Culture medium for enhancing cordycepin content using hermetia illucens adult insects and method for extracting cordycepin using same

Abstract

Provided is a culture medium for enhancing the content of cordycepin from Cordyceps militaris, wherein the culture medium comprises Hermetia illucens adult insects. According to the present invention, the culture medium can ensure a significantly higher cordycepin yield than conventional grain-based media or pupa-based media by means of a method for extracting cordycepin from Cordyceps militaris cultured using Hermetia illucens adult insects as a host.

Description

Culture medium for increasing cordycepin content using adult larvae of Dongae and method for extracting cordycepin using the same

[0001] The present invention relates to a technology for cultivating and extracting effective components of Cordyceps militaris, and more specifically, to a culture medium and a cordycepin extraction method for increasing the cordycepin content by culturing pupae of Cordyceps militaris using adult Hermetia illucens.

[0002] Cordycepin (3'-deoxyadenosine) is a representative bioactive substance derived from certain medicinal mushrooms, including Cordyceps militaris. Its anticancer, anti-inflammatory, antiviral, immunomodulatory, and antioxidant properties have been reported. In particular, cordycepin is attracting attention as a candidate for various tumor treatments due to its cell growth inhibition and apoptosis-inducing effects. Consequently, active development of industrial mass production technologies is underway.

[0003] Traditionally, Cordyceps militaris was cultivated primarily using rice, silkworm pupae, and brown rice as hosts. Cordycepin was then extracted and purified using organic solvents. However, these traditional cultivation and extraction processes have the following drawbacks.

[0004] - Variation in cordycepin yield: Cordycepin content is low or inconsistent due to limitations in medium components.

[0005] - Extraction conditions not established: Quantitative extraction is difficult due to lack of optimization of variables such as solvent ratio, extraction time, and temperature.

[0006] - Non-standardization of the purification process: Only partial separation is possible when the active ingredients are mixed, and it is difficult to secure high-purity cordycepin.

[0007] Various methods have been attempted to extract cordycepin from living organisms, and it is known that the production of cordycepin varies greatly depending on the isolation region of the Cordyceps militaris strain, the type of medium, and the culture conditions. In the past, 3.41, 2.72, and 1.85 mg / g of cordycepin were produced from fruiting bodies after culturing pupae of Cordyceps militaris using rice, silkworm pupae, and wheat as media, respectively. Cases of extracting cordycepin after culture using artificial media have also been reported. In a specific cultivation method, a cordycepin content of up to 18110.27 mg / kg was measured from fruiting bodies cultured for a total of 42 days, including a culture step that performed repeated low-temperature shock cycles. In addition, 28.338 and 20.445 mg / g of cordycepin were measured from fruiting bodies cultured after preparing a spore suspension containing insect extract and inoculating it into rhinoceros beetles.

[0008] However, existing cordycepin extraction and production methods have several limitations. Some host materials (e.g., silkworm pupae) require full importation, which hinders their substantial contribution to domestic farm income. Furthermore, there has been a persistent need to further improve cordycepin production yield, purity, and economic feasibility for industrial application.

[0009] Accordingly, the problem to be solved by the present invention is to provide a new extraction method for effectively increasing the content of cordycepin, mycelia for the method, and an extract with a high content of cordycepin extracted therefrom.

[0010] The present invention relates to a culture medium for increasing cordycepin content, wherein the culture medium is characterized in that it includes adult Hermetia illucens as a host for culturing pupae of Cordyceps militaris.

[0011] In one embodiment of the present invention, the adult mosquitoes are provided in a dried form and function as a carbon source and lipid source in a culture medium.

[0012] In addition, the present invention relates to a method for extracting cordycepin from Cordyceps militaris cultured using adult Cordyceps militaris as a host, comprising the steps of (a) extracting Cordyceps militaris mycelia using ethanol as a solvent; (b) concentrating the extract of step (a); and (c) isolating and purifying cordycepin from the concentrate of step (b).

[0013] In one embodiment of the present invention, the Cordyceps militaris mycelia can be cultured by (a) inoculating a pupa of Cordyceps militaris into a culture medium containing adult Cordyceps militaris; and (b) culturing under temperature and light conditions of 20 to 25°C for 6 to 11 weeks.

[0014] In one embodiment of the present invention, the ethanol is 50% ethanol, and the extraction can be performed under the following conditions: the extraction solvent drainage is 5 to 8 times; the extraction temperature is 50 to 80°C; the extraction time is 1 to 4 hours; and the number of extractions is 1 to 3 times.

