KR102297056B1 - Culture broth of Porostereum sp.(KCTC18837P) and composition comprising the same for preventing or treating diabetes mellitus - Google Patents

Abstract

The present invention relates to a composition for preventing and alleviating diabetes, the composition comprising a Porostereum sp. (KCTC18837P) mycelium culture as an active ingredient. The Porostereum sp. (KCTC18837P) mycelium culture contains a lot of active ingredients such as inorganic ingredients, amino acids, vitamins, and the like in addition to beta-glucan and extracellular polysaccharide effective for antidiabetics. It was confirmed that these cultures are safe as natural materials and have excellent antidiabetic effects. Accordingly, the Porostereum sp. (KCTC18837P) mycelium culture of the present invention is expected to be useful for the prevention and alleviation of diabetes.

Description

A new strain Porostereum sp. (KCTC18837P) mycelium culture and a composition for preventing and treating diabetes comprising the same as an active ingredient {Culture broth of Porostereum sp. (KCTC18837P) and composition comprising the same for preventing or treating diabetes mellitus}

The present invention relates to a Porostereum sp. (KCTC18837P) mycelium culture and a composition for preventing and treating diabetes comprising the same as an active ingredient.

Diabetes mellitus is an endocrine disorder caused by the failure of insulin to function normally. Globally, it is estimated that there are 347 million people with diabetes, or about 5% of the world's population. In particular, diabetes is accompanied by long-term complications such as arteriosclerosis, high blood pressure, cerebrovascular infarction, diabetic nephropathy, diabetic retinopathy, pyoderma, eczema, gangrene, and oral disease. It is predicted that it will rank 7th in mortality. Diabetes treatment drugs are mainly aimed at managing blood sugar and delaying complications, and use oral blood sugar enhancing agents such as insulin, sulfonylurea, biguanide, and alpha-glucosidase inhibitors. and limitations of treatment. Accordingly, research on the development of therapeutic agents derived from natural products that have fewer side effects and are safe for long-term administration is continuing.

Among the natural resources discovered so far, the available resources with widely known utilization have decreased in scarcity due to excessive competition.

Mushrooms are rich in carbohydrates, proteins, lipids, minerals, vitamins, etc., and contain a large amount of physiologically active substances including beta-glucan and extracellular polysaccharides. In addition, as a significant part of the ingredients and physiological activity mechanisms of mushrooms are scientifically proven, research such as discovering new mushrooms, developing a mass propagation method, and searching for functionality is needed.

Mushrooms can be divided into mycellium and fruit body. The mycelium contains various useful substances like the fruiting body. Induction of the fruiting body takes a long time to cultivate and technology development takes a long time, whereas the liquid culture method of the mycelium uses a hygienic and automated facility to stably and mass-produce a large amount in a short time. It has the advantage that it can be cultivated.

Therefore, most mushrooms recently made it possible to culture mycelium containing various physiologically active substances through bioconversion using nutrients contained in liquid medium. In addition, since mushroom mycelium has different culture characteristics depending on the medium composition, culture conditions, or mushroom mycelium type, suitable culture conditions for each mushroom must be found in order to improve the liquid culture efficiency of the mycelium. Depending on the nutrient composition and culture conditions, various metabolites having anticancer and antioxidant activities can be obtained (Lee Wi-Young, et al., 2008).

The genus Porostereum belongs to the genus Basidiomycota, Agaricomycetes, Polyporales, Phanerochaetaceae, and Porostereum. . (National Arboretum National Standard Mushroom List)

Twenty-one species of the genus Porostereum have been registered in the International Database (Index Fungroum) so far, and Porsterum crassum and Porostereum spadiceum belong to this genus. Muhammad Hamayun et al. confirmed that Porostereum spadiceum AGH786 could be used as a fungal biofertilizer in soybeans under salt stress (Muhammad H. et al., 2017). However, industrialization research on the genus Orifolia genus strain is still incomplete.

Regarding the genus Porostereum, Korean Patent Registration No. 1401887 describes that Porostereum spadiceum (KUC8602) has the ability to decolorize liquid dyes, but the genus Porostereum sp. KCTC 56671) and Specific species are different, and there is a difference from the use of the present invention in that it is used for dyeing wastewater treatment.

Korea Patent Registration No. 1401887, composition for removing chromaticity of dyeing wastewater and method of removing chromaticity of dyeing wastewater, registered on May 26, 2014

Wi-Young Lee, Jin-Kwon Ahn, Gang-Hyeon Ga, Eung-Jun Park, Liquid culture of mushroom mycelium and production of useful substances, 2008, Publication No.: 11-1400377-000243-01, National Academy of Forest Sciences. Muhammad Hamayun1, Anwar Hussain, Sumera A. Khan, Ho-Youn Kim, Abdul L. Khan, Muhammad Waqas, Muhammad Irshad, Amjad Iqbal, Gauhar Rehman, Samin Jan and In-Jung Lee, Gibberellins Producing Endophytic Fungus AGH786 Rescuberellins Producing Endophytic Fungus Poroster spa of Salt Affected Soybean, Frontiers in Microbiology, April 2017, (8); article 686. Cheol Soo Choi et al., The mechanism of development of insulin resistance in type 2 diabetes mellitus, 2002, Korean Journal of Medicine, 63(6): 613-624.

It is an object of the present invention to provide a composition for the prevention and improvement of diabetes comprising a culture of Porostereum sp. (KCTC18837P) mycelium as an active ingredient. Another object of the present invention is to provide a composition for preventing and improving diabetes comprising a processed product or extract of Porostereum sp. (KCTC18837P) mycelium culture as an active ingredient.

In order to achieve the above object, the present invention provides optimized conditions of the medium composition and culture environment for mass production of Porostereum sp. (KCTC18837P) mycelium culture.

In addition, in the present invention, the culture medium produced under the above conditions can produce beta-glucan, extracellular polysaccharide, inorganic components, amino acids, vitamins, and the like. In addition, the mycelium culture produced under the above conditions of the present invention may be effective in preventing and improving diabetes. In addition, the present invention provides a food, health functional food and pharmaceutical composition for preventing and improving diabetes by processing or extracting the culture medium produced under the above conditions.

The mycelium culture can be obtained by culturing the mycelium derived from the Porostereum sp. (KCTC18837P) strain.

The present invention also, based on the total amount of the liquid medium, starch 0.2 ~ 2% (w / v), sucrose 0.2 ~ 2% (w / v), glucose 0.2 ~ 2% (w / v), soybean powder 1.5% (w) /v), FeSO ₄ 0.001% (w/v), KH ₂ PO ₄ 0.001% (w/v), MgSO ₄ 0.003% (w/v), vitamin B7 0.003% (w/v), vitamin B6 0.002% (w/v) The new strain Porostereum sp. ( KCTC18837P) or a method for mass production of a mycelium culture thereof is provided.

Hereinafter, the present invention will be described in detail. The present invention relates to a composition for preventing and treating diabetes comprising Porostereum sp. (KCTC18837P) mycelium culture as an active ingredient. The Porostereum sp. (KCTC18837P) mycelium culture can produce complex polysaccharides and beta-glucan as well as vitamins, magnesium, calcium, and the like. The magnesium and calcium are known to be effective in improving insulin resistance, promoting insulin secretion, and lowering fasting blood sugar as inorganic elements.

