KR20050036495A - MANUFACTURING METHOD OF SOLUBLE β-GLUCAN - Google Patents

Abstract

Manufacturing method for improving fermentation conditions and yield of functional macromolecules for the cultivation of superior strains by physiological activity, providing water-soluble properties, and cultivating superior strains using Korean basidiomycetes To provide. The solubilization method uses sulfur to substitute an element of the insoluble β-glucan skeleton to prepare a water-soluble β-glucan. Finally, it is more easily soluble than natural biosynthetic insoluble β-glucan, which is easier to formulate, and is physicochemically stable It develops water-soluble β-glucan with little side effects and excellent physiological activity, and is used as a raw material for functional foods, immune enhancing pharmaceutical materials, cosmetic moisturizers and the like.

Description

Manufacturing method of water-soluble beta-glucan bioactive substance {MANUFACTURING METHOD OF SOLUBLE β-GLUCAN}

The present invention relates to a solubilization method of the physiologically active polysaccharide β-glucan, and particularly to the mass production of the non-aqueous β-glucan, which is a representative polysaccharide of basidiomycetes, and its physicochemical excellence according to its solubility method and solubility. The present invention relates to a water-soluble polysaccharide having excellent pharmacological activity.

In general, β-glucan of basidiomycetes is a functional food material, Biological Response Modifiers (BRM) that have an immune enhancing effect, and is a natural polysaccharide that is currently used as an immune chemotherapy agent in clinical practice.

Studies on the polysaccharides of Korean basidiomycetes began by revealing that the hydrothermal extracts of fruiting bodies such as cloud mushrooms, shiitake mushrooms, and oyster mushrooms have a strong deterrent to Sarcoma 180.

Carbohydrates in mushrooms are mainly made up of low molecular sugars such as trehalose, mannitol, and arabinitrol, and fluorinated phytofibers such as β-glucan, heteroglucan, and chitin. Among them, β-glucan is produced in all mushrooms such as Ganoderma lucidum, and is a bioactive polysaccharide that is synthesized as extracellular polysaccharide in mycelium.

Anti-cancer polysaccharides have been studied separately from natural resources such as higher plants, fungi, yeast, and bacteria.Since Linder et al. It has been done extensively.

The anticancer components of these basidiomycetes have also been studied for their immunity.Maeda et al. (1971) found that Lentinan acts as an immune-stimulating agent for cellular immunity, and Dennert et al. (1973) It has been demonstrated that lentinane acts as a T-cell adjuvant.

In particular, β-glucan is not only effective for host diseases caused by bacteria, viruses, fungi, parasites, etc., but also serves to block or change cancer progression. As a result, anti-cancer drugs using β-glucan, which is a reaction modulator, have been developed.

Response modulators play a regulatory role in antiviral action, cell proliferation inhibitory activity, macrophage activity, natural killer cell activation, antibody production and cancer cell gene expression. The advantage of such reaction modulators is that they exhibit anticancer activity due to a different mechanism from chemotherapy agents, and therefore, anticancer activity can be expected to increase when used in combination with chemotherapy agents. Therefore, the development of immune chemotherapy using reaction modulators has attracted attention as an excellent method for treating cancer.

However, an important obstacle to the clinical use of β-D-glucan is that it is not water-soluble. Williams' study shows that unrefined (1 → 3) -β-D-glucans isolated from yeast are insoluble microparticulate polysaccharides (~ 1-2 μm) (Williams et al., 1985), and if they are injected intravenously Lower limb injury or endotoxin shock was reported. On the other hand, it was reported that no toxicity was observed when applied to topical or focal lesions.

Therefore, the most problematic problem in the conventional research and development as described above is the insoluble problem of β-glucan. That is, all of the bioactive structures of these polysaccharides are β-glucan, but they have in common water-insoluble properties, and studies on improving physical properties of basidiomycete polysaccharides have not yet been made at home and abroad.

Therefore, in order for β-glucan to exhibit excellent pharmacological activity, a substance having excellent solubility while maintaining the original skeleton should be developed, and in order to develop various bioactive products in industry, mass production must be possible first, and β-glucan The problem of solubility of the substance has to be solved. The success of this research will be applied to industrial, medical, and pharmaceutical fields, and will be the basis for developing various products with domestic technology.

The present invention provides a water-soluble β-glucan as a functional biologically active polysaccharide as a biologically new material that is fermented and cultured in Ganoderma lucidum to produce mycelium polysaccharides and to accept β-glucan which is an insoluble polysaccharide having excellent physiological activity. It is for that purpose.

In another aspect, the present invention provides a substance having superior physiological activity and water-soluble properties than the conventional β-glucan polysaccharides using Korean basidiomycete.

In order to achieve the above object, the water-soluble β-glucan of the present invention is prepared by accepting a water-soluble β-glucan by substituting with an element of an insoluble β-glucan skeleton using a sulfur (S) element.

The solubilization method is characterized by dissolving urea in methylsulfoxide, adding insoluble glucan thereto, and slowly adding an acidified methylsulfoxide solution to the β-glucan-Me ₂ SO-urea solution and heating it.

In the present invention, basidiomycetes are used to obtain water-soluble β-glucan. Basidiomycetes are very advantageous for obtaining large amounts of bioactive polysaccharides among many organisms. That is, it is possible to mass culture using a fermentation tank, the cell internal and external polysaccharides can easily obtain a high purity polysaccharide through a series of process.

Polysaccharides present in mushrooms are mainly β-glucans. These are classified into α and β-glucan forms according to the binding method. Fungal glucans are mostly water-insoluble linear polymers in which glucose binds to β-1,3. The solubility difference of polysaccharides is caused by polymer size, structural difference and chain packing, and it is reported that alkali insoluble glucan is covalently bonded with chitin.

The composition of the polysaccharides that make up the cells of the mushroom was well studied in the Schizophyllum commune , and the cell wall of the mycelium is divided into the alkali soluble fraction (AS-glucan) and the alkali insoluble fraction (AR-glucan). AS-glucans are (1 → 3) -α-D-glucans branched into (1 → 6) -α-D-glucans and include xylose and mannose. AR-glucans are (1 → 3) -β-, (1 → 6) -β-D-glucans with chitin and protein. Alkaline insoluble glucans of cloud mushrooms are polysaccharides of β-1, 3 and β-1, 6 bonds covalently bound to chitin.

When the insoluble polysaccharide is substituted with a hydrophilic element, it can be converted to water solubility. In the present invention, a water-soluble functional polysaccharide is prepared through a sulfate method using sulfate (S).