[0015] In one embodiment of the present invention, the separation and purification of cordycepin is performed using chromatography, and includes a step of selectively purifying and obtaining a fraction having a high cordycepin content among fractions fractionated multiple times.

[0016] In addition, the present invention provides a mycelium of Cordyceps militaris used in the above extraction method, which is a mycelium cultured using adult Cordyceps sinensis as a host, and further provides an extract having a cordycepin content of 700 mg / g or more produced by the above method.

[0017] According to the present invention, a method for extracting cordycepin from Cordyceps militaris cultured using black soldier flies as a host can secure a significantly higher cordycepin yield than conventional grain-based media or pupa-based media. The extract according to the present invention has a cordycepin content of 700 mg / g or more, which is more than three times higher than that of conventional cultures widely used commercially, and thus has very high industrial value. In addition, by including a chromatography-based purification step, it is possible to obtain high-purity cordycepin, and the method of selectively purifying only the portion containing a high concentration of the active ingredient among the fractions greatly increases the possibility of its use in various application fields such as functional materials and pharmaceutical raw materials. Furthermore, the invention is also a technology that can effectively convert black soldier flies, which were previously used as feed or waste, into the production of high-value-added physiologically active substances, and thus has the effect of increasing the industrial utilization value of insect-derived by-products.

[0018] Figure 1 shows the results of thin layer chromatography analysis.

[0019] Figure 2 is a step diagram of an extraction method according to one embodiment of the present invention.

[0020] Figure 3 is a step diagram of a method for separating and purifying cordycepin, an indicator substance, from Cordyceps militaris.

[0021] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the attached drawings. However, the present invention is not limited to the following embodiments, and it will be apparent to those skilled in the art that various modifications or variations can be made within the spirit and scope of the present invention. Terms or words used in this specification and claims should not be interpreted as limited to their conventional or dictionary meanings, but should be interpreted with meanings and concepts consistent with the technical spirit of the present invention based on the principle that the inventor can appropriately define the concept of a term to best explain his or her invention. In addition, repeated descriptions of technical structures and operations identical to those of the prior art will be omitted.

[0022] Additionally, whenever it is stated throughout this specification that a component "includes" another component, it may mean that any other component may be included, rather than excluding any other component, unless specifically stated otherwise.

[0023] Meanwhile, it should be noted that the description of the third component or means may be omitted.

[0024] The present invention involves extracting cordycepin from Cordyceps militaris cultured in a medium containing adult Cordyceps militaris. The cordycepin content is significantly increased in the mycelia or fruiting bodies of Cordyceps militaris grown in a medium containing adult Cordyceps militaris, which serves as a carbon and lipid source. This will be described in more detail below.

[0025] The Cordyceps militaris fungus used in the present invention was performed using a pupa Cordyceps militaris fungus as a test strain, but is not limited thereto.

[0026]

[0027] Example 1

[0028] Preparation of complex media, inoculation and cultivation of Cordyceps militaris pupae

[0029] Preparation of larvae and adults in Dongae-deung

[0030] The artificially reared adult scorpion fly used in the present invention dies after laying eggs when placed in a spawning chamber, allowing only the adult scorpion fly to be effectively separated from the scorpion fly collected on the floor. The larvae and adults that developed after laying eggs were dried in a desiccator and then used as samples. Furthermore, the supply of larvae and adults can be continuously maintained through a continuous spawning system.

[0031]

[0032] Preparation and cultivation of inoculum (Cerdyceps militaris)

[0033] The Cordyceps militaris fungus used as an inoculum in the present invention was, but is not limited to, Cordyceps militaris pupae. The Cordyceps militaris pupae fungus was inoculated onto PDB medium and used as an inoculum after culturing for 7 days.

[0034]

[0035] Preparation and inoculation of test media

[0036] As a preliminary experiment, a medium was prepared by adding sterilized distilled water to adult worms (30 g) and brown rice (30 g) in a 1:1 (w / v) ratio, and sterilized at 121°C and 1.2 atm for 15 minutes. 10 ml of the culture suspension containing the inoculum prepared in the above 1-2 was inoculated onto the adult worm and brown rice medium, and cultured at 20°C under light conditions for 11 weeks.

[0037] In this experiment, 100 ml, 100 ml, 105 ml and 105 ml of sterilized distilled water were added to adult larvae (50 g, Example 1), brown rice (100 g, Comparative Example), adult + larvae (35 g each, Example 2) and adult + larvae (15 g and 45 g each, Example 3) of Dongae-deung, respectively, to prepare media, and sterilize at 121°C and 1.2 atm for 15 minutes. 20 ml of the culture suspension containing the inoculum prepared in the above 1-2 items was inoculated onto the adult larvae and brown rice media, and cultured under light conditions at 25°C for 8 weeks.

