KR100204982B1 - The method of culturing liquid starter of basidomycetis and liquid starter - Google Patents

Abstract

The present invention relates to a liquid spawn cultivation method of basidiomycetes and a liquid spawn thereof, and more particularly to a method capable of producing a large amount of liquid spawn useful for the cultivation of various mushrooms of high quality and a liquid spawn obtained by the method.

The present invention is a liquid culture method of common basidiomycetes, the pre-culture step (a) 2 ~ 7% (w / v) carbon source, 0.1 ~ 1.0% (w / v) nitrogen source, KH ₂ PO ₄ 0 ~ 0.15 Inoculate the plated basidiomycetes in the preculture consisting of% (w / v), MgSO ₄ · 7H ₂ O 0-0.15% (w / v), and the remaining distilled water, and (b) the inoculated preculture Incubated at 80 ~ 200 rpm, 22 ~ 30 ℃ shaking culture in 6 to 8 days; The above liquid culture process was carried out by (a) carbon source 2-7% (w / v), nitrogen source 0.1-1.0% (w / v), KH ₂ PO ₄ 0-0.15% (w / v), MgSO ₄ 7H ₂ O 0 ~ 0.15% (w / v), and prepare a liquid culture consisting of the remaining distilled water, (b) aseptic auto-inoculation of the pre-inoculation source to the liquid culture solution, (c) the inoculated The liquid culture consists of aeration of the culture medium at 22-28 ° C. with sterilized air on the order of 0.1-1.0vvm for 5-12 days.

The present invention is characterized in that it is composed of a series of steps which are inseparably connected to the preculture process and the liquid culture process under stringent conditions, respectively, after collecting the prokaryote of basidiomycetes.

Description

Liquid spawn culture method of basidiomycetes and liquid spawn by the method

1 is a schematic diagram of an inoculation device developed to efficiently perform sterile inoculation at the inoculation stage of pre-culture inoculation.

2 is a schematic diagram of the aeration culture mechanism of the liquid culture developed to efficiently perform the input, culture, and discharge process in the aeration culture stage of the liquid culture.

3 is a schematic view showing another embodiment of the aeration culture apparatus of the liquid culture of FIG.

* Explanation of symbols for main parts of the drawings

10: inoculation device 11: inoculation container

12: stopper 13: pre-culture inoculation

14: air injection line 15: filtration means

16: air supply means 18: discharge line

20: Aeration culture 21: Liquid spawn incubator

22: lid 23: liquid culture

24: air injection pipe 25a, 25b: filtering means

26: inoculation tube 27: liquid flow meter

28: air discharge pipe 29: discharge pipe of liquid spawn

31: connecting means 32a, 32b, 32c, 32d: locking device

[Purpose of invention]

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a liquid spawn cultivation method of basidiomycetes and a liquid spawn thereof, and more particularly to a method capable of producing a large amount of liquid spawn useful for the cultivation of various mushrooms of high quality and a liquid spawn obtained by the method.

[Background of invention]

Mushrooms are microorganisms belonging to basidiomycetes in the classification system of living organisms, and most of them are used for food except for toxicity. These mushrooms are expected to replace meat in the future as a high protein food, and according to various recent reports, glucan (glucan) and its derivatives contained in mushrooms are known to have anticancer activity. [Study on Anticancer Components of Korean Basidiomycetes. Korean Journal of Mycology, 8: 107 (1980); Chiraha et al., Fractionation and purification of the polysaccharides with marked antitumer activity from Lentinus edodes (Berk.). Cancer Res. 30: 2776-2781 (1970); Kang, Chang-Jin et al., Studies on the Anticancer Compounds of Korean Basidiomycetes, Mycelial Growth and Anticancer Constituents of Rhizome. Korean Journal of Biochemistry, 14: 101 (1981); Yamamura et al., A selective inhibitor of myxovirus from shiitake (Lentinus edodes). Mushroom Sci. 9 (1): 495-507 (1976)], and mushrooms have attracted much attention as health foods as well as normal food substitutes.

These basidiomycetes (mushrooms) differ in their cultivation methods or cultivation conditions for each type, but usually the first progenitors are collected and cultured in agar medium, and then cultured in a liquid or solid for a certain period of time to obtain a mushroom spawn, These mushroom spawns are grown in general farmhouses and the final mushrooms are harvested.

By the way, the harvesting and cultivation of the original bacteria is carried out in a conventional method, while a new method for culturing mushroom spawns has emerged day by day. As for the mushroom spawn cultivation method, a solid spawn cultivation method is usually prepared by injecting sawdust as a main ingredient, rice bran, and the like as a subsidiary material, and then inoculating mycelia of basidiomycete. However, this method has the trouble of crushing spawns in the cultivation container when lumberwood, rice straw, or bottle cultivation is used, and furthermore, the myceliums intertwined in the process of crushing spawns are severely damaged by sawdust spawning. It was given, which caused a significant drop in the vitality of sawdust spawn. In addition, special attention should be paid to the mixing of various germs, and the spawn masses must be mixed evenly, and a large amount of culture vessels are required in a small growing area. The problem was seriously emerging.

