KR20090054114A - Method for producing high amount of cordycepin from the cultivated cordyceps militaris - Google Patents

Abstract

The present invention relates to a method for stably producing a high content of cordycepin during artificial culture of pupa caterpillar fungus, inducing fruiting body formation ability and cordycepin content by inducing fruiting body of monocythes isolated from fruiting body of cordyceps collected or artificially cultured in nature. After the selection of this excellent strain, the present invention relates to a method comprising combining and breeding spores of selected strains and spores of other strains to screen for Cordyceps fungi having high fruiting body forming ability and cordycepin content.

Cordycepin, cordyceps, combination sarcoma

Description

Method for producing high amount of cordycepin reliably during artificial cultivation of pupa cordyceps mushrooms {Method for producing high amount of Cordycepin from the cultivated Cordyceps militaris}

The present invention chrysalis cordyceps fungi ( Cordyceps militaris) (Linnaeus: Fries) Link.) Is a method of stably producing high content of cordycepin in artificial cultivation.

Cordyceps spp. Are among the ascus gyunmun (Ascomycota), nuclear gyungang (Pyrenomycetes), Claviceps purpurea neck (Clavicipitales), and Claviceps purpurea (Clavicipitaceae), Cordyceps sinensis in (Cordyceps). Cordyceps militaris (Linnaeus: Fries) Link. Is a kind of Entomopathogenic fungi, mainly orange larvae, formed by the larva or pupa of Lepidoptera. Within the cylindrical vesicles are filaments of filamentous filaments.

In addition to the method of collecting fruiting bodies of Cordyceps sinensis, in addition to the method of collecting fruiting bodies in the natural state, spawn culture and cultivation methods using silkworm chrysalis and mass culture methods using various artificial media were developed. For example, a method of mass cultivation of cordyceps mushrooms using insects (Korean Patent No. 10-179455) and a method of producing fruiting bodies in grain medium by plate culture and liquid inoculation of the fungi of Cordyceps sinensis (Korean Patent No. 10-266082). No.), characterized in that the dermis is mixed in the animal medium such as pupa, silkworm, slugs (Korean Patent No. 10-0432472), or the method of cultivation by adding gluten, soy protein and brewer's yeast ( Korean Patent No. 10-0644243).

Conventional Cordyceps sinensis production method is to induce fruiting bodies by separating tissues or spores collected from nature, and then select strains with good fruiting body formation. There is a disadvantage that the weakening does not form the fruiting body.

It has been found that pupa cordyceps sinensis contains components that enhance immune function, thereby promoting cell immunity and humoral immunity and reinforcing immunity functions to increase resistance to various tumors and viral infections. Korean Patent Publication No. 10-2001-0093364 discloses that the extract of Cordyceps sinensis mycelium has anticancer activity that induces apoptosis of DNA in tumor cells such as Jurkat, U937, HL-60 and kills it. It is described. In addition, Cordyceps sinensis is known to have antidiabetic effect (Oh, Young-Beom, 2003, Master's Thesis, Hallym University “Study on the efficacy of Cordyceps sinensis on diabetes-induced diabetes, hepatotoxicity and lipid metabolism”)

Cunningham et al. Revealed a useful pharmacological component such as cordycepin (3-deoxyadenosine), an isomer of quinic acid, which is contained in a large amount of C. militaris mushrooms (Cunningham, KG et al., J.). Som., P. 2299-3000, 1951).

                          Cordycepin

Cordycepin has been disclosed that it can be used as a medicine and health functional food for the treatment and prevention of diabetes through Korean Patent Publication No. 10-2005-0063164, Korean Patent No. 10-0516026 in red sackcloth Cordyceps ( C It describes a composition for preventing and treating vascular restenosis comprising as an active ingredient cordycepin produced from pruinosa ).

Patent applications related to the production of cordycepin include Korean Patent Application No. 10-2006-0064175, which describes a method for producing antiviral cordycepin by culturing pupa cordyceps mycelia using adenosine as a biotransformation substrate. Considering that the recovery rate (drying) of mycelium is 0.2-1% in liquid cultivation of pupa puertococcus pneumoniae fungi, and that the cordycepine should be purified by crushing the liquid cultured mycelium, the production cost of cordycepin by the method described in the above application It is estimated to be quite high. Therefore, there is a need to develop a method for producing a high content of cordycepin more efficiently and economically.