The vitamins are vitamin B2 and vitamin D, which are abundantly contained in mushrooms, which are known to improve insulin secretion. The Porostereum sp. (KCTC18837P) mycelium culture can be obtained by liquid culture of the mycelium derived from the Porostereum sp. (KCTC18837P) strain. The culture may be a culture medium containing both the mycelium and the culture medium, or may be a culture medium separated from the mycelium and the culture medium. Preferably, it is a culture medium in which the mycelium and the culture medium are separated. The separated culture solution may be concentrated by a non-heating method in order to increase the efficiency of the freeze-drying process. Preferably, a reverse osmosis (RO) group is used.

The culture solution may be powdered using a conventional drying method. Preferably, it is freeze-dried. The culture is a liquid culture medium, a culture medium powder dried by drying the culture medium, water, C1-C4 lower alcohol, acetone, n-hexane, one or more solvents selected from the group consisting of dichloromethane and ethyl acetate It may be an extract extracted by adding it, but is not limited thereto.

The C1-C4 lower alcohol may be methanol, ethanol, propanol, isopropanol, buntanol, or the like. The extraction method of the extract may be selected from any one of hot water extraction method, cold extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method, compression method, and the like. In addition, the desired extract may be further subjected to a conventional fractionation process, and may be purified using a conventional purification method.

The components of the liquid medium during the liquid culture may affect the growth of the strain and the production of active ingredients. Therefore, it is necessary to establish the components and content conditions of the liquid medium optimized for culturing the Porostereum sp. (KCTC18837P) mycelium of the present invention.

The liquid medium is a carbon source as maltose, glucose, lactose, starch, dextrin, sucrose, fructose, galactose, mannose (Galactose), mannose ( Mannose) and sugar (Oligosaccharide) may be used, but is not limited thereto. Preferably it contains starch, sucrose and glucose, more preferably starch 0.2-2% (w/v) and sucrose 0.2-2% (w/v) and glucose 0.2-2% based on the liquid medium. % (w/v), most preferably starch 1% (w/v), sucrose 0.5% (w/v) and glucose 0.5% (w/v) based on the liquid medium. The liquid medium is a nitrogen source, soy flour (soy flour), yeast extract (yeast extract), L- glutamic acid (L-glutamic acid), soy peptone (soy peptone), malt extract (malt extract), ammonium (ammonium), nitric acid It may include one or more selected from the group consisting of calcium (cacium nitrate), potassium nitrate (potassium nitrate), sodium nitrate (sodium nitrate), and the like, but is not limited thereto. Preferably, 0.5 to 3% (w/v) of soybean powder is included based on the liquid medium. Most preferably, it contains 1% (w/v) soybean powder based on the liquid medium.

The liquid medium may include one or more selected from the group consisting _{of KH 2} PO ₄ , ZnSO ₄ , MgSO ₄ , CuSO ₄ , FeSO ₄ and CaCl _{2 as trace elements, but is not limited thereto.} Preferably FeSO ₄ , KH2PO ₄ , MgSO _{4 It} contains, more preferably based on the liquid medium 0.001 ~ 0.3% (w / v) FeSO ₄ , 0.001 ~ 0.3% (w / v) KH ₂ PO ₄ , 0.001 to 0.3% (w/v) MgSO ₄ Contained. Most preferably, it contains 0.001% (w/v) FeSO ₄ , 0.001% (w/v) KH ₂ PO ₄ , and 0.003% (w/v) MgSO ₄ , based on the liquid medium.

The liquid medium may contain vitamins. Preferably, vitamins B, C and E, but not limited thereto. More preferably, each vitamin is contained in an amount of 0.001 to 0.01% (w/v) based on the liquid medium. Preferably, it contains 0.005% (w/v) of vitamin B based on the liquid medium. Most preferably, it contains 0.003% (w/v) of biotin (B7) and 0.002% (w/v) of pyridoxine (B6).

The liquid medium may have a pH of 4.5 to 6.5. Preferably it is pH 5.5. The pH can affect the shape and activity of the protein by changing the charge of the amine group or carboxyl group of amino acids, which are units of enzyme proteins important for cell metabolism. In addition, changes in pH in the external environment may affect the ionization of microbial nutrients, thereby affecting microbial intake. In addition, when the pH of the liquid medium is less than 5, aerobic and acidic algae or archaea are easy to propagate, and when the pH is more than 7, alkaline actinomyces and mold fungi breed. It is not preferable because it is easy and the mycelium does not grow.

In the liquid medium, based on the liquid medium, the carbon source is starch 1% (w/v), sucrose 0.5% (w/v), glucose 0.5% (w/v), and the nitrogen source is soybean powder 1.5% (w/v) , Minerals are 0.001%(w/v) FeSO ₄ , 0.001%(w/v) KH ₂ PO ₄ , 0.003%(w/v) MgSO ₄ , Vitamins are Biotin (B7) 0.003%(w/v), Pyridoxine (B6) 0.002% (w/v), most preferably having a pH of 5.5.

The culture may use an agitated bioreactor, and the growth of the strain may be affected depending on the air supply amount, agitation speed, culture temperature and culture time, so that the Porostereum sp. It is important to establish optimal culture conditions for the (KCTC18837P) strain.

The air supply amount may be 0.02 ~ 1vvm, preferably 0.1 ~ 0.5vvm, more preferably 0.2vvm.

The stirring speed may be 25-100 rpm, preferably 50-100 rpm, more preferably 60 rpm.

The culture temperature may be 18 ~ 27 ℃, most preferably 23 ℃. When the temperature is less than 20 ℃, the activity of the mycelium is slowed and the incubation period is prolonged, resulting in lower productivity and a sharp increase in production cost. It is lowered to, and if it exceeds 28 ℃, the mycelium is undesirable to die.

The culture may have a pH of 4 to 7, preferably 5 to 6, and most preferably 5.5.

The incubation time is 5 to 7 days, more preferably 6 days. If the incubation time is less than 4 days, the amount of mycelium is small and the content of the active ingredient is low. If the culture time is more than 7 days, the content of the active ingredient no longer increases or gradually decreases, and if it exceeds 9 days, the content of the active ingredient is decreased. It is undesirable because it decreases rapidly.

The Porostereum sp. (KCTC18837P) mycelium culture should be separated from the mycelium for concentration. For this purpose, a filtering process must be performed, and the filtrate obtained by filtration with a filter press may have a sugar content of 0.5 to 1 brix, a viscosity of 2 to 10 Cp, and a moisture content of 95 to 98%.

The filtrate may be subjected to a concentration process in order to reduce the volume. Reverse osmosis (RO: reverse osmosis) can be used as a concentration method that prevents the reduction of active ingredients due to heat and has an excellent processing speed. The concentrated solution concentrated 12 times by performing RO may have a sugar content of 5 to 10 brix, a viscosity of 30 to 100 Cp, and a moisture content of 10 to 20%. The concentrate can be used as a food for diabetes prevention and improvement of diabetes, a material for health functional food, or added to other materials.

The concentrate may be powdered through freeze-drying. During freeze-drying, the sugar content of the concentrate should be maintained below 20 brix. If it exceeds 20 brix, a sugar film is formed and moisture is not evaporated, so drying is impossible. Preferably, it is to maintain 18 brix or less. In addition, during drying, the temperature of the heating plate should be maintained at 40° C. or less and the moisture content should be carried out until the moisture content becomes 0.1% or less. This is because, when the heating temperature is raised to 40° C. or higher to shorten the drying time, the protein and vitamin components are reduced due to heat damage. Preferably, drying is performed at 30° C. or less. Since the dried product after freeze-drying is in a swollen state like cotton, it must be pulverized using a grinder. Powder products can also be used as food for diabetes prevention and diabetes improvement, as a material for health functional food, or by adding to other materials.

