KR101969433B1 - Composition comprising product of two stage cultivation using Ganoderma applanatum and Ceriporia lacerata K1 mycelium for preventing or treating diabetes mellitus - Google Patents

Abstract

The present invention relates to a composition for preventing or treating diabetes including a product of two-stage cultivation using Ganoderma applanatum and Ceriporia lacerata K1 mycelium as ingredients. Based on a comparison and analysis result between the product of the two-stage cultivation using Ganoderma applanatum and Ceriporia lacerata K1 mycelium and a product of single-stage cultivation using Ganoderma applanatum or Ceriporia lacerata K1 mycelium, the content of β-glucan and exopolysaccharide in a two-stage cultivation product is remarkably increased compared with that of each single-stage cultivation product, and triterpenoid saponin is newly created and included. Moreover, based on a checking result of a diabetes curing effect of such two-stage cultivation product, an excellent effect is confirmed compared with the single-stage cultivation product. Thus, the product of the two-stage cultivation using Ganoderma applanatum and Ceriporia lacerata K1 mycelium can be useful in development of a diabetes preventing or treating medical product, and healthcare food for mitigating diabetes.

Description

[0001] The present invention relates to a composition for preventing or treating diabetes mellitus comprising as an active ingredient a two-stage cultured product obtained by using a mycelium of Kannabi broth and Sella liparia lacerata K1 (hereinafter referred to as " K2 mycelium for preventing or treating diabetes mellitus }

The present invention relates to the use of Ganderma applanatum and Ceriporia lacerata K1 K1) mycelium as an active ingredient. The present invention also relates to a composition for preventing or treating diabetes.

In addition, the present invention relates to a two-step culture method using Kannabibi bulrocho and Klebsiella la Serata K1 mycelium.

Mushrooms are rich in carbohydrates, proteins, lipids, minerals and vitamins, and contain large amounts of bioactive substances such as β-glucan and have long been used as edible and medicinal materials (Wani, BA, et al., 2010; Yu, HE, et al., 2006). (Kath, KS et al., 2006), cholesterol lowering (Caz, V., et al., 2015; Khatun, K. et al., 2007) (Padmavathy, M., et al., 2014) have been reported, and they have been used as various functional materials. Recently, , Environmental pollution and increased stress, and as a result, the mushrooms are also increasingly becoming an important health food or medicinal food.

Ganoderma applanatum ( Ganoderma applanatum ) is a medicinal mushroom, a perennial wood rot fungus that occurs mainly in broad-leaved trees. The mushroom shrub is semicircular horseshoe shaped and has a conical shape as a whole. The surface texture is covered with blackish brown hairs, and the surface of the mushroom is yellowish white. When it is rubbed, it is browned and becomes black by alkali. Manganese manganese has been used as an antidiabetic, anticancer and anti-diabetic drug in Korea, China and Japan for a long time. It contains an effective ergostaine steroid compound for diabetes treatment and polysaccharide such as β-glucan effective for cancer treatment. Was reported. In addition, in recent years, it has been reported that the extract of L. manganica has stem cell proliferation and skin cell proliferation effect (Korean Patent Publication No. 2017-0025352).

Ceriporia lacerata was first discovered in Japan in 2003 by a polyporales pigmented mushroom and Phanerochaetaceae mushroom, and its existence was reported to world academia (Shin, EJ, et al., 2016). It is a kind of white rot fungus which is a by-product of oak and chrysanthemum. It decomposes cellulose, hemicellulose, lignin and so on of wood. It is a kind of plant extract of Celiporria rera cerata (Korean Patent No. 1721836), anti-diabetic (Shin, EJ, et al., 2015; Shin, EJ, et al., 2016), liver function improvement (Korean Patent Publication No. 2016-0008425) Improvement of lipid metabolism (Korea Patent Publication No. 2016-0008431), and the like.

Recently, most mushroom cultivars have been capable of culturing mycelium containing various physiologically active substances through bioconversion using nutrients contained in liquid medium (Tang, Y.J., et al., 2007). Liquid culture of mushroom mycelium is to propagate mycelium, which is a nutrient generation in mushroom life, by liquid culture method. The liquid medium for mycelial cultivation contains a carbon source such as glucose and starch, a nitrogen source of various inorganic forms, a complex nitrogen source of an organic form, inorganic salts and trace elements, vitamins and the like which can grow mycelium in the medium. Since the culture characteristics of mushroom mycelium depend on the composition of the medium, the culture condition, and the type of mushroom mycelium, the culture conditions suitable for each mushroom must be determined in order to improve the liquid culture efficiency of the mycelium. In addition, a liquid culture containing mushroom mycelium can obtain various metabolites having activities such as anticancer and antioxidation depending on the nutrient composition and culture conditions of the medium (Lee, Y., et al., 2008).

In recent years, dual fermentation technology using microorganisms has been developed from the application of vinegar (acetic acid) fermentation. The vinegar is firstly hydrolyzed by glucose in yeast to produce alcohol, and the alcohol is oxidized by acetic acid bacteria to become vinegar. That is, the alcohol as a result of the primary fermentation is converted into a completely different substance called vinegar due to the secondary fermentation. Such a two-stage fermentation method of the food is a very useful technique for increasing the functionality of the food or changing the ingredient Respectively.

Although slightly different, two-stage cultivation of mushroom mycelium is also different from that of single culture. Furthermore, when two or more different mycelium of different mushrooms are successively cultured by two successive steps, the difference is even greater. Therefore, the two-stage culture or multi-stage cultivation method of the mycelium of mushrooms has the effect of fusing and enhancing the functional components of each mushroom and is also very effective for the purpose of producing a novel substance. However, no studies have been conducted on the mycelial cultivation of mycelia until now, and there have been no studies on component analysis and physiological activity of the products produced by the two-stage cultivation.

As a result of the single cultivation or double cultivation using the mycelia of Kannabibi bulrocho and Klebriella la Serratia K1 of the present invention, the content of the β-glucan and the extracellular polysaccharide was found to be a single culture of mycelium It was confirmed that triterpenoid saponin was newly produced in the two-stage cultures as compared to water. Further, as a result of confirming the diabetic therapeutic effect of such a two-stage cultured product, the present invention can be completed by confirming that it is superior to the sole culture product.

