KR20110017283A - NOVEL INONOTUS OBLIQUUS M5 HAVING THE ABILITY OF β-D-GLUCAN PRODUCTION - Google Patents

Abstract

PURPOSE: A novel inonotus Inonotus obliquus M5 having an ability of producing proteoglycan is provided to enhance productivity of proteoglycan and to produce a large amount of Inonotus Obliquus-derived proteoglycan. CONSTITUTION: A novel Inonotus obliquus M5(KACC93073P) has an ability of producing proteoglycan. The novel strain is prepared during regeneration of protoplast by treating UV to Inonotus obliquus mycelium. The proteoglycan is intracellular exopolysaccharide(IPS) or extracelluar exopolysaccharide(EPS). A method for preparing proteoglycan comprises a step of culturing Inonotus obliquus M5(KACC93073P) and producing proteoglycan from the culture liquid.

Description

Novel Inonotus obliquus M5 having the ability of β-D-glucan production}

The present invention relates to a new strain Inonotus obliquus M5 having a protein polysaccharide production ability, and more specifically, to form a protoplast after UV treatment of mycelia ( Inonotus obliquus ) mycelium and regeneration process of the protoplasts The present invention relates to a method for producing a protein polysaccharide, comprising the step of culturing the new strain Inonotus obliquus M5 and the new strain, and producing the protein polysaccharide from the culture.

Mushrooms, which are higher fungi, have long been used as foods because of their rich 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 and diabetic effects by immuno-stimulating action are water-soluble protein polysaccharides with β-D-Glucan structure. (Hereinafter referred to as “protein polysaccharide”). Among them , the protein polysaccharide derived from Chaga ( Inonotus obliquus ), which is one of the representative basidiomycetes, has been shown to have an excellent effect on the hypoglycemic effect by promoting insulin secretion and promoting insulin secretion, attracting attention as a new bioactive substance with diabetes improvement. have.

Chaga mushroom is known to have excellent antidiabetic effects, so it is difficult to breed because of the difficult growth conditions. 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. Recently, the anti-diabetic and immuno-enhancing effects of various mushrooms have been scientifically proven, and the component that shows such efficacy has been found to be a protein polysaccharide with β-D-glucan structure. Beta-glucan structured polysaccharide found to be anti-cancer and anti-diabetic component by immunopotentiation of Chaga mushroom ( Inonotus obliquus ) is a trace component having a content of less than 5% even in crude protein polysaccharide purified through a specially designed purification process.

Chaga-derived protein polysaccharides are composed of intracellular exopolysaccharides (IPS) present as cell wall components and extracelluar exopolysaccharides (EPS) secreted extracellularly. Since intracellular protein polysaccharide (IPS) is almost constant per unit cell, in order to secure economic feasibility of chaga-derived protein polysaccharide production, the cell growth rate is high and the extracellular protein polysaccharide (EPS) secretion is high. Development is essential. However, in the case of basidiomycetes that do not produce spores, it is impossible to obtain single colonies unless spores are obtained by inducing fruiting body formation, and thus, attempts to develop strains are very difficult.

An object of the present invention is to develop a new strain of mycelia by improving mycelia, and to produce a mass of protein polysaccharides that can be used in medicines or foods by using the mycelium. It is.

In order to solve the above problems, the present invention provides a new strain Inonotus obliquus M5 (KACC93073P) having a protein polysaccharide production capacity. In another aspect, the present invention provides a method for producing a protein polysaccharide, comprising the step of culturing the new strain Inonotus obliquus M5 and producing a protein polysaccharide from the culture solution.

The new strain Inonotus obliquus M5 of the present invention has higher cell growth rate and higher protein polysaccharide productivity than the chaga strain, which is a parent strain. Large quantities of chaga-derived protein polysaccharides are available.

The present invention provides a new strain Inonotus obliquus M5 (KACC93073P) having a protein polysaccharide production capacity.

In the present invention, the new strain may be prepared by UV treatment of Chaga mushroom ( Inonotus obliquus ) mycelium to form a protoplast and regeneration of the protoplast.

In the present invention, the protein polysaccharide may be an intracellular exopolysaccharide (IPS) or an extracellular secreted extracellular polysaccharide (extracelluar exopolysaccharide: EPS) that is a cell wall component.

The present invention also provides a method for producing a protein polysaccharide, comprising the step of culturing the new strain Inonotus obliquus M5 (KACC93073P) and producing a protein polysaccharide from the culture solution.