The freeze-dried powder may be used by separating and extracting only functional substances. As the extraction method, any one of methods such as hot water extraction method, cold extraction method, reflux cooling extraction method, solvent extraction method, steam distillation method, ultrasonic extraction method, elution method, compression method, etc. can be selected and used. In addition, the desired extract may be further subjected to a conventional fractionation process, and may be purified using a conventional purification method.

The extract may be beta-glucan, extracellular polysaccharide and other functional substances.

The composition comprising the Porostereum sp. (KCTC18837P) mycelium culture as an active ingredient may be a pharmaceutical composition for preventing and improving diabetes.

The Porostereum sp. (KCTC18837P) mycelium culture contains β-glucan and extracellular polysaccharide, which are known to have a therapeutic effect on diabetes, and inorganic substances and vitamins that help control blood sugar, so that it has an excellent blood sugar lowering effect.

The pharmaceutical composition may include the Porostereum sp. (KCTC18837P) mycelium culture and pharmaceutically acceptable excipients.

The Porostereum sp. (KCTC18837P) mycelium culture is preferably 0.001 to 50% by weight, more preferably 0.001 to 40% by weight, most preferably 0.001 to 30% by weight based on the total weight of the total pharmaceutical composition. can

The pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulating, it is prepared by using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient, for example, starch, It is prepared by mixing calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base of the suppository, witepsol, macrogol, tween-61, cacao butter, laurin, glycerogelatin, etc. can be used.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, weight, specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the judgment of the prescriber. Dosage determination based on these factors is within the level of one of ordinary skill in the art, and generally the dosage is in the range of about 1000-5000 mg/day. A more preferred dosage is 1000-3000 mg/day. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention may be administered to mammals such as mice, livestock, and humans by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intraperitoneal, intramuscular, subcutaneous, intrauterine dural or intracerebrovascular injection. In addition, since the pharmaceutical composition of the present invention is a composition derived from a natural product, it has almost no toxicity and side effects, so it is a drug that can be safely used even when taken for a long period of time for the purpose of prevention.

In addition, the composition comprising the Porostereum sp. (KCTC18837P) mycelium culture as an active ingredient may be a health functional food for preventing and improving diabetes.

The health functional food may include the Porostereum sp. (KCTC18837P) mycelium culture and food pharmaceutically acceptable food supplement additives.

The Porostereum sp. (KCTC18837P) mycelium culture is preferably added in an amount of 0001 to 90% by weight, more preferably 0001 to 70% by weight, and most preferably 0001 to 50% by weight based on the total weight of the total health functional food. can be

The intake of Porostereum sp. (KCTC18837P) mycelium culture in the health functional food is 2000 mg or less at a time, 5000 mg or less per day. Most preferably, it is to be ingested 3 to 4 times a day at 1000 mg at a time.

The health functional food of the present invention includes the form of tablets, capsules, pills or liquids, and the food to which the culture of the present invention can be added, for example, various foods, beverages, gum, tea, vitamins There are complex drugs and health functional foods.

The present invention provides a composition effective for preventing and improving the Porostereum sp. (KCTC18837P) mycelium culture and diabetes.

The present invention relates to a composition for preventing and improving diabetes comprising a Porostereum sp. (KCTC18837P) mycelium culture as an active ingredient. - In addition to glucan, it was confirmed that active ingredients such as inorganic components, amino acids, and vitamins were contained, and it was confirmed that these cultures were excellent in preventing and improving the onset of diabetes.

Through this, the Porostereum sp. (KCTC18837P) mycelium culture of the present invention is expected to be usefully used in the development of pharmaceuticals and health functional foods for the prevention or improvement of diabetes.

1 shows Porostereum sp. (KCTC18837P) is a copy of the depository certificate of the strain.
Figure 2 is a Porostereum sp. (KCTC18837P) is a phylogenetic diagram showing the taxonomic position of the strain.
3 shows significant changes in the number of pancreatic islets (islets/cm2), diameter (μm), and the ratio of zymogen granules per unit area of exocrine (zymogen granules %/mm2 of exocrine) by the test material as a result of autopsy of the experimental animals. It is a drawing that shows

The present inventors are rich in beta-glucan, extracellular polysaccharides, vitamins and minerals in the process of conducting research on industrially useful mushrooms and fungi, Porostereum sp. (KCTC18837P) strain was identified and optimal culture conditions were established, and was deposited at the National Institute of Biotechnology and Biotechnology Biological Resources Center (KCTC) (FIG. 1).

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided so that this disclosure will be thorough and complete, and will fully convey the spirit of the invention to those skilled in the art.

The active ingredient produced by the culture method according to the present invention is Porostereum sp. (KCTC18837P) produced by beta-glucan and extracellular polysaccharide. The extracellular polysaccharide and beta-Porostereum sp containing glucan. (KCTC18837P) Mycelium culture and its concentrates, dried products and extracts can be widely used as health functional foods and pharmaceutical materials to prevent and improve diabetes.

The present invention is Poostereum sp. (KCTC18837P) produced by Porostereum sp. (KCTC18837P) mycelium culture. Or it provides a method for preparing a dried product and extract thereof derived from a culture.

The composition of the present invention is Poostereum sp. (KCTC18837P) Contains extracellular polysaccharide and beta-glucan produced by mycelium.

In addition, the composition of the present invention comprises the extracellular polysaccharide and beta- Porostereum sp. (KCTC18837P) contains the mycelium culture solution and the concentrated solution of the culture solution. In addition, it contains the dried product and extract of the culture solution and the concentrate as an active ingredient.

Example 1. Establishment of optimal culture conditions of Porostereum sp. (KCTC18837P) strain

Example 1.1 Composition of nutrient medium for culture of Porostereum sp. (KCTC18837P) strain

The biological incubator used in this experiment was a 100ℓ biological incubator installed in CL Bio (Yeongdong-gun, Chungbuk). For each experiment, 30 liters of liquid medium containing nutrient medium was autoclaved at 123 ° C and 1.25 bar for 20 minutes, cooled to 23 ° C, inoculated with 600 ml of Porostereum sp. (KCTC18837P) strain, the temperature was 23 ± 1 ° C, and air supply Silver was cultured for 5 days while maintaining 0.2 vvm, 20% dissolved oxygen, and pH 5.5.

Example 1.1.1 Porostereum sp. (KCTC18837P) Determination of carbon source for culturing

The effects of the six most commonly used carbon sources in culturing this microorganism on the growth of Porostereum sp. (KCTC18837P) mycelia were tested. Based on the liquid medium, maltose, glucose, lactose, starch, sucrose, and fructose were added to each 2% (w/v). . Thereafter, 600ml of Porostereum sp. (KCTC18837P) strain was inoculated, and the temperature was 23±1℃, the air supply was 0.2 vvm, the dissolved oxygen was 20%, and the pH was 5.5. Table 1 shows.