The information described herein is merely intended to help the understanding of the technical background of the present invention, it is a natural that it can not act as a prior art for the present invention.

When artificial culture of pupa cordyceps sinensis, finding a method for producing a high content of cordycepin economically and stably is a technical problem to be achieved by the present invention.

The inventors of the present invention by selecting the strain excellent in fruiting body formation ability, cordycepin content and combination ability by inducing the fruiting body of the spores isolated from the fruiting body of Cordyceps sinensis collected or artificially cultivated in nature as a solution of the technical problem, selection The present invention provides a method for stably producing a high content of cordycepin, including selecting a Cordyceps fungi having high fruiting body forming ability and cordycepin content by combining and breeding spores of different strains.

The present invention is a method for increasing the content of cordycepin extracted from the pupa puertococcus fungi mushroom, can be utilized in fruiting bodies collected or artificially cultivated in nature, the fruiting body of pupa pupa fungi according to the present invention is maintained by the strain of the strain It is effective to stably produce pupa cordyceps fungi having a high content of cordycepin since the mutation is prevented.

The present invention relates to a method for stably producing a high content of cordycepin during artificial culture of pupa caterpillar fungus, inducing fruiting body formation ability, cordycepin content by inducing fruiting body of single spores isolated from the fruiting body of cordyceps harvested or artificially cultured in nature And selecting a strain having excellent combinatorial capacity, and performing a combination breeding of spores of the selected strain and spores of other strains to select Cordyceps fungi having high fruiting body forming ability and cordycepin content.

The inventor of the present invention isolates the spores from the pupa cordyceps collected from nature, and then cultured to form an artificial fruiting body of the five spores of the pupa as a medium to observe the respective fruiting body formation ability and measure the cordycepin content It was. Among them, the fruiting body was formed quickly and uniformly (FIG. 1), the cordycepin content (Table 2) was the highest, and the combination ability was also excellent (C06S05) was selected.

The spores of the selected strain (C06S05) and the spores of the other different strains were cultured together to form fruiting bodies, and a combination of strains (C06S05xC06S33, C06S05xC06S43) with rapid fruiting formation and high content of cordycepin was selected.

The selected strains are excellent in fruiting body formation ability and have a high content of cordycepin, and when cultured, a large amount of cordycepin can be obtained. In addition, when using this method, the selected combinations are F1, for example, C06S05 (♀) xC06S33, C06S43 (♂), which is stable. It can produce cepin.

In addition, the mycelial growth was found to be good at 20 ~ 25 ^o C as a result of incubation at various temperatures to find the proper mycelial culture temperature of selected strains (C06S05xC06S33).

Based on the above experimental results, the present invention provides a method for stably producing a high content of cordycepin as follows.

[1] (a) separating monospores from fruiting bodies of Cordyceps sinensis;

         (b) culturing the isolated single spores to induce fruiting bodies, and selecting strains having excellent fruiting body forming ability, cordycepin content, and combination ability;

         (c) stably producing a high content of cordycepin from Cordyceps sinensis comprising the step of combining the spores of the strain selected in step (b) with spores of other strains and selecting Cordyceps fungi having high fruiting body forming ability and cordycepin content. How to.

[2] The method of [1], wherein the cordyceps is pupa cordyceps.

[3] The method of [1], which maintains the incubation temperature of Cordyceps sinensis at 20-25 ^° C.

[4] pupa cordyceps, produced by the method of any one of [1] to [3], filed with a varietal protection application (application number 2007-125) at the National Seed Administration.

Example

Hereinafter, the present invention will be described in more detail with reference to the following examples. As these Examples are only for illustrating the present invention, it is obvious that the scope of the present invention should not be construed as being limited by these Examples.

< Example 1 > Of a spore  detach

The spores were isolated from pertidish with PDA (potato dextrose agar) cultured from fresh fruit of chrysalis of Choonggi-dong Chungchocho mushrooms collected at Okgye-ri, Deoksan-myeon, Deoksan-myeon, Chungcheongnam-do. In the same manner as above, 44 monospores were separated and numbered C06S01 to C06S44. Separated spores were passaged in YMA (yeast malt agar) media and used for testing.