      Water-soluble β-glucan obtained in the present invention can be a new material that can develop a variety of products. In particular, as an ideal material that can create high added value in various industries, its production method has the advantage that it can be produced in a continuous efficient fermentation method.

The method for solubilizing the physiologically active polysaccharide β-glucan of the present invention is to ferment culture from basidiomycetes to mass produce a water-insoluble β-glucan, which is a functional polysaccharide, and to increase the functionality by accepting the water-insoluble β-glucan obtained therefrom. , To summarize the steps,

First step: Mass production of functional polysaccharides from basidiomycetes.

Second Step: Improve the solubility that must be solved for industrial use of the water-insoluble polysaccharides of most polysaccharides of basidiomycetes. In the method, an insoluble β-glucan was converted into a hydrophilic polysaccharide using an element such as sulfur (S).

Step 3: Analyze the physical properties and structure of the partially substituted β-glucan.

Step 4: An acute toxicity test is performed to confirm the expression of unwanted antigenic or toxic of water-soluble β-glucan.

Step 5: Physiological activity tests were performed in vivo and in vitro to see if the immune activity of the accommodated β-glucan was superior or equivalent to the original polysaccharides. As a physiological activity test, macrophages that could be targets of anticancer test, anticomplement test and β-glucan were used. In detail, the activity of nitric oxide production of macrophages and the amount of tumor necrosis factor (TNF-α) were confirmed. The final goal was to develop β-glucan, an immune enhancer that is soluble in water.

Hereinafter, the water solubilizing method of the water-soluble β-glucan which is the bioactive polysaccharide of the present invention will be described in more detail.

1. Strain culture and material extraction

1-1. Strains and Cultures

The main basidiomycete used in the present invention is ganoderma lucidum ( G. lucidum ), and the mycelium storage medium uses potato dextrose agar (potato dextrose agar) medium. Culture medium was IYSM medium containing 2% water-soluble starch, 0.2% yeast extract, 0.5% K ₂ HPO ₄ , 0.001% KH ₂ PO ₄ , 0.1% MgSO ₄ · 7H ₂ O. IYSM medium was placed in a fermenter (COBIOTECH, 3L), adjusted to pH 4.5, and autoclaved at 2 atmospheres for 20 minutes. Herein, 10% (v / v) of G. lucidum mycelium precultured in potato dextrose medium was inoculated. At this time, the working capacity was 3L, the stirring speed was 300 rpm, the aeration amount was 1 vvm and incubated for 6 days at 27 ℃. 0.5N NaOH was used as a pH adjusting solution during the culture, and a silicone resin was used as the antifoaming agent.

1-2. Mycelial growth measurement

      The growth of mycelium was shown by filtration of the culture solution incubated for 7 days with Toyo No. 2 filter paper, followed by drying at 105 ° C. for 8 hours, and measuring the dry weight of the mycelium.

1-3. β-glucan extraction

Ethanol purification by alkaline extraction of β-glucan was performed by the following method.

NaOH was added (final 2N) to 3 L mycelial culture incubated for 6 days, and stirred at room temperature to extract alkali-soluble polysaccharides. To the extract was neutralized by adding glacial acetic acid, followed by centrifugation (3000 x g) to separate the alkali soluble and insoluble fractions again, to which ethanol was added to precipitate polysaccharides. All mycelial fractional polysaccharides precipitated in ethanol were collected by centrifugation (6000 xg), added with a small amount of distilled water, and then placed in a dialysis membrane bag (MW cut off: 10,000). Dialysis for days. The dialyzed fractions are centrifuged at 6000 x g to separate the aqueous and non-aqueous fractions and then lyophilized to obtain insoluble β-glucan.

1-4. Production condition of β-glucan

It is necessary to maintain a constant content of sugar and protein according to the production of β-glucan, but there is a difference in the content and solubility of sugar, protein according to the production lot. Therefore, experiments were conducted to compare the various extraction conditions, analyze the causes, and improve the problems. The results were as follows.

end. Mycelial Culture

The culture medium was inoculated with 10% of the seed (seed) in a sterilized 3 L fermenter and 1 L Erlenmeyer flask and incubated at 27.5 ° C. for 5 days before being used for the experiment.

I. Effect of Extraction Temperature

Based on the experimental basis that the sugar content was different depending on the extraction temperature, alkali extraction was performed for 24 hours at room temperature (25), 40, 60 and 80 ° C., respectively, and the change of physical properties of the sample was investigated.

As a result, as shown in Table 1, there was no change in yield according to the extraction temperature, but as the extraction temperature increases, the sugar content increased and the protein content decreased. Solubility in water showed the best solubility as extracted from room temperature. These results indicate that the temperature is increased during the cleavage of protein and carbohydrate by alkali, which promotes hydrolysis of the protein, lowers the molecular weight, and is filtered off during ultrafiltration, thereby reducing the amount of protein in the sample and the total amount of carbohydrate It is believed to increase. In addition, the increase in carbohydrate in the sample is believed to decrease the solubility due to the increase of β-glucan, which is mostly insoluble.

Table 1 Composition Changes of β-Glucan with Extraction Temperature

Extraction temperature (℃) Extraction time (hr) Β-glucan yield based on the culture (%) Total Carbohydrate (%) Solubility in water (%) 25 24 0.151 ± 0.011 62.2 ± 2.6 70.4 ± 2.3 40 24 0.152 ± 0.014 68.0 ± 5.2 52.2 ± 2.3 60 24 0.156 ± 0.020 79.4 ± 3.7 40.2 ± 1.1 80 24 0.172 ± 0.022 78.8 ± 3.5 32.0 ± 1.5

All. Effect of Extraction Time at Constant Temperature

The temperature was kept constant at 60 ° C. and the extraction time was 6, 12, 24 and 48 hours, and the alkali was extracted, respectively.

As a result, as shown in Table 2, there was no change in yield according to the extraction time, but as the extraction time became longer, the sugar content was found to increase. In addition, the solubility in water decreased as the extraction time was longer at 60 ° C. than at room temperature and 24 hours, which is a standard process.

As a result, the extension of extraction time in the process of the cleavage of protein and carbohydrate by alkali can sustain the decomposition of the hydrolyzed protein in the alkaline solution state, and thus the chance of lower molecular weight than the carbohydrate. It is believed that the amount of protein in the sample is decreased and the total amount of carbohydrate is relatively increased due to the deviation during ultrafiltration. Eventually, the increase in carbohydrate of β-glucan is believed to decrease in solubility due to the increase of β-glucan which is mostly insoluble in water.