As shown in Table 1 below, when starch, glucose, and sucrose were used as carbon sources, the average weight of mycelium increased by 18.6% more than when fructose, lactose, and maltose were used as carbon sources. Therefore, starch, glucose, or sucrose was used alone or in combination as a carbon source of Porostereum sp. (KCTC18837P), and 1% (w/v) starch based on the liquid medium in consideration of the mycelium yield under the above alone or mixed conditions. , sucrose 0.5% (w/v) and glucose 0.5% (w/v) were used in subsequent experiments.

culture water 30 liters composition ratio 2% (W/V) carbon source sucrose glucose starch fructose lactose maltose mycelium weight
(g/100ml) 20.31g 20.36g 21.30g 18.62g 16.23g 17.41g

Example 1.1.2 Porostereum sp. (KCTC18837P) Determination of nitrogen source for culturing

The effects of the six most frequently used nitrogen sources in culturing microorganisms on the growth of Porostereum sp. (KCTC18837P) mycelia were tested. Based on the liquid medium, soybean powder, bovine peptone, yeast extract, ammonium, calcium nitrate, and sodium nitrate were added to each 1% (w/v). Thereafter, 600ml of Porostereum sp. (KCTC18837P) strain was inoculated, and the temperature was 23±1℃, the air supply was 0.2 vvm, the dissolved oxygen was 20%, and the pH was 5.5. Table 2 shows.

As shown in Table 2 below, when soybean powder was used as a nitrogen source, the mycelium weight of Porostereum sp. (KCTC18837P) increased the most, and about 3 times more mycelium was obtained than when ammonium was used.

culture water 30 liters carbon source Starch 1%, sucrose 0.5%, glucose 0.5% (W/V) nitrogen source
composition ratio 1% (W/V) Soybean Powder Soipeeptone yeast extract ammonium calcium nitrate sodium nitrate mycelium weight
(g/100ml) 46.74g 37.45g 44.91g 15.65g 18.43g 17.25g

Example 1.1.3 Mineral crystals for culturing Porostereum sp. (KCTC18837P)

The effects of 6 types of minerals most commonly used in culturing microorganisms on the growth of Porostereum sp. (KCTC18837P) mycelia were tested. Based on a liquid medium containing 1% starch, 0.5% sucrose, 0.5% glucose (W/V), and 1% (W/V) soybean powder, after, KH ₂ PO ₄ , MgSO ₄ , CaCl ₂ , FeSO ₄ , ZnSO ₄ and CuSO ₄ were added to be 0.005% (w/v), respectively, and 600 ml of Porostereum sp. (KCTC18837P) strain was inoculated. The temperature was 23±1℃, the air supply was 0.2 vvm, and the dissolved oxygen was 20%. , cultured for 5 days while maintaining pH 5.5, the mycelium was harvested and the weight was measured and shown in Table 3.

As shown in Table 3 below, KH ₂ PO ₄ , MgSO ₄ , CaCl ₂ , FeSO ₄ , ZnSO ₄ and CuSO ₄ Among Porostereum sp. (KCTC18837P), the increase in mycelium weight was greatest in KH ₂ PO ₄ , MgSO ₄ , and FeSO ₄ conditions, followed by MgSO ₄ 0.003, KH ₂ PO ₄ 0.001%, FeSO ₄ 0.001% (W/V). It was used by adding it to the medium.

culture water 30 liters carbon source Starch 1%, Sucrose 0.5%, Glucose 0.5% (W/V) nitrogen source Soybean powder 1% (W/V) mineral
composition ratio 0.005% (W/V) KH ₂ PO ₄ MgSO ₄ CaCl ₂ FeSO ₄ ZnSO ₄ CuSO ₄ mycelium weight
(g/100ml) 47.65g 48.44g 45.32g 47.11g 46.33g 46.07g

Example 1.1.4 Porostereum sp. (KCTC18837P) Vitamin crystals for culture

Table 5 shows the results of experiments on the effects of the six most commonly used vitamins in culturing microorganisms on the growth of Porostereum sp. (KCTC18837P) mycelium. Other experimental conditions were the same as in Examples 1.1.1 to 4.

As shown in Table 5 below, the increase in mycelium was the largest in vitamin B6 or vitamin B7 conditions among vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B7, and vitamin C.

culture water 30 liters carbon source 2% (W/V) nitrogen source 0.03% (W/V) mineral MgSO ₄ 0.003, KH ₂ PO ₄ 0.001%, FeSO ₄ 0.001% (W/V) vitamin
composition ratio 0.005% (W/V) vitamin A vitamin B1 vitamin B2 vitamin B6 vitamin B7 vitamin C mycelium weight
(g/100ml) 48.44g 48.74g 48.52g 49.03g 49.11g 47.88g

As a result of the experiment, a preferred embodiment for liquid culture of Porostereum sp. (KCTC18837P) mycelium in step (a) is 1% starch (w/v), 0.5% sucrose (w/v), 0.5% glucose as a carbon source (w/v), nitrogen source is soybean powder 1% (w/v), minerals are MgSO ₄ 0.003% (w/v), KH ₂ PO ₄ 0.001% (w/v), FeSO ₄ 0.001% (w/v) ), vitamins are biotin (B7) 0.003% (w / v), pyridoxine (B6) 0.002% (w / v) weight %.

Example 2. Setting of environmental conditions for culture of Porostereum sp. (KCTC18837P) strain

In order to confirm the optimal culture conditions of the Porostereum sp. (KCTC18837P) mycelium of the present invention, starch 1% (w / v), sucrose 0.5% (w / v), glucose 0.5%(w/v), soybean powder 1%(w/v), MgSO ₄ 0.003 %(w/v), KH ₂ PO ₄ 0.001 %(w/v), FeSO ₄ 0.001 %(w) /v), inoculate 600ml of Porostereum sp. (KCTC18837P) strain into a culture medium containing biotin (B7) 0.003 % (w/v) and pyridoxine (B6) 0.002 % (w/v) for vitamins, and the air volume is 02 vvm maintained at 23 °C for 5 days.

Example 2.1. Determination of the agitation rate of the incubator

In mushroom mycelium culture, the rotational speed of the stirrer during culturing of mushroom mycelium is a very important factor in terms of the metabolic activity of the mycelium. In order to investigate the effect of the rotational speed of the stirrer on the mycelium growth, the results of measuring the mycelium amount after culturing while maintaining the agitation speed of the incubator differently are shown in Table 5.

Stirring speed (rpm) Mycelium weight (g/ℓ) 25 5.7 50 7.9 60 8.8 70 8.5 80 7.4 100 5.9

As a result of the experiment, the weight of Porostereum sp. (KCTC18837P) mycelium increased the most at a stirring speed of 60-70 rpm. Accordingly, the stirring speed of Porostereum sp. (KCTC18837P) culture was set to 60-70 rpm.

Example 2.2. Determination of the air supply of the incubator

In order to investigate the effect of the air supply on the growth of Porostereum sp. (KCTC18837P) mycelium, the results of measuring the amount of mycelium after culturing while maintaining the air supply in the incubator differently are shown in Table 6 below.

Air supply (vvm) Mycelium weight (g/ℓ) 0.05 8.5 0.1 8.7 0.2 8.8 0.5 8.6 One 8.4 2 7.9

As a result of the experiment, the increase in the mycelium weight of Porostereum sp. (KCTC18837P) was the largest at an air supply of 0.1 to 0.5 vvm, and then Porostereum sp. (KCTC18837P) was cultured at a stirring speed of 60 to 70 rpm.

Example 2.3. Determination of the incubation temperature of the incubator

In order to investigate the effect of the culture temperature on the mycelium growth, the results of measuring the mycelium amount after culturing while maintaining the temperature of the incubator differently are shown in Table 7 below.