In view of these circumstances, the method developed for obtaining more efficient mushroom spawn is a method for preparing liquid spawn. However, this method uses pure liquid as a culture medium, unlike the solid spawn culture method, and thus requires a high level of technology for cultivating progeny to a certain level. In recent years, research is being conducted [Park Youngjae, how to make a medium and spawn culture method. Ganoderma lucidum, shiitake, louse. Publishers both inside and outside. 308-322 (1993); Korean Patent Publication No. 95-26976. Reference]

By the way, the liquid culture method briefly introduced above [Park Youngjae, how to make a medium and spawn culture method. Ganoderma lucidum, shiitake, louse. Publishers both inside and outside. 308-322 (1993)] is just a brief introduction of the liquid composition as a laboratory culture method, another liquid culture method [Korean Patent Publication No. 95-26976] describes the technical content in more detail, This also has to go through repetitive gelatinization and sterilization, which is cumbersome, requires a large amount of energy, and each product has to be repeated individually, there is a risk of mixing of germs, It was difficult to achieve uniformity, and mass production was impossible.

[Purpose of invention]

The present invention was developed to solve the problems of the conventional liquid spawn production method as described above, it is possible to prevent the mixing of various germs during the manufacturing process, it is possible to uniformize the product, mass production is possible by a series of processes It is an object of the present invention to provide a liquid spawn and a liquid spawn prepared by the method.

The present invention is characterized in that it is a method of producing a liquid spawn consisting of a series of steps which are irreversibly connected to each other in strict conditions under the preculture process and the liquid culture process after collecting the prokaryote of basidiomycetes. In the present invention, basidiomycetes include edible mushrooms such as oyster mushroom, black scale mushroom, mandala mushroom, willow mushroom, shiitake mushroom, mushroom mushroom, bokyeong, ganoderma lucidum mushroom, and situation mushroom.

[Configuration of Invention]

[Technical composition of the invention]

In order to achieve the above object, the present invention is a liquid of the basidiomycetes consisting of the process of receiving the protococci of the basidiomycetes, plate culture, preculture of the plate cultured inoculum, and liquid culture of precultured inoculum In the culturing method, the pre-culture step (a) 2 ~ 7% (w / v) carbon source, 0.1 ~ 1.0% (w / v) nitrogen source, 0 ~ 0.15% (w / v) KH ₂ PO ₄ , MgSO ₄ · 7H ₂ O 0 ~ 0.15% (w / v), and the preparation step of the pre-culture consisting of the remaining distilled water, (b) inoculating the plate cultured basidiomycete in the pre-culture, (c) above The inoculated preculture is incubated for 6 to 8 days in a shaker at 80 to 200 rpm and 22 to 30 ° C .; The liquid culture process is (a) 2 to 7% (w / v) carbon source, 0.1 to 1.0% (w / v) nitrogen source, 0 to 0.15% (w / v) KH ₂ PO ₄ , MgSO ₄ · 7H ₂ O 0 ~ 0.15% (w / v), and preparing a liquid culture consisting of the remaining distilled water, (b) aseptic inoculation of the pre-inoculation source to the above liquid culture solution, but the liquid culture solution of the above Inoculating the pre-inoculation source = 20 to 200: 1, (c) aerated culture of the inoculated liquid culture with the sterilized air of 0.1 to 1.0vvm at 22 ~ 28 ℃ for 5 to 12 days It consists of a step.

In addition, the present invention provides a liquid spawn of basidiomycete cultured through the above-mentioned general process.

Preferred Embodiments of the Invention

Hereinafter, preferred embodiment of this invention is described in detail.

(A) Probiotics collection and plate culture

The present invention is used to obtain the original bacteria of basidiomycetes from the Rural Development Administration or the Forest Service, or to separate the tissue from fresh fresh tissue of edible mushrooms of the wild (small fungi). The cultured or tissue-separated progeny are plate-cultured in a Petri dish and expanded to early incubation inoculum. Plate culture in a petri dish can be performed by a conventional method.

(B) Pre-cultivation process of basidiomycetes

Escherichia coli grown in a petri dish is then transferred to the preculture process. The pre-cultivation process comprises the steps of preparing a predetermined liquid medium, inoculating the expanded cultured prokaryote into the liquid medium, and culturing the inoculated culture under stringent conditions.

(a) Preparation of Preculture

Probiotics expanded in Petri dishes should be sufficiently cultured in the liquid medium according to the present invention. The liquid medium according to the present invention has a carbon source of 2 to 7% (w / v), a nitrogen source of 0.1 to 1.0% (w / v), KH ₂ PO ₄ 0 to 0.15% (w / v), MgSO ₄ 7H ₂ O 0 ˜0.15% (w / v) and the remaining distilled water.

As a carbon source, one or more selected from the group consisting of sugar, maltose, fructose, glucose, sucrose, malt extract, and syrup are used. At 2% (w / v) or less, it is not suitable because it causes a mycelial growth, and at 7% (w / v) or more it is uneconomical as an excess of other nutrients.

