KR20180110367A - Culture method for mass producing Tricholoma matsutake mycelium - Google Patents

Abstract

The present invention relates to a method for mass-producing pine mushroom mycelium, capable of mass-culturing cells after isolating fresh cells from wild pine mushroom. According to the present invention, the mycelium production method enables replacement and reproduction of wild pine mushroom which is difficult to artificially cultivate by mass-producing cell culture products of pine mushroom. In addition, by processing the mass-produced cell culture products of pine mushroom, it is possible to apply the same as cosmetic compositions and various foods.

Description

[0001] The present invention relates to a method for mass production of mycelia of mung bean {Tricholoma matsutake mycelium}

The present invention relates to a mass production method of pine mushroom mycelia and a process for producing pine mushroom processed foods and cosmetic compositions containing Pine Mushroom Mycelium.

Tricholoma matsutake (Tricholoma matsutake) is an exogenous symbiotic organism belonging to the wormwood mushroom, which forms symbiotic bacteria in the roots of pine trees and obtains nutrients from pine trees. It is widely collected in Korea, North Korea, Japan and China East Asia. These pine mushrooms are large in appearance and rich in meat quality, and have been known as the best edible mushrooms for a long time with their unique flavor and taste.

The importance of pine mushroom in Korea has been remarkable since the export of pine mushroom in 1967, and the annual income of pine mushroom is estimated to be about 40 billion won annually. These income can be an important source of income for the farmers in the pine forests without special facility investment as a by-product of forestry, and it can be said that economic value is very high considering the effect of economic stabilization of mountain villagers.

However, according to the records of the past 30 years, the pine mushroom has not been constant every year, showing a tendency to alternate between abundant and hungry years. In recent years, the pine mushroom damage caused by pine forests, the decrease of pine tree forest due to forest fires, the vegetation of lower vegetation due to the neglect of mt. This is because the production of pine mushroom is decreasing as a result of the accumulation of organic matter.

So far, many researches have been conducted to produce sustainable pine mushroom by artificial efforts and further artificial cultivation of pine mushroom. However, the formation of fruiting body through artificial cultivation has not been realized yet, and the method of controlling the weather condition suitable for the pine mushroom generator has not yet been established yet, because the pine mushroom characteristically forms fruiting body only when it is in symbiosis with the pine root .

In addition, most mushrooms are germinated from dead trees and are parasitic. However, mushrooms are a living tree, a unique seed that is only parasitized on pine trees. For artificial cultivation, it is necessary to create the same environment as living pine trees. It has been studied continuously but it is still very sensitive to temperature, humidity, soil and environment so that it can not succeed in artificial cultivation.

Therefore, there is a growing interest in pine mushroom mycelium, which can replicate wild pine mushroom and reproduce it in the same way. Pine mushroom mycelium contains the same ingredients as the wild pine mushroom, and its efficacy is known to be the same. However, the study on the production of mycelium is merely a review of general cultivation conditions of mycelium. Therefore, it is urgent to develop mass production technology of mycelium by domestic technology in order to secure the pine mushroom-related market in potential domestic and international markets.

Accordingly, the inventors of the present invention confirmed that fresh pine mushroom can be replaced and reproduced by separating fresh cells from wild-purchased pine mushroom and culturing the cells in a large amount to complete the present invention.

It is an object of the present invention to mass produce pine mushroom mycelium.

It is still another object of the present invention to provide a processed food of pine mushroom produced by processing the mycelia obtained through the production method and a cosmetic composition containing the same as an active ingredient.

(A) culturing mycelia by inoculating part or the whole of the mushroom fruiting body into a culture medium, (b) pre-culturing the mycelium grown after culturing in the medium I, c) culturing the mycelium of the first pre-cultured medium in a culture medium II, ii) pre-culturing the mycelium in the culture medium II in the culture medium, wherein the pH is higher than the pH of the medium I and the pH of the medium III is higher than the pH of the medium II.

In the present invention, the pH of the media I to III may be in the range of 4.5 to 6.5, more preferably the pH of the medium I is 4.8 to 5.2, and the pH of the medium II is 5.3 to 5.7 , And the pH of the medium III is 5.8 to 6.2.

In addition, the present invention provides a pine mushroom processed food produced by processing the mushroom mycelium produced through the above production method.

