KR101119143B1 - Composition for the culturing of Inonotus obliquus - Google Patents

Abstract

The present invention is chaga mushroom ( Inonotus) showing anti-diabetic effect obliquus ) relates to a liquid medium composition for producing high concentration of chaga mushroom mycelium for producing a protein polysaccharide and a method of cultivating chaga mycelium using the same. More specifically, the present invention relates to a culture medium composition for chaga mushroom liquid culture containing oil or oleic acid having high efficiency as a carbon source, and a method for culturing mycelium using the same. The present invention relates to chaga mushroom mycelium and A composition of the new industrial liquid medium with about 1.5-fold increase in intracellular protein polysaccharide.

Chaga, Oil, Oleic Acid, Inototos Oblique, Liquid Medium

Description

Medium composition for cultivating chaga mycelium {Composition for the culturing of Inonotus obliquus}

The present invention is a chaga ( Inonotus) producing a protein polysaccharide obliquus ) relates to a liquid medium composition for mass production of mycelium and a method for culturing chaga mycelium using the same. More specifically, the liquid medium composition is a culture medium composition for chaga mycelium liquid culture medium added with oleic acid, which is a very efficient oil or oil as a carbon source, it is possible to grow chaga mycelium in a liquid phase at high speed per unit time Let's do it.

Mushrooms, which are higher fungi, have been used as foods for a long time since they are rich in taste and nutrition and have been widely used for medicinal purposes. In particular, Basidiomycetes mushrooms, which are the most noticeable natural products effective in the treatment and prevention of diseases, are widely used in oriental medicine because of their wide pharmacological action. Recently, the anticancer effect of medicinal mushrooms from basidiomycetes has been scientifically proved. Among the extracts of medicinal mushrooms, physiologically active substances exhibiting excellent anticancer efficacy and diabetes improvement effect by immune enhancing effect are water-soluble proteins having β-D-Glucan structure. It was found to consist of polysaccharides (hereinafter termed "protein polysaccharides"). Among them, the protein polysaccharide derived from Chaga (Inonotus obliquus ), which is one of the representative basidiomycetes, has been shown to be excellent as a new bioactive substance with diabetes improvement, as it has been found to have an excellent effect on lowering blood sugar by promoting insulin secretion as well as an immune enhancing effect. have.

On the other hand, conventional methods of using basidiomycetes include a method of collecting or growing fruiting bodies, and a method of using a mycelium by separating the mycelium from the fruiting bodies. Existing methods of collecting and using wild fruiting bodies not only have problems of resource depletion and destruction of ecosystems, but also cannot secure the required amount, and many constraints such as fruiting through cultivation take a long time to grow. It is not preferable as the practical use method.

Chaga mushroom, which is known for its excellent diabetic effect, is also difficult to grow due to difficult growth conditions and wild ones are too expensive and rare to purchase. Recently, the fruiting body is produced by artificial cultivation, but due to the long production period and low productivity, it is difficult to sell at low price desired by consumers.

Chaga-derived protein polysaccharides consist of intracellular exopolysaccharides (IPS) present as cell wall components and extracellular protein polysaccharides (extracelluar exopolysaccharides (EPS)) secreted extracellularly. Among them, the intracellular protein polysaccharide (IPS) is a trace component having a content of 5% or less even in the crude protein polysaccharide purified through a specially designed purification process, and since the ratio present per unit cell is almost constant, the high concentration in the final liquid culture process In order to obtain protein polysaccharides, it is essential to develop an industrial liquid medium capable of producing a large amount of chaga mycelium.

In other words, chaga mushroom mycelium culture using the conventional liquid medium composition has a problem that the growth rate per unit time is not only low, but the cell mass that can be obtained even after long-term culture is extremely limited. In addition, the yield of the final product showed a large difference depending on the medium composition used, the production medium composition was managed by the confidentiality of each manufacturer is not known to the outside, the medium composition determination was a very time-consuming and labor-intensive process. In addition, since the ratio of the medium price in the production of the product is more than 40%, it was necessary to reduce the price ratio of the medium using the necessary medium composition and concentration except for unnecessary components.

Accordingly, the present inventors have made diligent research efforts to overcome the problems of the prior art, the method of culturing chaga mushroom mycelium using the medium composition of the present invention using oil or oleic acid as a carbon source is chaga mycelium and intracellular protein polysaccharide is weak It confirmed that 1.5 times higher, and completed this invention.