The carbon source is used as a constituent that forms the backbone of molecules required for the metabolism of mushrooms, and is also used as an energy source for cell synthesis and metabolism. Originally, mushroom fungi are heterotrophic organisms that live on the basis of dead or aged plants or organic matters derived from them. Therefore, their main components are cellulose, hemicellulose, and lignin. On the basis of this fact, in the normal mushroom cultivation method, sawdust, rice bran (rice bran), rice straw or the like is used as a nutrient medium for basidiomycetes. However, in the case of using this chamber, in order to live the mycelium of the original bacteria, the above sawdust and the like must be decomposed again and converted into a form that is easily absorbed. Therefore, another energy is required and the amount of absorption is not large. On the other hand, in the Republic of Korea Patent Publication No. 95-26976 is used as a carbon source, rice flour, wheat flour, corn flour, starch, etc., but also to make it easy to use mycelium mycelium as a nutrient source, it must be luxury There is a disadvantage in that a large amount of energy is required in the process. In addition, even after gelatinization, polysaccharides cannot escape the form, so in order for the mycelium to absorb it, it is necessary to secrete and decompose the enzymes. All of the absorbed carbon sources are not used for the growth of the mycelium, and some of them are used as energy sources. It is a disadvantage. Therefore, in the present invention, one or two or more selected from the group consisting of monosaccharides or disaccharides such as glucose, maltose, sugar, fructose, lactose, etc., which are the most easily absorbed forms of mycelium mycelium without the need to decompose the carbon source any more, are used. . Since they are all soluble in water, they do not need to undergo a separate gelatinization process as in the prior art, and since they can be absorbed in the state, the mycelium does not need to use separate energy to decompose it. to be.

As a nitrogen source, either or both of soy flour and peptone are used together, and it is used within the range of 0.1 to 1.0% (w / v). If soy flour or peptone is used at 0.1% (w / v) or less, it is not preferable because the nitrogen source of mycelium is insufficient. Not preferred in Nitrogen is an indispensable nutrient source for the synthesis of genetic material such as DNA of mushrooms and the production of enzymes necessary for the synthesis and degradation of proteins. Nitrogen sources commonly used for the cultivation of mycelia include organic nitrogen sources such as yeast extract and inorganic nitrogen sources such as ammonium chloride (NH ₄ Cl) nitrate (KNO ₃ ). However, they only provide a single nitrogen source of mycelium and do not provide any further function. Therefore, it is necessary to provide other nutrients for the normal growth of mycelium. On the other hand, soybean meal is rich in protein and other nutrients compared to other foods (plants), so it is the optimal nutrient source for mycelial growth. There is an advantage of not being. In addition, soy flour can be easily obtained anywhere, anytime, and has the advantage of being very cheap compared to other organic nitrogen sources. In addition, the soy flour is fine but functions as an agglutinating nucleus in which the mycelium aggregates in the liquid medium to form the mycelium sphere. Therefore, the mycelium grows in a significantly smaller amount than the carbon source, and thus, the organic nitrogen source and other It is believed to be very effective at absorbing nutrients. On the other hand, since peptone is a low molecular protein component, the mycelia have an advantage of absorbing nitrogen sources more easily. It was observed that it is most preferable to use the nitrogen source in the range of about 1/10 of the said carbon source.

As inorganic salts, KH ₂ PO ₄ can be used as a source of phosphorus, and MgSO ₄ · 7H ₂ O can be used as a source of magnesium. These inorganic salts differ from the above-mentioned prior patent application (Korean Patent Publication No. 95-26976) in that they can be selectively used for liquid medium depending on the type of basidiomycetes. For example, it is preferable not to use these inorganic salts in mushrooms and mushrooms, but in the case of oyster mushrooms, black scale mushrooms, mandala mushrooms, willow mushrooms, shiitake mushrooms, and bokyeong, desirable. However, even when these inorganic salts were used, since it was observed that the growth of the mycelium would be hindered when exceeding 0.15% (w / v), it is preferable to limit it to 0.15% (w / v) or less, respectively.

And the rest uses distilled water or sterile drinking water. When using ordinary drinking water, it must be sterilized under high temperature and high pressure.

(b) Inoculation step of basidiomycetes

The preculture solution configured as described above is placed in a preculture vessel, and inoculated with the plate cultured basidiomycetes in that state to obtain a preculture. The preculture vessel is preferably a conical flask in laboratory culture, but is not necessarily limited to mass production. The inoculation time is when the mycelium of the plate incubator has grown to about 70% of the plate area, and the mycelia fragments are sterilely inoculated into the preculture in the preculture vessel using coke or platinum ball. The inoculation method itself can be carried out by conventional methods. At this time, it was observed that the more mycelia inoculated, the shorter the culture days of the inoculator, and the higher the density of the inoculated mycelium, the smaller the diameter of the mycelia (pellet-shaped mycelia) formed after the culture.

