KR100936176B1 - A crude exopolysaccharides produced from Leatiporus sulphureus var. miniatus having hypoglycemic activity and preparation method of the same - Google Patents

Abstract

The present invention is an extracellular polysaccharide produced through the optimal liquid culture conditions of the red myrtle mushroom (Leatiporus sulphureus var. Miniatus) mycelium and the proliferation of insulin secreting cells by these extracellular polysaccharides, the effect of increasing insulin secretion, and streptozotocin (streptozotocin) By confirming the effect of repairing the insulin secretion cells damaged by) can ultimately improve the glycemic control of diabetic patients, in particular, it provides an extracellular polysaccharide that can improve the insulin secretion disorder caused by pancreatic abnormalities.

Description

Red duck leg mushroom (Lｅａｔｉｐｏｒｕｓ ｓｕｌｐｈｕｒｅｕｓ ｖａｒ. Ｉｉｉｉｔｕｓ) Extracellular polysaccharide having antidiabetic activity derived from mycelium culture medium and a method for producing the same. miniatus having hypoglycemic activity and preparation method of the same}

The present invention relates to an extracellular polysaccharide having antidiabetic activity obtained from a mycelium of red duck mushroom (Leatiporus sulphureus var. Miniatus) and a method for producing the same.

The red duck mushroom (Leatiporus sulphureus var.miniatus), which belongs to the fungus of the basidiomycetes, is native to Mt. Baekdu, Wolchulsan, Jirisan, and Valwangsan, and is distributed throughout the tropics of Japan and Asia. This is a 1 year-old heartwood brownish fungi that regenerate on the conifers, trees, and stumps from spring to summer. Red duck mushrooms have long been reported to be effective in controlling function, improving health, and anti-disease, but the exact pharmacological effects are not known at all.

Red-legged mushroom fruiting bodies include N-methylated tyramine derivatives (Lee et al. 1975; List 1958; Rapior et al.), Polysaccharides, many lanostane triterpenoids, and reti. Laetiporic acids and various other mixtures are included (Weber et al. 2004; Davoli et al. 2005).

Over the last decade there has been an emphasis on the biological activity of polysaccharide and sugar-peptide mixtures derived from red duckling fruiting bodies. However, research on secreted metabolites through mycelial culture of the mushrooms is insufficient.

       In addition to insulin, various types of oral diabetes treatments have been used to treat diabetes, but various side effects have emerged. Therefore, more and more patients are trying to use anti-diabetic substances derived from natural products without side effects.

Today, there are approximately 33 types of herbal medicines that have been used mainly in folk medicine prescriptions to clinically treat diabetes and its complications. Antidiabetic agents in herbal medicines include polysaccharides, terpenoids, flavonoids, sterols and alkanoids. In many countries, efforts have been made to find natural antidiabetics derived from various medicinal plants.

In addition, various medicinal and edible mushrooms are representative natural herbal sources with antidiabetic activity. In spite of the large lack of scientific evidence, many researchers have found that anti-antibodies from fruiting bodies or mycelium of various edible / medical fungi, including Tremella aurantia , Lentinus edodes , Phellinus baumii , etc. Efforts have been made to study the effects of diabetes.

 For example, the medical applications of secreted polysaccharides through mushroom mycelium culture have been studied, and in particular, studies on the anti-diabetic effect of mushroom fruiting bodies and mycelium culture have been accumulated, and also the antidiabetic effect of secreted polysaccharides through the diabetic animal model. Research is underway (Wasser 2002; Hu et al. 2006; Kiho et al. 1994, 1995, 1996; Hong et al. 2007; Hwang et al. 2005; Cho et al. 2007).

Studies on new antidiabetic compounds include Agaricus, Viscum albuam , Sambuscus nigra , Averrhoa bilimbi , Studies have been carried out on the effect of insulin secretion using medicinal plants such as Oimum canum , Nigella sativa, and Urtica dioica . In order to understand the mechanism of antidiabetic activity by mushroom-derived products, studies on the stimulation and insulin secretion effects of pancreatic beta-cells were carried out.