As a result, the extraction temperature and extraction time under alkaline solution in the manufacturing process of β-glucan may be a technical indicator that can significantly affect the physical properties of β-glucan. When β-glucan was changed in extraction process (room temperature, 40, 60 and 80 ° C.), there was an increase in sugar content, a decrease in protein content, a decrease in solubility, and no change in yield. In addition, when the extraction time was extended at a constant temperature (extraction temperature: 60 ℃) during the manufacturing process of β-glucan, the sugar content gradually increased and the protein content gradually decreased, but the yield was largely changed as the extraction time increased. There was no.

Table 2 Effect of β-glucan on Extraction Time at Constant Temperature

Extraction temperature (℃) Extraction time (hr) Β-glucan yield based on the culture (%) Total Carbohydrate (%) Solubility in water (%) 25 24 0.150 ± 0.01 62.4 ± 2.6 70.4 ± 2.3 60 6 0.148 ± 0.01 65.1 ± 1.3 50.4 ± 2.8 60 12 0.157 ± 0.007 67.0 ± 2.2 44.5 ± 1.6 60 24 0.156 ± 0.02 79.4 ± 3.7 40.2 ± 1.1 60 48 0.153 ± 0.012 87.1 ± 2.1 32.4 ± 1.3

2. Solubilization of Insoluble β-Glucan

2-1. Water Solubilization of Insoluble Glucan (Sulfation Process of β-Glucan)

The method for preparing a water-soluble β-glucan sulfate by solubilizing insoluble glucan of ganoderma is as follows.

Urea was first dissolved in methylsulfoxide (Me ₂ SO), and then insoluble glucan was added thereto. After mixing until complete melting, a solution of concentrated sulfuric acid (acidified Me ₂ SO) was added to methylsulfoxide again. The acidified solution was slowly added to the β-glucan-Me ₂ SO-urea solution and then heated at 100 ° C. to induce the reaction. After the reaction was diluted with cooled water and the unreacted material was filtered through a filter. Ultrapure deionized water was added to the filtered pale orange solution, concentrated using an ultrafiltration apparatus, and the concentrated solution was lyophilized to obtain water-soluble glucan.

In order to prepare a water-soluble β-glucan sulfate by solubilizing insoluble β-glucan of Ganoderma lucidum, various samples as shown in Table 3 were tested.

Sulfate the direct extract obtained through direct hydrothermal extraction process yielded the highest yield of 0.578%, and the sample obtained by ultrafiltration of the alkaline extract (Alkali Ex-UF) yielded 0.084% and alkali for higher purity. After extraction, the sample precipitated with ethanol and dialyzed (Alkali-EtOH-DI) yielded a yield of 0.094%. In particular, the water-soluble β-glucan obtained through the solubilization process of 100% insoluble β-glucan was obtained in a yield of 85%.

Table 3 Yield of water-soluble β-glucan according to the extraction method of insoluble β-glucan

Way Sample number β-glucan yield (%) Water Soluble β-Glucan Yield (%) Direct extract A 1.445 0.578 B 1.466 Alkali Ex-UF A 0.211 0.084 B 0.21 Alkali-EtOH-DI A 0.22 0.094 B 0.25 Insoluble β-glucan A 100 85

3. Physicochemical Properties of Water-soluble β-Glucan

3-1. Solubility

100 mg of β-glucan partially substituted with sulfate was added to 10 ml (1%) of distilled water, and mixed vigorously to dissolve. The solution was centrifuged at 6000 rpm for 10 minutes to remove the supernatant, and then dried to measure the weight and the solubility relative to the initial weight. As a result, more than 95% of the solution was dissolved. The color was dark brown, but after being solvated it turned yellow brown.

Table 4 Solubility of Water-Soluble β-Glucan

characteristic Insoluble β-glucan Water-soluble β-glucan color dark brown Yellow brown yield(%) - 85 Solubility (%) 5% 〈 〉 95

3-2. Sulfur (S) content of water-soluble glucan

After dissolving 10 mg of water-soluble β-glucan in distilled water, 90% of formic acid was added so that the concentration of the sample was 1 mg / ml at the final 60%. This was put in a sealed test tube to dissolve well, and then hydrolyzed at 100 ° C. for 8 hours. The hydrolyzed sample is dried under vacuum conditions and then dissolved in 0.5 ml of distilled water (500 µg / ml). 3.8 ml of reagent A (solution of 4 g of TCA in 100 ml of distilled water) and 1 ml of reagent B (in 0.2 ml of hydrolyzate and standard solution (1.813 g of K ₂ SO ₄ dissolved in 100 ml of distilled water)) Gelatin / BaCl ₂ solution; 2.0 g of gelatin was dissolved while incubating 400 ml of distilled water at 60-70 ° C. The gelatinous solution was cooled and left at 4 ° C. for at least 6 hours, then 20 g of BaCl ₂ was added for 15 minutes, and then absorbance was measured at 500 nm. The sulfate content in the sample was calculated from the straight line obtained from the absorbance according to the concentration of K ₂ SO ₄ solution.

Table 5 shows the results of quantifying the amount of substituted sulfur contained in the water-soluble β-glucan obtained through the solubilization of insoluble polysaccharide through sulfate. In the case of insoluble β-glucan without the sulfate process, sulfur content was 0.2%, but the water-soluble β-glucan was 14.9% polysaccharide with sulfate process. The ratio of SO ₃ ⁻ / monosaccharide (%) to insoluble polysaccharide was 80: 48,600, one per 607 monosaccharide residues, but one soluble β-glucan was substituted for every nine residues. In addition, the ratio of SO ₄ / monosaccharide (%) was 1 molecule for every 506 residues in insoluble β-glucan, and 1 SO ₄ was found for every 7.5 monosaccharide residues in water-soluble β-glucan.

Table 5 Composition of sulfur compounds of insoluble β-glucan and sulfated water-soluble β-glucan

characteristic Insoluble β-glucan Water-soluble β-glucan Sulfate (%) 0.2 14.9 SO ₃ ^- Molecular ratio per unit (%) (80: 180 = 2.35) 0.2 / 54 = 1: 270 (80: 48,600) 607 1 SO ₃ ^- per residue 15/60 = 1: 4 (80: 720) 1 SO ₃ per nine ^residues SO₄ Molecular weight per unit (%) (96: 180 = 1.88) 0.2 / 54 = 1: 270 (96: 48,600) 506 1 SO ₄ per residue 15/60 = 1: 4 (96: 720) 7.5 1 SO ₄ per residue

3-3. Sugar content and protein content analysis

The sugar content of β-glucan was measured using the phenol-sulfuric acid method and the protein content using a BCA protein assay kit (piece company), and the results are shown in Table 6.