(c) preculture stage of basidiomycete

The preculture inoculated with mycelium is transferred to the shake culture in a state filled with the preculture vessel. Shake culture is incubated in a rotating shaker or reciprocating shaker incubator at a rotation speed of about 80 ~ 200 rpm. When the preculture is rotated at 80 rpm or less, the grown mycelium is not evenly distributed in the preculture and is entangled with each other, which also prevents sufficient supply of oxygen required for the growth of the mycelia in the preculture. Thereafter, high quality liquid seed cannot be obtained in the liquid culture step. On the other hand, when 200 rpm or more is sufficient to supply oxygen, it is not preferable because the growth of the mycelium is reduced or the mycelium is cut due to excessive rotational force. This phenomenon is remarkable early in the mycelial culture, and the effect is reduced when the mycelium is adapted to the environment in which the mycelium is cultured. Therefore, low speed rotation is preferable at the beginning of the preculture, and may be rotated at high speed within the above range as time passes. In the above rotational speed range, when the mycelium grows to a certain length while growing in the liquid medium, it is estimated that the mycelium is broken by the rotational force to form a new mycelium sphere and grow continuously. However, if it is outside the above range, it is not preferable because not only mycelium that has already grown but also mycelium that still needs to be grown are broken off from each other, so that mycelium spheres are not properly formed in the liquid medium.

On the other hand, the shaking culture can be appropriately selected according to the culture temperature conditions of basidiomycetes. Usually, it incubates for 6 to 8 days in the range of 22-30 degreeC. In the same basidiomycetes, it was observed that the incubation period tends to be shorter as the temperature of the culture solution approaches the optimum temperature condition of the mycelium.

(C) Cultivation process of liquid spawn

The mycelium precultured in the shake incubator is then transferred to the liquid spawn culture process. The culturing process of the liquid spawn comprises the steps of preparing a predetermined liquid culture solution, inoculating precultured mycelium, and culturing the inoculated culture under stringent conditions.

(a) Preparation of liquid culture liquid

Mycelium precultured in a shaker culture should be sufficiently cultured in the liquid culture solution according to the present invention. Liquid culture liquid according to the present invention is 2 ~ 7% (w / v) carbon source, 0.1 ~ 1.0% (w / v) nitrogen source, KH ₂ PO ₄ 0 ~ 0.15% (w / v), MgSO ₄ · 7H ₂ O 0 ˜0.15% (w / v), with the remainder consisting of a liquid culture consisting of distilled water.

As a carbon source, one or two or more selected from the group consisting of sugar, maltose, fructose, glucose, sucrose, malt extract, and starch syrup are used. At 2% (w / v) or less, it is not suitable because it causes a mycelial growth, and at 7% (w / v) or more it is uneconomical as an excess of other nutrients. The reason for the selection is as described above (hereinafter, the same).

As the nitrogen source, either one or both of the peptones are used together, and they are used within the range of 0.1 to 1.0% (w / v). If soy flour or peptone is used at 0.1% (w / v) or less, it is not preferable because the nitrogen source of mycelium is insufficient. If it is used at 1.0% (w / v) or higher, there is no significant difference in the growth of mycelia. Not preferred in In this case, yeast extracts commonly used as auxiliary components of nitrogen sources may be used.

As inorganic salts, KH ₂ PO ₄ can be used as a source of phosphorus, and MgSO ₄ · 7H ₂ O can be used as a source of magnesium. These inorganic salts can also be selectively used in the liquid culture liquid according to the type of basidiomycete. In this respect, it differs from the above-mentioned prior patent application (Korean Patent Publication No. 95-26976). For example, in the cultivation process of liquid spawn, it is preferable not to use these inorganic salts for oyster mushroom, black scale mushroom, mandala mushroom, willow mushroom, mushroom mushroom, and situation mushroom. It is preferable to use inorganic salts. It differs from the said preculture process in this respect. However, even when these inorganic salts were used, since it was observed that the growth of the mycelium exceeds 0.15% (w / v), it is preferable to limit the concentration to 0.15% (w / v) or less, respectively.

And the rest uses distilled water or sterile drinking water. When using ordinary drinking water, it must be sterilized under high temperature and high pressure.

In the present invention, the culture medium is dissolved in water to prepare a liquid culture solution. In order to prepare a liquid culture solution, the above-mentioned media components are weighed into water and mixed uniformly in a mixer with an impeller (stirring blade) attached thereto. At this time, in order to remove the bubbles generated during the cultivation of the liquid spawn, it is preferable to add a defoamer to the culture medium is mixed. As the antifoaming agent, various animal oils and vegetable oils which form a thin film on the surface of the liquid culture liquid can be used. As the vegetable oil, for example, one or two or more of the group consisting of household cooking oil, cottonseed oil, castor oil, olive oil, palm oil and the like can be used. The addition amount should be such that it can be spread throughout the upper portion of the culture, for this purpose it is preferably 0.1 to 1.0% (w / v) relative to the liquid culture. Since the antifoaming agent forms a thin film on the surface of the culture medium, there is no problem in the system for forcibly supplying the air (oxygen) to the liquid culture solution as in the aeration liquid culture method of the present invention, but the air is not forcibly supplied to the liquid culture medium. It should be noted that in systems that do not use oxygen, which is essential for the growth of mycelium, it cannot be supplied. Therefore, it can not be used in the pre-cultivation process, it can be used only in the culture process of liquid spawn in a forced aeration method. (However, if the above pre-cultivation process is converted to a forced aeration method, it is possible to use a defoamer.)

