Medium composition for cultivating cordyceps militaris and cordyceps militaris culture

Abstract

A medium composition for cultivating Cordyceps militaris, according to one embodiment of the present invention, comprises red ginseng marc, sugar, roasted soybean powder, and peanut powder, and contains oleic acid in an amount of 2.0 wt% or more on the basis of the total weight of the medium composition. A Cordyceps militaris culture, according to one embodiment of the present invention, is cultivated by inoculating Cordyceps militaris spores into the medium composition, and the content of cordycepin in the Cordyceps militaris culture is 10 mg / g or more.

Description

Composition of medium for cultivation of Cordyceps militaris and Cordyceps militaris culture

[0001] The present invention relates to a medium composition for cultivating Cordyceps militaris and a Cordyceps militaris culture.

[0002] Cordyceps militaris is called Cordyceps militaris because it lies dormant in the bodies of insects in the winter, absorbs nutrients, and kills them. Then, when the environment is right in the summer, it produces mushrooms like grass. It belongs to the Ergotomycetes family of the Ascomycota class.

[0003] Cordyceps sinensis and Cordyceps militaris are representative examples of Cordyceps militaris whose pharmacological effects have been previously discovered.

[0004] Cordyceps militaris is a mushroom that, in nature, primarily forms its fruiting body using insect pupae as a host. It contains the physiologically active ingredient cordycepin, which is known to have excellent pharmacological effects, including antidiabetic properties. Cordyceps militaris is known to be the only organism that synthesizes cordycepin.

[0005] Currently, the population of wild Cordyceps militaris has decreased significantly due to global warming and environmental pollution, and as a result, the collection of wild Cordyceps militaris is very limited.

[0006] Therefore, Cordyceps militaris cultivation methods are becoming increasingly important for securing a reliable supply, and grains such as brown rice and insects are widely used as substrates for Cordyceps militaris cultivation. However, commercial insects are significantly more expensive than grains, preventing them from becoming widespread as a substrate for Cordyceps militaris.

[0007] Compositions or health functional foods containing Cordycepin components to utilize the efficacy of cordycepin have been introduced, and cultivation methods to increase cordycepin content are being studied, but there is a limitation that the cordycepin content is not sufficient to exhibit pharmacological efficacy.

[0008] Meanwhile, during the red ginseng manufacturing process, various new bioactive compounds beneficial to the body are created. Red ginseng contains various special bioactive compounds, including maltol and ginsenosides, which are unique to red ginseng and not found in other types of ginseng, such as fresh or white ginseng. Maltol has anti-aging properties, while ginsenosides have various health benefits, including boosting immunity, alleviating fatigue, improving blood flow by inhibiting platelet aggregation, and enhancing memory. The byproduct left after red ginseng processing is called red ginseng marc, and is usually discarded rather than recycled.

[0009] One embodiment of the present invention provides a Cordyceps militaris culture and composition having a high concentration of cordycepin content.

[0010] One embodiment of the present invention provides a Cordyceps militaris culture and composition containing cordycepin and ginsenoside as active ingredients.

[0011] One embodiment of the present invention is to provide an environmentally friendly Cordyceps militaris culture and a method for producing the same by producing a Cordyceps militaris culture using discarded waste resources.

[0012] One embodiment of the present invention provides a method for producing a Cordyceps militaris culture with excellent economic efficiency.

[0013] In addition to the above-mentioned tasks, embodiments according to the present invention can be used to achieve other tasks not specifically mentioned.

[0014] A medium composition for cultivating Cordyceps militaris according to one embodiment of the present invention comprises red ginseng powder, sugar, roasted soybean powder, and peanut powder, and has oleic acid in an amount of 2.0 wt% or more based on the total weight of the medium composition.

[0015] Based on the total weight of the badge composition, oleic acid may be at least 3.5 wt%.

[0016] The badge composition may include a ginsenoside component.

[0017] According to one embodiment of the present invention, a Cordyceps militaris culture is cultivated by inoculating Cordyceps militaris spores into the medium composition.

[0018] The content of cordycepin in the Cordyceps militaris culture may be 10 mg / g or more.

[0019] Cordyceps militaris culture may contain ginsenoside components.

[0020] A composition for preventing or improving diabetes according to one embodiment of the present invention comprises the Cordyceps militaris culture.

[0021] A health functional food for preventing or improving diabetes according to one embodiment of the present invention includes the Cordyceps militaris culture.

[0022] A method for producing a Cordyceps militaris culture according to one embodiment of the present invention comprises a first step of culturing a Cordyceps militaris strain at 25°C to obtain a solid spawn inoculum, a second step of preparing a soybean powder-sugrose-liquid medium, adding the solid spawn inoculum to the soybean powder-sugrose-liquid medium and homogeneously crushing it, and then culturing the liquid spawn, a third step of pulverizing undried red ginseng marc, mixing the pulverized red ginseng marc, sugar, and roasted soybean powder to prepare a mixture, and mixing peanut powder into the mixture to prepare a red ginseng marc medium, a fourth step of inoculating the liquid spawn into the red ginseng marc medium, and a fifth step of culturing the red ginseng marc medium inoculated with the liquid spawn to obtain a Cordyceps militaris culture.

[0023] In the third step, the mixing ratio of the mixture and peanut powder can be 10:1 (w / w) to 10:5 (w / w).

[0024] In the fourth step, the ratio of the inoculated liquid spawn and red ginseng extract medium may be 1:10 (v / v).