Table 6 Total Carbohydrates of Insoluble β-Glucan and Sulfated Water Soluble β-Glucan

characteristic Insoluble β-glucan Water-soluble β-glucan Total carbohydrates 62.4 ± 2.9 74.2 ± 0.05 Total protein 12.5 ± 0.03 9.5 ± 0.26

3-4. Per Composition Analysis

10 mg of the sample was dissolved in 0.1 N HCl to fill the nitrogen, and then hydrolyzed at 100 ° C. for 5 hours. After adding three times the amount of ethanol and left overnight at 4 ° C., the supernatant obtained by centrifugation was concentrated under reduced pressure at 50 ° C. to completely remove moisture. 100 μl of saturated pyridine was added to the hydrolyzed monosaccharide to remove water and completely dissolved. In the case of control monosaccharide, α-glucose, β-glucose, arabinose, fructose, galactose, mannose, ribose, and xylose are dissolved in 10 ml of 5 ml vials and 1 ml pyridine is added. 20 μl of hexamethyl disilazone and 10 μl of trimethyl chlorosilane were added to each vial to complete dissolution. After reacting at 80 ° C. for 30 minutes, the sugar analysis in the reaction solution was analyzed using gas chromatograph (GLC). The analysis conditions are as follows. column; 3% OV-17 (80-100 mesh Shimalite) 3 mm (D) x 3 m (L) boron silicate glass column, column temperature; 150-180 ° C. gradient, Detector temperature; 240 ° C., Injection temperature; 240 ° C., carrier gas; He, Flow rate; N ₂ -40 mL / min, air-60 min, Detector; FID, Attenuation; 10 ² x 2 ¹ afs

As a result, as shown in Table 7, 80.8% of glucose was composed of glucose, 34.9% of α-linked glucose and 35.9% of β-glucose. Sigma's super reagents β-glucan, mannan, laminarin and gimosan A were used as control polysaccharides. As a result, manna was composed of 89% mannose, and high purity β-glucan was 42.7% α-glucose. , 47% β-glucose.

Table 7 Monosaccharide Content of Water-soluble β-Glucan

Polysaccharides Constituent monosaccharides Ribose Xylose Fructose Mannos Galactose α-glucose β-glucose Water-soluble β-glucan - - 6.4 17.9 4.8 34.9 35.9 Insoluble β-glucan - 1.3 4.26 4.24 9.4 44.8 23.8 Met - - 9.7 88.7 - 1.6 - Laminarin - - 2.2 - 8.1 42.7 47.0 Jimosan 0.6 - - 37.0 1.4 25.7 35.6

3-5. Hexosamine content

Each carbon source was used to determine the hexosamine content in the extracted and separated polysaccharides. 1 ml of 3N HCl was added to 5 mg of each sample, and the mixture was filled with nitrogen, hydrolyzed at 100 ° C. for 15 hours, and concentrated by filtration and concentrated under reduced pressure. 1 ml of distilled water was added to the dried sample to completely dissolve it, 0.5 ml of the resultant was placed in a test tube, and reagent A-1 (1.5 ml of acetylacetone and 50 ml of 1.25N Na ₂ CO ₃ mixed solution) was used for the sample. Reagent A-2 (1.5 ml of acetylacetone and 50 ml of 0.5N Na ₂ CO ₃ mixture) for standard glucosamine was added to each test tube containing polysaccharide and standard glucosamine, and reacted at 96 ° C. for 1 hour. After cooling the reaction solution at room temperature, 10 ml of 96% ethanol and 1 ml of reagent B (solution of 1.6 g of p-dimethylaminobenzaldehyde dissolved in 30 ml of concentrated hydrochloric acid and 30 ml of 95% ethanol) were added for 1 hour. After standing, the absorbance at 535 nm was measured. The total hexamine content in the sample was obtained from a standard curve prepared using standard glucosamine.

3-6. Infrared spectrum

After pulverizing well by adding 400-fold potassium bromide to a small amount of β-glucan, a disc was made and measured using FT-IR (Bruker, IFS-48). The IR spectrum was measured by scanning eight times for each sample. The results are shown in FIGS. 3 and 4.

Infrared spectra showed that the carbonyl group (C = O) in the carbohydrates had absorption wavelengths around 1725-49 cm ^-1 and the acetamido group was around 1648 cm ^-1 . . In the monosaccharide in the ring form, carbon 1 and carbon 5 form an ether bond (COC). This structure shows absorption wavelengths at 1150 to 1080 cm ^-1 (ring form) and 1150 to 1060 cm ^-1 (alkyl ether), and most of the samples showed peaks in this range of wavelengths.

3-7. NMR Analysis

The binding morphology and structural properties of the water-soluble β-glucan were analyzed by ¹³ C-NMR spectra using a 200 MHz spectrophotometer (Bruker, AM-200, Germany). The solvent was measured by dissolving 30 mg of water-soluble glucan and standard in 1 ml of Me ₂ SO-d ₆ or D ₂ O. Laminarin from Laminaria digitata (Sigma) was used as a control of the β-1,3 linked triple-helical glucopyranose.

The results are shown in Table 8. It was found that β-glucan was β-glucan due to the same peak at each carbon, although the purity was not higher than that of laminarin, which showed more than 95% purity of Sigma.

Table 8 Water soluble β-glucan and laminarin ¹³ C-NMR chemical shift

Sample C-1 C-2 C-3 C-4 C-5 C-6 Laminarin 106.2 76.5 88.5 71.5 78.8 63.3 Water-soluble β-glucan 105.2 76.0 86.0 73.5 78.0 63.5

3-8. Molecular Weight Measurement

10 mg of sample was dissolved in 1 ml of 0.3N NaOH and eluted on a sepharose CL-4B column. At this time, the column size was 16mm (D) x 530mm (L), the flow rate was set to 6 mL / hr, fraction 2 mL. The eluate obtained in each column chromatography was reacted using the phenol-sulfonic acid method and the absorbance was measured at 490 nm. Standard dextran used dextran with molecular weights of Sigma, 2,000, 124, 70 and 9.3 kD, respectively.

Each sample was eluted to obtain a chromatogram as shown in FIG. The main peak showed the largest peak in fractions 45-46, indicating that their average molecular weight was 9.3 kD polysaccharide.

4. Mass Production of β-Glucan

4-1. badge

Slope medium for storing the strain was using potato dextrose agar (PDA), all other media used IYSM medium.