Conventionally, Korean Patent No. 1444614 discloses a mixed culture using seripolylaceratara. However, it is a method of producing a fermented product in which GABA is enhanced through mixed culture with lactic acid bacteria, There is a difference in the constitution and effect from the culture obtained by the two-step cultivation using the Mycelium sp. Kanabi rosso and the three cultivars K1 mycelia and the triterpenoid saponin newly produced therefrom. Korean Patent No. 1406123 discloses a method for producing a functional fermentation product in which an active ingredient is produced through two-stage fermentation using microorganisms. Korean Patent No. 1106668 discloses a method for producing a fermentation product using two-stage fermentation of plant lactobacillus, Bacillus subtilis and lactic acid bacteria or vegetable lactic acid bacteria are used as microorganisms, and the constitution is different from that of the two-stage cultivation using the kanamycin of Kjellmaniella ceratata and the Kjellariella ceratata mycelium of the present invention.

Korean Patent Laid-Open Publication No. 2017-0025352, composition for improving skin, 2017. 03. 08. disclosed. Korean Patent No. 1721836, a composition for prevention and improvement of various cancers and cancer, which contains an active ingredient of a culture medium of Sellapori racerata, and a method for producing the composition, 2017. 03. 27. Registration. Korean Patent Laid-Open Publication No. 2016-0008425, a composition for improving liver function containing an extracellular polysaccharide derived from an extract of a culture medium of Sera liparia lacerata as an active ingredient. Korean Patent Laid-Open Publication No. 2016-0008431, a composition for improving lipid-related metabolism containing an extracellular polysaccharide derived from a culture extract of Sellapori racerata as an active ingredient. Korean Patent No. 1444614, a method for producing a fermented product in which GABA is promoted by mixed cultivation of Seripolyla lucerata and lactic acid bacteria. 2014. 09. 18. Registration. Korean Patent No. 1406123 discloses a method for producing a fermented product in which γ-PGA and GABA are enhanced through two-stage fermentation using Bacillus subtilis and lactic acid bacteria. 2014. 06. 03. Registration. Korean Patent No. 1106668, Process for the production of natural Gabba fermented product by two-stage fermentation of vegetable lactic acid bacteria having protease producing ability and GABA production ability. Jan. 10, 2012 Registration.

Liquid culture of mushroom mycelium and production of useful materials, National Forestry Academy Research Report 08-07, National Forestry Academy, 2008. Caz, Z., et al., Modulation of Cholesterol-Related Gene Expression by Dietary Fiber Fractions from Edible Mushrooms, J. Agric. Food Chem., 63 (33), 7371-7380, 2015. Khatun, K., et al., Oyster mushroom reduced blood glucose and cholesterol in diabetic subjects, Mymensingh Med. J., 16 (1), 94-99, 2007. Kidd, P. M., The use of mushroom glucans and proteolycans in cancer treatment, Altern. Med. Rev., 5 (1), 4-27, 2000. Lee, YS, et al., Analysis of Nutritional Components of Lepista nuda, Korean Journal of Food Preservation, 13 (3), 375-381, 2006. Padmavathy, M., et al., Antimicrobial Activity of Mushrooms against Skin Infection Causing Pathogens, Research in Biotechnology, 5 (2), 22-26, 2014. Shin, E.J., et al., Hypoglycemic effects of submerged culture of Ceriporia lacerata mycelium, Korean J. Food. Preserv., 22 (1), 145-153, 2015. Shin, E. J., et al., Effect of Submerged Culture of Ceriporia lacerata Mycelium on Insulin Signaling Pathway in 3T3-L1 Cell., Journal of Life Science, 26 (3), 325-330, 2016. Tang, Y. J., et al., Submerged Culture of Mushrooms in Bioreactors-Challenges, Current State-of-the-Art, and Future Prospects, Food Technol. Biotechnol., 45 (3), 221-229, 2007. Wani, B. A., et al., Nutritional and medicinal importance of mushrooms, J. Med. Plants Res., 4 (24), 2598-2604, 2010. Yu, H.E., et al., Screening of bioactive compounds from mushroom Pholiota sp., The Korean Journal of Mycology, 34 (1), 15-21, 2006.

An object of the present invention is jannabi bulrocho (Ganderma applanatum) and three other reports Ria la sera K1 (Ceriporia lacerata K1) mycelium as an active ingredient. The present invention also provides a composition for preventing or treating diabetes.

It is also an object of the present invention to provide a two-step cultivation method using Kannabibi bulrocho and Klebsiella lucerata K1 mycelium.

The present invention relates to the use of Ganderma applanatum and Ceriporia lacerata K1 K1) mycelia as an active ingredient, and to a health functional food for improving or preventing diabetes.

The cultured product obtained by the two-step cultivation may be a culture obtained by inoculating K1 mycelium with K1 mycelium into a primary culture obtained by inoculating mycelium of Mycobacterium tuberculosis.

The cultured two-stage culture may contain triterpenoid saponin.

The present invention also provides a method for preparing a liquid culture medium for culturing Mycobacterium tuberculosis mycelium; A second step of inoculating and cultivating the Mycobacterium tuberculosis mycobacteria in the liquid medium of step 1 to secure a culture medium of Mycobacterium tuberculosis mycelium; 3) adding a carbon source and a nitrogen source to cultivate Celpolaris la Serratia K1 mycelium and culturing the medium for sterilization and cooling to prepare a liquid culture medium for the secondary culture; And a fourth step of inoculating and culturing the mycelium of Seripolyla cerarata K1 in the liquid medium of step 3; And a method for two-step cultivation of K1 mycelium broth and Celporia la Serratia K1 mycelium.

Hereinafter, the present invention will be described in detail.

The present invention relates to a culture obtained by two-stage cultivation of mycelia broccoli and mycelium of Cerporaria la Serera K1.