[0025] According to one embodiment of the present invention, a Cordyceps militaris culture and a composition for preventing or improving diabetes may have a high concentration of cordycepin content and may simultaneously include cordycepin and ginsenoside as active ingredients.

[0026] A method for producing a Cordyceps militaris culture according to one embodiment of the present invention can be environmentally friendly and economically efficient by producing a Cordyceps militaris culture using discarded waste resources.

[0027] Figure 1 is a graph showing the cordycepin content measured for samples manufactured according to Preparation Examples 1 to 5.

[0028] Figure 2 is a graph showing the carbohydrate, protein, and fatty acid contents of samples manufactured according to Preparation Examples 1 to 4.

[0029] Figure 3 is a graph showing the correlation between the carbohydrate, protein, fatty acid and cordycepin contents contained in samples manufactured according to Preparation Examples 1 to 4.

[0030] Figure 4 is a graph showing the contents of palmitic acid, linoleic acid, and oleic acid in samples manufactured according to Preparation Examples 1 to 4.

[0031] Figure 5 is a graph showing the correlation between the contents of palmitic acid, linoleic acid, oleic acid and cordycepin contained in samples manufactured according to Preparation Examples 1 to 4.

[0032] Figure 6 is a graph showing the relative transcription levels of the cns1 and cns2 genes of samples prepared according to Preparation Example 1, Preparation Example 2, and Preparation Example 5.

[0033] Figure 7 is a graph showing the relative transcription levels of cns1 and cns2 genes when oleic acid or linoleic acid was added to a sample prepared according to Preparation Example 1.

[0034] Figure 8 is a photograph showing the manufacturing process of a Cordyceps militaris culture manufactured according to Example 1.

[0035] FIG. 9 is a graph showing the cordycepin content measured for Cordyceps militaris culture samples prepared according to Examples 1 to 6 and Comparative Examples 1 to 4.

[0036] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings so that those skilled in the art can easily practice the present invention. The present invention may be implemented in various different forms and is not limited to the embodiments described herein. In the drawings, parts irrelevant to the description are omitted to clearly explain the present invention, and the same reference numerals are used throughout the specification for identical or similar components. In addition, detailed descriptions of widely known and publicly known technologies are omitted.

[0037] In order to clearly represent various layers and regions in the drawings, the thicknesses are enlarged. When a layer, membrane, region, plate, etc. is said to be "over" another part, this includes not only the case where it is "directly over" that part, but also the case where there is another part in between. On the other hand, when a part is said to be "directly over" another part, it means that there is no other part in between. Conversely, when a layer, membrane, region, plate, etc. is said to be "under" another part, this includes not only the case where it is "directly under" that part, but also the case where there is another part in between. On the other hand, when a part is said to be "directly under" another part, it means that there is no other part in between.

[0038] Throughout the specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise stated.

[0039] Throughout the specification, “composition” means a substance in which two or more components are uniformly mixed, and is a concept that includes not only finished products but also intermediate materials for manufacturing finished products.

[0040]

[0041] The Cordyceps militaris culture according to the present invention contains a high concentration of cordycepin, and thus can be used for the prevention or improvement of diabetes. Furthermore, the Cordyceps militaris culture additionally contains cordycepin and ginsenoside, a saponin component, thereby exhibiting various effects, including enhanced immunity, alleviation of fatigue, improved blood flow by inhibiting platelet aggregation, and improved memory.

[0042] The Cordyceps militaris culture according to the present invention can be economically efficient and environmentally friendly by utilizing red ginseng residue, a waste product generated after processing red ginseng products, as a medium.

[0043] Below, a method for producing a Cordyceps militaris culture, a medium composition for cultivating Cordyceps militaris, and a Cordyceps militaris culture are described in detail.

[0044] A method for producing a Cordyceps militaris culture comprises a first step of culturing a Cordyceps militaris strain to obtain an inoculum, a second step of culturing liquid spawn, a third step of preparing a red ginseng marmalade medium, a fourth step of inoculating the red ginseng marmalade medium with liquid spawn, and a fifth step of obtaining a Cordyceps militaris culture through culturing.

[0045] The strain used in the method for producing a Cordyceps militaris culture may be a Cordyceps militaris strain, and may be the deposited Cordyceps militaris JW001 strain.

[0046] First, step 1 is performed.

[0047] In the first step, the Cordyceps militaris strain is cultured at about 20 to 30°C for about 20 to 40 minutes, and the Cordyceps militaris strain is inoculated onto a YMA (Yeast Mannitol Agar) medium. Then, the culture is performed in a dark room at about 20 to 30°C for about 5 to 9 days to obtain solid spawn (monospore colonies).

[0048] Next, the inoculated YMA plate is cut using a cutting device such as a cylindrical cutter to prepare an inoculum. At this time, the diameter of the inoculum may be approximately 1 to 3 cm.

[0049] Next, the second step is performed. The second step may be performed simultaneously with the first step, or may be performed after the first step.

[0050] In the second step, prepare a soybean flour-sucrose-liquid (SSB) medium, add the inoculum to the liquid culture, homogenize with a homogenizer, and culture the liquid inoculum for about 4 to 6 days while supplying air to the culture bottle in a dark room at room temperature.

[0051] Here, the soybean flour-sucrose-liquid (SSB) medium may contain roasted soybean flour, sugar, KH2PO4 and MgSO4·7H2O, and the growth rate of the liquid starter culture may be improved due to the SSB medium.