4-2. Spawn culture

The mycelium is separated from the slope medium, added to a small amount of liquid culture medium, homogenized with a homogenizer and dispensed into a 500ml culture flask containing a 100ml liquid culture medium, and shaken at 25 ° C for 120 days at 120 rpm. Incubated.

4-3. Present culture

10 ml of the homogenized spawn culture solution was inoculated into a 500 ml culture flask containing 100 ml of the liquid culture medium, followed by shaking culture at 25 ° C. for 6 days.

4-4. Fermenter culture

Inoculate 10% (v / v) into the fermenter (COBIOTECH, 3L) in the culture medium containing the cells, homogenizing the entire culture solution. Incubated at 25 ° C. for 6 days. 0.5N NaOH was used as a pH adjusting liquid, and silicone resin was used as an antifoamer.

4-5. Preparation of β-glucan

Mass production of β-glucan was performed by the following method.

After incubating the mycelia, the alkaline extract was filtered and concentrated through an ultrafiltration apparatus (Satorius. Co. SM 17521) to separate the polysaccharide of the polymer. The pore size of the membrane was a membrane of molecular weight M.W 10,000. Ultrafiltration was concentrated by diluting the glucan extract while applying an air pressure of 2 bar at room temperature, and then continuously added 1,000 times deionized water to the concentrate. The concentrated polysaccharide solution was harvested through lyophilization.

4-6. Yield according to manufacturing process

NaOH, which is used to extract glucan from cells and culture filtrates, may be an important factor depending on the amount added. Therefore, the results as shown in Table 9 were obtained when NaOH was treated from 0.1M to 3.0M, respectively. As shown in Table 9, the proportion of Wet mycelium obtained from the culture solution treated at each concentration decreased as the concentration increased. It is believed that the higher the concentration of sodium hydroxide, the more active the hydrolysis process that can act on the mycelium, thereby disrupting the cell structure of the mycelium. However, the concentration of sodium hydroxide does not significantly affect the yield of Wet mycelium from 1.5M.

Therefore, it is considered that it is most economical to add 1.5 M to 2.0 M NaOH during the process at the pilot scale, and there is no significant difference compared to the case where the sodium hydroxide is directly added so that the final concentration is 2.0 M.

The cultured mycelium is mixed with a mixer, placed in each beaker, and left at room temperature for 1 day after addition of 2M NaOH, followed by neutralization with acetic acid. As a result of examining the ratio of glucan recovered after adding 2 v / v, 2.5 v / v and 3 v / v ethanol to the neutralized culture, the yield of polysaccharide gradually increased according to the amount of ethanol added, but there was no significant difference. In addition, when the neutralized culture solution was used for the ethanol precipitation method (sample 2), the recovery rate of the polysaccharide was 0.25%. In the control experiment, the filtrate was separated by ultrafiltration (MW size; 10,000), and about 0.1% of the polysaccharide was recovered from the concentrate having a molecular weight of 10,000 or more.

Table 9 Yield according to extraction method

Sample number Purification Method Discharge amount (L) Glucan (g) yield(%) AI AS One Ultrafiltration (UF) 2.9 4.10 0.144 2 Ultrafiltration (UF) 2.5 0.85 2.08 0.117 3 EtOH precipitation 1.0 4.10 3.17 0.250 4 When extracting using UF: 0.1N NaOH 1.0 3.90 0.390 5 UF: 0.5N NaOH extraction 1.0 4.40 0.440 6 UF: 1N NaOH extraction 1.0 2.34 0.234 7 UF: 1.5N NaOH extraction 1.0 2.05 0.205 8 UF: 2N NaOH extraction 1.0 2.11 0.211 9 UF: 3N NaOH extraction 1.0 1.61 0.161 10 UF: 10N NaOH extraction 1.0 2.34 0.340 11   2V EtOH 1.0 1.18 0.118 12 2.5V EtOH 1.0 2.17 0.217 13   3V EtOH 1.0 2.07 0.207 14 Heat extract (freeze drying) 1.4 sup / 1.9 23.94 1.260 15 Thermal extract (EtOH precipitation) 2.4 sup / 2.7 4.44 0.164

Abbreviation-AI; Alkali soluble, alkali insoluble, AS; Soluble in centrifugation

            Part, sup; Supernatant

5. Safety of Water-Soluble β-Glucan

5-1. Acute Abdominal Toxicity Test

end. Test animal and administration method

      Five-week-old ICR (♂, ♀) rats were grouped by random method in a week-long breeding room. The male and female groups were separated according to the dosage and one group was composed of 5 rats. Dosage setting was based on the Acute Abdominal Toxicity Test of the National Institute of Safety, and the control group, group 1 (1140 mg / kg), group 2 (1520 mg / kg), group 3 (2020 mg / kg), and It was set as 4 groups (2700 mg / kg). The powdered test substance (Table 10) was suspended in physiological saline and forced intraperitoneal administration using a 1 ml syringe, and the test period was 7 days (Table 11). After administration of the test substance, weight changes, clinical symptoms, and dead animals were checked, and final autopsy was performed on the 7th day of the end of the experiment to observe internal organ changes.

Table 10 Characteristics of Acute Toxicity Tested Samples

Sample number characteristic Remarks Sample 1  Alkaline Extract → Ultrafiltration Concentration → Insoluble Part → High Temperature and High Pressure Sterilization → Insoluble β-Glucan Route of administration: intraperitoneal administration Sample 2  Sample 1 → Sulfate Solubilization → Ultrafiltration Concentration → Soluble Glucan → High Temperature High Pressure Sterilization → Water Soluble β-Glucan Route of administration: intraperitoneal administration Sample 3  Water soluble β-glucan-40 mg / kg (i.v) 40 mg / kg intravenously

Table 11 Acute Toxicity Test Methods for Water Soluble β-Glucan

Test group gender The number of animals Total dose (mg / kg) Dosing frequency Dosage amount (mg / kg) Examination Day contrast ♂ 5 - One 20 7 ♀ 5 - One 20 7 G1 ♂ 5 1140 One 20 7 ♀ 5 1140 One 20 7 G2 ♂ 5 1520 One 20 7 ♀ 5 1520 One 20 7 G3 ♂ 5 2020 One 20 7 ♀ 5 2020 One 20 7 G4 ♂ 5 2700 One 20 7 ♀ 5 2700 One 20 7

I. Inspection items

(1) Clinical symptoms and death animals

Clinical symptoms and deaths due to the test substance immediately after administration and during the test period were recorded. The specific clinical symptoms of animals due to the test substance during the test period showed decreased activity, abnormal gait, nipples, and ptosis after 1 day after administration, and gradually returned to normal. Death was insoluble β-glucan in Table 12, water-soluble β. -Glucan is shown in Table 13. In addition, there were no clinical signs and deaths of animals when intravenous administration of test substance water-soluble β-glucan.