< Example  2> Of a spore  Selection

The pupa 80g was added to a 1100cc culture bottle and sterilized. Five strains of the isolated spores were inoculated by 20ml each, and cultured in a culture chamber at 20 ± 2 ° C. for 60 days to form an artificial fruit body.

The strain C06S01 had a faster fruiting body formation period, but the fruiting body was not uniform. The strain C06S02 had a slower fruiting body, but the fruiting body had a uniform characteristic. Also, the inside of the fruiting body is full. The strain C06S03 was slower and more heterogeneous than the strain C06S02, and was characterized by unevenness of white mycelia at the end of the head of the fruiting body. Strain C06S04 did not form fruiting bodies. The C06S05 strain showed a uniform, highly repellent, and full-bodied fruiting body (Figure 1 and Table 1).

Protein, sugar content and cordycepin content of the artificial fruiting body formed by the monospores culture were investigated.

Protein and sugar content were analyzed according to AOAC method ( 1995, Official Method of Analysis (16th Edition) Association of Official Analytical Chemist, Washington, DC). Crude protein was analyzed by Kjeldahl method and sugars were analyzed by Anthron method. 1 g of the dried sample was decomposed using a mixture of nitric acid, perchloric acid and nitric acid, and hydrochloric acid sequentially, diluted with a predetermined amount, filtered, and quantified by atomic absorption spectrometry using an ICP analyzer (GBC integra XMP, Australia).

Cordycepin was extracted using hot water and 80% ethanol and analyzed using HPLC. After accurately measuring 5 g of the sample, 100 g of water was added thereto, followed by extraction for 6 hours at 90 ° C., followed by filtration. The extraction was repeated by the above method, and the filtrate was concentrated to 20 mL. Four times of ethanol was added to the concentrate, and the mixture was left overnight. After centrifugation, the concentrate was concentrated to 50 mL. This fixed solution was filtered through a membrane filter of 0.45㎛ and analyzed by HPLC. The HPLC analyzer used was a Waters 510 (USA) instrument. The column was analyzed by Novapak C ₁₈ (3.9 × 300 mm) and the mobile phase was CH ₃ CN: H ₂ O (18:82, v / v). The flow rate was 0.75 mL / min, and the detector was measured at 254 nm by UV and compared with a standard cordycepin (Sigma, USA).

The C06S05 strain was selected as a candidate for the combination breeding to be performed in the next step in consideration of the results of the culture and the content of cordycepin, such as culture characteristics and biological efficiency (%).

< Example  3> Spores  Combination ability test

In order to investigate the combination ability using the separated spores, add 80 g of pupa to the 1100cc culture bottle, sterilize and inoculate 20ml of the cultured combination cultured liquid, and incubate in the culture room at 20 ± 2 ℃ for 60 days. Induction of formation was investigated.

The strain C06S01xC06S02 is fast to ripen and the fruiting body is long. The fruiting bodies of the fruiting bodies are medium, the occurrence of the fruiting bodies is not uniform, and there are secondary fruitings. The strain C06S01xC06S03 has a fast maturity and a long fruiting body. As the fruiting body is thin, the fruiting body falls to the side during harvesting, and the occurrence of fruiting body is not uniform. The strain C06S01xC06S04 had slow growth of hyphae in the pupa, the host, and the formation of fruiting bodies was weak, resulting in poor fidelity. C06S01xC06S05 strain is fast ripening, and the thickness of the fruiting body is also thick. There is growth of secondary fruiting bodies, but it can be used as an early seed strain. The strain C06S02xC06S03 has a slow development of fruiting bodies and severe secondary pruning. The fruiting body is a hollow form. Strain C06S02xC06S04 showed slow growth of mycelia and poor fruiting. The strain C06S02xC06S05 was medium in length and mature, and the fruiting body was full in shape and had good uniformity. Strain C06S03xC06S04 had no fruiting body.

After the artificial fruiting body was formed in a 1100cc culture bottle using a pupa 80g medium, the protein, sugar content, and cordycepin content of each combination were examined by the method described in Example 2.

In consideration of the fruiting body formation ability, cordycepin content, and the combination ability through Example 2 and Example 3, C06S05 was selected as the assay parent for the next combination breeding. C06S05 was fast and uniform mushroom development.