Temperature (℃) Mycelium weight (g/ℓ) 18 6.5 19 6.8 20 7.6 22 8.5 23 8.8 24 8.7 25 8.4 26 6.2 27 3.3 28 1.2

As a result of the experiment, the increase in the mycelium weight of Porostereum sp. (KCTC18837P) was the largest at 22~24℃ in culture temperature. Most preferably, it was 23°C.

Example 2.4. Determination of culture pH in the incubator

When the medium is prepared as in the preferred embodiment, the pH is 6 at this time. In order to investigate the effect of pH on mycelial growth, the amount of mycelium was measured after culturing with sodium hydroxide and glacial acetic acid so that each pH was 4-7. The results are shown in Table 8 below.

pH Mycelium weight (g/ℓ) 4 3.2 4.5 6.1 5 8.6 5.5 8.8 6 8.5 6.5 5.3 7 2.8

As a result of the experiment, the optimal pH of the mycelium of Porostereum sp. (KCTC18837P) was 5-6, and most preferably pH 5.5. The optimum pH for liquid culture of general mushroom mycelium is known to be 4 to 6, but in the case of Porostereum sp. (KCTC18837P) mycelium, at pH 4, the weight of Porostereum sp. wrote Therefore, by establishing the optimal culture pH of Porostereum sp. (KCTC18837P), rapid mass culture of Porostereum sp. (KCTC18837P) became possible.

Example 2.5. Determination of culture period of Porostereum sp. (KCTC18837P) strain

In order to investigate the effect of the incubation period on the mycelium growth, After setting the medium and environmental conditions as in the preferred embodiment, and adjusting the pH to 5.5, the amount of mycelium according to the incubation period was measured. The results are shown in Table 9 below.

incubation period Mycelium weight (g/ℓ) 3 3.7 4 6.1 5 8.6 6 8.8 7 8.7 8 7.9 9 7.2

As a result of the experiment, as shown in Table 9, it was confirmed that the mycelium amount of Porostereum sp. (KCTC18837P) increased rapidly from the 4th day, reached a peak on the 6th day, and then gradually decreased from the 7th day. Therefore, the incubation period is preferably 5-7 days, and most preferably 6 days.

Example 3. Preparation of concentrates and lyophilisates and extraction of active ingredients

The Porostereum sp. (KCTC18837P) mycelium culture was completed, filtered, concentrated, and dried.

Example 3.1 filter press

The culture medium itself is too bulky and easily contaminated with various germs, so it has many problems during commercialization and distribution. Therefore, in order to reduce the volume, it is necessary to go through a process of concentration or drying, and when high-temperature heat is applied in this process, the protein and vitamin components contained in the culture medium are reduced, so a non-heating method must be used. As a non-heating concentration method with high efficiency, there is a method using reverse osmosis (RO). In order to use the reverse osmosis machine, the mycelium in the culture medium and the culture medium must be separated, and for this purpose, a filter press process must be performed. The standard of the filter cloth mounted on the filter press is 0.5 ~ 2.0 ㎛ pore size, and in the case of Porostereum sp. (KCTC18837P) mycelium culture, 1.0 ㎛ pore size is preferable. The filtrate may have a sugar content of 0.5 to 1 brix, a viscosity of 2 to 10 Cp, and a moisture content of 95 to 98%.

Example 3.2 Reverse Osmosis (RO)

By using reverse osmosis, a significant portion of the pure water contained in the culture can be removed, and the culture can be concentrated up to 12 times without applying heat. Before freeze-drying, the volume of the object should be reduced as much as possible. This is because a large-capacity freeze dryer is uncommon, the usage fee is too high, and it takes a long time to dry. The 12-fold concentrate may have a sugar content of 5 to 10 brix, a viscosity of 30 to 100 Cp, and a moisture content of 10 to 20%.

Example 3.3 Lyophilization

The concentrate may be powdered through freeze-drying. The freeze-dried powder is usually obtained in an amount of 1.2 to 1.8% of the weight of the culture medium. Preferably, it is 1.5% by weight of the weight of the culture medium, and the moisture content may be 0.01 to 0.03%.

Example 3.4 Extraction of active ingredients

When it is difficult to directly use the Porostereum sp. (KCTC18837P) mycelium culture or the dried culture material obtained above as a cosmetic material, after extraction with a solvent, extracellular polysaccharide or beta-glucan, which is an active ingredient of the culture according to the present invention, is isolated and a method for preparing it.

The dry powder was stirred by adding 100 ml of distilled water per 2 g of the powder, centrifuged (10,000 rpm, 20 minutes), and then ethanol corresponding to 3 times the amount was added to the supernatant and refrigerated conditions (2-8 ° C). After standing for 16 hours, centrifugation (10,000 rpm, 20 minutes) of the above-mentioned fixed material, the precipitate is taken and freeze-dried to extract extracellular polysaccharide (EPS).

The dry powder was suspended by adding 50 ml of distilled water per 10 g of the powder, and then the pH was adjusted to 10.0 using 20% sodium carbonate at room temperature, and then left for about 10 hours to sufficiently soften the powder sample. To this softened sample solution, α-amylase, which is stable at high temperature, is added, stirred in a constant temperature water bath at 60° C. for 2 hours, treated with enzyme, and then cooled to room temperature. This solution was adjusted to pH 5 and then stirred with amyloglucosidase in a constant temperature water bath at 60° C. for 2 hours. Then, after adjusting the pH to 4.5 in order to inactivate the enzyme, it is stirred in a constant temperature water bath at 95° C. for 30 minutes and then cooled.

The cooled extract is centrifuged (30 min × 8000 rpm), only the supernatant is taken, and then concentrated under reduced pressure at a temperature of 40°C. After adding 4 times the amount of ethanol to this concentrate, leaving it for about 4 hours, and then centrifuging, the precipitate obtained is dissolved again in water, the pH is adjusted to 4.5, and the enzyme is re-treated with amyloglucosidase at 58°C for 2 hours. .

After reprocessing, the enzyme is inactivated at 95°C for 30 minutes, then cooled and centrifuged to take the supernatant and reprecipitate with ethanol to extract crude beta-glucan (β-glucan).

The extract of the mycelium culture of the present invention is not limited to the above method, but according to a conventional method known in the art, hot water extraction, room temperature extraction, warming extraction, ultrasonic extraction, supercritical extraction, etc. It can be prepared by a method.

Example 4. Analysis of main component content of Porostereum sp. (KCTC18837P) mycelium

Porostereum sp. (KCTC18837P) mycelium culture cultured according to the preferred embodiment not only contains complex polysaccharides and beta-glucan, but also contains 5-hydroxy-6,7-dimethoxyphthalide, vitamins, magnesium, Calcium can be produced. Component analysis was conducted at the Korea Functional Food Research Institute, and the results are shown in Table 10.

ingredient content beta glucan 34.23 (%/g) extracellular polysaccharide 43.11 (%/g) 5-hydroxy-6,7-dimethoxyphthalide 29.51 (μg/g) vitamin 1.34 (%/g) magnesium 0.52 (%/g) calcium 0.74 (%/g)

Through this, the Porostereum sp. (KCTC18837P) mycelium culture of the present invention contains a large amount of physiologically active substances such as beta-glucan extracellular polysaccharide, 5-hydroxy-6,7-dimethoxyphthalide, vitamins, magnesium, and calcium. confirmed that. It has been reported that many kinds of mushroom-derived polysaccharides have anticancer and antioxidant activities, and the present inventors confirmed the antidiabetic effect of Porostereum sp. (KCTC18837P) mycelium culture containing a large amount of physiologically active substances.