Table 12 Death and LD50 Death of Insoluble β-Glucan

gender Capacity (mg / kg) Number of deaths by date after dosing Mortality rate LD50 (mg / kg) 0 One 2 3 4 5 6 7 ♂ 0 0/5 2216.9 1140 0 0/5 1520 0 0/5 2020 0 One One 2/5 2700 2 2 4/5 ♀ 0 0/5 1662.6 1140 0 0/5 1520 0 2 2/5 2020 0 3 One 4/5 2700 One 4 5/5

Table 13. Death and LD50 Deaths of Water-soluble β-Glucan

gender Capacity (mg / kg) Number of deaths by date after dosing Mortality rate LD50 (mg / kg) 0 One 2 3 4 5 6 7 ♂ 0 0/5 2700.0〉 1140 0 0/5 1520 0 One 1/5 2020 0 One 1/5 2700 One 1/5 ♀ 0 0/5 2700.0〉 1140 0 0/5 1520 0 0/5 2020 One 1/5 2700 One 1/5

(2) weight change

Body weight changes were recorded on the day of dosing, 1, 3 and 7 days after dosing. Body weight change between the control group and the test substance-administered group was observed on the day of administration, 1 day, 3 days after the administration and the autopsy day.

(3) autopsy findings

At the end of the test all animals were killed with ethyl ether to record changes in internal and external organs due to the test substance. Hepatic, gastrointestinal and spleen adhesions were observed in the overall cases of dead and surviving individuals after administration of Test Sample 1 (insoluble glucan). In addition, no abnormal findings were observed in the individuals who died after the intravenous administration of Test Substance Sample 2 (soluble β-glucan) and in surviving individuals (see Tables 12 to 13).

(4) Acute Toxicity

In order to evaluate the stability of water-soluble β-glucan in development as an adjuvant, non-soluble polysaccharides were isolated from mass-cultured Ganoderma lucidum mycelium β-glucan, and partially substituted with sulfate. (ICR, ♂♀) The acute toxicity test was performed on the abdominal cavity and the following results were obtained.

First, β-glucan mixed with insoluble and soluble moieties is a non-soluble part of mass-cultured β-glucan, which was 1822.0 mg / kg in females based on clinical symptoms, autopsy findings, and half-value (LD ₅₀ ). Males showed 1691.0 mg / kg. Intestinal adhesion was noted, which is thought to be due to the inflammatory action of the insoluble part of β-glucan.

Water-soluble β-glucan, which partially substituted the insoluble portion of β-glucan with sulfate, showed excellent stability in terms of clinical symptoms, autopsy findings, and half lethal dose (LD ₅₀ ). When soluble β-glucan was injected by intravenous route, the clinical symptoms and autopsy findings showed stability at this concentration.

In conclusion, as described in Table 14, the LD50 value of the acute toxicity test in mouse intraperitoneal administration of water-soluble β-glucan was very stable.

Table 14 Acute Toxicity Studies of Water Soluble β-Glucan and WI-β-Glucan During Mouse Intraperitoneal Administration

gender Insoluble β-glucan (mg / kg) Water-soluble β-glucan (mg / kg) ♂ 2216.9 2700〉 ♀ 1661.6 2700〉

6. Physiological Activity of β-Glucan

6-1. Anticancer Activity of β-Glucan and Related Polysaccharides Against Solid Cancer S-180

end. Laboratory Animals and Tumor Cells

Mice were purchased from samyuk livestock with ICR males of 20-25 g. Sarcoma 180, a cancer cell, was used by the Korea Cell Line Bank.

I. Tumor cell transplantation

Sarcoma 180 cells, which were transplanted and preserved at weekly intervals in the abdominal cavity of ICR mice, were used as experimental tumor cells. Sarcoma 180 cells cultured intraperitoneally in experimental animals for 7 days were taken with ascites, ice-cold and sterilized physiological saline was added, and centrifuged at 400 x g for 3 minutes to separate cell precipitates. The separated cells were washed three times with ice-cold, sterilized saline and then diluted to 1 × 10 7 cells / ml. 0.1 ml of each suspension (1 x 106 cells / mouse) was implanted subcutaneously in the left inguinal part of the experimental animal.

All. drug injection

Tumor cells were transplanted into groups of 10 mice each, and drug administration was started after 72 hours, but was intraperitoneally injected once daily for 10 consecutive days. The control group was infused with physiological saline at a concentration of 20 mg / kg in the positive control group and 20 mg / kg in physiological saline at the treatment group, and the injection dose was 0.1 ml.

la. Judgment of anticancer test result

On the 30th day after the tumor cell transplantation, the experimental animals were killed and the solid tumors induced were removed, and the average tumor weight was obtained by measuring the weight. The percentage of inhibition was compared to the control group administered with saline (Percent). inhibition ratio = IR (%)) was calculated.

I.R. (%) = (Cw-Tw) / Cw × 100

Cw: mean tumor weight of control group

Tw: mean tumor weight of treatment group

hemp. Anticancer test results

Solubility is one of the problems to be solved in order to industrially use β-glucan in various ways. Developed to solve this solubility is water-soluble β-glucan. The results of comparing the anticancer activity between the water-soluble β-glucan and the water-insoluble β-glucan are shown in Table 15. In other words, the anticancer activity of insoluble β-glucan was 52.5%, while the anticancer activity of water-soluble β-glucan was 80.5%. This was more than 20% cancer blocking effect than the control group Crestine (PS-K).

6-2. Immune activity test

end. Reagents, Laboratory Animals, and Controls

RPMI 1640, Dulbecco's modified Eagle's medium (DMEM) medium and fetal bovine serum were purchased from GIBCO (Grand Island, N. Y), streptomycin, penicillin, LPS ( E. coli 0127: B8), IFN-γ, NaNO ₂ Laminarin, β-glucan and the like used as a control polysaccharide was purchased from Sigma (St. Louis, MO). The ICR male mice used in the experiments were purchased from Samyuk Livestock, weighing 20-24 g at 8-10 weeks of age. As the breeding conditions of the mice, the experimental animal room temperature was maintained at 22 ± 2 ℃, the humidity was 55 to 60%, and the solid feed and water for the mice were supplied without restriction.

In order to test the immunological activity of the water-soluble glucan, the control material was INF-γ (Sigma), LPS (Sigma) and biological polysaccharides.