In the production method of the present invention, the culture is obtained by germinating spores of chaga ( Inonotus obliquus ) to obtain a mycelium, the mycelium may be a liquid culture in a liquid medium.

In the preparation method of the present invention, the protein polysaccharide may be an intracellular exopolysaccharide (IPS) or an extracellular secreted extracellular polysaccharide (extracelluar exopolysaccharide: EPS).

Hereinafter, a new strain Inonotus obliquus M5 having a protein polysaccharide production ability and a method for producing a protein polysaccharide using the same will be described in detail.

The present invention relates to a new strain Inonotus obliquus M5 which is an improved strain of chaga mushroom with excellent protein polysaccharide productivity. The present invention also relates to a method of culturing the new strain and producing a protein polysaccharide from the culture.

Inotos obliquus M5 of the present invention (hereinafter referred to as the new strain) is a mycelium obtained from the fruiting body of chaga (chaga mushroom collected in the wild), which is a production strain of protein polysaccharides having high immune enhancement, anticancer and antidiabetic effects. Mycelium of the following strains are called mycobacterial strains to induce UV mutations and isolate the single genes and select the strains found to produce the most protein polysaccharide among the protoplast-formed and regenerated strains. It was deposited with the Institute of Engineering on May 26, 2009. The accession number is KACC93073P.

The protein or polysaccharide produced by the new strain or the strain prepared according to the present invention may be provided as a medical or food use.

The present inventors have developed a new strain M5 which biosynthesizes high concentrations of protein polysaccharides using mutagenesis and protoplast formation and regeneration. The present invention has been made to overcome the limitations of the prior art, the object of the present invention is to significantly increase the productivity of protein polysaccharides compared to wild-type chaga mushroom that is a fungus strain Inonotus obliquus ( Inonotus obliquus ) To develop new strains.

Hereinafter, examples and experimental examples of the present invention will be described. However, these Examples and the like are only for illustrating the present invention in more detail, the scope of the present invention is not limited by these Examples and the like.

Example 1 Liquid Culture of Chaga Mycelia

1. The process of germinating spores of chaga 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. Cha mycelia were subcultured every 11-15 days at 24 ℃ temperature in passage agar medium (malt extract 20 g, yeast extract 2 g, agar 20 g, distilled water 1L). Political culture was performed using a political culture medium (22.5 g of glucose, 2 g of yeast extract, 2 g of peptone, 2 g of diphosphate, 0.6 g of magnesium sulfate, 1 L of distilled water). A 20% glycerol stock was made and stored at -80 ° C. When needed, the stock stored was taken out and inoculated in a solid passage medium to be incubated.

2. The process of liquid culture of chaga mycelium in liquid medium

The mycelia grown in the stationary medium of 1. were aseptically collected and then inoculated to 5% (v / v) in the liquid medium. Cell wall polysaccharide (IPS) production medium and liquid medium for extracellular polysaccharide (EPS) production include glucose 30 g, yeast extract 8 g, peptone 4 g, diphosphate 2 g, magnesium sulfate 2 g, distilled water 1 L, pH 5.0 Media adjusted to were used. Liquid culture of the mycelium was cultured for 7-10 days at 28 ℃, 150rpm in a shaker, or cultured for 3 to 5 days and then passaged to another flask.

3. Determination of Extracellular Protein Polysaccharide and Intracellular Protein Polysaccharide in Chaga Culture

1) Cell concentration measurement and recovery of extracellular protein polysaccharide (EPS)

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., and then at 4,000 rpm. It was centrifuged for 10 minutes. After that, the supernatant was removed, and the remaining pellet was dried in a 90 ° C. oven for 12 hours and weighed.

2) Quantitative Analysis of Protein Polysaccharide (IPS) in Cells Using HPLC

For the quantitative analysis of intracellular protein polysaccharide (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 for 12 hours at 90 ℃ 25g pulverized dry mycelium suspended in 50ml of distilled water, extracted with steam bath for 2 hours, and again 40ml of distilled water was added for 6 hours. Thereafter, after performing two filtration processes using 0.45 μm filter paper, 15 ml of the filtrate was transferred to a new tube and precipitated in 2 times of 99.9% ethanol for 6 hours. After centrifugation for 10 minutes at 2,800rpm, the supernatant was removed and lyophilized, and analyzed using a gel chromatography column and 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 to the above HPLC The method was analyzed.