One object of the present invention is to provide an optimized culture composition and culture conditions of chaga mycelium for the mass production of chaga mycelium and protein polysaccharides having immuno-enhancing, anti-cancer, anti-diabetic effect.

Another object of the present invention is glucose, oil, or a type of oil, oleic acid, casein, soytone peptone, KH ₂ PO ₄ And to provide a liquid medium composition for mass production of chaga (Inonotus obliquus) mycelium mass comprising distilled water.

It is still another object of the present invention to provide an optimum liquid medium composition and culture conditions including a specific content of each composition in the liquid medium composition for mass production of chaga mycelium.

Still another object of the present invention is to provide a method for culturing chaga mycelium comprising the optimized liquid medium composition.

The present invention is a liquid medium for the production of mycelia (Inonotus obliquus) mycelium, characterized in that consisting of glucose (oil), oil (casein), soytone peptone, KH ₂ PO ₄ and distilled water It relates to a composition.

The oil of the present invention is corn oil (corn oil), wheat germ oil (wheat germ oil), soybean oil (soybean oil), cotton seed oil (cotten seed oil), sunflower oil (sunflower oil), lard oil (lard oil), Olive oil, peanut oil, almond oil, sesame oil, oleic acid.

The oil of the invention is preferably oleic acid.

In the present invention, chaga mycelium culture composition is characterized by using an oil or oleic acid, the composition ratio of the composition is also significantly different from the conventional one.

Specifically, the composition of the present invention is glucose (glucose) is 14 to 22 g / l, oleic acid is 25 to 30 g / l, casein is 2 to 6 g / l, Soytone peptone is 6 to _{10 g / l, KH 2 PO} 4 is preferably composed of 3 ~ 6g / l.

More preferably, 1 g of distilled water contains 22 g of glucose, 30 g of oleic acid, 6 g of casein, 10 g of soyton peptone, and 3 g of KH ₂ PO ₄ .

Oleic acid, which is a main component of the medium of the present invention, is a kind of oil, which was used as a carbon source, and the oil used as a carbon source has different cell mass (Y _{x / s} ) (production yield) of the mycelium produced per unit mass of oil consumed. In addition to being higher than the carbon source, the oil acts as an antifoaming agent in the fermenter culture with oil added, so it is very easy to scale-up the culture process in the culture of higher fungi, which generate a lot of foam during fermenter culture.

In one embodiment of the present invention, as a result of checking whether the chaga mycelium can be used as a carbon source by decomposing the oil, chaga mycelium has a relatively excellent secretion ability of the lipase (lipase), which is an oil degrading enzyme to decompose the oil It was confirmed to be used as a carbon source. In one embodiment of the present invention, cell productivity was compared with various types of oils in order to find an oil that can be used as an optimal carbon source. As a result, it showed a high productivity in lard oil (wheat oil), wheat oil (wheat oil), corn oil (corm oil), it can be seen that oleic acid in particular has a greater effect on the growth of chaga mycelium than other oils. It can be seen that oleic acid oil has a greater effect on mycelial growth when the required amount of glucose is supplied.

Determination and optimized content of the culture medium composition for chaga mycelium liquid culture medium added with oil as the carbon source is made through a partial factorial design experiment which is a statistical method. The medium optimization experiment using statistical method is a method that can confirm the effect of various media composition on the result through dozens of experiments through statistical treatment and optimize the optimal concentration of the medium composition. By removing media components that do not affect or adversely affect the result, and maximize the chaga mycelium concentration can ultimately increase the efficiency of the process.

The liquid medium composition of the present invention may be prepared by sterilization at 121 ° C. for 15 minutes by adjusting to pH 6 and dispensing the flask and incubator.

Another aspect of the invention, the step of subcultured chaga mycelium in agar medium; Static culture of the passaged mycelia in the step of culture medium; And a liquid culture step of culturing the subculture of the mycelium in the liquid phase in a liquid medium. The method of culturing the mycelium mycelium, wherein the liquid culture step is characterized in that the difference is achieved by the optimized liquid medium composition. It relates to a culture method of mushroom mycelium.