I. Invitro test

(1) Measurement of Blood Tumor Necrosis Factor (TNF-α) Following Oral and Intravenous Administration of Mouse

① Quantitative method of TNF-α

Quantification of TNF-α in serum and culture was determined by solid-phase ELISA using the multiple antibody sandwich principle. Serum preparation for measuring TNF-α in mouse serum was performed as follows. 20-25 g of ICR mice were continuously administered with water-soluble β-glucan (20 mg / kg), LPS (500 μg / kg) and β-glucan and Crestine ^TM (20 mg / kg) for 5 days. Four days after drug administration, mice were anesthetized with ether and the abdomen was excised to collect blood from the abdominal aorta. The collected blood was left for 2 hours, then centrifuged to separate serum and -70 It was stored frozen at ° C and used for the experiment.

TNF-α quantification in macrophages and monosite cultures was performed as follows. After activating factors including water-soluble β-glucan to the macrophage culture medium and incubated for 24 hours, the culture supernatant was harvested for ultracentrifugation. Supernatants obtained through centrifugation were -70 Cryopreservation was used for the experiment at ℃. Simplification of quantitation of TNF-α in serum and culture supernatant was performed by placing 50 μl of dilution buffer in a mono-coated anti-mTNF-α precoated 96-well microtiter plate and activating with β-glucan. 50 μl of the culture supernatant of the prepared mouse serum and macrophages is added to bind the mouse TNF-α present in the test sample. After the sample was added, it was reacted at 37 ° C. for 3.5 hours, and then washed four times with a washing buffer to remove unbound material. Peroxidase-conjugated polyclonal anti-mTNF-α (HRP-conjugate) is added to bind to the already bound mTNF-α. After reacting at 37 ° C. for 1 hour, the plate is washed four times to remove unbound material, and the substrate solution is added to induce a catalysis reaction with peroxidase. 100 μl of reaction stopper was added to stop the reaction of the enzyme. Within 30 minutes after the reaction was stopped, absorbance was measured at 450 nm using an ELISA reader (Bio-Tek instrument, Inc. Ceres UV. HDi). The amount of mTNF-α in the sample was calculated as a standard straight line obtained by plotting the absorbance obtained according to the concentration of the standard mTNF-α.

② Result of blood tumor necrosis factor during oral administration

Increasing the production of tumor necrosis factor in mouse blood by soluble β-glucan showed a 15.5-fold increase in the concentration of TNF in the water-soluble β-glucan than the control group, as shown in Table 16, and 1.4 times more tumor necrosis than insoluble β-glucan. The promoting effect of the factor was seen.

(2) Measurement of Tumor Necrosis Factor from Monoocytes of Rats

① Monosite Cell Culture

Monosite was collected from rat bone marrow and cells were centrifuged at 200 xg for 10 min at 4 ° C. 1 L of 40 mM glutamine, 50 ml of endotoxin free heat inactivated (58 ° C., 30 min) fetal calf serum, 100 IU penicillin and 100 μg streptomycin containing DMEM medium were washed and then 100 mm tissue Aliquots were placed in a culture dish (Cornning 25020) and incubated for 2 hours in a 5% CO ₂ incubator at 37 ° C. After 2 hours, the supernatant of the culture dish was removed to discard non-adherent cells, and then fresh DMEM medium was added and cultured to obtain adherent cells. This was harvested and used for the experiment.

② monosite activity

Bone marrow monosite cells were aliquoted to 2 x 10 ⁶ cells / ml cells in a Costa Petri dish and pre-cultured at 37 ° C and 5% CO ₂ . Non-adherent cells were removed from the cultured macrophages and washed with fresh DMEM medium maintained at 37 ° C. before the experiment. The harvested monosite cells were stained with tryphan blue and used for experiments with 95% cell viability. The cell number was adjusted to 2 x 10 ⁶ cells / ml using a hemocytometer, and then 200 µl aliquots (4x10 ⁵ cells / well) were put into 96 wells and incubated at 37 ° C. and 5% CO _{2 for} 90 minutes. . After 90 minutes non-adherent cells were removed and fresh 180 μl DMEM medium was dispensed into each well. 20 μl of the medium containing the water-soluble β-glucan to be tested was added thereto, followed by incubation for 24 hours at 37 ° C. and 5% CO ₂ to measure the activity of macrophages.

③ Monosite activity result

As a result of activating the monosite of rats by water-soluble β-glucan, the production of tumor necrosis factor was increased by 6.3 times as compared with the control group.

(3) Serum TNF-α concentration measurement after intravenous administration of water-soluble β-glucan

Monokine, TNF, is a protein that mediates anticancer activity and is secreted by activated macrophages. In addition, TNF can induce the regulation of neutrophil cell function, promote differentiation of myloid cells, proliferation of B cells, and activity of macrophages. In particular, TNF-α has the ability to recognize and kill cells infected with tumors, salmonellosis, and viruses regardless of normal cells. In this sense, the promotion of the production of tumor necrosis factor by anticancer polysaccharides can be an indicator of the promotion of immune response in vivo. After administration of water-soluble β-glucan, blood was collected from mice to separate serum, and the amount of TNF-α was measured by the ELISA method described above.

The degree of TNF-γ production by intravenous administration (tail tail vein) is as follows. Water-soluble β-glucan was dissolved in distilled water and sterilized, and then administered to the tail caudal vein (5 mg / 0.2 ml / kg). The results are shown in Table 18.

(4) Determination of nitric oxide from macrophages

Nitric oxide (NO) produced by macrophages is present for 6 to 8 seconds, after which it spontaneously oxidizes and accumulates in the NO ₂ ⁻ and NO ₃ ⁻ states. Therefore, the amount the reactive nitrogen intermediates (RNI) is generated NO ₃ ^- must be accurately converted to a reducing element, but usually NO ₂ ^- was indirectly quantified according to the method, such as by color coding them (Ding) Since most of the. Briefly, 100 μl of culture and the same amount of Greases reagent (1% sulfonylamide / 0.1% naphthylenediamine dihydrochloride / 2.5% H ₃ PO ₄ ) are mixed and left at room temperature, followed by ELISA reader. Measurement was made at 540 nm. The concentration of NO ₂ ^- was obtained by diluting sodium nitrate and measuring the absorbance to create a standard curve.

Bone marrow was collected from the femoral rats and centrifuged at ficoll-paque to collect monosite in the bone marrow. Collected monocytes were cultured in RPMI1640, the number of cells to 2 × 10 ⁶ / ㎖, and after the sample was processed 48 hours after the measurement of nitric oxide is shown in Table 19.