The present invention relates to a medicinal mushroom which has been used as an antidiabetic, anticancer and anticancer drug, and which has been distributed at the National Institute of Agricultural Science and Technology (National Institute of Agricultural Science and Technology) (Accession No. KACC 50174).

The above-mentioned Sera liparia la Serratta K1 was obtained by cross-breeding method using conventional Sella liparia la Serratia strains as a parent strain, and the inventors of the present invention have found that the Sella lipara serarata strain CL-KU and CL-KS as the parent strains, and deposited with the National Institute of Agricultural Science and Technology, Microorganism Bank under accession number KACC 83018BP.

The cross breeding method is not limited, and various crossing methods known in the summer and those skilled in the art can be used. Preferably a mononuclear-mononuclear crossing method and a binuclear-mononuclear crossing method. This monoclonal-monoclonal crossing method is a method of replacing two different hybrid mononuclear mycelia by a distance of about 2 to 3 cm and allowing hyphae grown on both sides to cross each other, The di-mono crossing method is a method in which the nucleus mycelium and the mononuclear mycelium of another strain are substituted with each other and the mononuclear mycelium migrates into the nucleus mycelium to perform crossing.

The CL-KU was collected on July 24, 2013 at the Sohwang-ri, Kumgang-Song, Uljin-myeon, Gyeongsangbuk-do. The fruiting bodies are yellowish-red.

The CL-KS was collected on September 15, 2010 in Sogong-ri, Hadong-myeon, Sangju, Gyeongsangbuk-do. Fruiting body is reddish white.

The growth rate of mycelial growth of mycelial growth factor K1 was more than 30% higher than that of the parent strain and the aging speed was more than 2 times slower. In addition, it is highly resistant to contamination of bacteria during culture of the strain, and it is possible to overcome the low culture success rate due to the contamination caused by the germs in the cultivation of the existing mycelium, and thus it is possible to industrialize highly economically.

The mycelium is formed by entanglement of hyphae in the process of mycelium continuing to feed nutrients, and contains various useful substances. The mycelium derived from the novel strain of Seroliaracea K1 of the present invention does not show a floating strain in the mycelial growth step, and the mycelium is thick and dense.

The 'two-step cultivation' of the present invention is a two-stage cultivation process using microorganisms of a useful component. The two-stage culture is used to fuse and increase the functional components of each microorganism, .

The two-stage cultivation is carried out by dividing the mycelium of different matures by two stages. Preferably, the mycelium is cultivated by inoculating mycelium of Mycobacterium tuberculosis and inoculated with mycelium of Klebriela rera rata K1 to secondary culture.

More specifically, the two-step culture comprises a first step of preparing a liquid medium for culturing mycelium of Mycobacterium tuberculosis; A second step of inoculating and cultivating the Mycobacterium tuberculosis mycobacteria in the liquid medium of step 1 to secure a culture medium of Mycobacterium tuberculosis mycelium; 3) adding a carbon source and a nitrogen source to cultivate Celpolaris la Serratia K1 mycelium and culturing the medium for sterilization and cooling to prepare a liquid culture medium for the secondary culture; And a fourth step of inoculating and culturing the mycelium of Seripolyla cerarata K1 in the liquid medium of step 3; . ≪ / RTI >

The liquid medium of step 1 may be a medium which is optimized for growth of Mycobacterium tuberculosis mycelium.

The liquid medium of step 1 may be at least one selected from the group consisting of sucrose, glucose and starch, but is not limited thereto. Preferably glucose, and more preferably 2% [w / v] glucose, based on the liquid medium.

The liquid medium of step 1 may be at least one selected from the group consisting of tryptone, yeast extract, L-glutamic acid and peptone as a nitrogen source, , But is not limited thereto. Preferably contains L-glutamic acid, more preferably 1% [w / v] L-glutamic acid, based on the liquid medium.

The liquid medium of step 1 may include at least one 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 at least one selected from the group consisting of ZnSO ₄ , CuSO ₄ and FeSO ₄ , more preferably 0.02% [w / v] ZnSO ₄ , 0.01% [w / v] includes CuSO ₄ and 0.01% [w / v] FeSO 4.

The liquid medium of the first step may include at least one selected from the group consisting of Ca, P, Na, K, Cl, Cu, I, Mn, Zn, . Preferably, K, Mn, Zn, Ca and Co may be contained in an amount of 0.005% or more based on the liquid medium, [w / v].

The liquid medium of step 1 may have a pH of 4.5 to 6.5. It is preferably pH 6. pH can affect the morphology or activity of proteins by changing the charge of amino groups or carboxyl groups of amino acids, the unit of enzymatic proteins that are important for cell metabolism. In addition, changes in pH in the external environment can affect the ionization of microbial nutrients, which can affect microbial nutrient intake. If the pH of the liquid medium is less than 4.5, fungi which are acidophilic bacteria grow better than mycelium, and when the pH is higher than 6.5, archeophilic bacteria of Bacillus spp. Grow better.

Liquid medium of the first step is glucose 2%, based on the liquid medium [w / v], 1% L- glutamate [w / v], ZnSO ₄ 0.02% [w / v], CuSO 4 0.01% [w / v], FeSO 4 It is most preferable that it contains 0.01% [w / v] and 0.005% [w / v] each of K, Mn, Zn, Ca and Co.

The two-stage culture can be performed using an agitated bioreactor, and the growth of the strain may be influenced by the amount of air supplied, the stirring speed, the amount of dissolved oxygen, the incubation temperature, and the incubation time. Condition must be provided.

The culture in the second step may have an air supply of 0.5 to 3 vvm, preferably 1 to 2 vvm, and more preferably 1 vvm.

The above two-step culture may be carried out at a stirring speed of 50-175 rpm, preferably 100-125 rpm, and more preferably 125 rpm.

The culture in the second step may have a dissolved oxygen amount of 5 to 20%, preferably 10 to 15%, and more preferably 10%.

The culture in the two-step culture may be performed at 12-18 days, preferably 15 days.