[0052] Next, the third step is performed.

[0053] The third step is to prepare a red ginseng extract medium by crushing the undried red ginseng extract, mixing the crushed red ginseng extract, sugar, and roasted soybean powder, and then mixing peanut powder into the mixture.

[0054] Red ginseng residue is a waste product generated after processing red ginseng products, and the Cordyceps militaris culture according to the examples is manufactured using this waste product, so it can be economically efficient and environmentally friendly.

[0055] The red ginseng marc used in the third step may be undried (unprocessed) red ginseng marc containing about 70% or more moisture.

[0056] Red ginseng marc is rich in fibers such as cellulose and lignin, so when used in a culture medium, it increases the internal space (pores) to ensure sufficient oxygen supply to the inside of the medium. This internal space can be secured by using undried red ginseng marc, which contains a lot of moisture. Pores can be formed as moisture evaporates during the high-temperature and high-pressure sterilization process of the red ginseng marc medium described below. Due to this sufficient pore space, an adequate supply of oxygen can be provided during the culture process, which can increase the cordycepin content in the Cordyceps militaris culture, and the content of other physiologically active ingredients (active ingredients) such as ginsenosides can also be increased. In addition, since culture occurs not only on the surface of the medium but also inside the medium, the culture speed and yield can be greatly increased.

[0057] The average size of the crushed red ginseng marc can be about 30 to 10 mesh, and within this range, a desirable porosity is formed, so that the cultivation speed and yield can be further increased, and at the same time, the physiologically active components contained in the red ginseng marc, such as ginsenosides, can be included in large quantities in the Cordyceps militaris.

[0058] The mixing ratio of crushed red ginseng powder, sugar, and roasted soybean powder can be about 10:1:1 (w / w / w).

[0059] A mixture containing crushed red ginseng powder, sugar, and roasted soybean powder is mixed with peanut powder.

[0060] Here, the cns1 and cns2 genes are involved in the biosynthesis of cordycepin. Adenosine or its 2',3' cyclic monophosphate (2',3'-cAMP) is dephosphorylated into 3'-AMP by the cns2 product, and cordycepin is then synthesized from 3'-AMP by the cns1 product.

[0061] The inventors of the present invention have discovered that fatty acids are highly correlated with the expression and transcription of the cns1 and cns2 genes, with oleic acid showing a particularly high correlation. When the oleic acid content in the medium composition is increased to a certain level, the cordycepin content in the culture can significantly increase. This is because oleic acid significantly promotes the expression of the cns1 and cns2 genes.

[0062] Peanuts are one of the foods with the highest oleic acid content among various foods, for example, the oleic acid content in peanuts averages 20 to 25 wt%.

[0063] In addition, peanuts are a food product with a low purchase price, and in the case of examples utilizing these peanuts, the cost of producing culture material can be reduced, thereby improving economic feasibility.

[0064] Among the insects used in the cultivation of Cordyceps militaris, the larvae of the longicorn beetle also show an average oleic acid content of about 10 to 12 wt%, but because the purchase price is high, if used as a medium composition, the economic feasibility may be reduced.

[0065] The method for producing a Cordyceps militaris culture according to the examples can greatly increase the cordycepin content of the culture by utilizing peanut powder having a very high oleic acid content.

[0066] The mixing ratio of the mixture containing the crushed red ginseng powder, sugar, and roasted soybean powder to the peanut powder may be about 10:1 (w / w) to 10:5 (w / w). Within this range, the cordycepin content of the culture can be significantly increased.

[0067] In addition, when the mixing ratio of the mixture containing the crushed red ginseng powder, sugar, and roasted soybean powder and the peanut powder is about 10:2 (w / w) to 10:5 (w / w), the cordycepin content in the culture can be more significantly increased.

[0068] Based on the total weight of the red ginseng marc medium composition, the content of oleic acid may be about 2.0 wt% or more, and within this range, the cordycepin content of the culture may be significantly increased. In addition, based on the total weight of the red ginseng marc medium composition, the content of oleic acid may be about 3.5 wt% or more, and within this range, the cordycepin content of the culture may be more significantly increased.

[0069] The red ginseng extract medium composition contains various types of ginsenosides, and the culture also contains ginsenosides, so it may exhibit a pharmacological synergistic effect together with cordycepin.

[0070] The third step involves high-pressure sterilization of the prepared red ginseng extract medium. For example, the red ginseng extract medium can be placed in an autoclave and sterilized for approximately 50 to 70 minutes at approximately 110 to 130°C and approximately 100 to 110 kPa.

[0071] At this stage, as the moisture contained in the undried red ginseng marc evaporates, pores can be created, which allows for a smooth supply of oxygen during cultivation, and active cultivation can be achieved not only on the surface area of ​​the red ginseng marc medium but also in the internal area.

[0072] Next, the fourth step is performed, in which the red ginseng seed medium is cooled and distributed into polypropylene boxes, and then inoculated with liquid starter.

[0073] Here, the ratio of liquid starter and red ginseng extract medium can be 1:10 (v / v), and efficient cultivation can be achieved at this ratio.

[0074] Inoculation can be performed using a metering pump.

[0075] Next, step 5 is performed.

[0076] The fifth step is to obtain a Cordyceps militaris culture by culturing the red ginseng paste medium inoculated with liquid spawn.