In addition, the present invention provides a cosmetic composition comprising, as an active ingredient, a mushroom mycelium produced through a production method.

According to the present invention, it is possible to produce cultivated pine mushroom cells in large quantities, and it is possible to substitute and reproduce low-cost natural pine mushroom which is difficult to artificially cultivate. In addition, the pine mushroom cultivated in large quantities can be processed into various food groups and cosmetic compositions even in areas where pine mushrooms are not available due to their high price.

1 is a process diagram for a mass production method of mycelia of pine mushroom according to the present invention.
Fig. 2 is a photograph of a cultured mycelium at the time of primary pre-culture. Fig.
Fig. 3 is a photograph showing a cultured mycelium at the time of the second pre-culture.
Fig. 4 is a photograph of the appearance of a biological incubator during the present culture.
5 is a graph showing the growth of mycelium in each culture step.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In one aspect, the present invention provides a method for producing a mushroom comprising: (a) culturing a mycelium by inoculating part or all of the mushroom fruiting body into a medium; (b) primary culturing the mycelium grown in culture medium in medium I; (c) secondarily pre-culturing the mycelium that has been pre-cultured in medium II; (d) culturing the second pre-cultured mycelium and culturing the medium in culture medium III in a biological culture medium, wherein the pH of medium II is higher than the pH of medium I, and the pH of medium III is higher than that of medium II pH higher than the pH of the mushroom mycelium.

Mushrooms have no distinction of root-stem leaves in the body and usually consist of hyphae (fungi). They do not have chlorophyll and live with the nutrients made by other creatures. And the breeding consists of spores. In other words, when spores are sprayed and germinated, hyphae are formed, and when these hyphae are infested, fruiting bodies that produce spores are formed again. The occurrence of mushrooms can be properly determined by temperature, humidity, humidity of soil, light, nutrients in soil, etc. The range of conditions varies depending on the type of mushroom.

The fleshy body consists of gods and sacks. The upper side of the god is dark brown, and the lower side has many wrinkles. Spores occur on both sides of the wrinkles. When they are ripe, they are blown in the wind and germinate in suitable places.

The present invention relates to a method for producing pine mushroom mycelium in large quantities by separating fresh cells from the pine mushroom fruiting body and gradually increasing the pH of the medium according to the culture step. In the present invention, a large amount of cell mass is produced at a low pH during the first preculture, and then the pH is gradually increased to obtain a cell mass in a larger form.

Since the method of producing pine mushroom mycelium according to the present invention can also be applied to a mass bioreactor of ton unit, it is possible to dramatically increase the production amount of mycelium produced in units of liter (liter) by ton (ton).

In the present invention, the pH of the media I to III may be within the range of 4.5 to 6.5, preferably the pH of the medium I is 4.8 to 5.2, the pH of the medium II is 5.3 to 5.7, 5.8 to 6.2.

In the present invention, the pH of the medium I was 4.8-5.2 at the time of the first pre-culture, and a large amount of cell mass was formed at a relatively low pH. Then, the pH of the medium II was raised to 5.3-5.7 during the second pre- The lumps can be led to a larger shape. Then, by increasing the pH of the culture medium III to 5.8 to 6.2 at the time of the present culture, the volume of the cell culture can be ultimately increased.

In one embodiment of the present invention, the maximum amount of mycelium production is 30.62 g / L (Dry Cell Weight, DCW) by raising the pH of medium I to about 5, pH of medium II to about 5.5 and pH of medium III to about 6, .

In one embodiment of the present invention, large-scale cultivation was performed at a time using a 5-ton incubator, and it was possible to produce mycelium of 107.17 kg (DCW) with a working volume of 3.5 tons. Furthermore, when the number of culture tanks in an incubator is increased, the amount of the mycelium can be a multiple of 107.17 kg (DCW).

In one embodiment of the present invention, a total of 428.68 kg (DCW) of mycelium was produced by operating four culture tanks.

As described above, the mycelium culturing method according to the present invention is applicable to a large scale culture unit of ton unit, and thus it is advantageous to mass culture of mycelium at once.

The present invention also provides an environment suitable for mycelial growth through the conditions of an optimal culture medium composition.