The culture in the two-step culture may be carried out at a temperature of 24 to 28 ° C, preferably 26 ° C. When the temperature is lower than 24 ° C, the activity of the mycelium of Mycobacterium tuberculosis sharply decreases so that the purpose of culturing can not be achieved. When the temperature is above 28 ° C, the mycelium of Mycobacterium tuberculosis is killed.

The liquid medium of step 3 may be a medium which is optimized for the growth of mycelium of Seripolla la Serera K1, which is the second mycelium of the two-stage culture of the present invention.

The liquid medium of step 3 may be one obtained by adding components necessary for culturing Mycelia marcera K1 mycelium to the culture solution of the step 2 mycelium broth culture and sterilizing it.

The sterilization may be performed not only to prevent microbial contamination in the liquid medium of step 3 but also to kill the microbial flour mycelia contained in the culture liquid of the step 2 microbial flora. In the case where the mycelium of Mycobacterium tuberculosis is not killed, the cultivation of Mycelium mycelium, which is the second mycelium of the two-stage culture, is interfered with, so that the two-stage cultivation using the mycelium of the present invention may not be performed.

The liquid medium of step 3 may include at least one selected from the group consisting of sucrose, glucose and starch as a carbon source in the culture medium of step 2 of the above-mentioned method. It does not. Preferably sucrose and starch are added, more preferably 1% [w / v] sucrose and 0.5% [w / v] starch are added, based on the liquid medium.

The liquid medium of step 3 is prepared by adding soybean flour, tryptone, yeast extract, L-glutamic acid, and peptone (hereinafter, referred to as " peptone) may be added, but the present invention is not limited thereto. Preferably, the soy flour is added, and more preferably 1% [w / v] soy flour is added based on the liquid medium.

The pH of the liquid medium of step 3 may be 4.5 to 6.5. Preferably pH 5.5. pH can affect the morphology or activity of proteins by changing the charge of amino groups or carboxyl groups of amino acids, the unit of enzymatic proteins that are important for cell metabolism. In addition, changes in pH in the external environment can affect the ionization of microbial nutrients, which can affect microbial nutrient intake. If the pH of the liquid medium is less than 4.5, fungi which are acidophilic bacteria grow better than mycelium, and when the pH is higher than 6.5, archeophilic bacteria of Bacillus spp. Grow better.

The culture in the above step 4 can be performed using an agitated bioreactor and the growth of the strain may be influenced by the amount of air supplied, the stirring speed, the amount of dissolved oxygen, the incubation temperature and the incubation time, The optimal culture conditions of the mycelium should be provided.

The culture in the above four stages may have an air supply of 0.5 to 3 vvm, preferably 1.5 to 2.5 vvm, and more preferably 2 vvm.

The culture in the above four steps may have a stirring speed of 50-175 rpm, preferably 50-125 rpm, and more preferably 75 rpm.

The 4-step culture may have a dissolved oxygen content of 5 to 30%, preferably 15 to 25%, and more preferably 20%.

The culture in the above-mentioned four stages may be carried out for 7 to 15 days, preferably 10 days.

The culture at the above four stages may be carried out at a culture temperature of 18 to 25 캜, preferably 21 캜. When the temperature is lower than 18 ° C, the activity of the mycelium of Celapiora rera cerata is drastically decreased, so that the purpose of culture can not be achieved. When the temperature exceeds 25 ° C, The production of the physiologically active substance is decreased, which is undesirable.

In the two-step culture of the present invention, the order of culturing the microorganisms is important. When the culture sequence is changed, the growth of the two-step inoculation strain may occur due to the inherent growth characteristics of the two different microorganisms, and the two-stage culture may not occur.

In the two-step cultivation using the heterogeneous mycelium of the present invention, the mycelium having the physiological activity better than that of the inoculum mycelium in the first stage should be selected as the mycelium in the second stage, and the mycelium which can survive in the first stage mycelium should be selected.

The two-stage cultivation of Kannabibi bulrocho and Sellapori la Serera K1 mycelium is carried out by culturing mycelium of Mycenae subsp. Mycelia and culturing mycelium of Klebriela rera ceratata K1. The content of the resulting β-glucan in the cultured product is not less than The amount of extracellular polysaccharide can be 1.9 ~ 2.0 times higher than that of cultivated mycelium broth or K1 mycelium lyceratta alone. In addition, the two-stage cultures of kanamabi broth and Sera liparia la Serera K1 mycelium can be newly produced and contained in the triterpenoid saponins which are not detected in the sole culture of kanamabi brook or cultivar Sera liparia ceratata K1.

The triterpenoid saponin is one of the saponin classes, and is a component contained in the hinoki tree and the bellflower, and is known to have a blood glucose lowering effect and a liver damage treatment effect.

When culturing the mycelia of S. saprophyticus mycelium in one step and culturing the mycelium of Mycobacterium tuberculosis in two steps during the two-step culturing, the mycelia of S. minnifolia of the present invention is cultured in one step, The newly produced triterpenoid saponin is not produced in the two-step cultured K1 mycelium in the two-step culture, and the content of? -Glucan and extracellular polysaccharide is also low, which is undesirable.

The two-stage cultures of Kannabibi brooch and Klebsiella reratera K1 mycelium may include both mycelium and culture medium, or the mycelium and the culture medium may be separately isolated from the culture medium. Preferably, both the mycelium and the culture medium are included.

The cultured product may be cultivated in a liquid culture medium, a culture liquid in which the culture liquid is pulverized, a culture liquid in water, C1-C4 lower alcohol, acetone, n-hexane, dichloromethane and ethyl acetate And then extracting the extract. Preferably, it is a culture medium and a culture medium powder.

The culture liquid obtained by culturing the liquid culture can be pulverized using a conventional drying method. Preferably, it is lyophilized and powdered.

The C1 to C4 lower alcohols may be methanol, ethanol, propanol, isopropanol, butanol, and the like.

The method for extracting the above extract may be selected from a method such as hot water extraction, cold extraction, reflux cooling, solvent extraction, steam distillation, ultrasonic extraction, leaching, and pressing. In addition, the desired extract may be further subjected to a conventional fractionation process or may be purified using a conventional purification method.