[0077] Stage 5 may include a dark culture stage and a light culture stage.

[0078] The dark culture stage can be carried out in a dark room at room temperature for about 6 to 8 days using a red ginseng seed medium inoculated with liquid spawn.

[0079] The light culture stage can be performed after the dark culture stage, and can be performed by irradiating the cells with white light plus blue light for approximately 3 to 5 weeks in a light culture room at room temperature. During this time, the illumination level can be maintained at, for example, approximately 2000 lux.

[0080] The fifth stage may further include hot air drying and grinding steps.

[0081] The culture that has gone through the dark and light culture stages can be dried with hot air at about 50 to 70°C for about 45 to 55 hours, and then ground into fine powder.

[0082] The Cordyceps militaris culture thus prepared may contain a high concentration of cordycepin. For example, the content of cordycepin in the Cordyceps militaris culture may be about 10 mg / g or more.

[0083] Additionally, Cordyceps militaris culture may contain ginsenoside components.

[0084] The pharmaceutical composition for preventing or improving diabetes according to the embodiments may include the Cordyceps militaris culture described above.

[0085] Cordycepin can exhibit excellent effects in preventing or improving diabetes by promoting the synthesis of insulin, and the culture according to the examples can exhibit excellent effects on diabetes by containing a very high content of cordycepin.

[0086] Health functional foods for preventing or improving diabetes according to the embodiments may include the Cordyceps militaris culture described above. Here, the health functional food may be any known form of health functional food.

[0087]

[0088] Hereinafter, the present invention will be described in more detail with examples and experimental examples. However, the present invention is not limited by the examples and experimental examples.

[0089] Silkworm moth (Bombyx mori, B. mori) larvae, white-spotted flower chafer (Protaetia brevitaris, P. brevitaris) larvae, brown mealworm (Tenebrio molitor, T. molitor) larvae, and longicorn beetle (Allomyrina dichotomy, A. dichotomy) larvae used in the following preparation examples, examples, comparative examples, and experimental examples were purchased in dried form from an insect farm, brown rice was purchased in dried form from an online shopping mall, and red ginseng marc was supplied by Punggi Ginseng Agricultural Cooperative.

[0090] The Cordyceps militaris strain used in the following preparation examples, examples, comparative examples, and experimental examples is 'Cordyceps militaris JW001' (Accession No.: KACC 83049BP), which has been deposited with the KACC as an international patented microorganism.

[0091]

[0092] Preparation Example 1

[0093] The Cordyceps militaris JW001 strain stored at -80°C was cultured at 25°C for 30 minutes, and the Cordyceps militaris strain was inoculated onto a potato-dextrose-agar (PDA) slope containing 20.0 g / L of glucose, 3.0 g / L of KH2PO4, and 1.5 g / L of MgSO4·7H2O, and then cultured in a dark room at 25°C for 1 week.

[0094] The prepared culture was cultured on PDA medium at 25°C for 13 days and then stored at 4°C for subculture.

[0095] Next, the PDA plate inoculated with the strain was cut into pieces with a diameter of 1 cm using a sterilized cylindrical cutter to prepare an inoculum.

[0096] 500 mL of potato-dextrose liquid culture (PDB) was prepared, the inoculum was added to the liquid culture, homogenized with a homogenizer, and the liquid spawn was cultured in a dark room at 25°C for 5 days.

[0097] Meanwhile, longicorn beetle larvae were placed in a polypropylene bottle and sterilized at 121°C for 30 minutes using a high-pressure sterilizer, and the polypropylene bottle was cooled to room temperature.

[0098] Next, the prepared liquid spawn and longicorn beetle larvae were inoculated at an inoculation ratio of 1:2 (v / w).

[0099] Afterwards, the inoculated longicorn beetle larvae were cultured in the dark at 25°C and 70% relative humidity for 6 days to obtain longicorn beetle larvae completely covered with white mycelia. Subsequently, these were cultured in the light at 20°C and 90% relative humidity for 49 days.

[0100] Next, the cultured Cordyceps militaris fruiting bodies and mycelia were dried with hot air at 60°C for 48 hours, then ground into fine powder to obtain a sample, which was then stored at -80°C.

[0101]

[0102] Preparation Example 2

[0103] Samples were obtained and stored in the same manner as in Preparation Example 1, except that silkworm larvae were used instead of longicorn beetle larvae.

[0104]

[0105] Preparation Example 3

[0106] Samples were obtained and stored in the same manner as in Preparation Example 1, except that white-spotted flower chafer larvae were used instead of longicorn beetle larvae.

[0107]

[0108] Preparation Example 4

[0109] Samples were obtained and stored in the same manner as in Preparation Example 1, except that brown beetle larvae were used instead of longicorn beetle larvae.

[0110]

[0111] Preparation Example 5

[0112] Samples were obtained and stored in the same manner as in Preparation Example 1, except that brown rice was used instead of longicorn beetle larvae.

[0113]

[0114] Experimental Example 1 - Measurement of cordycepin content

[0115] 1 g of the sample prepared according to Preparation Examples 1 to 5 was extracted in 10 ml of 80% methanol at room temperature.

[0116] The extract was filtered through a 0.45 μm PTFE filter, the solvent was removed by vacuum evaporation, and the dried extract from which the solvent was removed was diluted to 10 mg / ml in methanol.

