efficiency of mushroom cultivation.

First, according to the present invention, the mixing ratios of water or distilled water: standard gold nanocolloid solution (based on gold nanoparticle content of 6.0 g / l) are respectively 1: 0.00015 (i.e., 20: 3 ml) 1: 0.0002 : 4 ml), 1: 0.0005 (i.e. 20 l: 10 ml), 1: 0.002 (i.e. 20 l: 40 ml), 1: 0.0025 (800 dozen / times) cultivated 5 times in total, and the syngas contamination rate (dozen / total 800 dozen contaminated with germs) of caterpillar fungus cultivated 5 times according to the conventional method at the same time are shown in Table 1 Respectively.

Comparison of germicidal contamination rates of common Chinese cabbage cultivation methods and Chinese cabbage cultivation methods using gold nanocolloid solution division Common caterpillar fungus
How to grow How to grow gold nano Cordyceps 1: 0.00015 1: 0.0002 1: 0.0005 1: 0.002 1: 0.0025 1st round 128 (16.00%) 126 (15.76%) 62 (7.75%) 18 (2.25%) 15 (1.88%) 0 Second round 109 (13.63%) 110 (13.76%) 60 (7.50%) 16 (2.00%) 13 (1.63%) 0 Three times 214 (26.75%) 212 (26.50%) 58 (7.25%) 16 (2.00%) 15 (1.88%) 0 Four times 156 (19.50%) 158 (19.75%) 65 (8.13%) 16 (2.00%) 15 (1.88%) 0 5 times 140 (17.50%) 136 (17.13%) 62 (7.75%) 16 (2.00%) 15 (1.88%) 0

As shown in Table 1, when the water or distilled water 1: gold nanocolloid solution is mixed with 0.00015, the germicidal performance of the gold nanoparticles is insignificant and the contamination rate of the germ bacteria is not greatly improved. However, the gold nanocolloid solution (1: 0.0002 ~ 0.002), it can be seen that the contamination rate of germs is significantly reduced compared to the conventional method of cultivating Cordyceps. This is because the germs that inhibit the growth of Cordyceps bacillus are selectively killed by the gold nanoparticles. However, when mixed with water or distilled water 1: gold nano colloid solution 0.0025, the pollutant contamination rate is 0, but it has a negative effect on the growth of Cordyceps, due to the killing of ciliothermophilus species together with germs.

Comparison of Mycelial Growth, Sagittarius and Cryptophyte Formation of Common Cordyceps and Cultivation of Cordyceps with Gold Nano Colloid Solution division
Common caterpillar fungus
How to grow How to grow gold nano Cordyceps 1: 0.00015 1: 0.0002 1: 0.0005 1: 0.002 1: 0.0025 Mycelium agar 7 to 10 days 7 to 10 days 8 to 9 days 6 to 7 days 9th 0 days Mycorrhizal infiltration day 13-15 days 13-14 days 12-13 days 11 to 12 days 12-13 days 0 days Date of formation 21 to 24 days 21-25 days 20 to 21 days 18th ~ 19th 20 to 21 days 0 days Date of conization 35-40 days 35-40 days 33-35 days 29 to 31 days 32 to 34 days 0 days Fruit body (5cm) formation date 50 to 60 days 50 to 59 days 48 to 51 days 46 to 47 days 48 to 50 days 0 days

As shown in Table 2, when the water or distilled water 1: gold nanocolloid solution is mixed with 0.00015, the germicidal performance of the gold nanoparticles is insignificant and the contamination rate of the germ bacteria is not greatly improved. Therefore, the growth period of the Chinese cabbage larvae is hardly shortened and water or distilled water 1 : 0.0025, it was not possible to grasp the growth period. However, when the cucumber pickling was grown using the gold nanocolloid solution (1: 0.0002 ~ 0.002) according to the present invention, It is possible to shipment the Cordyceps in an early stage because the growth period of the Cordyceps spp. Is shortened most remarkably in the case of mixing with 1: 0.0005. It is understood that this is due to the selective destruction of germs that competitively inhibit the growth of Cordyceps mushroom by gold nanoparticles, contributing to stable formation of mycelium, and subsequent ingestion of the nutrients of the culture medium.

60 days after cultivation, comparison of fruiting bodies of caterpillar fungus using common caterpillar fungus and gold nanocolloid solution division Common caterpillar fungus
How to grow Gold Nano Cordyceps 1: 0.00015 1: 0.0002 1: 0.0005 1: 0.002 1: 0.0025 Fruit body average
Length (cm) 9-11 10 to 11 12-13 14-16 12-13 0 Fruit body average weight (g) 20-25 22-25 29-30 34 ~ 38 30 to 32 0 Fruit body average
Effective Object Quantity () 60 to 75 59 ~ 75 72 ~ 79 80 ~ 87 72 ~ 79 0

As shown in Table 3, when the mixture was mixed with water or distilled water 1: gold nanocolloid solution 0.00015, the germicidal performance of gold nanoparticles was insignificant and the quality (average length, average weight) and productivity (effective population) , Water or distilled water at a ratio of 1: 0.0025, the quality and productivity of Cordyceps was not determined, but when the Cordyceps is grown using the gold nanocolloid solution (1: 0.0002-0.002) according to the present invention, The quality and productivity of Cordyceps is improved compared with the method of cultivation of Cordyceps, and the quality and productivity of Cordyceps is remarkably improved in the case of mixing 1: 0.0005. Therefore, it is possible to mass produce good quality Cordyceps. It is understood that this is due to the selective destruction of germs that competitively inhibit the growth of Cordyceps mushroom by gold nanoparticles, contributing to stable formation of mycelium, and subsequent ingestion of the nutrients of the culture medium.