The invention related to the culturing method uses a conventional method of cultivating chaga mushroom mycelium, but the composition, such as using oil or oleic acid as a carbon source in the liquid phase culturing step, or using a liquid medium composition whose composition ratio is significantly different from the conventional one It relates to a method of culturing mycelium comprising a liquid phase culture step. When cultured using a liquid medium composition for mass production of chaga mushroom mycelium obtained through the embodiment of the present invention, chaga mushroom mycelium productivity and intracellular protein polysaccharide concentration increased 1.5 times per unit time compared to those cultured in a conventional chaga mushroom liquid remediation medium. You can see that.

In the case of culturing chaga mycelium using the oil-added liquid medium composition for mass production of chaga mycelium mass according to the present invention, the growth rate per unit time and intracellular protein polysaccharide are about 1.5 times higher than that of the conventional composition. Mass production and supply of protein polysaccharides that show augmentation, anticancer, and anti-diabetic efficacy are possible.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are only for illustrating the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples. .

< Example  1> Chaga Mushroom  Mycelium Liquid Culture Method

1-1. Chaga  Germinating Spores to Obtain Mycelium

The mycelium of chaga was used as a mycelium obtained by germinating spores obtained from the fruiting body of chaga from wild. Chaga mycelium was passaged every 11 to 15 days at 24 ℃ temperature in agar medium for passage (malt extract 20 g, yeast extract 2 g, agar 20 g, distilled water 1L). Political culture was used for the culture medium (glucose 22.5 g, yeast extract 2g, peptone 2g, diphosphate 2g, magnesium sulfate 0.6 g, distilled water 1L), strain storage was stored at 4 ℃ mycelium A 20% glycerol stock was prepared and stored at -80 ° C. When necessary, the stock stored was taken out and inoculated in a solid passage medium to be incubated.

1-2. Chaga Mushroom  Mycelium In a liquid medium  Incubation step

The mycelia grown in the above medium were aseptically collected, and then inoculated so as to be 1 to 10%, preferably 5% (v / v), in the liquid medium. Liquid medium for cell wall polysaccharide (IPS) production is liquid medium for chaga mycelium (8 g of glucose, 26 g of fructose, 3.5 g of casein, 7.5 g of soytone peptone) , 6 g of KH 2 PO 4, 3 g of MgSO 4, 1 L of distilled water, pH of 3.5 to 6.0, preferably adjusted to 5.0, were good in terms of mycelial growth (IPS) and protein polysaccharide productivity. In the present invention, all cultures were performed in a culture volume of 30 ml in a 250 ml flask. Liquid culture of the mycelium was incubated for 7-10 days at 28 ℃, 150rpm in a shaking incubator, or cultured for 3 to 5 days and then passaged to another flask.

1-3. Chaga Mushroom  In culture Extracellular protein polysaccharide  And Intracellular protein polysaccharide  Quantification Step

1-3-1. Cell concentration  Measure and Extracellular protein polysaccharide ( EPS ) Recovery

Cell concentration was measured by mycelium dry weight (DCW). The mycelium was recovered uniformly from the culture solution, and 20 ml of cells were centrifuged at 4,000 rpm for 10 minutes. The supernatant was recovered and transferred to a 1.5 ml microtube to measure the remaining sugar. After removing the remaining supernatant, the remaining sugar, insoluble substance or salts were removed through three washing processes. Thereafter, put in a weighing dish and dried at 90 ℃ for 12 hours to confirm that there is no change in the weight of the dry cell concentration, the dry weight was measured and expressed in terms of cell concentration per 1L. For extracellular protein polysaccharide (EPS), 5 ml or 10 ml of the supernatant was taken from the culture medium centrifuged for the first 10 minutes to measure dry cell concentration, and then doubled 100% ethanol and left overnight at -20 ° C. Centrifuged for 10 minutes. Thereafter, the supernatant was removed, and the remaining pellets were dried in a 90 ° C. oven for about 12 hours and then weighed.