The present invention is to provide an extracellular polysaccharide having the effect of insulin secretion cell proliferation, insulin secretion ability, and streptozotocin (Streptozotocin) damaged in the production of mycelium culture medium of Red Duck Mushroom (Leatiporus sulphureus var. Miniatus) .

The present invention also provides a method for producing extracellular polysaccharide derived from mycelium culture medium of red duck mushroom.

In an embodiment of the present invention provides an extracellular polysaccharide derived from the mycelium culture medium of red duck mushroom (Leatiporus sulphureus var. Miniatus) having antidiabetic activity.

The extracellular polysaccharide according to the embodiment of the present invention has insulin cell proliferation, insulin secretion effect, and repair of insulin secretion cell damaged by streptozotocin.

The extracellular polysaccharide according to the embodiment of the present invention may be one containing a carbohydrate and a protein. At this time, the carbohydrate is glucose as a main component, and the protein may include aspartic acid, threonine, and serine.

In another embodiment of the present invention comprising the steps of preparing a mycelium liquid culture solution of Red Duck Mushroom (Leatiporus sulphureus var. Miniatus); Centrifuging the liquid culture solution; Taking the supernatant and adding alcohol; Centrifuging; And it provides a method for producing an extracellular polysaccharide derived from red duck mushroom mycelium culture medium comprising the step of lyophilizing the precipitate.

In the production method according to the embodiment of the present invention, the preparing step of the liquid culture solution may be performed under a culture medium containing deep sea water.

According to the present invention, the extracellular polysaccharide produced through the optimum liquid culture conditions of the red mycelium (Leatiporus sulphureus var. Miniatus) mycelium is the insulin damaged by the growth of insulin secreting cells, the effect of increasing insulin secretion, and streptozotocin (streptozotocin) By having the effect of restoring secretory cells can ultimately improve the glycemic control of diabetic patients, in particular can improve the insulin secretion disorder caused by pancreatic abnormalities.

The present invention will be described in more detail as follows.

The present invention obtains the extracellular polysaccharide from the mycelium culture medium of red duck mushroom (Leatiporus sulphureus var. Miniatus), using the insulin secretion cells RINm5F cells derived from the rat insulinoma (insulinoma), insulin cell proliferation, insulin secretion effect and streptoto Zotocin (hereinafter referred to as STZ) treatment confirmed the effect of repairing damaged insulin cells.

Specifically, the extracellular polysaccharide derived from the mycelium culture medium of red duck mushroom (Leatiporus sulphureus var. Miniatus) includes carbohydrates and proteins, carbohydrates are mainly glucose, and aspartic acid, threonine and threonine It includes serine. More specifically, carbohydrates include glucose, mannose, and galactose as main ingredients, and as proteins, aspartic acid, threonine, serine, glutamic acid ( glutamic acid, cysteine, valine, methionine, isoleusine, lysine, leucine, tyrosine, phenylalanine, lysine and arginine And the like.

The specific method of obtaining such extracellular polysaccharide is first prepared a mycelium liquid culture solution of red duck leg mushroom. Culturing by adding deep sea water to 50% to 80% of the volume of the culture in the liquid culture preparation step; Centrifuging the liquid culture solution; Taking the supernatant and adding alcohol; Centrifuging; And it provides a method for producing an extracellular polysaccharide derived from red duck mushroom mycelium culture medium comprising the step of lyophilizing the precipitate.

In the production method according to the embodiment of the present invention, the preparing step of the liquid culture solution may be performed under a culture medium containing deep sea water.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.