In the present invention, the culture medium I contains 2 g / L of soypeptone, 3 g / L of yeast extract, 10 g / L of glucose, 10 g / L of starch, It may include (L-arginine) 0.5 g / L, thiamine _{(thiamine) 0.02ppm, KH 2 PO} 4 1 g / L, MgSO 4 0.5 g / L.

In the present invention, the medium I and medium II have the same composition and pH may be different.

In one embodiment of the present invention, medium I and medium II are those wherein composition is "Soypeptone 2 g / L, Yeast extract 3 g / L, Glucose 10 g / L, L-arginine 0.5 g / L, Thiamine 0.02 ppm, KH ₂ PO ₄ 1 g / L and MgSO ₄ 0.5 g / L ", and the pH of medium I Was about 5, while the pH of medium II was raised to about 5.5, and only pH conditions were different.

As a result, the amount of mycelium production, which was 13.27 g / L in the first preculture, increased to 21.56 g / L through the second preculture, and the cell mass obtained in the first preculture proliferated in a larger form Respectively.

In the present invention, the medium III is prepared by mixing 2 g / L of soypeptone, 3 g / L of yeast extract, 15 g / L of glucose, 5 g / L of starch, 0.5 g / L of L-arginine, 0.02 ppm of thiamine, 1 g / L of KH ₂ PO ₄ , 0.5 g / L of MgSO _{4 and} 0.3 g / L of CaCl ₂ .

In the step (d), when the carbon source of the medium III is exhausted, the medium IV may be further supplied.

As a method of analyzing whether the carbon source has been exhausted from the medium, there is a method of analyzing the content of total sugar (glucose, starch, maltose, etc.) initially injected and measuring the amount of carbohydrate in the culture solution by the total sugar amount method (Phenol-sulfuric acid method) And the DNS method for the measurement.

In one embodiment of the present invention, the carbohydrate in the culture was quantitated using the Phenol-sulfuric acid Method.

The medium IV may include maltose and malt extract. Preferably, the medium IV contains 36 g / L of maltose and 18 g / L of malt extract. And the pH may be 6.

In order to increase the production amount of mushroom mycelium and to shorten the incubation period, it is possible to carry out a fed-batch culture in which malt extract, which is a nitrogen source, is supplied in an intermittent and continuous manner together with maltose as a carbon source.

This is to further induce the growth of mycelium by additionally supplying glucose (other than glucose) used as an energy source. As a result of the preliminary test, it was confirmed that cell growth in the two components had a significant effect at the corresponding pH condition Respectively.

As described above, the present invention establishes the optimum medium composition capable of maximizing the growth of mycelium of Pine Mushroom during the primary culture, the secondary culture, and the main culture, and at the same time, This invention relates to a method for producing pine mushroom mycelium.

In the present invention, (e) the step of removing the supernatant from the culture broth to obtain a mycelium may be further included.

At this time, when removing the supernatant, only mycelium can be separated using a filtration membrane.

In another aspect, the present invention relates to a pine mushroom processed food produced by processing the pine mushroom mycelium produced through the production method.

Pine mushroom has been reported to have various physiological functions such as excellent anti-cancer, antiviral, immunity and blood cholesterol lowering effect, and it has excellent prevention and improvement effect on adult diseases such as stroke and heart disease, Has increased significantly.

Therefore, by processing pine mushroom and using it in various food groups, it can contribute to human health.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to produce pine mushroom mycelia in large quantities, and thus the present invention can be applied to various fields that have not been utilized due to high price.

In the present invention, the pine mushroom mycelium may be processed by, for example, freeze-drying, low-temperature drying or the like, but is not limited thereto. Freeze drying has the advantage of minimizing the destruction of nutrients in mushrooms.

The pine mushroom processed foods may be health functional foods, health supplement foods, preference foods, and the like, but are not limited thereto. For example, the present invention relates to a food, a drink, a tea, a confectionery, a seasoning, a drink and a drink, a vitamin complex, a dairy product, a candy, a jelly, an ice cream and a frozen dessert, a morning cereal, , Cereal products, pasta, soups, sauces and dressings, confectionery products, oil and fat products, dairy drinks, tea, soy milk, frozen foods, cooked foods and alternative foods, meat products, cheese, yogurt, breads, cakes, And so on.

In another aspect, the present invention relates to a cosmetic composition comprising the mushroom mycelium produced through the above production method as an active ingredient.