1-3-2. HPLC Using In mycelium Protein Polysaccharide  ( IPS Quantitative analysis

For quantitative analysis of cell wall polysaccharides (IPS) having a β-D-glucan structure from chaga, the cultured mycelium was centrifuged to remove the supernatant and then washed three times using distilled water. After drying at 90 ℃ for 12 hours, 25g of the pulverized dry mycelium suspended in 50ml of distilled water, extracted with steam bath for 2 hours, and extracted again 40ml of distilled water for 6 hours. Thereafter, two filtration processes were performed using 0.45 μm filter paper, and 15 ml of the extract filtrate was transferred to a new tube and precipitated in 2 times of 99.9% ethanol for 6 hours. After centrifugation at 2,800 rpm for 10 minutes, the supernatant was removed and lyophilized, and analyzed using a gel chromatography column and an ELSD detector for HPLC.

   On the other hand, the analysis of the extracellular polysaccharide (EPS) is pulverized extracellular protein polysaccharide recovered by the above method using a mortar and then dissolved in an appropriate amount of distilled water, and finally obtained by using a 0.45μm filter (filter) It was analyzed by the above HPLC method.

Sugar analysis

The sugar analysis in the culture solution was centrifuged for 10 minutes at 12,000rpm, and then the supernatant was taken, and centrifuged three times for 10 minutes at 12,000rpm, and only the supernatant was taken and filtered using 0.45μm HPLC filter paper. Sugar analysis conditions using HPLC are as follows:

Column A: RS tech, NH ₂ column (250mm x 46mm)

  Column No: 091264AE

  Column temperature: 40 ℃

  Mobile phase: 75:25 (acetonitrile: water)

  Flow rate: 1.2 ml / min

  Detection: RI

< Example  2> Chaga Mushroom  Mycelium Liquid Culture As a badge  Optimal Oil Selection

Chaga mycelium in order to use the oil as a medium for the liquid culture medium must first determine whether chaga mycelium can be used as a carbon source by decomposing the oil. As a result of the experiment, the production strain was found to have a relatively good ability to secrete lipase, an oil degrading enzyme, to decompose oil and use it as a carbon source. In this example, by examining the tendency of the production medium composition change when oil is added to the basic liquid medium, it was intended to present the results of the optimization of the production medium again.

In this example, various oils were additionally added based on the liquid medium for chaga mycelium presented in Example 1 to select the optimal oil to be added to the liquid medium for chaga mycelium. As a method, 5, 10 ml of each oil was added to a liquid medium for chaga mycelium to prepare a medium, and then cultured by inoculating chaga mycelium. Table 1 shows the types of oils used in this example.

Types of oil for adding liquid medium for cultivating chaga mycelium Oil One Corn oil 7 Olive oil 2 Wheat germ oil 8 Peanut oil 3 Soybean oil 9 Almond oil 4 Cotton seed oil 10 Sesame oil 5 Sunflower oil 11 Oleic acid 6 Lard oil

As a result of two repeated addition experiments, 5 ml and 10 ml of 11 oils were added, respectively, and the overall productivity was higher than that of the control group Chaga mushroom mycelium liquid medium, especially in wheat germ oil and lard oil. (See FIG. 1).

Therefore, wheat germ oil and lard oil, which showed high productivity, and corn oil, which had relatively high productivity, were used to perform the addition concentration experiment in more detail. In other words, since 5ml and 10ml were added arbitrarily, the productivity was improved when the lower or higher concentration was shown to increase the concentration to 2-20 ml / L. Chaga mushroom mycelium liquid medium was prepared by the method described in Example 1, and each oil was added according to the concentration, followed by culturing chaga mushroom mycelium. For lard oil, 11 ml / L (19 g / L), wheat Embryonic oil 5 ml / L (18 g / L), corn oil 11 ml / L (16.5 g / L) was found to show the highest productivity (Fig. 2). In liquid culture, oil or oleic acid is present in a very reduced state, so it contains excess energy compared to other carbon sources, so even if only a small amount is added as a medium component, the amount of mycelium produced per unit mass of oil consumed (Y _{x / s} ) (production yield) was higher than other carbon sources. In addition, oleic acid or oil also serves as an antifoaming agent, which facilitated the culturing process in the case of the culture of higher fungi, which generate a lot of foam, especially in fermenter culture. That is, the foam generated during the cultivation is not only to reduce the volume of the culture solution when it is leaked to the outside of the fermenter, but also cause contamination, it is very important to develop a process for suppressing the foam generation during the culture. In addition, when oil or oleic acid was used without using silicone as an antifoaming agent, it was possible to facilitate the separation and purification of the protein polysaccharide which is a subsequent process. Silicone antifoams remain in the culture medium because they are not available to microorganisms, making it difficult to separate and purify protein polysaccharides.Oil or oleic acid does not remain in the culture solution after the culture is finished because the production strains are both energy and carbon sources. Therefore, it had no effect on the separation and purification process.