(5) anti-complement activity test

Anticomplementary activity was measured by modifying the method of Yamada et al. 150 μl of GVB ²⁺ buffer in 10 ml test tubes (gelatin veronal buffered saline: 0.15 mM CaCl ₂ , 0.5 mM MgCl ₂ , 1.8 mM sodium barbital, 3.1 mM barbituric acid, 141 mM NaCl and 0.1% gelatin, pH 7.4 ) And 50 [mu] l of each sample at a concentration of 250 [mu] g / ml, and then 50 [mu] l (100 U / ml) of guinea pig serum as a complement and reacted at 37 [deg.] C. for 30 minutes, followed by 4.75 ml of GVB. ²⁺ buffer was added to adjust the final concentration of complement to 1 unit / ml. Dispense the adjusted complement mixture into each test tube at 1.0 unit, 1.2 unit and 1.6 unit, then add anti-sheep hemolysin (2MHU / mL) and the same amount of SRBC (5x10 ⁸ cells / mL). After mixing and sensitizing at room temperature for 30 minutes, 2 ml of sensitized SRBC was added to the test tube and adjusted with GVB ²⁺ buffer to a final 5 ml. Each test tube is allowed to react at 37 ^° C for 60 minutes. To stop the reaction, 70 μl of 0.5M EDTA solution was added and mixed, and then the absorbance of the supernatant obtained by centrifugation at 400 × g for 5 minutes was measured at 541 nm. Anti-complement activity is indicated by the inhibition of TCH ₅₀ , ITCH ₅₀ compared to control (50% of total complement hemolysis, TCH ₅₀ ).

% Inhibition = (Control Total Complement Hemolysis-Sample Treatment Total Complement Hemolysis) /

             Control Total Complement Hemolysis × 100

     The immune system is a series of highly complex reaction systems, of which the complement system is known to play an important role in defense immunity and tumor immunity, such as defense against infection, one of the immune functions in vivo. Since the complement system is activated by polysaccharides having anticancer effects, anticomplement activity was searched using the same sample used for anticancer activity screening.

As shown in Table 20, the activity of the complement system showed concentration-dependent anti-complement activity for insoluble β-glucan and was 22.4% at 25 μg / ml, but about 43.7% of the anti-complement increased at 200 μg / ml. Activity was shown. On the other hand, water-soluble β-glucan showed a relatively low 13.2% anticomplementary activity, which is thought to be due to the low molecular weight of the polysaccharide of the sulfate. As a control, crestine showed an anticomplement activity of 20.9%.

Table 15 Anticancer Activity Comparison

Sample Mg / ml Tumor Weight (g) statistics Anticancer activity One 2 3 4 5 6 7 8 9 10 Average SD Water-soluble β-glucan 10 0.1 1.5 0.7 2.1 0.0 0.0 0.0 0.0 0.0 0.0 0.55 0.77 80.5 Insoluble β-glucan 10 0.0 0.4 1.8 3.0 2.3 0.5 2.0 2.0 0.1 0.0 1.34 1.04 52.5 Crestine (PS-K) 10 0.0 0.0 1.6 1.4 0.0 3.0 2.7 0.0 1.4 0.0 1.12 1.12 60.3 Saline 10 3.6 3.6 3.5 3.4 0.9 3.8 3.2 3.4 1.4 0.9 2.77 1.19 0 Saline 10 2.3 3.7 2.4 2.9 3.5 2.9 2.0 1.9 4.2 0.0 2.87 0.80 0

Table 16 TNF-γ Production in Mouse Serum Following Oral Administration of Water-Soluble β-Glucan

sample TNF-γ (pg / ml)  Negative control 4.55  Water-soluble β-glucan 70.60  Insoluble β-glucan 49.00

Table 17 Production of water-soluble β-glucan treated TNF-γ in rat myeloid monosite cultures

sample TNF-γ (pg / ml)  Water-soluble β-glucan 1320.9  Insoluble β-glucan 1462.0  LPS 2441.8  IFN-γ 1995.7  Negative control 209.7

Table 18 TNF-γ production by intravenous administration (tail tail vein) (mouse)

time TNF-γ (pg / ml) 1 hours 68.43 4 hours 83.63 6 hours 132.17 12 hours 115.27 24 hours 92.03 27 hours 29.67 48 hours 16.17

Table 19 Nitric Oxide Production Capacity of Bone Marrow Cells According to Water Soluble β-Glucan Concentration

Water Soluble β-Glucan Concentration Nitric Oxide (Micro M) Control 6.51 ± 0.84 1 μg / ml 15.94 ± 1.10 5 μg / ml 45.16 ± 10.38 10 μg / ml 53.02 ± 2.66 20 μg / ml 82.26 ± 2.78

Table 20 Comparison of Complement System Activation Effects of β-Glucan and Similar Products

Sample Concentration (µg / ml) Anti-complement activity (ITCH 50%)  Insoluble β-glucan (Ganoderma lucidum mycelium extract) 2550100200 22.428.638.643.7  Water-soluble β-glucan 50 13.2  Crestine 50 20.9

The present invention provides a water-soluble β-glucan which is a functional biologically active polysaccharide as a biological new material that is widely used industrially by accepting β-glucan, an insoluble polysaccharide having excellent physiological activity.

In another aspect, the present invention provides a substance having superior physiological activity and water-soluble properties than the conventional β-glucan polysaccharides using Korean basidiomycete.

1 is a graph showing a gas chromatograph pattern and a monosaccharide composition of the water-soluble β-glucan of the present invention.

Figure 2 is a graph showing the gas chromatograph pattern and monosaccharide composition of insoluble β-glucan.

3 is a graph showing the IR spectrum of the water-soluble β- glucan of the present invention.

4 is a graph showing the IR spectrum of insoluble β-glucan.

5 is a ¹³ C-NMR spectrum analysis graph of the water-soluble β-glucan of the present invention.

6 is a molecular weight distribution chromatograph of the water-soluble β-glucan of the present invention.

Claims (4)

A method for producing a water-soluble β-glucan, wherein insoluble β-glucan is substituted with an element of an insoluble β-glucan skeleton using a sulfur element. The aqueous solution according to claim 1, wherein after dissolving urea in methylsulfoxide, an insoluble glucan is added thereto, and an acidified methylsulfoxide solution is slowly added to the β-glucan-Me ₂ SO-urea solution and heated. Method for preparing β-glucan. The method for producing a water-soluble β-glucan according to claim 1, wherein the sulfur content of the water-soluble β-glucan is 14.9%. The method for producing a water-soluble β-glucan according to claim 1, wherein the insoluble β-glucan is obtained using Korean basidiomycetes.