Example 5. Antidiabetic efficacy test

Diabetes is a collective term for a series of metabolic diseases characterized by high blood sugar. Diabetes is caused by a complex interaction of genetic factors and environmental factors, although the etiology differs according to the detailed classification. Mechanisms leading to hyperglycemia include impaired insulin secretion, impaired glucose utilization in peripheral tissues, and excessive gluconeogenesis in the liver. The two largest classes of diabetes are type 1 and type 2 diabetes. Type 1 diabetes is characterized by an absolute deficiency of insulin due to destruction of pancreatic beta cells, and type 2 diabetes is characterized by varying degrees of beta cell dysfunction on top of insulin resistance. In diabetes, disorders of various metabolic pathways including glucose cause various microvascular complications such as kidney, retina, and nerve, as well as complications such as coronary artery disease and stroke (Cheol Soo Choi et al., The mechanism of development of insulin resistance in type) 2 diabetes mellitus, 2002, Korean Journal of Medicine, 63(6): 613-624).

Currently used blood sugar-lowering drugs are divided into oral preparations and injections. Oral drugs include sulfonylureas and metiglitinides that promote insulin secretion, metformin that inhibits hepatic glucose production and improves peripheral insulin sensitivity, α-glucosidase inhibitor that inhibits carbohydrate absorption in the intestinal tract, and mainly improves insulin sensitivity. These include thiazolidinediones, which use incretin hormones to inhibit DPP-4, and SGLT-2 inhibitors to inhibit glucose reabsorption in the kidneys. In addition, injections contain GLP-1 receptor agonists similar to insulin and incretin hormones (Guidelines for the treatment of type 2 diabetes, 2017, Korean diabetes association, 1-15).

This test was carried out to evaluate the antidiabetic effect after repeated oral administration of the dried Porostereum sp. (KCTC18837P) mycelium culture to db/db mice for 3 weeks.

The efficacy evaluation test was conducted at Chemon Nonclinical Research Institute, which is a non-clinical GLP testing institution of the Ministry of Food and Drug Safety.

Example 5.1 Test Preparation

Example 5.1.1 Preparation of materials and test substances

The test substance was prepared by weighing the amount of the test substance in the high-dose group and adding an excipient to the suspension. The intermediate dose group and the low dose group were diluted stepwise to prepare a suspension. The control substance administration group weighed the control substance and added an excipient to prepare suspension. Methods for preparing test materials, control materials and excipients are shown in Table 11 below.

substance name supplier test substance Porostereum sp. culture dry matter CL Bio Co., Ltd. control material Diabex Tablets (500mg) Daehan Pharmaceutical Co., Ltd. excipient sterile water Daehan Pharmaceutical Co., Ltd.

Example 5.1.2 Test system and breeding environment

Specific pathogen-free (SPF) db/db mice and C57BLKS/J-db/db were used as test animals (Table 12). The animals were acclimatized for 12 days after acquisition, and general symptoms were observed at least once a day. During the acclimatization period, the buoyancy method using red oily magic was used for identification. During the administration and observation period, it was identified using the unbuoyed method using black oily magic. An individual identification card for distinguishing capacity by color was attached to the breeding box, and a unique number was assigned to the breeding box. At the entrance to the breeding room, an animal room usage record was attached. Animals are kept at a temperature of 23 ± 3 °C, relative humidity of 55 ± 15 %, ventilation frequency of 10 - 20 times/hr, illumination time of 12 hours (lights at 8 am - lights out at 8 pm), and illuminance of 150-300 Lux. They were bred in room 1 of the animal breeding area of the center. Temperature and relative humidity were measured every hour using a computer system, and the number of ventilation and illuminance were measured regularly. There were no abnormalities that could affect the test results during the experiment period.

species lineage Pathogen-free (SPF) db/db mice, C57BLKS/J-db/db supplier Central Laboratory Animals Co., Ltd. (Seoul) number of animals 50 males week old 7 weeks old weight range 32.66 - 39.90 g

For the feed, solid feed for rodents (Teklad certified irradiated global 18 % protein rodent diet; 2918C, ENVIGO, UK) was supplied from Dooyeol Biotech (Seoul Metropolitan City) and allowed to be freely consumed. As for water, tap water disinfected with an ultraviolet sterilizer and microfiltration device was placed in a polycarbonate drinking water bottle and allowed to be consumed freely. The water quality test was conducted by the Gyeonggi-do Institute for Health and Environment (Gyeonggi-do) and was suitable for drinking water quality standards. The rug was used after autoclaving. As a result of the pollutant test, there were no abnormalities that could affect the test results. Breeding box and breeding density Animals were housed in a polycarbonate breeding box (W 170 × L 235 × H 125 mm) containing an appropriate amount of rug at 2 animals/ breeding box during all test periods.

Example 5.1.3 Test group composition, group separation and substance administration

Group separation of animals was carried out as shown in Table 13 below. After specifying and ranking the weights of animals, they were randomly distributed so that the average weight of each group was distributed as evenly as possible according to the ranked weights. The frequency of administration was once a day, 7 times a week for 3 weeks. The dose amount was calculated as 10 mL/kg/day based on the measured body weight.

army number of animals animal number dose amount
(mL/kg/day) Dosage
(mg/kg) dosing substance route of administration G1 male 10 1-10 10 0 excipient oral administration G2 male 10 11-20 10 500 Porostereum sp. (KCTC18837P) G3 male 10 21-30 10 750 Porostereum sp. (KCTC18837P) G4 male 10 31-40 10 1,000 Porostereum sp. (KCTC18837P) G5 male 10 41-50 10 400 Diabex Tablet 500mg

* G1: excipient control group, G2-G4: test substance administration group, G5: control substance administration group Example 5.2. conduct of the test

Example 5.2.1 Observation and Inspection

For the observation of general symptoms, the status of death, type of general symptoms, onset date, and severity of symptoms were observed once a day during administration and observation period and recorded for each individual. The administration start date was set to Day 1. Body weight was measured at the time of acquisition, at the time of group separation, at the start of administration, and at the time of autopsy. Otherwise, it was measured at least once a week. Feed intake was measured once a week during the administration period. As for the measurement method, the difference was calculated by measuring the remaining amount of feed in units of breeding boxes the day after quantitative feeding.

Example 5.2.2 Blood glucose measurement

Animals were fasted (water provided) for about 6 hours prior to blood glucose measurement. Blood glucose was measured using a blood glucose meter (G-Doctor, Green Cross) on the measurement day, and was measured once a week.

Example 5.2.3 Oral glucose tolerance test (OGTT)

(1) Fasting (water provided) for about 16 hours the day before OGTT.

(2) Fasting body weight was measured on the day of OGTT measurement, and blood glucose was measured before administration of the test substance (-30 min).

(3) After 30 minutes of oral administration of the test substance, blood glucose was measured immediately before glucose administration (0 min).

(4) After oral administration of glucose at a dose of 2 g/10 mL/kg, blood was collected from the caudal vein at 30, 60, 90, 120 and 240 min, and blood glucose was measured using a blood glucose meter.