Comparison of preservation period of Cordyceps sinensis using general ciclosporic acid and gold nanocolloid solution division
Common caterpillar fungus
How to grow Gold Nano Cordyceps 1: 0.00015 1: 0.0002 1: 0.0005 1: 0.002 1: 0.0025 Preservation at room temperature (20 ℃, room) 5 to 7 days 6 to 7 days 40 to 45 days 55 to 60 days 42 to 49 days 0 days Slow cooling (10 ° C, indoor) 8-12 days 25 to 29 days 40 to 43 days 49-56 days 41 to 44 days 0 days

As shown in Table 4, when the mixture was mixed with water or distilled water 1: gold nanocolloid solution 0.00015, the germicidal performance of the gold nanoparticles was insignificant, so that the preservation period of room temperature or slow cooling of the Chinese cabbage was not greatly improved. However, since the preservation period of the Chinese caterpillar fungus was not determined, the growth period of the Chinese caterpal fungus was continued by using the gold nanocolloid solution (1: 0.0002 ~ 0.002) according to the present invention. In contrast, the preservation period of Cordyceps sinensis was improved most remarkably (continuous growth was observed even when stored at 5 캜). In particular, when the mixture was mixed at 1: 0.0005, the preservation period of Cordyceps is remarkably improved, And the like.

The reason for this is thought to be that the germs inhibiting the growth of the Chinese cabbage plants are selectively killed by the gold nanoparticles and thus the nutrients of the culture medium are maintained for a long time. It was found to be more advantageous to keep. However, in order to preserve the water during the preservation of the Chinese caterpillar, the storage container should be partially sealed.

As can be seen from Tables 1 to 4, the dilution ratio of water or distilled water: gold nanocolloid solution was 1: 0.0002 to 0.002 or less. From the confirmed data and the actual production process, The most efficient mixing ratio until the stage of growth of the fruiting body after the composition step was 0.0005 of water or distilled water 1: gold nanocolloid solution.

Although the present invention has been described with reference to the embodiment of the method for cultivating insect hallow, the present invention is not limited to the artificial mycelium cultivation method without any change of the technical idea or essential features of the present invention. In addition to other mushrooms of other fungi that are difficult to activate, they are also widely applied to mushroom cultivation methods such as mushroom and oyster mushroom which form exogenous mycorrhiza at the time of cultivation and fruiting body grows at the mycelium of exogenous mycorrhiza It is expected to be able to become. It is therefore to be understood that the above-described embodiments are illustrative and not restrictive in every respect.

Claims (5)

a step of isolating the collected mushroom or collecting the mushroom bacterium, b) culturing the mushroom bacterium in large quantities as a solid or liquid seed, c) preparing a growth medium for growing the mushroom by inoculating the seed bush, d) inoculating the growth medium into a growth medium and cultivating the mushroom of Cordyceps mushroom,
In step b), at least one of gold nanoparticles, gold nanocolloid solution, or a mixed solution of gold nanocolloid and water or distilled water is added to the culture medium or the culture medium to sterilize the germs,
Gold nano-colloid solution is a gold nanoparticle prepared by reducing HAuCl _{4 with} phytochemical or gold nanoparticles coated with a polymer to ensure pH stability in vivo.
Characterized in that the mixing ratio of water or distilled water: standard gold nano-colloid solution is 1: 0.0002 to 0.002, by volume, when the content of gold nanoparticles in the gold nanocolloid solution is 6.0 g / l as a standard.
How to grow mushrooms from. a step of isolating the collected mushroom or collecting the mushroom bacterium, b) culturing the mushroom bacterium in large quantities as a solid or liquid seed, c) preparing a growth medium for growing the mushroom by inoculating the seed bush, d) inoculating the growth medium into a growth medium and cultivating the mushroom of Cordyceps mushroom,
In step c), at least one of gold nanoparticles, gold nanocolloid solution or mixed solution of gold nanocolloid and water or distilled water is added to the growth medium to sterilize the germs,
Gold nano-colloid solution is a gold nanoparticle prepared by reducing HAuCl _{4 with} phytochemical or gold nanoparticles coated with a polymer to ensure pH stability in vivo.
Characterized in that the mixing ratio of water or distilled water: standard gold nano-colloid solution is 1: 0.0002 to 0.002, by volume, when the content of gold nanoparticles in the gold nanocolloid solution is 6.0 g / l as a standard.
How to grow mushrooms from. a step of isolating the collected mushroom or collecting the mushroom bacterium, b) culturing the mushroom bacterium in large quantities as a solid or liquid seed, c) preparing a growth medium for growing the mushroom by inoculating the seed bush, d) inoculating the growth medium into a growth medium and cultivating the mushroom of Cordyceps mushroom,
In step d), immediately before transferring the growth medium to the general culture chamber, at least one of gold nanoparticles, gold nanocolloid solution or gold nanocolloid and a mixed solution of water or distilled water is sprayed on the fruiting body,
Gold nano-colloid solution is a gold nanoparticle prepared by reducing HAuCl _{4 with} phytochemical or gold nanoparticles coated with a polymer to ensure pH stability in vivo.
Characterized in that the mixing ratio of water or distilled water: standard gold nano-colloid solution is 1: 0.0002 to 0.002, by volume, when the content of gold nanoparticles in the gold nanocolloid solution is 6.0 g / l as a standard.
How to grow mushrooms from.
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