[0038]

[0039] Experimental Example 1

[0040] Ethanol extraction from Cordyceps militaris mycelia

[0041] ethanol extraction

[0042] In the above example, the cultivated Cordyceps militaris mycelia were extracted with ethanol (1:10, w / v) in a shaking incubator (25°C / 200 rpm) for 24 hours. Each extract was filtered using Whatman No. 1 (GE Healthcare, Buckinghamshire, UK) filter paper, and the solvent was evaporated using a rotary evaporator (EYELA NN, Tokyo, Japan) and concentrated in distilled water (1 / 10, v / v).

[0043]

[0044] Thin layer chromatography (TLC) of ethanol extract

[0045] Commercially purified cordycepin (Merck Sima, Germany) was purchased, and 1 μg (Lane 1) and 10 μg (Lane 2), and 10 mg of Cordyceps militaris mycelia extract cultured on brown rice medium and 10 mg of Cordyceps militaris mycelia extract cultured on adult culture medium (Lane 4) were analyzed by TLC silica gel 60 F. 254 The plate (Merck Millipore, Germany) was treated and thin layer chromatography was performed for 3 hours in a developing solvent (chloroform:methanol=1:1, v / v).

[0046] Figure 1 shows the results of thin layer chromatography analysis.

[0047] Referring to Figure 1, the results of the primary sample experiment showed that lane 4, which was treated with 10 mg of Cordyceps militaris mycelia extract cultured on adult insect culture medium, had a higher cordycepin content than lane 3, which was treated with 10 mg of Cordyceps militaris mycelia extract cultured on brown rice medium. However, the spot intensity was observed to be weaker than that of 10 μg of pure cordycepin, which was an indicator substance.

[0048] As a result of the secondary sample experiment, 10 mg of Cordyceps militaris mycelia extract cultured on brown rice medium (lane 3) showed the lowest content. In addition, 10 mg of Cordyceps militaris mycelia extract cultured on adult black fly medium (lane 4) showed a higher cordycepin content than 10 mg of Cordyceps militaris mycelia extract cultured on adult + larvae (3:1, w / w) mixed medium (lane 5) and 10 mg of Cordyceps militaris mycelia extract cultured on adult + larvae (1:1, w / w) mixed medium (lane 6). In addition, the spot intensity was observed to be stronger than that of 10 μg of pure cordycepin, which was the indicator substance.

[0049]

[0050] High-performance liquid chromatography (HPLC) analysis of ethanol extracts

[0051] To obtain certification, primary sample analysis (Certificate No. 2404S0281) and secondary sample analysis (Certificate No. 2405S0393) were requested from the Korea Institute of Science and Technology (KIST, Seoul, Korea).

[0052] Analysis of the primary sample using high-performance liquid chromatography showed that the content of cordycepin, an effective ingredient extracted from mycelia cultured on a medium containing adult larvae, increased 7.3-fold compared to the mycelia extract harvested from Cordyceps militaris cultured on brown rice medium.

[0053] Table 1 below shows the results of comparing the cordycepin content extracted with ethanol from the mycelia of Cordyceps militaris cultured on brown rice medium and medium containing adults.

[0054] Sample name (classification) Unit test item and test result comparison example ppm (mg / ℓ) Cordycepin 103.66 Example 1 ppm (mg / ℓ) Cordycepin 752.15

[0055] Another experiment was conducted to compare the effects of using a medium containing a mixture of adult and larval Cordyceps militaris on the content of cordycepin. To this end, Cordyceps militaris strain KACC44455 was used to culture Cordyceps militaris in a medium containing adult and adult + larvae, and brown rice was used as a control. The results of quantitative analysis of cordycepin, a major component of the cultured Cordyceps militaris, are shown in Table 2 below.

[0056] Test items and test results for each medium Brown rice medium ppm (mg / ℓ) Cordycepin 531.14 Adult medium ppm (mg / ℓ) Cordycepin 2717.35 Adult + larvae (1:1, w / w) ppm (mg / ℓ) Cordycepin 2428.14 Adult + larvae (3:1, w / w) ppm (mg / ℓ) Cordycepin 2638.62