3) 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 were 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> Preparation of the new strain Inonotus obliquus M5

1. Induction of Mutation of Chaga Mushrooms by UV

In order to obtain a mutant strain with improved protein polysaccharide content and productivity from the probiotic strain, the mycelia of the probiotic strain were collected and used for the mutation treatment. UV treatment was applied to the agar plate in 0.1 ml by irradiating two UV lamps of 254 nm in the dark for 0 to 380 seconds (about 97% kill conditions). The colonies that survived were counted to obtain a percentage of the untreated UV control, and the UV treatment time of about 90% mortality was used as the variation condition. Based on the results, UV mutation experiments were performed at 320 seconds, the minimum inhibitory concentration (MIC) of UV chaga fungi. Mycelium subjected to the UV mutation was incubated for about 7 days in order to prevent photoreactivation phenomenon that the UV mutation is inactivated by visible light.

2. Formation of Protoplasts for Chaga Mushroom Single Colony Acquisition

Basidiomycetes, such as chaga, are characterized by having a multinuclear structure in which several nuclei exist together in the same mycelium. Therefore, when mutations are induced in a gene through the mutation treatment, nuclei having different genotypes exist together in the same mycelium. Therefore, in order to obtain genetically identical single colonies, it is essential to form protoplasts to separate single nuclei and then regenerate them into cells.

The mutant mycelium grown after the mutagen treatment step 1. was isolated and cultured in agar medium for passage for 7 days, and then collected with 20% glycerol to collect a minimal liquid medium (30g glucose, 5g ammonium sulfate, 1g potassium monophosphate). , 0.2 g of potassium chloride, 1 L of distilled water) was inoculated and the cells were shaken at 28 ° C. and 230 rpm for 36 hours to obtain cells required for protoplast formation. The culture solution and mycelium in the flask were passed through a filter having a pore size of 20 to 25 μm (Watman No. 4) to filter out residues such as media components to recover only the mycelium. The mycelium obtained through filtration was washed twice with 0.6 M magnesium sulfate solution and 1 g of the mycelium was used for protoplast formation.

To form protoplasts, 1 g of bacteria and 0.05 ml of Viscozyme L were added to 5 ml of a protoplast-forming solution (0.6 M magnesium chloride, 10 mM phosphate buffer pH 5.8) for 28 hours at 28 ° C. and 80 rpm. After the reaction, the formed protoplasts were carefully transferred to a 50ml conical tube and centrifuged at 3,000rpm for 20 minutes to remove the supernatant, leaving only about 5ml and 10ml of the protoplast formation buffer. Thereafter, the supernatant was removed by centrifugation at 3,000 rpm for 20 minutes. About 2 ml of remaining amount was used for protoplast regeneration.

3. Regeneration of Protoplasts

The protoplasts prepared by the method of 2. were suspended in protoplast formation buffer to prepare a solution of 1 × 10 ⁵ protoplasts / ml concentration. And 0.1 ml of these protoplast solutions were added to 5 ml of a minimum of agar medium and layered on a solid minimum agar medium. After culturing for about 7 days, the incubated protoplasts were measured and inoculated in a minimal medium without osmotic stabilizer to determine the protoplast formation rate, and colony formation was compared and the photograph is shown in FIG. See FIG. 1). The formed protoplasts were separated from each other using a self-made 0.5mm agar cylinder to prevent them from touching the surrounding mycelium, and then transferred to an already prepared subculture medium to be used for comparison of various physiological characteristics including protein productivity.

4. Selection of Chaga Mushroom-derived Protein Polysaccharide High Productivity

In this example, a large number of mutant strains having excellent production capacity of chaga-derived total protein polysaccharides (IPS, EPS) among the protoplasts of the above-mentioned UV mutation were attempted. According to the previous research results of the present inventors, chaga mycelium was observed that the content of intracellular polysaccharides (IPS) contained almost constant amount per cell weight. In addition, the production of extracellular polysaccharides (EPS) should be prioritized in the production of high concentration mycelium. Therefore, we tried to select mutants growing rapidly through protoplast formation of mutated mycelium.

On the other hand, in the present embodiment, since the pigment produced by the microorganism in the production of useful substances through the mass culture of microorganisms is likely to cause problems in the product manufacturing process, it was intended to obtain a pigment low-productive strain. Protein polysaccharide productivity and growth rate of the mutant strains obtained through a series of procedures were examined and the method shown in Example 1 was used. Eighteen mutants were selected, and these were grown at 28 ° C., 250 rpm for 7 days in liquid phase, and compared to growth rates (see FIG. 2). Growth rate was measured using the dry cell mass confirmation method shown in Example 1 after the 7-day incubation.