<Materials and Methods>

Microbe and Medium

Mushroom mycelium culture was maintained at 4 ℃ on malt extract agar (hereinafter referred to as MEA) slant (2% malt extract, 1.8% agar) and subcultured at two weeks intervals. Liquid culture was carried out in a 250 ml Erlenmeyer flask with 50 ml MCM (Mushroom Complete Medium: 20 g / L glucose, 2.0 g / L meat peptone, 2.0 g / L yeast extract, 0.46 g / L KH ₂ PO ₄ , 1.0 / LK ₂ HPO ₄ and 0.5 g / L MgSO ₄ .7H ₂ O) medium was incubated at 150 rpm for 7 days at 25 rpm.

Deep sea water (DSW)

DSW was provided by the WaterVis company. This deep seawater was pulled up more than 1,100 meters deep in the East Sea of Korea, and then separated into desalted and concentrated seawater by reverse osmosis secondary filter. Various kinds of minerals separated from concentrated seawater were added to demineralized seawater to make various types of DSW, and the results of the component analysis of these DSW using Inductively Coupled Plasma Atomic Emission Spectroscopy and ion chromatography were analyzed. It is shown in Table 1 below. Sterile DSW was used to add 50-70% of the culture medium volume.

Element ^a Desalinated water (mg ml ¹ ) Hardness of deep sea water (mg ml ¹ ) Hardness 500 Hardness 700 Ca 0.9 40.3 56.4 Mg 2.8 96.7 135.4 K 1.0 27.4 38.4 Na 4.3 20.6 28.9 Cl 17.9 268.4 375.7 SO ₄ 3.6 183.3 256.7

^{In a} component analysis, Ca, Mg, K, and Na were analyzed by inductive plasma element emission spectroscopy, and Cl and SO _4. were analyzed by ion chromatography.

Flask liquid culture of mycelium

Flask liquid culture of mycelium was cut by cutting the edge of mycelium cultured in MEA Petri dish to 5 mm using a self-made apparatus, and then implanted 4% by volume in a 250 ml Erlenmeyer flask containing 50 ml of MCM medium. Incubated at 11 ° C. for 11 days,

Mycelial growth in a bioreactor

The mycelium cultured in the flask was inoculated at 4% in a 5L stirred-tank incubator containing 3L MCM medium and cultured under the following culture conditions; Temperature-25 ° C, Aeration rate-2.0 vvm, Agitation speed-150 rpm, Initial pH-2.0, Working volume-3L

Dry weight and extracellular polysaccharide concentration of mycelium

The cultured mycelium was centrifuged at 12,000 × g for 20 minutes to recover the mycelium, and then washed repeatedly with distilled water and dried at 90 ° C. for 18 to 24 hours to determine the weight of the mycelium.

On the other hand, after centrifugation of the culture medium, the supernatant of the culture medium is taken, and mixed with ethanol in a volume of 4 times the volume. The mixture is left well at 4 ° C for 18 hours to 24 hours, and then centrifuged at 10,000 × g for 20 minutes to discard the supernatant. Freeze-dried polysaccharides were weighed secreted polysaccharides. The total equivalent of secreted polysaccharide was measured according to phenol-sulfuric acid method (Dubois et al. 1956) using glucose as a standard.

RINm5F Cell Culture

RINm5F insulinoma cell lines were purchased from the American Type Culture Collection (ATCC).

Cell culture was incubated in RPMI1640 medium containing 10% fetal bovine serum, 100unit / ml penicillin, 100ug / ml streptomycin, and was cultured at 37 ℃ with 5% carbon dioxide. The culture medium was replaced with fresh medium every three days, and after two weeks of culture, the cells were trypsin-treated and diluted to suit the experiment.

RINm5F Cell Proliferation Assay

RINm5F cell proliferation assay was performed according to MTT (3- (4,5-dimethylthiazol-2yl) -2,5-diphenyl tetrazolium bromide) assay method. In short, the proliferated cells were detached using trypsin, diluted with RPMI1640 culture medium containing 10% FBS to a concentration of 1 × 10 ⁴ / ml cells, and divided into 96-well cultures. The cells were cultured in an incubator fed with% carbon dioxide. After 24 hours, the cells were washed with PBS, replaced with fresh medium containing crude polysaccharides, and cultured for 24 hours, followed by incubation for 4 hours by adding 20ul of MTT solution (5mg MTT / ml in PBS). After washing, 100ul DMSO was added to measure the cell proliferation effect by measuring the number of living cells by absorbance at 540 nm using a microtiter plate reader (Microtiter plate reader, Thermo Electron Co., Vantaa, Finland).