In the present invention, it is preferable to uniformly mix the liquid culture solution in the same manner as usual when preparing the liquid culture solution, and then sterilize it in a high-temperature autoclave. This is to prevent the liquid culture liquid from being contaminated by other various bacteria to prevent the use of the liquid medium of the desired basidiomycetes in advance. However, in the case of mass production, the liquid culture solution may be sterilized in a separate container, and the liquid culture solution may be prepared by filling each incubator of the sterilized liquid seed.

(b) Inoculation step of pre-inoculation center

The present invention inoculates the pre-culture inoculation shaken in sterile liquid culture solution to prepare a liquid culture of the mycelia. The vaccination process must be performed aseptically. In the present invention, an inoculation device is used for this purpose.

1 is a conceptual diagram schematically showing the inoculation device 10 described above. The inoculation mechanism 10 is provided with an air injection line 14 and a discharge line 18 of the pre-culture inoculation source 13 at the stopper 12 of the inoculation container 11. The air injection line 14 is provided with filtration means 15 for filtering various germs in the air, one end of which is located at the bottom of the inoculation vessel 11, the other end of which is provided with an external air supply means 16. (E.g., air compressor). At this time, the filtering means 15 to filter the various bacteria in the air to prevent the liquid culture liquid and the liquid culture is contaminated by the various bacteria. The filtration means 15 may be a filter for microbial culture, an air bactericidal filter, a cotton plug filter, or the like. On the other hand, the discharge line 18 of the pre-inoculation source is located on the liquid surface of the pre-inoculation source 13, the other end is in the inoculation tube 26 of the liquid seedlings installed in the liquid seed incubator 21 [See also Figure 2].

Therefore, in order to inoculate the pre-inoculation source contained in the inoculation container 11 into the liquid seed culture incubator 21, after completion of the shaking culture step, the stopper of the shaking culture container is opened in a sterile phase and a predetermined amount of pre-incubation is performed. Inoculate the inoculation vessel 11 into the inoculation vessel 11, connect the air injection line 14 of the inoculation device, which has been injected, to the air supply means 16, and inoculate the discharge line 18 of the pre-inoculation source. When the inoculation vessel 11 is inverted and injected at a pressure higher than atmospheric pressure, the pre-inoculation source 13 contained in the inoculation vessel 11 is connected to the pipe 26. 18) and to move along the inoculation tube 26, both into the liquid seed incubator 21. At this time, if the liquid flow rate measuring device 27 is installed between the discharge line 18 and the inoculation tube 26 of the pre-culture inoculation source, the added amount of the pre-culture inoculation source 13 can be more accurately measured. See also]. If you want to move the pre-inoculation source 13 in the state in which the inoculation vessel 11 is left as it is, one end of the air injection line 14 and the discharge line 18 may be installed oppositely. .

In the present invention, the above liquid culture solution: the pre-inoculation source = 20 to 200: 1 to inoculate the ratio. More preferably, it is the ratio of 50-100: 1. Too little inoculation of the pre-inoculation source increases the likelihood that the growth of mycelium will slow down or fail, whereas inoculation in excess of the above amount is not economically desirable.

On the other hand, in Figure 1 above, the pre-cultured pre-inoculation source in the inoculation vessel (11), and then described the method of proceeding to the above-mentioned process, but in a more mass production method shaking culture vessel inoculation vessel ( 11) may be directly inoculated aseptically into the liquid seed incubator (21).

(c) Aeration culture step of liquid culture

In the present invention, after inoculating a shaker cultured mycelium (pre-inoculation source) in the liquid seed culture incubator, the liquid culture inoculated with the mycelium with a sterilized air of about 0.1 to 1.0 vvm at 22 ~ 28 ℃ 5 to 12 days Aeration for days. This results in a liquid seed which is the final object of the present invention. In the present invention, the aeration culture step of the liquid culture is efficiently performed using the aeration culture device of the predetermined liquid culture.