[0117] The content of cordycepin was quantified by HPLC analysis. Cordycepin was quantified using an HPLC system equipped with a Waters 600 Q-pump, a 996 photodiode array detector, and Waters Empower software. Separation was performed on an RP-C18 column (5 μm, 10 mm × 150 mm) using isocratic elution with a mixture of methanol and water (12, 88, v / v). 10.0 μl of sample was injected. The solvent flow rate was set to 2 mL / min throughout the analysis. All separations were performed at room temperature with a detection wavelength of 260 nm and a run time of 40 min.

[0118] The measured cordycepin content is shown in Fig. 1 and Table 1 below.

[0119] Cordyceps militaris preparation medium Example 1 Preparation Example 2 Preparation Example 3 Preparation Example 4 Preparation Example 5 Main ingredients A. dichotomy B. mori P. brevitaris T. molitor Brown rice Cordycepin content (mg / g) 8.95 0.23 4.72 0.36 0.12

[0120] Referring to FIG. 1 and Table 1, it can be confirmed that the cordycepin content of the sample according to Preparation Example 1 utilizing a medium composition including longicorn beetle larvae is significantly higher than the cordycepin content of the samples according to Preparation Examples 2 to 5.

[0121]

[0122] In order to determine the relationship between the nutritional components of insects (Preparation Examples 1 to 4) and the cordycepin content, the contents of the nutritional components of samples prepared according to Preparation Examples 1 to 4 were determined, and the relationship between the nutritional components and the cordycepin content is shown in FIGS. 2 and 3 and Table 2 below.

[0123] At this time, the carbohydrate content of each sample was measured by the phenol-sulfuric acid method, the protein content was measured by the Karl Fischer method, and the fatty acid content was measured by the fatty acid analysis method using GC-MS.

[0124] Cordyceps militaris medium preparation example 1 preparation example 2 preparation example 3 preparation example 4 main ingredient A. dichotomy B. mori P. brevitaris T. molitor cordycepin content (%) 8.95 0.23 4.72 0.36 carbohydrate content (%) 35.6 110.31 12.72 8.17 protein content (%) 32.9 36 8.67 58.77 6 1.58 fatty acid content (%) 20.48 4.02 12.05 5.09

[0125] Referring to Figures 2, 3 and Table 2, each insect contained high levels of protein and fatty acid, but the content varied among each insect.

[0126] For example, in the case of the sample according to Preparation Example 2, the protein content was the highest (68.67%), and in the case of the sample according to Preparation Example 1, the protein content was the lowest (32.93%).

[0127] In contrast, the content of fatty acids was highest in the sample according to Preparation Example 1 (20.48%) and lowest in the sample according to Preparation Example 2 (4.02%).

[0128] In the case of carbohydrates, the order was sample according to Preparation Example 1, sample according to Preparation Example 3, sample according to Preparation Example 2, and sample according to Preparation Example 4.

[0129] Referring to Figure 3, which shows the correlation between nutritional components and cordycepin, the content of fatty acids showed the highest correlation with the content of cordycepin. The higher the content of fatty acids, the higher the content of cordycepin.

[0130] Conversely, proteins, previously known to play a significant role in increasing cordycepin content, showed the lowest correlation with cordycepin. As protein content increased, cordycepin content decreased.

[0131] From this, it was confirmed that fatty acids play an important role in the synthesis of cordycepin.

[0132] In order to determine which fatty acid among the fatty acids increases the content of cordycepin, the correlation between the content of palmitic acid, linoleic acid, and oleic acid and the content of cordycepin in the samples prepared according to Preparation Examples 1 to 4 was determined, and the results are shown in FIGS. 4 and 5 and Table 3 below.

[0133] At this time, the palmitic acid content, linoleic acid content, and oleic acid content of each sample were measured by fatty acid analysis using GC-MS.

[0134] Cordyceps militaris medium preparation example 1 preparation example 2 preparation example 3 preparation example 4 main ingredient A. dichotomy B. mori P. brevitaris T. molitor cordycepin content (%) 8.95 0.23 4.7 20.36 palmitic acid (%) 1.92 0.55 1.89 0.57 linoleic acid (%) 0.00 2.21 0.00 0.00 oleic acid (%) 10.89 0.46 7.90 2.31

[0135] Referring to FIGS. 4, 5 and Table 3, in the case of oleic acid, the sample according to Preparation Example 1 showed the highest content (10.89%), the sample according to Preparation Example 3 showed the next highest value (7.90%), and the sample according to Preparation Example 2 and the sample according to Preparation Example 4 showed very low oleic acid contents.

[0136] For linoleic acid, it was included only in the case of samples according to Preparation Example 2.

[0137] Referring to Figure 5, which shows the correlation between nutritional components and cordycepin, the content of oleic acid showed the highest correlation with the content of cordycepin. The higher the content of oleic acid, the higher the content of cordycepin.

[0138] On the other hand, linoleic acid showed the lowest correlation with the content of cordycepin, and when linoleic acid was included, the content of cordycepin was very low.

[0139] From this, it was confirmed that oleic acid plays an important role in the synthesis of cordycepin, and linoleic acid was confirmed to inhibit the synthesis of cordycepin.

[0140]

[0141] Experimental Example 2 - Genetic Analysis

[0142] The key genes involved in the biosynthesis of cordycepin are cns1 and cns2, and adenosine or its 2',3' cyclic monophosphate (2',3'-cAMP) is dephosphorylated into 3'-AMP by the product of cns2, and then cordycepin is synthesized from 3'-AMP by the product of cns1.