Analysis of insulin secretion cell repair effect of extracellular polysaccharide

Insulin secreting cells were cultured in a 37 ° C. thermo-hygrostat supplied with 5% CO ₂ using RPMI 1640 medium containing 10% fetal bovine serum (FBS). After 24 hours of incubation with 2 × 10 ⁴ / well in a 96 well plate, 2 mg / mL of the extracellular polysaccharide is treated for 24 hours before or after 2mM streptozotocin for 30 minutes. MTT assay was then followed according to the cell proliferation assay.

All experimental results were analyzed statistically by one-way analysis of ANOVA test using SPSS program. All data are expressed as mean ± standard deviation ( p <0.05). When measured according to the technique of protective LSD, the mean value was considered to be significant difference at p <0.05.

Insulin Secretion Analysis

After treatment with the extracellular polysaccharide obtained according to the cell proliferation assay method, washed with Tris buffer (pH 7.0) and then lysis buffer (7M urea, 2M thiourea, 4% CHAPS, 1mM PMSF, 20 mM DTT, 2% IPG buffer) ) 5 times for 30 seconds, the cells were disrupted with a cell crusher, and insulin secretion was measured using a Rat Insulin Kit (Shibayagi, Gunma, Japan).

Immunofluorescence Cell Staining

The cells were treated with 4% p -formaldehyde for 15 minutes, fixed, washed three times with PBS, diluted with insulin guinea pig polyclonal primary antibody at a ratio of 1: 200, and treated with a secondary antibody attached with fluorescent material. Insulin-stained RINm5F cells were magnified 400 times with an inverted fluorescence microscope (Olympus Corp., Japan).

Example 1: Effect on Initial pH and Temperature in Flask Culture

In order to confirm the effect of initial pH and temperature on mycelial growth and extracellular polysaccharide production, the mycelia were inoculated and incubated in a 250 ml flask containing 50 ml of medium composed of various pHs, and the results are shown in FIG. 1.

As can be seen from the results of Figure 1 (A), mycelium growth and secretion polysaccharide production peaked at pH 2.0, which is unusually very acidic. In addition, (B) of Fig. 1 shows the results of confirming the effect of the culture temperature on the mycelia growth and extracellular polysaccharide production bar, it can be seen that the maximum value when the culture temperature is all 25 ℃.

Example 2: Influence of Carbon and Nitrogen Sources in Flask Culture

To examine the effect of carbon sources on mycelial growth and extracellular polysaccharide production, the flasks were cultured in a medium to which 20 g / L of various carbon sources were added as shown in Table 2 below, and the results are shown in Table 2 below. From the results in Table 2, maltose is the most effective carbon source. On the other hand, as a result of further experiments, the optimum maltose concentration in the mycelial growth and secretion polysaccharide production was 30 g / L.

In order to determine the effect of the carbon source on the mycelial growth and extracellular polysaccharide production, it was cultured in a medium containing various nitrogen sources as shown in Table 3 and the results are shown in Table 3 below. From the results in Table 3, when soy peptone or polypepton was added, the efficiency was good. Similarly, further experiments resulted in maximum mycelial growth and production of secreted polysaccharides at 2 g / L soy peptone.