2 is a schematic view of the aeration culture mechanism 20 of the liquid culture developed to more efficiently perform the aeration culture step of the liquid culture according to the present invention. The aeration culture device 20 is composed of a liquid spawn incubator 21 for receiving a liquid culture and a lid portion 22 thereof, and an air injection tube 24 for injecting air from the outside into the lid portion 22 thereof. ), An inoculation tube 26 into which the liquid culture liquid is introduced or inoculated with the mycelium in the liquid culture liquid, an air discharge tube 28 which discharges air from the inside of the liquid seed incubator 21, and a liquid after aeration culture. A discharge tube 29 for liquid spawn for discharging spawn is provided. However, the inoculation tube 26 and the discharge tube 29 may be integrated into one to perform two functions simultaneously. The air inlet tube 24 is provided with filtration means 25a capable of filtering various bacteria in the air, one end of which is located at the bottom of the liquid seed incubator 21, and the other end of which is provided with an external air supply means. (16) (e.g., air compressor). In addition, one end of the inoculation tube 26 is located at the bottom of the liquid spawn incubator 21, and the other end thereof is connected to another connecting tube (not shown) to receive the liquid culture solution or the preculture inoculation source. It is preferable to provide a liquid flow rate measuring device 27 inside the other end. In addition, the air discharge pipe 28, the one end is located on the surface of the liquid culture 23, the other end is open to the outside atmosphere, it is preferable to provide the filtering means 25b at the other end. . At this time, the filtering means (25a) filters the bacteria in the air supplied from the outside, while the other filtering means (25b) is detoxifying harmful bacteria that may have occurred in the liquid spawn incubator 21 to the outside Prevent release. On the other hand, the discharge tube 29 of the liquid spawn is located at the bottom of the liquid spawn incubator 21, the other end is open to the outside, it is preferable to install the liquid flow rate measuring device 27 on the other end That's right. The liquid flow rate measuring device 27 may accurately measure the amount of the liquid culture solution or the preculture inoculation source flowing into the liquid seed incubator 21 and the amount of the liquid seed flowing out of the liquid seed incubator 21. The other end of the air injection pipe 24, the inoculation pipe 26 and the discharge tube 29 of the liquid spawn is provided with a connecting means 31 for efficient connection with other members, respectively, and the fluid flowing therein (air Or locking devices 32a, 32b, 32c and 32d, respectively, for the control of the liquid).

Therefore, when using the aeration culture mechanism 20, it is possible to efficiently perform the aeration culture step as follows. First, the mycelium is inoculated by injecting the liquid culture solution prepared in the preparation step of the liquid culture solution and the preculture inoculation source prepared in the preparation step of the preculture solution into the liquid seed culture incubator 21 in order through the inoculation tube 26. Make a liquid culture (23). At this time, the mixing ratio of the liquid culture solution and the pre-culture inoculation source may be made through a culture vessel of a predetermined capacity, but in the case of mass production, it can be easily solved through the liquid flow rate measuring device 27. Subsequently, when air is forcibly introduced through the air injection pipe 24, bubbles are generated in the liquid culture 23, and oxygen flows into the liquid culture 23 as the bubbles move upward. At the same time as it is supplied, the liquid culture 23 continuously and continuously flows to be uniformly mixed. As for the injection amount of air, about 0.1-1.0 vvm is preferable like usual. When the amount of air is 0.1 vvm or less, supply of sufficient oxygen to the liquid culture 23 is difficult and the mixing effect is small. On the other hand, when the amount of air injection is 1.0 vvm or more, the supply of oxygen is sufficient but the liquid culture (23 This is because it is not possible to obtain a high quality liquid seed by excessively rocking) and causing unnecessary foaming, resulting in loss of culture solution. In addition, this culturing process is carried out at 22 ~ 28 ℃, because the mycelium of basidiomycete has the optimum growth environment in this temperature range.

When cultured under these conditions for about 5 to 12 days, each mycelium present in the liquid culture 23 gradually grows to form small mycelial spheres, and these mycelium spheres grow innumerably and evenly, thereby requiring high quality required by the present invention. It becomes the liquid spawn of.

When the desired liquid spawn is obtained, the liquid spawn is automatically discharged aseptically through the following procedure. First, when the lock device 32c of the air discharge pipe 28 is closed (in this case, the lock device 32b of the inoculation pipe 26 is closed), and the lock device 32d of the discharge pipe 29 is opened. , By the air pressure introduced through the air inlet tube 24, the liquid spawn in the liquid spawn incubator 21 (that is, the liquid culture completed with the aeration culture) is automatically discharged to the outside through the discharge pipe 29. Lose.

EXAMPLE

Hereinafter, the present invention will be described in more detail with reference to Examples. The conditions presented in this embodiment are merely to illustrate the technical spirit of the present invention in more detail. Accordingly, the invention is not limited to the conditions set forth in the following examples and should not be construed in that sense.

(A) Liquid spawn culture of the mushroom

When the mycelium of Pleurotus eryngii grown in Petri dishes grows about 70%, three pieces of mycelium are inoculated into the liquid medium using platinum sphere.

Inoculate the mycelium to the above liquid medium to obtain a preculture, the preculture was shaken at 100 ± 5rpm, about 25 ℃ for 7 days to obtain a pre-culture inoculation, and then placed in a separate inoculation device. Thereafter, a liquid culture liquid having the composition shown in Table 1 is prepared, and the liquid culture liquid is filled in all of the aeration culture apparatus according to the present invention. 200 ml of the pre-inoculation source was inoculated with 10,000 ml of the liquid culture solution, and the sterilized air at about 25 ° C. was continuously supplied for 6 days at a rate of 0.4 vvm to obtain a liquid seed. Thereafter, the cultured liquid spawn is uniformly sprayed on rice straw medium to grow oyster mushrooms.