[0143] In order to analyze the effect of oleic acid, which showed a high correlation with the content of cordycepin in the above Experimental Example 1, on the expression of cns1 and cns2 genes, qRT-PCR analysis was used to measure the transcription levels of cns1 and cns2 in samples of Cordyceps militaris cultured on longicorn beetle larvae (Preparation Example 1) with the highest cordycepin content, silkworm larvae (Preparation Example 2) with the lowest cordycepin content, and brown rice (Preparation Example 5).

[0144] The primers used in qRT-PCR are listed in Table 4 below.

[0145] NameDNA SEQUENCE (5' to 3')CNS1-FCGCTTGATGAACCACCCTCTCNS1-RCTAGCATCATGCCTCCTCCGCNS2-FGCCATGGAAGACGCACAAAACNS2-RTCGTACATGTCGATGTGGGC

[0146] The experimental results are shown in Fig. 6 and Table 4 below, and the relative levels are shown using the sample according to Preparation Example 5 as a control group.

[0147] Cordyceps militaris medium preparation example 1 preparation example 2 preparation example 5 (control) main ingredient A. dichotomy B. mori Brown rice cns1 transcript level (x times) 43.50 1.631 cns2 transcript level (x times) 15.00 1.251

[0148] Referring to Fig. 6 and Table 5, it was confirmed that the cns1 and cns2 transcript levels of Cordyceps militaris of Preparation Example 1, which was cultivated using longicorn beetle larvae as the main ingredient of the medium, were remarkably high.

[0149]

[0150] Experimental Example 3 - Genetic Analysis

[0151] When oleic acid or linoleic acid was added to Cordyceps militaris (Preparation Example 1) grown using longicorn beetle larvae as the main ingredient of the medium, the relative expression levels of cns1 and cns2 genes were measured, and the results are shown in Fig. 7 and Table 6 below.

[0152] Additives Brown rice and oleic acid Brown rice and linoleic acid Control group (Control) Oleic acid Linoleic acid cns1 transcript level (x times) 13.36 0.98 cns2 transcript level (x times) 11.9 3 0.97

[0153] Referring to Figure 7 and Table 6, it was confirmed that the level of cns1 significantly increased when oleic acid was added compared to when no oleic acid was added. In addition, it was confirmed that the level of cns2 also increased approximately 1.9-fold due to the addition of oleic acid.

[0154] On the other hand, when linoleic acid was added, it was confirmed that the transcription levels of cns1 and cns2 were slightly reduced.

[0155] From this, it can be confirmed that oleic acid can significantly amplify the expression of cns1 and cns2 genes, which can significantly increase the production of cordycepin.

[0156]

[0157] Example 1

[0158] The Cordyceps militaris JW001 strain stored at -80°C was cultured at 25°C for 30 minutes, and the Cordyceps militaris strain was inoculated onto YMA (Yeast Mannitol Agar) medium, followed by culture in a dark room at 25°C for 1 week (see (d) of Figure 8).

[0159] Next, the YMA plate inoculated with the strain was cut into pieces with a diameter of 2 cm using a sterilized cylindrical cutter to prepare an inoculum (see (e) of Fig. 8).

[0160] A soybean powder-sucrose-liquid (SSB) medium containing 10.0 g of roasted soybean powder, 15.0 g / L sugar, 1.0 g / L KH2PO4, and 1.0 g / L MgSO4·7H2O was prepared, and the inoculum was added to the liquid culture medium, homogenized with a homogenizer, and then the liquid spawn was cultured for 5 days in a dark room at 25°C while supplying air through a 0.45 ㎛ PTFE filter to the culture bottle (see (f) of Fig. 8).

[0161] Meanwhile, red ginseng marc was placed in a grinder and ground (see (a) and (b) of Fig. 8, where (a) is the received red ginseng marc and (b) is the ground red ginseng marc). The average size of the ground red ginseng marc was approximately 20 mesh.

[0162] A mixture was prepared by mixing crushed red ginseng marc, sugar, and roasted soybean powder in a ratio of 10:1:1 (w / w), and peanut powder was added to the mixture in a ratio of 10:1 (w / w) to prepare a red ginseng marc medium. Next, the red ginseng marc medium was placed in an autoclave and sterilized at 121°C and 103 kPa for 60 minutes (see Fig. 8 (c)). Next, the red ginseng marc medium was slowly cooled to 25°C, and the sterilized red ginseng marc medium was dispensed into a transparent polypropylene box equipped with a silicone stopper for air entry.

[0163] The previously cultured liquid spawn and red ginseng marc medium were inoculated onto the red ginseng marc medium using a quantitative pump at a ratio of 1:10 (v / v) (see (g) of Figure 8).

[0164] Next, the red ginseng seed medium inoculated with liquid spawn was cultured in a dark room at 25°C for 7 days (see (h) of Fig. 8), and then irradiated with white light plus blue light for 4 weeks in a light culture room at 25°C (illuminance was maintained at 2000 lux) (see (i) of Fig. 8).

[0165] Next, the Cordyceps militaris mycelia culture cultured on a red ginseng paste medium was dried with hot air at 60°C for 48 hours and then ground into fine powder to obtain a culture sample, which was then stored at -80°C.