The two-stage cultures of Kannabi broth and Sella lipylaceratata K1 mycelia have increased β-glucan and extracellular polysaccharide components known to be effective for treating diabetes, and newly produced triterpenoid saponin, which is known to have a hypoglycemic effect , And thus the blood glucose lowering effect or the insulin secretion promoting effect is remarkably superior to that of the single cultured mycelium broth or C. lipolasera K1 mycelium.

In addition, the present invention relates to a pharmaceutical composition for preventing or treating diabetes comprising as an active ingredient a two-tine culture of Mycobacterium tuberculosis and Sella lipylacerata K1 mycelium.

The pharmaceutical composition may comprise the Janavia pyrochlore and the Cefolaria la Serera K1 mycelial cultures and a pharmaceutically acceptable excipient.

The pharmaceutical compositions may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to conventional methods. Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations may contain at least one excipient in the two-stage cultures of Janpanibiroloblast K1 and Selipporia lacerata of the present invention, For example, it is prepared by mixing starch, calcium carbonate, sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include witepsol, macrogol, tween-61, cacao butter, laurin, glycerogelatin and the like.

Preferably, the two-stage cultured product of K. nabi broth and Cryptococcus sp. K1 is 0.001-50 wt%, more preferably 0.001-40 wt%, and most preferably 0.001-30 wt% based on the total weight of the total pharmaceutical composition %. ≪ / RTI >

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, body weight, the specific disease or condition to be treated, the severity of the disease or condition, the route of administration, and the judgment of the prescriber. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from about 0.1 to 2000 mg / day. A more preferable dosage is 0.1 to 1000 mg / day. The administration may be carried out once a day or several times. The dose is not intended to limit the scope of the invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intraperitoneal, muscle, subcutaneous, intra-uterine or intracerebral injection.

Further, since the pharmaceutical composition of the present invention is a composition derived from a natural product, it has little toxicity and side effects, and therefore can be safely used for long-term use for preventive purposes.

The pharmaceutical composition may promote blood glucose depression or insulin secretion in serum of a diabetic patient.

The present invention also relates to a health functional food for diabetes mellitus comprising the above-mentioned two-stage cultured product of Kannabibi bulrocho and Sella liparia la Serratia K1 as an active ingredient.

Said health functional food may comprise said Janavia < RTI ID = 0.0 > Floro < / RTI > and Cefoliaria la Serera K1 mycelial biotreatment cultures and food acceptable food supplementary additives.

The cultivars of Kannabibi broth and Sella liparia lacerata K1 mycelium are preferably 0.001-90% by weight, more preferably 0.001-70% by weight, and most preferably 0.001-50% by weight, based on the total weight of the whole health functional food By weight.

The ingestion amount of Janabi Fluorosaccharide and Sellapora Racerata K1 mycelial two-stage culture in the above-described health functional food is 2000 mg or less per day and 6000 mg or less per day. Most preferably 1000 mg once a day, three to four times a day.

The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids. Examples of the foods to which the two-stage culture of the present invention can be added include various foods, beverages, gums, tea, Vitamin complex, and health functional foods.

The health functional food may promote blood glucose lowering or insulin secretion in serum.

The present invention relates to the use of Ganderma applanatum and Ceriporia lacerata K1 K1) mycelium and a composition for preventing or treating diabetes mellitus comprising the herbal cultures prepared by the method as an active ingredient. The present invention relates to a composition for preventing or treating diabetes mellitus, which comprises firstly culturing mycelium of Mycobacterium tuberculosis, The two-stage culture obtained by inoculating the mycelium of Serratia K1 and the cultured product obtained by culturing the cultured Mycelium broth or K1 mycelium lera cerata alone, respectively, showed that the content of? -Glucan and extracellular polysaccharide As compared with the single culture of each of the mushroom mycelium, the content in the two-stage culture was remarkably increased and it was confirmed that triterpenoid saponin was newly produced and contained. Further, as a result of confirming the therapeutic effect of diabetes on diabetic cultures, it was confirmed to be superior to that of single cultures.

Thus, it is expected that the two-step cultures using the mycelium of Kannabibi and Klebsiella lera cerata of the present invention can be usefully used for the development of medicines for the prevention or treatment of diabetes, and health functional foods for improvement.

FIG. 1 shows the results of confirming the secretion promoting effect of C-peptide reflecting the insulin secretion dynamics of the two-stage cultures of Janpanibiollochos and Sellapori Racerata K1 mycelium of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, the intention is to provide an exhaustive, complete, and complete disclosure of the principles of the invention to those skilled in the art.

≪ Example 1 > Two-step cultivation of mycelium broth and three cultivars K1 mycelium>

Gamoderma applanatum) was used to breed (Accession No. KACC 50174) received pre-sale at the National Academy of Agricultural Sciences Agricultural Genetic Resources Center, three repositories Ria La Serra Other K1 (Ceriporial lacerata K1) is a three lipoic new strains are bred breeding present invention the inventors Riaraserata K1 (Accession No. KACC 83018BP) was used. Liquid culture was carried out using a 500 L agitated biological incubator installed at Gyeongbuk Marine Biotechnology Research Institute.

In the first step culture, 2% [w / v] glucose as a carbon source, 1% [w / v] L-glutamic acid as a nitrogen source and 0.02% [w / v] ZnSO ₄ as a trace element were added to purified water, , 0.01% [w / v] of CuSO _{4 and} 0.01% [w / v] of FeSO ₄ and 0.005% [w / v] of K, Mn, Zn, Ca and Co as mineral complexes And sterilized and cooled. 400 ml of the mycelial flour mycelia was inoculated in a sterilized liquid medium and cultured for 15 days at a culture temperature of 26 캜, an air supply amount of 1 vvm, a stirring speed of 125 rpm, a dissolved oxygen amount of 10%, and a pH of 6.0.