On the other hand, except the glucose and fructose which are the most easily used carbon source among the basic chaga mycelium liquid medium components, experiments were conducted to investigate the growth rate of chaga mycelium when using only oil as a carbon source. The oils shown in Table 1 were added at a concentration of 30 ml / L, and the remaining media components were kept the same as in Example 1. The results of this experiment are shown in FIG. 3, and the results show that, when only oil is used as a single carbon source without the addition of sugar components, the overall cell yield was low (see FIG. 3). Notably, oleic acid was found to have a greater effect on chaga mycelium growth than other oils. As a result of this study, it can be inferred that the presence of sugar components acts as a major factor to replenish nutrients lacking in oil. In particular, oleic acid was expected to have a better effect than wheat germ oil when the required amount of glucose was supplied. In addition, oleic acid was a pure substance composed of the same molecule, whereas wheat germ oil was not clear, so it was thought that oleic acid would have a better effect in the separation and purification.

To confirm this, an experiment was conducted to investigate the concentration of glucose in the production medium to which oleic acid and lard oil were added. As shown in Table 2, the oil-to-glucose ratio was changed from 5 g / L to 30 g / L of glucose, and 10 ml and 20 ml of 30 ml of oil were added to investigate the mutual effect between glucose and oil. The culture method was performed in the same manner as in Example 1.

Table of Experimental Design for Determination of Medium Component for Liquid Culture of Chaga Mycelium Glucose 0,5,10,15,20,25,30 g / L Lard oil,
Oleic acid 10,20,30ml / L Casein 3.5g / L Soytone peptone 7.5g / L KH2PO4 6g / L MgSO4 3g / L

Figure 4 shows the chaga mycelia concentration and residual glucose amount for each condition. When 30 ml / L of oleic acid and 15-20 g / L of glucose were added, chaga mycelium productivity was found to be 19 g / L, which was confirmed to be the highest, and there was almost no amount of residual glucose. It is important to note that there were many culture results that showed much higher cell productivity compared to the amount of glucose added to the production medium. From these results, it can be seen that the chaga mushroom mycelium used in this example is not only consumed by the sugar component, which is relatively easy to use, but also at the same time grows very smoothly using the oil component added together. there was. Therefore, it was determined that the presence of oil with a certain ratio of glucose would have the best effect on the growth of chaga mycelium.

< Example  3> As a carbon source  Oiled Chaga Mushroom  Mycelium Liquid Culture Badge composition  For decision Fractional factorial design  Experiment

As a result of Example 2, it was recognized that there is a small degree of correlation between oleic acid and glucose, and thus, a fractional factorial design (FFD) experiment, which is a statistical method for determining correlation between two factors, was performed to investigate more accurately. The medium optimization experiment method using statistical method is an experiment method that can confirm the effect of various media composition on the result through dozens of experiments through statistical treatment and optimize the optimal concentration of the medium composition. It was possible to remove media components that did not affect or adversely affect the results and to maximize chaga mycelium concentration, ultimately increasing the efficiency of the process. Statistical design and analysis of results were done using Design Expert 6.0, a statistical analysis program. Focusing on the concentration of the high productivity in the concentration experiment of Example 2, as shown in Table 3, as defined in the range of high concentration and low concentration, the culture was carried out under 37 culture conditions, 21 ~ 26.7g Mycelium productivity up to / L was obtained (Table 4).