The present invention can be utilized as a cosmetic composition for improving wrinkles, improving skin elasticity, whitening effect, and the like by using the mushroom mycelia produced in large quantities according to the present invention.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

One. Mushroom  Separate selection

After sterilization of surface of fruiting body with sterilized alcohol (70% concentration) after sterilization of natural pine mushroom, it is turned over and the middle of the sack portion is cut with a sterilized scalpel and torn with two hands with two hands to remove a part of fruiting body into small pieces, (PDA, Potato Dextrose agar) and cultured at 25 ° C for about 2 to 4 weeks to obtain pure mushroom cells (mycelium).

2. Storage of purely isolated cells

The mushroom cells cultivated in the artificial medium (PDA) were cut into 0.5 cm × 0.5 cm size with sterilized scalpel and placed into a cryovial vial containing 20% of Glycerin (cryoprotectant) for 1 block Here, 'block' refers to the shape of a cell mass taken when a fungus such as mold, mushroom, etc. is transferred to another medium.) 2 blocks were placed and allowed to stand at room temperature for about 15 minutes, and then stored in an ultra-low temperature freezer at -80 ° C for a long period. At this time, the glycerin protects the mushroom cell tissue from damage during ice crystal formation during freezing.

3. Primary mushroom cells Pre-culture (batch culture)

In order to derive the cell block obtained from the above-mentioned solid medium (PDA) in the first step into various tissues, the first pre-culture was performed. Mushroom cells grown sufficiently in artificial medium (PDA) (about 2 to 3 weeks) were cultured in liquid medium and medium 1 [soypeptone 2 g / L, yeast extract 3 g / L, glucose 10 g / L, starch (starch) 10 g / L, L- arginine (L-arginine) 0.5 g / L, thiamine _{(thiamine) 0.02ppm, KH 2 PO} 4 1 g / L, MgSO 4 0.5 g / L] (Total volume 250 ml flask, pH 5.0) under sterilization conditions of high pressure at 121 캜 for 30 minutes. Then, mushroom cell block Block 5ea was inoculated and cultured with shaking at 25 캜 and 140 rpm for 2 weeks (Fig. 2).

The present inventors have found that, in the composition of the medium I, "2 g / L of soypeptone, 3 g / L of yeast extract, 10 g / L of glucose, 10 g / L of starch, L-arginine 0.5 g / L, Thiamine 0.02 ppm, KH ₂ PO ₄ 1 g / L, and MgSO ₄ 0.5 g / L ". That is, the composition of the medium I was established by the present inventor by studying optimal conditions for growth of the mushroom cells.

4. Secondary of mushroom cells Pre-culture (batch culture)

Secondary liquid culture was carried out to further increase the mycelium of the primary culture medium. The primary cell culture broth was homogenized at 25,000 rpm for 15 seconds using a homogenizer (tissue homogenizer) and cultured at 121 ° C for 60 minutes under sterile conditions at a high pressure of medium II [soypeptone 2 g / L, yeast extract Yeast extract, 10 g / L glucose, 10 g / L starch, 0.5 g / L L-arginine, 0.02 ppm thiamine, KH ₂ PO ₄ (1 g / L, MgSO ₄ 0.5 g / L) was inoculated into a 15 L culture vessel (total volume 20 L, pH 5.5) and incubated at 25 ° C for 2 weeks in an aeration volume of 0.1 vvm (aeration volume / medium volume / minute).

Medium II in the second preincubation process was increased from pH 5 to 5.5 while using the same medium composition as the first. As a result, it was confirmed that the volume of the cell mass obtained in the primary pre-culture was proliferated in a larger form (FIG. 3).

5. Pine mushroom cells Cultivation (Fed-batch culture)

The second pre-culture solution was added to a 100 L inoculum (homogenizer function), and the homogenizer was operated at 10,000 rpm for 1 minute to break up the cell mass into small particles and then inoculated into a 5-ton tank (bio-incubator) (Fig. 4). The inoculator is provided with a blade inside to drive the motor to crush the cell culture mass finely. The sterilizing and drying air pressure is controlled and the mycelium is moved from the inoculator to the culturing tank.