[0166]

[0167] Example 2

[0168] A culture sample was prepared in the same manner as in Example 1, except that a mixture was prepared by mixing crushed red ginseng powder, sugar, and roasted soybean powder in a ratio of 10:1:1 (w / w), and a red ginseng powder medium was prepared by mixing peanut powder in a ratio of 10:2 (w / w).

[0169]

[0170] Example 3

[0171] A culture sample was prepared in the same manner as in Example 1, except that a mixture was prepared by mixing crushed red ginseng powder, sugar, and roasted soybean powder in a ratio of 10:1:1 (w / w), and a red ginseng powder medium was prepared by mixing peanut powder in a ratio of 10:3 (w / w).

[0172]

[0173] Example 4

[0174] A culture sample was prepared in the same manner as in Example 1, except that a mixture was prepared by mixing crushed red ginseng powder, sugar, and roasted soybean powder in a ratio of 10:1:1 (w / w), and a red ginseng powder medium was prepared by mixing peanut powder in a ratio of 10:4 (w / w).

[0175]

[0176] Example 5

[0177] A culture sample was prepared in the same manner as in Example 1, except that a mixture was prepared by mixing crushed red ginseng powder, sugar, and roasted soybean powder in a ratio of 10:1:1 (w / w), and a red ginseng powder medium was prepared by mixing peanut powder in a ratio of 10:5 (w / w).

[0178]

[0179] Example 6

[0180] A culture sample was prepared in the same manner as in Example 1, except that a mixture was prepared by mixing crushed red ginseng powder, sugar, and roasted soybean powder in a ratio of 10:1:1 (w / w), and a red ginseng powder medium was prepared by mixing longicorn beetle larva powder in a ratio of 10:2 (w / w).

[0181]

[0182] Comparative Example 1

[0183] The Cordyceps militaris JW001 strain stored at -80°C was cultured at 25°C for 30 minutes, and the Cordyceps militaris strain was inoculated onto YMA (Yeast Mannitol Agar) medium, followed by culture in a dark room at 25°C for 1 week.

[0184] Next, the inoculated YMA plate was cut into pieces with a diameter of 2 cm using a sterilized cylindrical cutter to prepare an inoculum.

[0185] Soybean flour-sucrose-liquid (SSB) medium containing 10.0 g of roasted soybean flour, 15.0 g / L sugar, 1.0 g / L KH2PO4, and 1.0 g / L MgSO4·7H2O was prepared, and the inoculum was added to the liquid culture medium, homogenized with a homogenizer, and then the liquid spawn was cultured for 5 days in a dark room at 25°C while supplying air to the culture bottle through a 0.45 ㎛ PTFE filter.

[0186] Meanwhile, a brown rice medium consisting solely of brown rice was prepared. The brown rice medium was then placed in an autoclave and sterilized at 121°C and 103 kPa for 60 minutes. The brown rice medium was then slowly cooled to 25°C and dispensed into transparent polypropylene boxes fitted with air-permeable silicone stoppers.

[0187] The previously cultured liquid spawn and brown rice medium were inoculated onto the brown rice medium using a quantitative pump at a ratio of 1:10 (v / v).

[0188] Next, the brown rice medium inoculated with liquid spawn was cultured in a dark room at 25°C for 7 days, and then irradiated with white light plus blue light for 4 weeks in a light culture room at 25°C (illuminance was maintained at 2000 lux).

[0189] Next, the Cordyceps militaris mycelia culture cultured on brown rice medium was dried with hot air at 60°C for 48 hours and then ground into fine powder to obtain a culture sample, which was then stored at -80°C.

[0190]

[0191] Comparative Example 2

[0192] A culture sample was prepared in the same manner as in Comparative Example 1, except that brown rice medium was prepared by mixing brown rice with longicorn beetle larva powder at a ratio of 10:2 (w / w).

[0193]

[0194] Comparative Example 3

[0195] A culture sample was prepared in the same manner as in Comparative Example 1, except that brown rice medium was prepared by mixing brown rice and peanut powder in a ratio of 10:2 (w / w).

[0196]

[0197] Comparative Example 4

[0198] A culture sample was prepared in the same manner as in Example 1, except that a red ginseng marmalade medium was prepared by mixing crushed red ginseng marmalade, sugar, and roasted soybean powder in a ratio of 10:1:1 (w / w). Here, the red ginseng marmalade medium did not contain peanut powder or longicorn beetle larva powder.

[0199]

[0200] Experimental Example 4 - Measurement of cordycepin content

[0201] 1 g of the samples prepared according to Examples 1 to 6 and Comparative Examples 1 to 4 was placed in a 50 ml conical tube, and 20 ml of 50% ethanol was added to the conical tube.

[0202] A 50 ml beaker was placed on a hot plate, 300 ml of water was added, and a conical tube containing the sample was placed into the beaker (double boiler).

[0203] Next, an ultrasonic grinder was installed on the above-mentioned double boiler, and heating and ultrasonic extraction were performed for 1 hour at a temperature and frequency of 65 ℃ and 56 kHz.

[0204] Next, centrifugation was performed at 2000 rpm for 30 minutes, and the resulting supernatant was filtered through a 0.2 μm PTFE filter and transferred to a new tube. The culture extract samples thus prepared were stored at -80 °C.