(B) Culture of liquid spawn of shiitake mushrooms

When the mycelia of shiitake mushrooms grown in Petri dishes grow about 70%, three mycelium fragments are inoculated into the liquid medium using a platinum ball.

Inoculate the mycelium to the above liquid medium to obtain a preculture, the preculture is shaken at 90 ± 5 rpm, about 25 ℃ for 7 days to obtain a pre-culture inoculation, and then placed in a separate inoculation device. Thereafter, a liquid culture liquid having the composition shown in Table 2 is prepared, and the liquid culture liquid is filled in both the aeration culture apparatus according to the present invention. At this time, corn oil which is household cooking oil is used as the antifoaming agent. 100 ml of the pre-inoculation source was inoculated with 10,000 ml of the liquid culture solution, and the sterilized air at about 26 ° C. was continuously supplied for 6 days at a rate of 0.6 vvm to obtain a liquid seed. After that, the incubation is completed, the liquid seedlings are inoculated in a log medium to cultivate shiitake mushrooms.

(C) Culture of liquid seedlings of Bokryeong

When approximately 70% of the mycelium of Fuling expanded in Petri dishes was grown, 5 pieces of mycelium were inoculated into the liquid medium using the platinum sphere.

Inoculate the mycelium to the above liquid medium to obtain a preculture, the preculture was shaken at 120 ± 5 rpm, about 25 ℃ for 9 days to obtain a pre-culture inoculation, then placed in a separate inoculation device. Thereafter, a liquid culture liquid having the composition shown in Table 3 is prepared, and the liquid culture liquid is filled in all the aeration culture apparatus according to the present invention. At this time, soybean oil which is household cooking oil is used as a defoaming agent. 300 ml of the pre-inoculation source was inoculated with 10,000 ml of the liquid culture solution, and the sterilized air at about 27 ° C. was continuously supplied at a rate of 0.6 vvm for 9 days to obtain a liquid seed. After that, inoculate the medium after the completion of the culture of the liquid seed culture to cultivate Bokryong mushroom.

(D) Culture of liquid spawn of situation mushroom

When the mycelium of the situation mushrooms expanded in Petri dishes grew about 80%, five mycelium fragments were inoculated into the liquid medium using a platinum ball.

Inoculate the mycelium to the above liquid medium to obtain a preculture, the preculture is obtained by pre-incubation by shaking culture for 9 days at 120 ± 5 rpm, about 25 ℃. Thereafter, a liquid culture liquid having the composition shown in Table 4 is prepared, and the liquid culture liquid is filled in all the aeration culture apparatuses according to the present invention. At this time, soybean oil which is household cooking oil is used as a defoaming agent. 400 ml of the pre-inoculation source was inoculated with 10,000 ml of the liquid culture solution, and the sterilized air at about 27 ° C. was continuously supplied at a rate of 0.6 vvm for 10 days to obtain a liquid seed. Thereafter, the cultured liquid spawn is inoculated into the medium to grow the situation mushroom.

[Effects of the Invention]

Unlike the conventional method for producing a liquid spawn according to the present invention, since the whole process proceeds in a series of processes, mass production is possible, and since the series of processes are performed aseptically, the occurrence of defective products due to infection of various germs is prevented. There is an effect that is significantly reduced.

In addition, the liquid spawn prepared by the production method of the present invention, while mycelia of the basidiomycetes grew in the environment by severe fluctuations or rotations from the beginning of the preculture to the culture stage of the liquid spawn, unlike in the process Since it grew while absorbing the necessary nutrients directly without the enzymatic degradation process, the mycelia and the vitality is excellent. Therefore, when using the liquid spawn according to the present invention, when compared under the same cultivation conditions, it is possible to obtain a high-quality mushroom product compared to the conventional.

Preferred embodiments of the present invention have been described above with reference to the accompanying drawings, which have been described in detail with reference to the examples. However, this is only to explain the technical spirit of the present invention, and the scope of the present invention is not limited thereto. . Those skilled in the art to which the present invention pertains will be understood that various forms of imitation and modification are possible within the scope of the technical idea of the present invention based on the above specification. For example, those which add a conventional method to the cultivation method of a liquid seed | seat according to this invention can be mentioned.

(※ The present invention was developed by Prof. Jae-Mo Sung, a professor of agricultural and life sciences at Kangwon National University, and was promoted by `` Gangwon-do Rural Development Institute '' hosted by Gangwon-do. It was published as a publication in the seminar, and the present inventor intends to be subject to the new agenda pursuant to Article 30 (1) 1 of the Patent Act.)