Effects of Carbon Sources on Growth of Red Duck Mushroom Mycelia and Extracellular Polysaccharide Production in Flask Culture Carbon source (2%, wv ^-1 ) Mycelium dry weight (gl ^-1 ) Extracellular Polysaccharide Dry Weight (gl ^-1 ) Fructose 1.75 ± 0.24 ^b 0.13 ± 0.07 Glucose 4.05 ± 0.10 1.13 ± 0.22 Lactose 1.63 ± 0.21 0.56 ± 0.07 Maltose 4.26 ± 0.20 2.54 ± 0.39 Sorbitol 1.72 ± 0.34 0.19 ± 0.02 Sucrose 3.68 ± 0.59 0.68 ± 0.15

^a incubation was performed in shake flasks at 25 ° C. for 7 days.

^b The value is expressed as the average standard deviation value of three tests.

Effect of Nitrogen Sources on Growth of Red Duck Mushroom Mycelium and Extracellular Polysaccharide Production in Flask Culture Nitrogen source (0.4%, wv ^-1 ) Mycelium dry weight (gl ^-1 ) Extracellular Polysaccharide Dry Weight (gl ^-1 ) Control 2.27 ± 0.01 ^b 0.20 ± 0.00 Ammonium sulfate 0.25 ± 0.01 Nd ^c Corn steep liquor 2.39 ± 0.01 0.43 ± 0.14 Malt extract 0.45 ± 0.04 0.07 ± 0.03 Meat peptone 2.06 ± 0.00 0.13 ± 0.01 Polypepton 4.05 ± 0.44 0.51 ± 0.00 Soy peptone 4.09 ± 0.24 0.58 ± 0.11 Yeast extract 1.63 ± 0.18 0.22 ± 0.09

^a incubation was performed in shake flasks at 25 ° C. for 7 days.

^b The value is expressed as the mean standard deviation of three tests.

Example 3: Effect of Minerals in Flask Culture

In order to determine the effect of minerals on mycelial growth and extracellular polysaccharide production, as shown in Table 4, 2mM of various minerals were added to the flask culture, and the results are shown in Table 4 below. From the results of Table 4, it can be seen that the mycelial growth and the extracellular polysaccharide production effect is the best when 2mM MnSO ₄ · 5H ₂ 0 is added.

At this time, the mineral is a mineral separated from concentrated seawater as described in the above-mentioned 'ocean deep water' related item, and the deep sea water obtained by adding it to desalted sea water is added to the culture medium.

On the other hand, the effect on the mycelial growth and extracellular polysaccharide production according to the concentration of the deep sea water was evaluated and the results are shown in the following Figure 2, from the results of FIG. Production of secreted polysaccharides is increased, and the cell growth and extracellular polysaccharide growth are increased by 4 and 6.7 times, respectively, when the concentration of deep sea water is 70%.

Effect of Minerals on Growth of Red Duck Mushroom Mycelia and Production of Secretory Polysaccharides in Flask Culture Mineral Type (2 mM) Mycelium dry weight (gl ^-1 ) Extracellular Polysaccharide Dry Weight (gl ^-1 ) Control 4.34 ± 0.25 ^b 0.72 ± 0.19 FeSO ₄ ㅇ 7H ₂ O 4.62 ± 0.76 0.97 ± 0.57 K ₂ HPO ₄ 3.77 ± 0.18 0.83 ± 0.05 KH ₂ PO ₄ 3.01 ± 0.04 0.62 ± 0.36 MgSO ₄ ㅇ 7H ₂ O 3.07 ± 0.10 0.65 ± 0.14 MnSO ₄ ㅇ 5H ₂ O 5.11 ± 0.35 1.20 ± 0.29

^a incubation was performed in shake flasks at 25 ° C. for 7 days.

^b The value is expressed as the mean standard deviation of three tests.

Example 4: Mycelial Culture Using Incubator

Meanwhile, the mycelial growth and the production of cell polysaccharide in the incubator using the incubator over the course of the incubation time were evaluated and the results are shown in FIG. 3.

Figure 3 (A) is a result of measuring the mycelia and extracellular polysaccharide production after incubation under a basal condition in a stirred tank incubator, (B) is a mycelia and extracellular polysaccharide production under optimal culture conditions in a stirred tank incubator (C) is the result of measuring the mycelium and extracellular polysaccharide production under the optimal culture conditions in an airlift incubator.