[0057] Referring to Table 2, the results of analyzing the secondary sample by high-performance liquid chromatography (HPLC) showed that the content of cordycepin, an effective ingredient, extracted from the mycelia cultured in a medium containing adults was significantly increased compared to the mycelia extracts harvested from the medium containing adults + larvae (1:1, w / w) and adults + larvae (3:1, w / w). The present invention further provides a novel method for efficiently obtaining a Cordycepin extract containing a high content of cordycepin, and Cordyceps militaris for the same in the form of mycelia or fruiting bodies. In particular, the present invention extracts, concentrates, separates, and purifies cordycepin from the mycelia or fruiting bodies of Cordyceps militaris cultured using adult soldiers flies as a host, and as a result, cordycepin can be extracted at a significantly higher content than that of other conventional insects. That is, the present invention focused on the fact that Hermetia illucens is an insect that is easy to artificially rear and can be mass-produced, and its adults can be utilized as an alternative medium that can solve the problems of existing host materials that depend on imported silkworm pupae and ensure economic feasibility. In addition, the present invention focused on the novel point that the unique nutritional composition of Hermetia illucens adults, especially the high lipid content and oleic acid content, can increase the cordycepin production of Cordyceps militaris fungus. This will be explained in more detail in the experimental examples below.

[0058]

[0059] Example 2

[0060] The Cordyceps militaris mycelia used in the examples of the present invention are mycelia cultured by a specific culture method (by inoculating pupae of Cordyceps militaris into a culture medium containing adult Cordyceps militaris).

[0061] In one embodiment of the present invention, the mycelia were cultured by a method comprising the steps of (a) inoculating a pupa of Cordyceps militaris into a culture medium containing adult Cordyceps militaris; and (b) culturing under temperature and light conditions of 20 to 25°C for 6 to 11 weeks.

[0062] Thereafter, (a) the cultured mycelia are extracted using ethanol as a solvent, (b) the extract of step (a) is concentrated, and (c) cordycepin is separated and purified again from the concentrate of step (b).

[0063] Figure 2 is a step diagram of an extraction method according to one embodiment of the present invention.

[0064] Referring to Figure 2, the extraction in step (a) can be performed using 50% ethanol as a solvent. This is because cordycepin is a polar substance, but it also contains a purine ring structure, which significantly affects the ethanol concentration in terms of its non-polar properties, significantly affecting the extraction efficiency. This will be explained in more detail in the experimental examples below.

[0065] In the present invention, the extraction in step (a) was performed under the conditions of extraction solvent drainage 8 times, 70°C, 3 hours, and 2 extractions. However, the extraction may be performed with extraction solvent drainage 5 to 8 times, extraction temperature 50 to 80°C, extraction time 1 to 4 hours, and number of extractions 1 to 3 times. This range of conditions provides an optimal extraction environment for obtaining high content cordycepin.

[0066] The extract concentration in step (b) above can be performed using a general concentration method. For example, evaporative concentration using a rotary evaporator can be applied.

[0067] According to one embodiment of the present invention, the separation and purification of cordycepin in step (c) is performed using chromatography. Chromatography is an effective method used to separate and purify a specific component from a mixture, and is an essential step for securing high purity of cordycepin.

[0068] Figure 3 is a step diagram of a method for separating and purifying cordycepin, an indicator substance, from Cordyceps militaris.

[0069] Referring to FIG. 3, in one embodiment of the present invention, the cordycepin separation and purification in step (c) includes a plurality of fractionation processes, and includes a step of selecting and purifying a fraction (the fifth fraction) having a high cordycepin content among the fractionated fractions.

[0070] In one embodiment of the present invention, the following multiple rounds of chromatographic purification were performed.

[0071] In the first separation / purification step, the concentrate can be separated using RP-18 chromatography under methanol:water (0:100 → 100:0 (v / v)) conditions to obtain an active fraction.

[0072] In one embodiment of the present invention, selecting the fifth fraction among the seven fractions was advantageous in securing a fraction with a high cordycepin content.

[0073] In the second separation / purification step, a specific fraction from the active fraction obtained in the above step can be separated using silica gel chromatography in the condition of CHCl3:MeOH (10:1 → 1:1 (v / v)). High-purity cordycepin can be ultimately obtained by purification from the third fraction among the five fractions obtained in this step. Through these multiple fractionation and selective purification processes, the purity and yield of cordycepin can be maximized.

[0074]

[0075] Experimental Example 2

[0076] Comparison of extraction efficiency and content according to ethanol concentration

[0077] In this experimental example, an experiment was conducted to compare the cordycepin content according to the type of extraction solvent in order to establish a cordycepin extraction method.

[0078] In this experiment, 1 kg of Cordyceps militaris cultured by the applicant was used as a raw material, and the experiment was conducted under the same conditions as the type of extraction solvent (water, 20% ethanol, 50% ethanol, 80% ethanol, 100% ethanol), extraction solvent multiple (8 times), extraction temperature (70℃), extraction time (3 hours), concentration temperature (50℃), and number of extractions (2 times).