Example 5.2.4 Autopsy and histopathological examination

Autopsies were performed on all surviving animals. At the time of autopsy, the animals were anesthetized by inhalation with isoflurane, followed by laparotomy and blood collection from the posterior vena cava. After blood collection, the animals were killed by excision of the abdominal aorta and posterior vena cava. The thymus, spleen, and pancreas of the animal confirmed to have completely died due to exsanguination were removed and weighed. The pancreas was fixed in 10% neutral buffered formalin solution. At the time of autopsy, all organs were observed for abnormalities and autopsy findings were recorded.

For histopathological examination, the trimming step of pancreatic tissue fixed in 10% neutral formalin was performed as follows. For general histopathological examination, H&E stain was performed, and the number of pancreatic islet numbers (pancreatic islet numbers, islets/cm2 of pancreatic tissues) and the diameter of pancreatic islets (diameters, μm), the ratio of zymogen granules per unit area of the exocrine part (zymogen) granule %/mm2 of exocrine pancreas) was analyzed, and the image analysis program (iSolution EL ver 9.1, IMT i-solution Inc., Quebec, Canada) was used for image analysis.

Example 5.2.5 Clinical Pathology

1) blood collection

They fasted (water was provided) for about 16 hours the day before necropsy. More than 200 μL of blood collected was injected into a tube containing EDTA, an anticoagulant, and kept refrigerated until delivery, and then delivered for HbA1c analysis during blood biochemical tests. The remaining blood was placed in a vacutainer tube containing a clot activator and left at room temperature for more than 30 minutes, followed by centrifugation at 3000 rpm for 10 minutes. The serum obtained by centrifugation was transferred to a new tube and stored in a deep freezer until blood biochemical testing.

2) Blood biochemical test

Whole blood and serum separately stored for blood biochemical testing were tested for the following items using a blood biochemical analyzer. Triglycerides (TG), total cholesterol (TCHO), aspartate aminotransferase (AST), high-density lipoprotein (HDL), low-density lipoprotein (LDL), alanine aminotransferase (ALT), glycated hemoglobin (HbA1c).

3) Measurement of blood insulin and c-peptide

Insulin and c-peptide were quantitatively analyzed using ELISA kits (Shibayagi, AKRIN-011T and AKRCP-031) for each measurement in serum separated for analysis.

Example 5.3 Statistical Analysis and Results

For measurement results, SPSS statistics 22 for medical science was used, and Student's t-test and ONE-WAY ANOVA were used to check whether there was a difference between groups. Comparison between the excipient control group, the test substance administration group, and the control substance administration group was performed, and when the significance level was P<0.05, it was determined to be statistically significant.

The test results are as follows. As a result of observation of general symptoms during the test period, no abnormal symptoms or death animals were observed due to the test substance. However, in the control group (G5), 1 death on DAY 17 and 6 deaths on DAY 19 were observed.

As a result of body weight measurement, no significant change in body weight was observed by administration of the test substance and control substance compared to the excipient administration group (G1).

As a result of the measurement of feed intake, no significant change in feed intake was observed by administration of the test substance and control substance compared to the excipient administration group (G1).

As a result of blood glucose measurement, as shown in Table 14 below, a significant decrease (P<0.05) or a decrease trend was observed by the administration of the test substance compared to the excipient administration group (G1). A significant decrease (P<0.05) or a decreasing trend was observed on days 8, 15, and 21 in the control group (G5) compared to the excipient administration group (G1).

BLOOD GLUCOSE LEVELS (mg/dL) GROUPS 1 day 8day 15day 21day G1 378.5 ± 27.5 467.1 ± 24.4 563.7 ± 17.6 519.4 ± 12.5 G2 429.2 ± 26.1 448.7 ± 22.7 540.1 ± 19.5 486.8 ± 40.0 G3 347.1 ± 22.9 432.4 ± 13.7 527.0 ± 11.7 464.5 ± 14.4* G4 417.1 ± 40.7 416.7 ± 23.2 506.6 ± 14.5 430.1 ± 22.0* G5 355.0 ± 22.3 366.5 ± 36.9* 503.8 ± 23.3 438.7 ± 47.8* *: significantly different from G1, P <0.05

As a result of OGTT measurement, as shown in Table 15 below, a significant decrease in blood glucose was observed in the excipient administration group (G1) compared to the test substance 1000 mg/kg administration group (G4) (P<0.05). A significant decrease in blood glucose was observed in the control group (G5) compared to the excipient group (G1) (P<0.05 or P<0.01).

BLOOD GLUCOSE LEVELS (mg/dL) GROUPS 0min 30min 60min 90min 120min 240min G1 486.9±20 637.4±15 620.5±13 619.2±20 581.0±28 391.7±39 G2 556.6±22 678.6±13 617.2±22 594.2±23 456.9±18 388.2±30 G3 504.6±22 664.2±18 604.9±12 536.1±12 435.9±22 347.8±43 G4 573.5±29 705.9±19 575.7±11 533.1±12 389.1±15* 262.8±32* G5 369.4±10** 421.4±29** 334.2±23** 246.2±26** 206.8±18** 109.0±12** *: significantly different from G1, P <0.05
**: significantly different from G1, P <0.01

As a result of organ weight measurement, no significant change in weight was observed in both the test substance administration group (G2-G4) and the control substance administration group (G5) compared to the excipient administration group (G1).

3 shows significant changes in the number of pancreatic islets (islets/cm2), diameter (μm), and the ratio of zymogen granules per unit area of exocrine (zymogen granules %/mm2 of exocrine) by the test material as a result of autopsy of the experimental animals. It is a drawing that shows Histopathological examination results (Table 16), the number of pancreatic islets (islets/cm2), diameter in the test substance 500 mg/kg administration group (G2) and the test substance 750 mg/kg administration group (G3) compared to the excipient administration group (G1) (μm) and the ratio of zymogen granules per unit area of exocrine (zymogen granules %/mm2 of exocrine) were not observed to change significantly, but the number of pancreatic islets (islets/cm2) in the test substance 1000 mg/kg administration group (G4). ), a significant decrease in diameter (μm) and a significant increase in the ratio of zymogen granules per unit area of exocrine (zymogengranules %/mm2 of exocrine) were observed (P<0.01). Significant decrease in the number of pancreatic islets (islets/cm2) and diameter (μm) in the control group (G5) compared to the excipient group (G1) and the ratio of zymogen granules per unit area of the exocrine (zymogen granules %/mm2 of exocrine) A significant increase was observed (P<0.05 or P<0.01).

HISTOMORPHOMETRICAL ANALYSIS GROUPS PANCREATIC ISLETS ZYMOGEN GRANULES
(%/㎟ of exocrine pancreas) NUMBERS
(islets/cm2) DIAMETERS
(μm) G1 14.40±1.19 166.58±9.93 26.00±1.71 G2 14.10±1.29 165.75±13.71 28.07±1.91 G3 13.30±1.20 162.09±8.92 30.19±1.73 G4 6.10±0.57** 123.29±6.66** 35.25±1.35** G5 5.67±0.88** 117.72±10.26* 41.39±1.02** *: significantly different from G1, P <0.05
**: significantly different from G1, P <0.01

As a result of the blood biochemistry test, as shown in Table 17 below, a tendency to decrease HbA1c (%) was observed in the test substance administration group (G2), (G3) compared to the excipient administration group (G1), (P=0.076), TCHO, TG , LDL, and HDL were not observed, but a significant decrease in AST (P<0.01) and a decrease in ALT were observed.