Claims (11)

In the liquid culture method of basidiomycetes comprising the steps of receiving the protococcus of basidiomycetes, plate culture, preculture of plate cultured inoculum, and liquid culture of precultured inoculum, 1) The pre-culture step (a) 2 ~ 7% (w / v) carbon source, 0.1 ~ 1.0% (w / v) nitrogen source, 0 ~ 0.15% (w / v) KH ₂ PO ₄ , MgSO ₄ · 7H ₂ O 0 ~ 0.15% (w / v), and the step of preparing a pre-culture solution consisting of the remaining distilled water, (b) inoculating the plate cultured basidiomycete in the pre-culture solution, (c) the inoculated above Culturing the culture in a shaker at 80-200 rpm, 22-30 ° C. for 6 days to 8 days; 2) The above liquid culture process (a) 2 ~ 7% (w / v) carbon source, 0.1 ~ 1.0% (w / v) nitrogen source, 0 ~ 0.15% (w / v) KH ₂ PO ₄ , MgSO ₄ · 7H ₂ O 0 ~ 0.15% (w / v), and preparing a liquid culture consisting of the remaining distilled water, (b) aseptically inoculated the pre-inoculation source of the above liquid culture medium, but the liquid culture medium: Inoculating the pre-inoculation source = 20 to 200: 1 ratio, (c) the inoculated pre-culture at 5 ~ 12 days with the sterilized air of 0.1 to 1.0 vvm at 22 ~ 28 ℃ Culture method of liquid spawn of basidiomycete, characterized in that consisting of aeration culture. The method of claim 1, wherein 1) (a) .Preparation of the preculture and 2) (a) .In the preparation of the liquid culture, the respective carbon sources are sugar, maltose, fructose, glucose, sucrose, malt extract, and starch syrup. Cultivation method of the liquid spawn of basidiomycete, characterized in that selectively using one or two or more from the group consisting of. The method of claim 1 or 2, wherein 1) (a) .preparation of the preculture and 2) (a) .preparation of the liquid culture, the nitrogen source is one or both of soy flour and peptone. Cultivation method of liquid spawn of basidiomycete characterized in that. 2) (a). Liquid spawn of basidiomycete, characterized in that the addition of a defoamer to the mixed culture medium in order to eliminate the bubbles generated during the culture of the liquid spawn in the preparation step of the liquid culture solution. Cultivation method of. The method of culturing liquid spawn of basidiomycetes according to claim 4, wherein the antifoaming agent is various animal oils and vegetable oils. The method of cultivating a liquid spawn of basidiomycete according to claim 5, wherein the vegetable oil is used in the group consisting of household cooking oil, cottonseed oil, castor oil, olive oil, palm oil, and the like. The method of claim 1, wherein 2) (b) the automatic inoculation step of the liquid culture solution is characterized by using the inoculation device (10). According to claim 1, 2) (c) Aeration culture step of the liquid culture method of culturing the liquid spawn of basidiomycete, characterized in that using the aeration culture mechanism (20). 9. The aeration culture apparatus (20) according to claim 8, wherein the aeration culture device (20) is composed of a liquid seed incubator (21) for accommodating a liquid culture and a lid portion thereof (22). An air injection tube 24 for injecting, an inoculation tube 26 for injecting a liquid culture solution from the outside or inoculating a mycelium into the liquid culture solution, and an air discharge tube for discharging air from the inside of the liquid seed incubator 21 ( 28) and a liquid spawn culture method of basidiomycetes, characterized in that the discharge tube 29 of the liquid spawn for discharging the liquid spawn after the aeration culture is used. 10. The method of claim 9, wherein the aeration culture apparatus 20 is a method for culturing the liquid spawn of basidiomycete, characterized in that to perform two functions simultaneously by integrating the inoculation tube (26) and the discharge tube (29) into one. In the liquid spawn of basidiomycete cultivated through the process of receiving a culture of the progeny of basidiomycete, plate culture, preculture of plate cultured inoculum, and liquid culture of precultured inoculum, One). As the pre-culture step (a) 2 to 7% (w / v) carbon source, 0.1 to 1.0% (w / v) nitrogen source, 0 to 0.15% (w / v) KH ₂ PO ₄ , MgSO ₄ · 7H ₂ O 0 ~ 0.15% (w / v), and the preparation step of the pre-culture solution consisting of the remaining distilled water, (b) inoculating the plate cultured basidiomycetes in the pre-culture solution, (c) the inoculated pre-culture It is carried out to the step of culturing 6 to 8 days in a shaker at 80 ~ 200 rpm, 22 ~ 30 ℃; 2). As the above liquid culture process, (a) 2 to 7% (w / v) carbon source, 0.1 to 1.0% (w / v) nitrogen source, 0 to 0.15% (w / v) KH ₂ PO ₄ , MgSO ₄ · 7H ₂ O 0 ~ 0.15% (w / v), and preparing a liquid culture consisting of the remaining distilled water, (b) aseptic inoculation of the pre-inoculation source to the above liquid culture solution, but the liquid culture solution of the above Inoculating the pre-inoculation source = 20 to 200: 1, (c) aerated culture of the inoculated liquid culture with sterilized air at 0.1-1.0 vvm at 22-28 ° C. for 5-12 days Liquid spawn of basidiomycetes to be cultured, characterized in that the step is shifted to.

Images