LOW HIGH Glucose 14 22 Oleic acid 25 30 Casein 2 6 Soytone peptone 6 10 KH ₂ P0 ₄ 3 6

Experimental Table of Fractional Factorial Design for Determination of Liquid Culture Cultures of Chaga Mycelium Added Oil as Carbon Source and Chaga Mycelium Productivity for Each Condition Run Glucose
(glucose) (g / L) Oleic acid
(oleic acid) (g / L) casein
(casein) (g / L) Soytone peptone
(g / L) KH ₂ PO ₄ (g / L) D.C.W (g / L) One 22 30 2 6 6 17.03 2 22 25 6 10 6 17.68 3 18 27.5 4 8 4.5 14.36 4 22 25 2 6 6 14.53 5 22 25 6 10 3 15.88 6 22 30 2 10 6 16.83 7 14 25 2 10 3 17.93 8 14 25 2 6 6 15.35 9 14 25 2 6 3 15.38 10 22 25 2 10 6 17.82 11 22 30 2 10 3 18.93 12 22 30 6 10 6 19.34 13 18 27.5 4 8 4.5 18.19 14 22 30 6 6 3 23.13 15 14 30 6 6 3 21.23 16 14 25 2 10 6 14.92 17 14 30 2 10 3 18 18 14 25 6 10 6 16.46 19 14 30 2 6 3 18.17 20 18 27.5 4 8 4.5 15.19 21 22 30 6 10 3 26.66 22 18 27.5 4 8 4.5 17.51 23 14 30 2 10 6 16.42 24 14 25 6 6 6 14.5 25 14 30 6 6 6 16.42 26 22 25 2 6 3 14.4 27 22 30 2 6 3 17 28 22 25 2 10 3 18.35 29 14 30 6 10 3 16.45 30 18 27.5 4 8 4.5 16.69 31 14 30 2 6 6 13.97 32 14 25 6 10 3 15 33 22 25 6 6 6 21.3 34 14 25 6 6 3 19.04 35 22 30 6 6 6 17.27 36 14 30 6 10 6 17.3 37 22 25  6 6  3 17.33

Looking at the results, chaga mycelium concentrations vary widely depending on the conditions, but it is noteworthy that chaga mycelium productivity was very high at 26.7g / L in 21 combinations. Table 5 shows the main effect of the statistical analysis of the experimental results of this example. All of the four components except Soyton peptone showed high contributions, especially casein enzymatic hydrolysate with 11.73. In addition, since only KH ₂ PO ₄ showed a negative value and all other factors showed a positive effect, when KH ₂ PO ₄ was decreased, the concentration of KH ₂ PO ₄ was decreased. It was expected to have a good effect.

Analysis of the Contributions of the Compositions of the Fractional Factorial Design (FFD) Experiments Term Standized effect Sum squares % Contribution Intercept Glucose (A) 1.68 22.68 9.49 Oleic acid (B) 1.77 24.99 10.45 Casein (C) 1.87 28.05 11.73 Soytone peptone (D) 0.49 1.96 0.82 KH ₂ PO ₄ (E) -1.61 20.70 8.66 Glucose * Oleic Acid 0.60 2.83 1.18 Glucose * Casein 1.09 9.50 3.98 Glucose * Soytone Peptone 0.69 3.84 1.60 Glucose * KH ₂ PO ₄ 0.37 1.12 0.47 Oleic acid * casein 0.81 5.23 2.19 Oleic acid * soytone peptone 0.22 0.38 0.16 Oleic acid * KH ₂ PO ₄ -1.52 18.36 7.68 Casein * soytone peptone -1.18 11.07 4.63 Casein * KH ₂ PO ₄ -0.20 0.31 0.13 Soytone peptone * KH ₂ PO ₄ 0.31 0.74 0.31

ANOVA analysis was performed to confirm the significance of the experimental design and results of this example, which is shown in Table 6. The prob> F value was 0.0303, indicating that the overall significance was significant.

ANOVA Analysis of Fractional Factorial Design Experiments for the Determination of Medium Composition for Liquid Culture of Chaga Mycelium Added Oil as Carbon Source Response: D.C..W ANOVA for Selected Factorial Model
Analysis of variance table [Partial sum of squares] Source Sum of
Squares DF Mean
Square F Value Prob> F 　
　 Model 151.78 15.00 10.12 2.469133 0.0303 significant A 22.68 1.00 22.68 5.534334 0.029 B 24.99 1.00 24.99 6.098583 0.0227 C 28.05 1.00 28.05 6.84469 0.0165 D 1.96 1.00 1.96 0.478322 0.4971 E 20.70 1.00 20.70 5.052278 0.036 AB 2.83 1.00 2.83 0.691105 0.4156 AC 9.50 1.00 9.50 2.319333 0.1434 AD 3.84 1.00 3.84 0.936159 0.3448 AE 1.12 1.00 1.12 0.272692 0.6073 BC 5.2326125 One 5.2326125 1.276847 0.2719 BD 0.3828125 One 0.3828125 0.093413 0.763 BE 18.3618 One 18.3618 4.480592 0.047 CD 11.0685125 One 11.0685125 2.700906 0.1159 CE 0.31205 One 0.31205 0.076146 0.7854 DE 0.7442 One 0.7442 0.181597 0.6746 Curvature 5.353229797 One 5.353229797 1.306279 0.2666 not significant Residual 81.9614925 20 4.098074625 Lack of fit 71.8162125 16 4.488513281 1.769695 0.3085 not significant Pure Error 10.14528 4 2.53632 Cor total 239.0950973 36