As can be seen from the results of Figure 3, the dry weight of the mycelia and extracellular polysaccharides produced under optimal culture conditions as in (B) is 8.1g / L and 3.8g / L, which is 5.4g / L in the basal state This is a nearly double increase from 2.2g / L. On the other hand, as a result of culturing in a stirred-tank incubator (FIG. 3B) and an airlift incubator (C) in FIG. And the extracellular polysaccharide production is higher.

Example 5: Properties of Extracellular Polysaccharides

As a result of analyzing the components of the extracellular polysaccharide, it is shown in Table 5 and Figure 4, the carbohydrate was 95.15% by weight, protein was 4.85% by weight. The major component of the carbohydrate was glucose, and 16.58% by weight of 100% by weight protein consisted of 13 amino acids including aspartic acid, 15.44% by weight threonine and 14.47% by serine. In Table 5, the composition ratios of the components constituting the carbohydrate and the protein are expressed in weight percent with respect to 100% by weight of the carbohydrate and the protein.

Component analysis specifically, the extracellular polysaccharide component was dissolved in tertiary distilled water and injected into Sepharose CL-column (2.4 cm x 100 cm, Sigma Chemical Co., Ltd., USA). The column was eluted at the flow rate of 1.4 ml / min using the same buffer. The total carbohydrate content of the extracellular polysaccharide was measured by phenol-sulfuric acid method using glucose as a standard. Total protein was measured according to the Lowry method using albumin of urea serum as a standard. The protein portion of the extracellular polysaccharide was measured at 280 nm and the carbohydrate portion was measured at 480 nm.

Among them, the analysis results of the total carbohydrate are shown in FIG. 4.

ingredient Composition ratio (%, w / w) Carbohydrate (95.15% by weight of total composition) Glucose 91.44 Mannose 4.92 Galactose 1.81 Other 1.83     Protein (4.85% by weight of total composition) Aspartic acid 16.58   Throneine 15.44 Serine 14.47 Glutamic acid 12.82 Cysteine 1.59 Valine 11.26 Methionine 0.82 Isoleucine 7.22 Leucine 7.42 Tyrosine 4.06 Phenylalanine 4.73 Lysine 2.45 Arginine 1.14

Example 6: RINm5F Cell Growth and Insulin Secretion Effects by Polysaccharides

Results of confirming the effect of growth and insulin secretion of RINm5F cells according to the treatment concentration of the extracellular polysaccharide produced according to the present invention is shown in Figure 5 and 6, respectively, RINm5F cells 24 hours after 2mg / ml extracellular polysaccharide treatment Proliferation increased by 152% and insulin secretion increased. For these results, insulin-producing cells stained by immunofluorescence staining are shown in FIG. 7.

Example 7 Recovery of Cell Growth of Insulin Secreting Cells Damaged by STZ Treatment by Extracellular Polysaccharides

As a result of confirming the cell recovery effect of the insulin secretion cells damaged by STZ treatment by the extracellular polysaccharide produced according to the present invention is shown in Figure 8, 2mg / ml EPS treatment group before or after the 2mM STZ treatment cell recovery In particular, the cell group treated with EPS for 24 hours prior to STZ treatment showed about 190% cell repair efficiency compared to STZ alone.

1 is a graph confirming the effect of the initial pH and temperature on the red mycelium mycelium growth and extracellular polysaccharide production in the flask culture, (A) is the result according to the pH, (B) is the result according to the temperature.

FIG. 2 is a graph showing the effect of red duck fungus mycelium (Cell) growth and extracellular polysaccharide (EPS) production according to the depth of seawater treatment in flask culture. FIG.