[0205] The content of cordycepin was quantified by HPLC analysis. Cordycepin was quantified using an HPLC system equipped with a Waters 600 Q-pump, a 996 photodiode array detector, and Waters Empower software. Separation was performed on an RP-C18 column (5 μm, 10 mm × 150 mm) using isocratic elution with a mixture of methanol and water (12, 88, v / v). 10.0 μl of sample was injected. The solvent flow rate was set to 2 mL / min throughout the analysis. All separations were performed at room temperature with a detection wavelength of 260 nm and a run time of 40 min.

[0206] The measured cordycepin content is shown in Fig. 9 and Table 7 below.

[0207] Here, the average oleic acid content contained in the peanut powder was found to be about 22.5 wt%, and the average oleic acid content contained in the rhinoceros beetle larva powder was found to be about 11 wt% (see Table 3). In Table 7 below, the oleic acid content refers to the oleic acid content contained in the peanut powder or the rhinoceros beetle larva powder based on the entire medium composition, taking into account the average oleic acid content of the peanut powder or the rhinoceros beetle larva powder.

[0208] Sample No.Main ingredient (mixing ratio (w / w))Codycepin content (mg / g)Oleic acid content (wt%)Example 1Red ginseng marc mixture and peanut powder (10:1)10.942.04Example 2Red ginseng marc mixture and peanut powder (10:2)14.903.75Example 3Red ginseng marc mixture and peanut powder (10:3)14.925.19Example 4Red ginseng marc mixture and peanut powder (10:4)14.916.43Example 5Red ginseng marc mixture and peanut powder (10:5)14.897.5Example 6Red ginseng marc mixture and rhinoceros beetle larva powder (10:1)9.721.0Comparative example 1Brown rice (10:0)1.210Comparative example 2Brown rice and rhinoceros beetle Larva powder (10:2) 2.75 1.83 Comparative example 3 Brown rice and peanut powder (10:2) 6.3 3.75 Comparative example 4 Red ginseng powder mixture (10:0) 8.220

[0209] Referring to Fig. 9 and Table 7, it can be confirmed that the cordycepin content is relatively low in the culture samples according to Comparative Examples 1, 2, and 3 in which brown rice was used as a medium composition instead of red ginseng marc. In the case of the culture sample according to Comparative Example 1 in which only brown rice was used as a medium composition, the cordycepin content was significantly low.

[0210] The culture sample according to Comparative Example 4 showed a relatively high cordycepin content compared to Comparative Examples 1 to 3. From this, it was confirmed that when a red ginseng marc mixture was used in the medium, the amount of cordycepin produced increased compared to when brown rice was used in the medium.

[0211] On the other hand, in the case of culture samples according to Examples 1 to 6, it was confirmed that the cordycepin content was relatively high.

[0212] When comparing the culture samples according to Examples 1 to 5 using peanut powder as a medium composition, it can be confirmed that cordycepin increases significantly when the mixing ratio of the red ginseng marc mixture and peanut powder is 10:2 (w / w) or more (Examples 2 to 5).

[0213] From this, it was confirmed that when peanut powder containing rich oleic acid was mixed with a red ginseng powder mixture, the amount of cordycepin produced was significantly increased.

[0214] Additionally, it was confirmed that longicorn beetle larva powder containing rich oleic acid can also increase cordycepin production.

[0215]

[0216] Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims also fall within the scope of the present invention.

Claims

1. Regarding a medium composition for cultivating Cordyceps militaris, The above badge composition includes red ginseng powder, sugar, roasted soybean powder, and peanut powder. Based on the total weight of the above badge composition, oleic acid is 2.0 wt% or more. A composition of a medium for cultivating Cordyceps militaris.

2. In paragraph 1, Based on the total weight of the above badge composition, oleic acid is 3.5 wt% or more A composition of a medium for cultivating Cordyceps militaris.

3. In paragraph 1, The above medium composition is a medium composition for cultivating Cordyceps militaris containing a ginsenoside component.

4. A Cordyceps militaris culture cultivated by inoculating the medium composition of any one of clauses 1 to 3 with Cordyceps militaris spores.

5. In paragraph 4, A Cordyceps militaris culture having a cordycepin content of 10 mg / g or more.

6. In paragraph 4, The above Cordyceps militaris culture is a Cordyceps militaris culture containing ginsenoside components.

7. A composition for preventing or improving diabetes, comprising the Cordyceps militaris culture of Article 4.

8. Health functional food for preventing or improving diabetes containing Cordyceps militaris culture of Article 4.

9. The first step is to obtain a solid inoculum by culturing the Cordyceps militaris strain at 25°C. The second step is to prepare a soybean flour-sugrose-liquid medium, add the solid spawn inoculum to the soybean flour-sugrose-liquid medium, homogeneously crush it, and then culture the liquid spawn. The third step is to prepare a red ginseng marmalade by grinding the undried red ginseng marmalade, mixing the ground red ginseng marmalade, sugar, and roasted soybean powder to prepare a mixture, and mixing peanut powder into the mixture. The fourth step of inoculating the liquid starter into the red ginseng paste medium, and Step 5 of culturing the red ginseng paste medium inoculated with the above liquid spawn to obtain a Cordyceps militaris culture Including Method for producing Cordyceps militaris culture.

10. In paragraph 9, In the third step above, A method for producing a Cordyceps militaris culture, wherein the mixing ratio of the above mixture and the peanut powder is 10:1 (w / w) to 10:5 (w / w).

11. In paragraph 9, In the above 4th step, A method for producing a Cordyceps militaris culture in which the ratio of inoculated liquid spawn and red ginseng marc medium is 1:10 (v / v).

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