In addition, as a result of statistical analysis (ANOVA) for this example, the interaction between the factors (factor) can be seen by the following equation.

Chaga mycelial mass = +17.50 + 0.84A + 0.88 * B + 0.94C + 0.25D-0.80E + 0.30AB + 0.55AC + 0.35AD + 0.19AE + 0.40BC + 0.11BD-0.76BE-0.59CD-0.099CE + 0,15DE

Experimental results of this example, the addition of oleic acid for the liquid culture of chaga mycelium culture composition for the new liquid culture medium is as follows;

22 g of glucose, 30 g of oleic acid, 6 g of casein, 10 g of soytone peptone, 3 g of KH 2 PO 4, 1 L of distilled water.

Chaga mushroom mycelium obtained in this Example was compared with the growth of the liquid medium for rapid growth and Chaga mushroom mycelium productivity and intracellular protein polysaccharide concentration in the conventional culture medium chaga mushroom culture, the results are shown in Table 7. The culture method was carried out in the same manner as in Example 1, was cultured for 10 days to compare the dry cell weight.

Comparison of Chaga Mushroom Mycelial Productivity and Intracellular Protein Polysaccharide Concentrations in Chaga Mushroom Mycelia Base and Newly Developed Media Mycelium Productivity Intracellular Protein Polysaccharide Concentration Basic liquid medium 17.2 g / L 1.204 g / L New Development Badge 26.7 g / L 1.869 g / L

As shown in the table, the new development medium was found to increase the productivity of mycelium per day and intracellular protein polysaccharide (IPS) by 1.5 times compared to the basic liquid medium.

< Example  4> Medium composition preparation

The medium was sterilized for 15 minutes at 121 degrees sufficient to kill microorganisms through a moist heat sterilizer, and each medium was separated to prevent brown sedimentation reaction which occurs during simultaneous sterilization of carbon and nitrogen sources depending on the composition of the medium. The oil component oleic acid was also sterilized separately and the media components were mixed and used before each experiment. The amount of oil added was adjusted at the time of manufacture to suit each concentration and volume so that all cultures were carried out at the same volume.

1 shows a comparison of chaga mycelium productivity in a liquid medium to which various oils are added.

Figure 2 shows a comparison of chaga mycelium productivity in the liquid culture medium of chaga mycelium supplemented with lard oil, corn oil, wheat germ oil.

Figure 3 shows a comparison of chaga mycelium productivity in a liquid medium using oil as the sole carbon source.

Figure 4 shows the comparison of chaga mycelium productivity in liquid medium with various glucose (glucose) and oil added.

Claims (6)

Liquid for the production of chaga mycelium containing 14-22 g glucose, 25-30 g oleic acid, 2-6 g casein, 6-10 g soyton peptone, 3-6 g KH ₂ PO ₄ per 1 liter of distilled water Medium composition. According to claim 1, wherein the composition is a liquid medium composition for chaga mycelium production, characterized in that sterilized. delete The composition of claim 1, wherein the composition comprises 22 g of glucose, 30 g of oleic acid, 6 g of casein, 10 g of soyton peptone, and 3 g of KH ₂ PO ₄ with respect to 1 L of distilled water. Subcultured chaga mycelium on agar medium; Static culture of the passaged mycelia in the step of culture medium; And chaga mushroom mycelium culture method comprising the step of culturing the passage culture of the mycelium in a liquid medium, wherein the liquid culture step is cultured with the composition of any one of claims 1, 2 or 4. Chaga mushroom mycelium culture method characterized in that. delete

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