Figure 3 is a graph showing the mycelia growth and extracellular polysaccharide production (dry weight) according to the culture conditions and incubator, (A) is a result of culturing under basal conditions in a stirred incubator, (B) is the optimum conditions in a stirred incubator The result of the culture under the graph, (C) is the result of the culture under the optimum conditions in the air lift incubator graph, ● the mycelium dry weight, ■ the extracellular polysaccharide dry weight.

Figure 4 is the result of Sepharose CL-4B chromatographic elution of the crude extracellular polysaccharide, which is calculated by the absorbance measurement of the carbohydrate (●) at 480nm. A separate box, shown at the top right of FIG. 4, shows the results of gel permeation chromatography for molecular weight measurements. This is the value analyzed by absorbance measurement at 480 nm for carbohydrates, where Kav = (V _e -V ₀ ) / (V _t V ₀ ) (V ₀ : void volume; V _t : total volume; V _e : elution volume). Four groups of extracellular polysaccharides are indicated with ●.

Figure 5 is a graph showing the effect of red duck mushroom mycelium-derived extracellular polysaccharides on the proliferation of insulin secreting cells RINm5F cells.

Figure 6 is a graph showing the effect of red duck mushroom mycelium derived extracellular polysaccharide on insulin secretion effect by insulin secreting cells RINm5F cells, where □ is intracellular insulin secretion, ■ is extracellular insulin secretion.

7 is a photograph of insulin-producing RINm5F cells (x400) stained by immunofluorescence staining, A; Control, B; 0.5 mg / ml polysaccharide treatment group, C; 1.0 mg / ml polysaccharide treatment group, D; 2 mg / ml polysaccharide treatment group.

8 is a photograph of the cell repair effect by the extracellular polysaccharide for cells damaged by streptozotocin (STZ, streptozotocin) treatment, Conrol; Control, EPS; 2 mg / ml extracellular polysaccharide treatment group, STZ; 2 mM streptozotocin treated group, STZ (2 mM) + EPS (2 mg / ml); EPS (2 mg / ml) treated group, EPS (2 mg / ml) + STZ (2 mM) after STZ (2 mM) treatment; STZ (2 mM) treated group after EPS (2 mg / ml) pretreatment.

Claims (8)

Extracellular polysaccharide derived from mycelium culture medium of red duck mushroom (Leatiporus sulphureus var. Miniatus) having antidiabetic activity. The extracellular polysaccharide derived from the mycelium culture medium of red duck mushroom (Leatiporus sulphureus var. Miniatus) according to claim 1, which has an effect of proliferating insulin secreting cells. The extracellular polysaccharide derived from the mycelium culture medium of red duck mushroom (Leatiporus sulphureus var. Miniatus) according to claim 1, which has an insulin secreting effect. [Claim 2] The extracellular polysaccharide derived from the mycelium culture medium of Red Duck Mushroom (Leatiporus sulphureus var. Miniatus) according to claim 1, which has an effect of repairing insulin cells damaged by Streptozotocin treatment. The extracellular polysaccharide of claim 1, wherein the extracellular polysaccharide comprises a carbohydrate and a protein. The extracellular polysaccharide derived from the mycelium culture medium of Lepiporus sulphureus var. Miniatus. [6] The mycelium of claim 5, wherein the carbohydrate is glucose as a main component and the protein includes aspartic acid, threonine and serine. Extracellular polysaccharide derived from culture. Preparing a mycelium liquid culture solution of red duck mushroom (Leatiporus sulphureus var. Miniatus); Centrifuging the liquid culture solution; Taking the supernatant and adding alcohol; Centrifuging; And Method for producing an extracellular polysaccharide derived from mycelium culture medium of red duck mushroom (Leatiporus sulphureus var. Miniatus) comprising the step of lyophilizing the precipitate. [Claim 8] The method for preparing extracellular polysaccharide derived from the mycelium culture medium of Red Duck Mushroom (Leatiporus sulphureus var. Miniatus) according to claim 7, wherein the preparing of the liquid culture solution is carried out under a culture medium containing deep sea water.

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