A significant decrease ((P<0.01)) of HbA1c (%) was observed in the test substance administration group (G4) compared to the excipient administration group (G1), and changes in TCHO, TG, LDL, and HDL were not observed, but significant in AST and ALT. A significant decrease (P<0.01) was observed.

In the control group (G5) compared to the excipient-administered group (G1), a tendency to decrease HbA1c (%) (P=0.076), and a significant decrease in TCHO and HDL (P<0.01 or P<0.05) were observed, and TG, LDL, No significant differences were observed in AST and ALT.

CLINICAL BLOOD BIOCHEMISTRY GROUPS IN WHOLE
BLOOD IN SERUM HbA1c (%) TCHO
(mg/dL) TG
(mg/dL) LDL
(mg/dL) HDL
(mg/dL) AST
(U/L) ALT
(U/L) G1 7.6±0.2 135.8±2 128.6±1 27.2±1 105.5±2 120.9±8 72.2±6 G2 7.2±0.2 144.0±9 132.4±8 30.8±3 108.3±6 86.4±5** 58.0±4 G3 7.0±0.1 145.2±5 140.6±1 32.0±2 110.6±3 85.7±5** 55.7±4 G4 6.5±0.2* 130.6±5 154.8±1 26.0±1 100.8±4 75.0±6** 49.4±4** G5 6.8±0.5 115.3±1* 147.3±1 24.7±3 87.4±8** 115.6±3 71.2±6 *: significantly different from G1, P <0.05
**: significantly different from G1, P <0.01

As a result of blood insulin measurement, as shown in Table 18 below, a decreasing trend was observed in the excipient administration group (G1) compared to the 750 mg/kg administration group (G3) and the 1000 mg/kg test substance administration group (G4), but it was not significant. , A decreasing trend was observed in the control group (G5) compared to the excipient group (G1), but it was not significant.

GROUPS SERUM INSULIN LEVELS (ng/mL) G1 2.940 ± 0.503 G2 3.307 ± 0.319 G3 2.518 ± 0.304 G4 1.919 ± 0.269 G5 1.920 ± 0.528

As a result of blood c-peptide measurement, as shown in Table 19 below, a significant decrease was observed in the excipient administration group (G1) compared to the 750 mg/kg administration group (G3) and the 1000 mg/kg test substance administration group (G4) ( P<0.01 or P<0.05). A decreasing trend was observed in the control group (G5) compared to the excipient group (G1), but it was not significant (P = 0.07)

GROUPS SERUM C-PEPTIDE LEVELS (pg/mL) G1 1007.3 ± 146.8 G2 910.0 ± 77.4 G3 716.1 ± 78.9* G4 615.2 ± 77.3** G5 440.8 ± 129.2 *: significantly different from G1, P <0.05
**: significantly different from G1, P <0.01

Example 5.4. Considerations and Conclusions

This test was performed to evaluate the antidiabetic effect after repeated oral administration of Porostereum sp. (KCTC18837P) culture to db/db mice for 3 weeks.

In the histopathological examination results, in the group administered with the test substance 1000 mg/kg, the effect of inhibiting the morphological change of the pancreatic endocrine gland by a significant decrease in the number (islets/cm2) and diameter (μm) of the pancreatic islets and the enzyme source granules per unit area of the exocrine part It is considered that there is a possibility of improving the control of pancreatic digestive enzyme secretion by a significant increase in the ratio (zymogen granules %/mm2 of exocrine).

In the blood biochemical test results, a decrease in HbA1c (%) was observed in (G2) and (G3) of the test substance administration group, and a significant decrease was observed in the (G4) group, which was judged to have an effect of improving blood sugar. No changes in TCHO, TG, LDL or HDL were observed. However, as a significant decrease in AST and a decrease in ALT were observed, it is judged that the test substance does not affect liver function.

The tendency to decrease blood insulin in the test substance 750 mg/kg administration group and the test substance 1000 mg/kg administration group and the significant decrease tendency of blood c-peptide in the test substance 750 mg/kg administration group and the 1000 mg/kg test substance administration group A decrease was observed, which is judged to have improved insulin resistance by the test substance.

In conclusion, when Porostereum sp. (KCTC18837P) mycelium culture was repeatedly orally administered to db/db mice for 3 weeks, a significant decrease was observed in the test substance 1000 mg/kg administration group (G4) in blood glucose measurement and OGTT measurement. In addition, the long-term (21-day) efficacy of the test substance was observed to be superior to that of the short-term (24-hour experiment) efficacy, and on the 21st day, (G4) of the test group showed better blood sugar improvement than the control substance. In the pancreatic histopathological examination, a significant decrease in the number and diameter of pancreatic islets and a significant increase in the ratio of enzyme source granules per unit area of the exocrine part were observed in the test substance 1000 mg/kg administered group (G4), and the test substance 750 mg/kg/kg was observed. A decrease trend in serum insulin and a significant decrease in c-peptide were observed in the kg and 1000 mg/kg administration groups, and it is judged that the test substance has a diabetes improvement function.

As described above, in the case of the control substance administration group during the test period, 7 out of 10 test animals died, whereas in the Porostereum sp. As a result, it was confirmed that it was a very safe material. In addition, as a result of the 21-day long-term test, it was confirmed that (G4) of the test substance administration group had a better blood glucose improvement effect than the control substance. Therefore, it was confirmed that Porostereum sp. (KCTC18837P) mycelium culture was an effective agent for the prevention and improvement of diabetes.

Various modifications and applications of the present invention described above are possible by those skilled in the art to which the present invention pertains, and the scope of the technical idea according to the present invention should be defined by the following claims.

Claims (7)

New strain Porostereum sp. (KCTC18837P). Mycelium culture of the new strain Porostereum sp. (KCTC18837P). 3. The method of claim 2,
The mycelium culture is a mycelium culture of the new strain Porostereum sp. (KCTC18837P), characterized in that it comprises beta-glucan, extracellular polysaccharide, 5-hydroxy-6,7-dimethoxyphthalide. A pharmaceutical composition for preventing or improving diabetes, comprising the new strain Porostereum sp. (KCTC18837P) or a mycelium culture thereof as an active ingredient. 5. The method of claim 4,
The prevention or improvement of diabetes is a pharmaceutical composition for preventing or improving diabetes, characterized in that it comprises one of a decrease in HbA1c (%) in blood, a decrease in blood insulin, a decrease in blood c-peptide, a decrease in blood sugar, and a decrease in OGTT. A health functional food for preventing or improving diabetes, comprising a culture obtained by culturing the new strain Porostereum sp. (KCTC18837P) mycelium. Based on the total amount of liquid medium, starch 0.2 ~ 2 % (w/v), sucrose 0.2 ~ 2 % (w/v), glucose 0.2 ~ 2 % (w/v), soybean powder 1.5% (w/v), FeSO ₄ 0.001% (w/v), KH ₂ PO ₄ 0.001% (w/v), MgSO ₄ 0.003% (w/v), vitamin B7 0.003% (w/v), vitamin B6 0.002% (w/v) ), the new strain Porostereum sp, characterized in that in a liquid medium having a pH of 5 to 6, the air supply is 0.02 to 1 vvm, the stirring speed is 25 to 100 rpm, and the culture temperature is 18 to 27 ° C. for 5 to 7 days. .(KCTC18837P) or a method for mass production of a mycelium culture thereof.

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