Based on the mycelial culture of Zanabi fullocci cultured at the end of the first stage culture, a two - step culture of mycelium of. In the second step culture, 1% sucrose [w / v], 0.5% starch [w (w)] as a carbon source was added to the cultured mycelia of the fungus cultivated in the first step in order to obtain a liquid medium composition optimized for the growth of mycelium K1. / v] and 1% [w / v] of soybean powder as a nitrogen source. The sterilized liquid medium was inoculated with 400 ml of the mycelium of Celpolaris la Serera K1 and cultured for 10 days at a culturing temperature of 21 캜, an air supply amount of 2 vvm, a stirring speed of 75 rpm, a dissolved oxygen amount of 20%, and a pH of 5.5. In a two-stage cultivation of Mycobacterium tuberosum K1 and cultivated Mycobacterium tuberosum L., the culture was terminated by freezing, and 380 L was lyophilized to obtain 8.2 kg of a powder, which was then stored at 2 ° C.

≪ Comparative Example 1 > Mycelial cultivation of Mycelia broccoli mycelium &

And cultured alone using the mycelium broth of Example 1.

To the purified water was added 2% w / v of glucose, 1% w / v of L-glutamic acid, 0.02% w / v of ZnSO ₄ , 0.01% w / v of CuSO _{4 and} 0.01% w / v of FeSO ₄ 400 ml of a Sambrook et al. Strain was inoculated into 400 liters of a sterilized liquid medium containing 0.005% [w / v] of each of K, Mn, Zn, Ca and Co as a mineral complexing agent and then cultured at 26 ° C under an air supply of 1 vvm , A stirring speed of 125 rpm, a dissolved oxygen amount of 10%, and a pH of 6.0. After culturing, 20 L of the cultured Mycelia broth was centrifuged and 380 L was lyophilized to obtain 5.7 Kg of powder, which was then stored at 2 ° C.

≪ Comparative Example 2 > Culture alone of mycelium of Seripolyla cerasata K1 >

And cultured alone using the mycelium of Seripolylacerata K1 of Example 1 above.

To 400 liters of a sterilized liquid medium containing 1% [w / v] sucrose, 0.5% [w / v] starch and 1% [w / v] soybean powder in purified water, 400 ml of mycelia lera cerata K1 mycelia After inoculation, the cells were cultured for 10 days at a culturing temperature of 21 캜, an air supply amount of 2 vvm, a stirring speed of 75 rpm, a dissolved oxygen amount of 20%, and a pH of 5.5. 20 L of the cultured mycelium of C. lipolasera K1 was completely frozen and 380 L was lyophilized to obtain 5.8 Kg of powder, which was stored at 2 째 C.

 ≪ Comparative Example 3 > Two-step culture of mycelium of Seripolyla ceratata K1 and mycelium of Mycobacterium tuberculosis &

The culturing procedure of the mycelium of Example 1 was changed to prepare a two-stage cultured cell of Sacrifolia la Serratta K1 and S. minilifolia.

The first stage cultivation was performed by culturing the mycelia of Celpolia rera cerata K1. The culture broth was prepared by adding 1% [w / v] sucrose, 0.5% w / v starch and 1% w / v soybean powder as a nitrogen source , Cultured for 10 days at a culture temperature of 21 DEG C, an air feed rate of 2 vvm, a stirring speed of 75 rpm, a dissolved oxygen content of 20%, and a pH of 5.5, and the cells were cultured for 10 days. Respectively.

Based on the mycelial cultures of three lipolylacerate K1 cultured at the end of the first step cultivation, the two-step cultivated mycelium broth was cultured. In the two-step culture, 2% [w / v] glucose as a carbon source and 2% [w / v] L-glutamic acid as a nitrogen source were added to the culture medium of the three- ZnSO ₄ 0.02% [w / v], CuSO ₄ 0.01% [w / v] and FeSO ₄ 0.01% [w / v] as trace elements and K, Mn, Zn, 0.005% [w / v] of Ca and Co, respectively, and sterilized and cooled. 400 ml of the mycelial flour mycelia was inoculated in a sterilized liquid medium and cultured for 15 days at a culture temperature of 26 캜, an air supply amount of 1 vvm, a stirring speed of 125 rpm, a dissolved oxygen amount of 10%, and a pH of 6.0. 20 L of the two cultures of Celpolaris la Serratta K1 and Mycobacterium tuberosum L. cultivated at the end of the cultivation were frozen and 380 L were lyophilized to obtain 8.5 kg of powder and refrigerated at 2 ° C.

 ≪ Comparative Example 4 > Mixture of solely cultured Mycelia broccoli mycelium and single cultured Mycelia marcera K1 mycelium >

The lyophilized powder of the cultivated solitary broth of mycelia of Comparative Example 1 and the lyophilized powder of the cultivated solitary cultivar K1 of Comparative Example 2 were mixed in a weight ratio of 1: 1 to prepare Example 1 and Comparative Example 3 ≪ / RTI > was prepared.

≪ Example 2: Comparison of main component content of mycelial culture >

An exopolysaccharide (EPS) in a mixture of the lyophilized powder of the culture of Example 1 and Comparative Examples 1 to 3 and the cultured solitary broth of mycelium of Comparative Example 4 and the cultured mycelium of Seripolyla Serratia K1 alone ),? -glucan and triterpenoid saponin were analyzed. The results are shown in Table 1 below.

The content of the extracellular polysaccharide was obtained by mixing 20 g of the lyophilized powder or mixture of each culture with 400 ml of distilled water and centrifuging at 15,000 rpm for 20 minutes to separate the supernatant. Isopropyl alcohol corresponding to 4 times the volume of the supernatant was added to the separated supernatant, and the supernatant was allowed to stand at 4 ° C for 24 hours and then centrifuged at 15,000 rpm for 20 minutes to recover the precipitate. The recovered precipitate was lyophilized to determine the weight of the crude extracellular polysaccharide.

The content of? -Glucan was determined according to the test method for the functional food using 20 g of the lyophilized powder or mixture of each culture, and the content was calculated according to the formula 1 below.