The culture medium in the bio-culture medium contained medium III (Soypeptone 2 g / L, Yeast extract 3 g / L, Glucose 15 g / L, Starch 5 g / L, L- The working volume was adjusted to pH 6.0 with 0.5 g / L of arginine, 0.02 ppm of thiamine, 1 g / L of KH ₂ PO ₄ , 0.5 g / L of MgSO _{4 and} 0.3 g / L of CaCl _2. The medium was sterilized at a pressure of 1.2 bar and 121 ° C for 60 minutes at a standard of 3.5% of 70%, and then inoculated on the basis of the amount of cells inoculated at 2.8%.

Medium III increased the pH from 5.5 to 6.0 as compared to medium II of the previous stage, and as a result, it was confirmed that the volume of the cell culture finally increased greatly.

The culture medium (maltose 36 g / L and malt extract 18 g / L, pH 6.0) was added to 100 L of the medium (glucose, starch) after 10 days from the start of the culture And the cells were cultured with aseptic injection twice at intervals of 5 days. When the carbon source was exhausted, the carbohydrate content in the culture medium was quantified using the Phenol-sulfuric acid method. As a result, it was confirmed that the mycelial growth was greatly induced by supplying additional energy source when the energy source was exhausted.

5 and Table 1, the amount of the mycelium produced by the mycelial production method according to the present invention was 13.27 g / L in the first pre-culture, 21.56 g / L in the second pre-culture, 30.62 g / L.

[Production of mycelium by culturing step] step Mycelial production (Dry Cell Weight (g / L)) Primary Pre-culture 13.27 Secondary Pre-culture 21.56 Cultivation 30.62

The amount of the final cell culture produced by the present invention was 30.62 g / L at the maximum, and the amount of final mycelium reached to 107.17 kg (dry cell weight, DCW) when converted to 5 ton culture medium. The four culture tanks were able to produce up to a total of 428.68 kg (DCW) at the time of operation.

6. Recovery of mushroom cells

The culture supernatant was discarded using a filtration membrane and pure cell culture was obtained.

7. Processing

The recovered cell culture was lyophilized to obtain the final product.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

(a) culturing a mycelium by inoculating a part or the whole of the mushroom fruiting body into a medium;
(b) primary culturing the mycelium grown in culture medium in medium I;
(c) secondarily pre-culturing the mycelium that has been pre-cultured in medium II;
(d) culturing the mycelium secondary to the pre-culture and culturing the medium in medium III in a biological incubator,
Wherein the pH of the medium II is higher than the pH of the medium I and the pH of the medium III is higher than the pH of the medium II.
The method according to claim 1,
Wherein the pH of said media I to III is within the range of 4.5 to 6.5.
The method according to claim 1,
Wherein the pH of the medium I is from 4.8 to 5.2, the pH of the medium II is from 5.3 to 5.7, and the pH of the medium III is from 5.8 to 6.2.
The method according to claim 1,
Wherein said medium I and medium II are the same in composition and pH is different.
The method according to claim 1,
The culture medium I contained 2 g / L of soypeptone, 3 g / L of yeast extract, 10 g / L of glucose, 10 g / L of starch, L-arginine , 0.5 g / L of thiamine, 0.02 ppm of thiamine, 1 g / L of KH ₂ PO ₄ , and 0.5 g / L of MgSO ₄ .
The method according to claim 1,
The culture medium III contained 3 g / L of soypeptone, 3 g / L of yeast extract, 15 g / L of glucose, 5 g / L of starch, L-arginine , 0.5 g / L of thiamine, 0.02 ppm of thiamine, 1 g / L of KH ₂ PO ₄ , 0.5 g / L of MgSO _{4 and} 0.3 g / L of CaCl ₂ .
The method according to claim 1,
Wherein in the step (d), when the carbon source of the medium (III) is exhausted, the medium (IV) is further supplied.
8. The method of claim 7,
Wherein said medium IV comprises maltose and malt extract. ≪ RTI ID = 0.0 > 18. < / RTI >
8. The method of claim 7,
Wherein the culture medium IV contains 36 g / L of maltose and 18 g / L of malt extract and the pH is 6. The method for mass production of mushroom mycelium according to claim 1,
The method according to claim 1,
(e) removing the supernatant in the culture medium to obtain a mycelium.
A processed food of matsutake mushroom produced by processing the mycelium produced by the production method according to claim 1.
A cosmetic composition comprising the mycelium produced by the production method according to claim 1 as an active ingredient.

Images