[0079] After preparing the extracts according to each extraction condition, the extraction yield and cordycepin content were quantified according to the ethanol content, and the results are shown in Table 3 below.

[0080] Extraction ConditionsWeight (g)Dry Weight (g)Yield (%)Cordycepin Content (%)Water Extraction101.4114.13.8520% Ethanol101.3213.23.8950% Ethanol101.2212.24.3480% Ethanol101.0410.43.99100% Ethanol100.818.12.49

[0081] As shown in Table 3, the cordycepin content in the 50% ethanol extract was the highest at 4.34% compared to other ethanol extracts. This suggests that cordycepin, although a polar substance, also possesses some nonpolar properties, including a purine ring structure, and thus exhibits significantly high solubility in a 50% concentration of water and ethanol. Comparison of extraction efficiency and content by type of Cordyceps sinensis

[0082] In this experiment, in order to compare and analyze the effect of the type of insect used as a Cordyceps militaris medium on the cordycepin content, the cordycepin content of existing known insects was compared with the results according to the present invention.

[0083] Table 4 below shows the results of this comparison.

[0084] Insect medium cordycepin content (mg / g) References Adult black soldier fly (Hermetia illucens) 720-Rhinoceros beetle (Allomyrina dichotoma) 89.5 Turk et al., 2022 (Frontiers in Microbiology) Mealworm (Tenebrio molitor) 13.75 Ha et al., 2022 (Foods, 11(16), 2477) Larvae of white-spotted flower chafer (Protaetia brevitarsis) 2.932 Nguyen et al., 2023 (Fermentation, 6(4), 30) Silkworm (Bombyx mori) 2.63 Turk et al., 2022 (Frontiers in Microbiology) Grasshoppers (Caelifera spp.) 1.85 Kim et al., 2022 (ResearchGate dataset) Cricket (Gryllus bimaculatus) 1.65 Kim et al., 2022 (ResearchGate dataset)

[0085] As shown in Table 4, the content of cordycepin, the main substance of Cordyceps militaris, was very high at 720 mg / g when black soldier flies were used as a host. This is a significantly superior cordycepin content compared to Cordyceps militaris cultured in other insect hosts (e.g., 89.5 mg / g of rhinoceros beetles, 13.75 mg / g of mealworms, 2.63 mg / g of silkworms, etc.). The results described above demonstrate that each developmental stage of black soldier flies, especially adults, are very effective hosts for increasing cordycepin content, and this result is thought to be related to the high lipid content and oleic acid content of black soldier flies that act favorably on cordycepin production.

[0086] The present invention regarding cordycepin extraction is recognized as having industrial applicability.

Claims

1. A culture medium for increasing cordycepin content, characterized in that it includes adult Hermetia illucens as a host for cultivating pupae of Cordyceps militaris.

2. In paragraph 1, The above-mentioned adult insects are provided in dried form, and the culture medium for increasing cordycepin content is characterized in that it functions as a carbon source and lipid source in the culture medium.

3. A method for extracting cordycepin from Cordyceps militaris cultured using adult Cordyceps militaris as a host, characterized in that it comprises the following steps: (a) A step of extracting the Cordyceps militaris using ethanol as a solvent; (b) a step of concentrating the extract of step (a); and (c) A step of separating and purifying cordycepin from the concentrate of step (b).

4. In paragraph 3, A method for extracting cordycepin, characterized in that the Cordyceps militaris is cultured by a method comprising the following steps: (a) a step of inoculating Cordyceps militaris pupae into a culture medium containing adult worms; and (b) Culturing step for 6 to 11 weeks under temperature and light conditions of 20 to 25°C.

5. In paragraph 3, A cordycepin extraction method characterized in that the above ethanol is 50% ethanol and the extraction of step (a) is performed under the following conditions: - Extraction solvent drainage is 5 to 8 times; - Extraction temperature is 50 to 80℃; - Extraction time is 1 to 4 hours; and - The number of extractions is 1 to 3 times.

6. In paragraph 3, A cordycepin extraction method characterized in that the cordycepin separation and purification in step (c) above is performed using chromatography.

7. In paragraph 6, The separation and purification of cordycepin in step (c) above is as follows: A method for extracting cordycepin, characterized by including a step of purifying and obtaining a fraction having a high cordycepin content among fractions fractionated multiple times.

8. A cordycepin extraction method using Cordyceps militaris for extracting cordycepin according to any one of clauses 3 to 7, wherein the Cordyceps militaris is cultured using adult Cordyceps sinensis as a host.

9. An extract having a cordycepin content of 700 mg / g or more, prepared by the method according to any one of claims 3 to 7.

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