[Formula 1]

? -glucan content (mg / g) = C × a / S × 10 × 1 / 1,000 × 0.9

C is the glucose concentration (쨉 g / ml) in the test solution, a is the total amount (ml) of the test solution, 10 is the dilution factor, S is the sample weight (g) Is the? -Glucan conversion factor (162/180).

The content of triterpenoid saponin was determined by adding 400 ml of ethyl acetate to 10 g of the lyophilized powder or mixture of each culture, separating by ultrasonic treatment at 30 ° C for 2 hours, filtering it using a diatomaceous earth filter, and concentrating the filtrate under reduced pressure To obtain a concentrate. The obtained concentrate was dissolved in methanol to make 10 ml, and then analyzed by high performance liquid chromatography (HPLC) (YL9100 Plus HPLC system).

Culture β-glucan
(Mg / g) Extracellular polysaccharide
(% / g) Triterpenoid
Saponin
(Mg / g) Comparative Example 1 Solanaceous mycelium culture alone 13.21 4.49 - Comparative Example 2 Three mycelia of K1 mycelium
Sole culture 42.74 4.67 - Comparative Example 3 ≪ RTI ID = 0.0 > K1 < / RTI &
Two-stage culture of mycelium broth 45.51 5.12 - Comparative Example 4 Mycobacterium solani culture and
Three mycelia of K1 mycelium
Mixture of single cultures 27.46 4.56 - Example 1 Zanabi Floro and
Three mycelia of K1 mycelium
Double culture 63.12 8.87 0.11

As shown in the above Table 1, in the case of the two-stage cultures of Janabi Fluorosaccharide and Sellapori Racerata K1 mycelium of Example 1, the content of? -Glucan and extracellular polysaccharide was remarkably higher than that of the cultures or the mixtures of Comparative Examples 1 to 4 I confirmed a lot.

More specifically, in the case of the content of [beta] -glucan, compared with the culture of single mycelium broth of Comparative Example 1 alone or the cultured Mycelium of Cerporiallacerata K1 of Comparative Example 2, It was found that 1.4 times or 1.5 times as many cultures of Lacerata K1 mycelium as in Example 1 were obtained, and 1.4 times as much as that of the two cultures of the three cultivars of Comparative Example 3, which were cultured in the reverse order of the mycelial culturing procedure of Example 1, Times as much.

In the case of the extracellular polysaccharide content, the two-stage cultures of Janabi broth and Celpolyla sera K1 mycelium of Example 1 were cultured alone in the cultured mycelium of Mycobacterium tuberculosis mycelium of Comparative Example 1, 1.9 times, or 1.7 times as much as that of the cultured product or the two-stage cultured product of the three cultivars K1 and K. natto.

On the other hand, in the case of the mixture of the cultured solitary broth of mycelium of Comparative Example 4 alone and the cultured mycelium of Seripolylacerata K1 mycelium, the content of? -Glucan and the extracellular polysaccharide was less than that of the cultures of Comparative Examples 1 to 3 Similar.

On the other hand, in the case of the triterpenoid saponin content, no culture or mixture of the cultures or the mixtures of Comparative Examples 1 to 4 was detected, but only 0.11 mg / ml of the cultured product of Janpanobiocinospora and Sellapori Racerata K1 mycelium of Example 1, g. < / RTI >

Through the two-step cultivation of the mycelia of Kannabi broth and Celapiora la Serera of the present invention, the content of the components of β-glucan and extracellular polysaccharide having physiological activity is remarkably increased and a novel The material was found to be produced.

Furthermore, the two-stage cultivation of mushroom mycelium increases the functional content of each mushroom according to the type and culture sequence of the mushroom mycelium, Could know.

≪ Example 3: Confirmation of antidiabetic effect of mycelial culture >

Example 3-1. Check blood glucose lowering effect

Animal experiments were conducted to confirm the anti-diabetic effect of the two-stage cultures of Janpanibiollochos and Sellapori la Serratia K1 mycelium of the present invention. At this time, the clinical characteristics of type 2 diabetes due to mutation of the leptin receptor Db / db mice were used.

Male db / db mice were weighed before and after 150 mg / dl of body weight, and 150 mg / dl of blood glucose. After 3 days of adaptation period, body weight and blood glucose were measured Mice suitable for similar experiments were selected and classified into diabetic group and sample treatment group. At this time, mice with normal leptin receptor regulating appetite were used as normal group. The feeds were restricted to 5 g per day to allow the maintenance of blood glucose levels in commercially available laboratory animal feeds, and water was freely available. In order to confirm the antidiabetic effect, the other groups except for the normal group were orally administered for 42 days at the same time every day so that 40% glucose was 2 g / kg. In the sample treatment group, the cultures of Comparative Examples 1 to 3 and Example 1 were orally administered at the same time once per day at 300 mg / kg each. As a positive control, 300 mg / kg of metformin (MET), a hypoglycemic agent, was treated in the same manner as the above sample treatment. To measure blood glucose change, blood glucose was fasted for 12 hours on a fasting blood glucose meter (Accucheck Guide, Korea) The results are shown in Table 2 below.

Experimental group Blood sugar (㎎ / ㎗) 0 day Day 7 Day 14 Day 21 Day 28 35th day Day 42 Normal group 151 151 151 150 152 149 153 Diabetic group 150 200 235 400 445 570 600 Comparative Example 1 Treatment group 151 237 252 374 398 372 445 Comparative Example 2 [ 150 169 221 263 322 341 388 Comparative Example 3 Treatment group 150 169 220 261 311 335 374 Example 1 Treatment group 151 165 215 256 304 295 287 MET (positive control) 150 161 213 241 276 333 351

As shown in Table 2, the blood glucose level of all the groups before and after the sample treatment (0 day difference) showed a similar level before and after 150 mg / dl. However, in the diabetic group administered with only glucose, On the other hand, in the case of the positive control group treated with Comparative Example 1 to 3, Example 1 and metformin, the blood glucose level was lower and the blood glucose level was lower than that of the diabetic group even when glucose was administered. Particularly, when Example 1 was treated, it was confirmed that the blood glucose level was lower than that of the positive control group treated with metformin, which is a blood glucose lowering agent, after peaking at 28th day.