In spite of the strains derived from one species of parent strain, it was found that the mutation strains produced a very wide variety of characteristics by UV mutation. While the strains showed a dry cell weight of about 13 g / L, strains showing small growth rates of 7 g / L to 22 g / L were selected. It is noteworthy that strain 5, which showed a growth rate of 22 g / L after 7 days of incubation, is a low-dye strain, which is a great advantage in an industrial scale production process. Solid culture photographs of parent strains and mutant strains 5 are shown in FIG. 3 (see FIG. 3). The mutant strain 5 was found to have a pale off-white color due to the low pigment production, whereas the cultivars had a high pigment yield.

Comparing the total protein polysaccharide productivity of the 5 strains with about 1.7 times the growth rate compared to the probiotics, the results are shown in Table 1 below, and the HPLC results of the protein polysaccharides are shown in FIG. 4 (see FIG. 4). .

Table 1 Comparison of productivity of total protein polysaccharide (concentration: g / L) of strain 5

Day 0 Day 1 Day 2 3rd day Day 4 Day 5 Day 6 7th day I. obliquus
wild type IPS 0.035 0.07 0.14 0.21 0.42 0.63 0.84 0.91 EPS 0 0 0 0.4 0.8 1.0 1.6 1.8 I. obliquus
# 5 IPS 0.028 0.14 0.42 0.63 0.98 1.19 1.4 1.54 EPS 0 0.2 0.3 1.2 1.7 2.1 2.5 2.6

(In Table 1, the protein polysaccharide production medium contains glucose 30g, yeast extract 8g, peptone 4g, diphosphate 2g, magnesium sulfate 2g, distilled water 1L, pH 5.0, temperature 28 ℃, stirring speed 7 at 250rpm Analysis results after daily incubation.)

As shown in Table 1, the productivity of the intracellular protein polysaccharide (IPS) and the extracellular protein polysaccharide (EPS) of the new strain was 1.7 times and 1.4 times higher than that of the parent strain, respectively, and thus the total protein polysaccharide productivity was approximately 1.5 times. It was confirmed that the increase.

Thus, the mutant strain No. 5, which is a growth rate per unit time, total protein polysaccharide productivity, and a low tonality producing new strain, was named M5 and was deposited with the Institute of Agricultural Biotechnology and assigned the accession number KACC93073P.

The development method of the new strain of the present invention is not only a very essential method for developing industrial strains that produce high protein polysaccharides, but also a general technology applicable to other microorganisms, so that it may be directly applied to the production process of other materials. In addition, when the high production strain obtained through the present invention is used, the productivity of chaga mycelium and protein polysaccharide is maximized, which is expected to be a great help in product development and market entry.

Figure 1 shows a protoplast formation picture of the mutant strains by UV.

Figure 2 shows the dry cell mass obtained as a result of 7-day liquid culture of single colonies (new strains) separated by protoplast formation and regeneration of the mutated parent strains.

Figure 3 shows a photograph comparing the pigment production rate of the solid culture of the parent strain and the new strain Inonotus obliquus M5.

Figure 4 shows the protein polysaccharide HPLC analysis table produced in the pro strain and the new strain Inonotus obliquus M5.

Claims (6)

New strain Inonotus obliquus M5 (KACC93073P) with protein polysaccharide production capacity. According to claim 1, wherein the new strain is a myococcal strain ( Inonotus obliquus ) mycelium ( Inonotus obliquus ) M5 to form a protoplast and produced by the regeneration process of the protoplast after UV treatment mycelium ( Inonotus obliquus ) M5 . According to claim 1, wherein the protein polysaccharide is an intracellular exopolysaccharide (IPS) that is a cell wall component or an extracellular secreted extracellular protein (extracelluar exopolysaccharide: EPS) characterized in that the new strain Inotos obliques ( Inonotus obliquus ) M5. A method for producing a protein polysaccharide, comprising culturing the new strain Inonotus obliquus M5 (KACC93073P) of claim 1 and producing a protein polysaccharide from the culture medium. The method of claim 4, wherein the culturing is a method for producing a protein polysaccharide, characterized in that by germinating spores of chaga ( Inonotus obliquus ) to obtain a mycelium, the mycelium is cultured in a liquid medium. The method of claim 4, wherein the protein polysaccharide is an intracellular exopolysaccharide (IPS) or an extracellular secreted extracellular polysaccharide (extracelluar exopolysaccharide) (EPS).

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