Further, in the case of treating the two-stage cultures of the three cultures of Comparative Example 3, cultured in the same manner as in Example 1, except for culturing the culture of Comparative Example 2, It was confirmed that the effect of reducing the blood glucose level was exhibited.

As a result, it was found that the two-stage cultures of Janpanibirosophila and K. mycelium of the present invention inhibited the increase of blood glucose and showed a remarkably superior antidiabetic effect by decreasing the blood glucose level as compared with metformin, which is a blood glucose lowering agent, over time there was.

Example 3-2. C-peptide secretion confirmation

C-peptide (C-peptide) is secreted during the conversion of insulin precursor, proinsulin, to insulin. It has no physiological activity but reflects the secretion of insulin. Therefore, the content of C-peptide in serum is measured The activity of the insulin secretory function can be predicted.

Thus, the content of C-peptide in the mouse serum of day 42 of Example 3-1 was measured by using a radioimmunoassay method to confirm the antidiabetic effect of the two-step cultures of the kanamycin of K. nattoplasma And the results are shown in Table 3 and FIG.

Experimental group Serum C-peptide content (pg / ml) Normal group 13.72 Diabetic group 134.14 Comparative Example 1 Treatment group 140.44 Comparative Example 2 [ 147.18 Comparative Example 3 Treatment group 149.86 Example 1 Treatment group 188.65 MET (positive control) 173.54

As shown in Fig. 1 and Table 3, it was confirmed that the C-peptide content in the serum of the diabetic group and the group treated with the Example 1 was higher than those of the diabetic group and the control group of the Comparative Examples 1 to 3. In addition, the C-peptide content of the group treated with Example 1 was higher than that of the positive control group treated with metformin, which is a blood glucose lowering agent.

Thus, the two-stage cultures of Janabi Fluoroso and Cefoliaria la Serera K1 mycelium in Example 1 were significantly higher in the pancreas than in the single cultured Mycelium of Mycobacterium mycelium of Comparative Example 1 or of the Mycobacterium tuberculosis mycelium of Comparative Example 2 (Positive control group), which is used as a therapeutic agent for diabetes as a hypoglycemic agent, was significantly superior to that of Example 1. In addition, The two - stage cultures were found to have better antidiabetic activity.

Further, in the case of treating the two-stage cultures of the three cultures of Comparative Example 3, cultured in the same manner as in Example 1, except for culturing the culture of Comparative Example 2, Serum C-peptide content was measured.

As a result, it was found that the antifungal activity of both the kanamycin and the cultivar K1 mycelium of the present invention was excellent in antidiabetic activity.

≪ Formulation Example 1 >

Formulation Example 1-1. Manufacture of tablets

200g of the two-stage cultures of Janpanibiollochos and Sellapori la Serratia K1 mycelium of the present invention were mixed with 175.9g of lactose, 180g of potato starch and 32g of colloidal silicic acid. A 10% gelatin solution was added to the mixture, followed by pulverization and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.

Formulation Example 1-2. Injection preparation

1 g of the kanamycin of the present invention, and 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.

<Formulation Example 2: Preparation of Health Functional Food>

Formulation Example 2-1. Manufacture of Health Functional Foods

20 g Vitamin B acetate, 70 g of vitamin A acetate, 1.0 mg of vitamin E, 0.13 mg of vitamin B1, 0.15 mg of vitamin B2, 0.5 mg of vitamin B6, 0.5 g of vitamins A, 0.2 mg of B12, 10 mg of vitamin C, 10 g of biotin, 1.7 g of nicotinic acid amide, 50 g of folic acid, 0.5 mg of calcium pantothenate, an amount of an inorganic mixture, 1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 15 mg of potassium phosphate, 55 mg of dicalcium phosphate, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride were mixed to prepare granules. In addition, the composition ratio of the above-mentioned vitamin and mineral mixture may be arbitrarily modified, and the above components may be mixed according to a conventional health functional food manufacturing method.

Formulation Example 2-2. Manufacture of health functional drinks

The mixture was mixed with 3 g of the kanamycin of the present invention and 3 g of the cultured Mycelium lycerata K1 mycelium, 0.1 g of citric acid, 30 g of fructooligosaccharide and 300 ml of purified water, and the mixture was stirred, heated, filtered, sterilized and refrigerated A beverage was prepared.

Claims (7)

The Mycobacterium tuberosum obtained by inoculating mycelium of Ceriporia lacerata K1 (Accession No. KACC 83018BP) with the mycelium of Ganoderma applanatum and inoculated with the mycelium of Sacrifolia lacerata K1 (Accession No. KACC 83018BP) A pharmaceutical composition for the prevention or treatment of diabetes comprising a cultured product obtained by culturing mycelia of the other K1 strain as an active ingredient. delete The method according to claim 1,
The pharmaceutical composition for preventing or treating diabetes according to claim 1, wherein the culture obtained by the two-step culture comprises triterpenoid saponin. The Mycobacterium tuberosum obtained by inoculating mycelium of Ceriporia lacerata K1 (Accession No. KACC 83018BP) with the mycelium of Ganoderma applanatum and inoculated with the mycelium of Sacrifolia lacerata K1 (Accession No. KACC 83018BP) A health functional food for improving diabetes comprising a cultured product obtained by culturing mycelium of K1 strain in two stages as an active ingredient. delete 5. The method of claim 4,
Wherein said cultured two-stage culture comprises triterpenoid saponin. A first step of preparing a liquid medium for culturing Mycelia broccoli mycelium;
A second step of inoculating and cultivating the Mycobacterium tuberculosis mycobacteria in the liquid medium of step 1 to secure a culture medium of Mycobacterium tuberculosis mycelium;
3) adding a carbon source and a nitrogen source to cultivate Celpolaris la Serratia K1 mycelium and culturing the medium for sterilization and cooling to prepare a liquid culture medium for the secondary culture; And
Four steps of inoculating and culturing the mycelium of Celpolaris lacerata K1 in the liquid medium of step 3;
And culturing the two cultures of K1 mycelium broth and three K1 cells.

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