< Example  4> Selection of excellent strains of fruiting bodies

The selected C06S05 strains were cultured in YMB (yeast malt broth) medium for 4 days using chrysalis from C06S06 strains to C06S44 strains.

Fruiting Body Formation in Combining C06S05 and Other Strains  Strain combination Asymptomatic pyramidal formation Chair  Feet  Fruiting body (g / bottle) Biological Efficiency (%) Length (cm) Thickness (mm) Biomass  Dry weight C06S05 X C06S06 X 2 2.0 7.0 12.5 2.3 49.1 C06S05 X C06S07 X 2 5.0 9.8 23.5 4.4 125.6 C06S05 X C06S08 X 0.5 1.0 1.8 3.8 1.0 15.1 C06S05 X C06S09 0.0 0.0 0.0 0.0 C06S05 X C06S010 X One 5.0 7.5 12.5 2.8 43.7 C06S05 X C06S011 0 0 0.0 0.0 0.0 0.0 C06S05 X C06S012 X 4 5.5 15.3 30.0 6.6 120.0 C06S05 X C06S013 O 6.5 4.0 61.3 50.0 9.1 267.5 C06S05 X C06S014 O 3 1.2 160.0 28.8 4.9 132.3 C06S05 X C06S015 X 2.5 4.0 53.8 35.0 6.5 177.1 C06S05 X C06S016 X 3.5 2.0 10.3 12.0 2.1 48.1 C06S05 X C06S017 O 4 1.2 100.0 27.5 4.2 117.8 C06S05 X C06S018 0 0.0 150.0 2.3 0.4 9.7 C06S05 X C06S019 0 0.0 0.0 0.0 0.0 C06S05 X C06S020 X 2 2.0 35.0 17.3 2.6 78.2 C06S05 X C06S021 O 3 2.0 36.5 0.0 0.0 0.0 C06S05 X C06S022 X 3 2.0 175.0 39.0 6.7 194.2 C06S05 X C06S023 0 0.0 8.3 1.6 35.1 C06S05 X C06S024 0 0.0 0.0 0.0 0.0 C06S05 X C06S025 X One 2.0 15.0 0.0 0.0 0.0 C06S05 X C06S026 O 3 1.2 105.0 5.0 1.1 20.9 C06S05 X C06S027 0 0.0 0.0 0.0 0.0 C06S05 X C06S028 X 2 1.0 45.0 0.0 0.0 0.0 C06S05 X C06S029 0 0.0 0.0 0.0 0.0 C06S05 X C06S030 O 4 2.5 125.0 33.0 5.6 172.8 C06S05 X C06S031 X 6 3.0 31.3 35.3 5.7 284.3 C06S05 X C06S032 O 9 4.5 100.0 50.0 5.4 831.5 C06S05 X C06S033 O 9 5.0 100.0 66.6 9.2 759.1 C06S05 X C06S034 0 0.0 0.0 0.0 0.0 C06S05 X C06S035 X 7 7.0 5.5 22.5 5.8 144.4 C06S05 X C06S036 X 11 9.0 5.0 40.6 7.3 313.8 C06S05 X C06S037 O 6 5.0 60.0 23.0 3.5 147.2 C06S05 X C06S038 O 7 3.0 90.0 35.0 6.2 334.9 C06S05 X C06S039 O 7 3.0 100.0 41.6 6.4 329.6 C06S05 X C06S040 O 3.5 2.0 60.0 23.0 2.6 153.0 C06S05 X C06S041 O 5 2.3 68.0 25.5 3.0 174.7 C05S06 X C06S042 X 2 2.0 10.0 4.6 0.8 25.5 C06S05 X C06S043 X 6 5.0 18.0 41.6 7.5 334.2 C06S05 X C06S044 X 4.5 4.0 15.0 38.3 6.8 283.2

From these results, C06S05xC06S13, C06S05xC06S22, C06S05xC06S31, C06S05xC06S32, C06S05xC06S33, C06S05xC06S36, C06S05xC06S43 were selected and the content of cordycepin was measured.

Considering the content of cordycepin, strains C06S05 × C06S33 and C06S05 × C06S43 were selected. Cordycepin content of C06S05 strain was 246mg%, and C06S05 × C06S33 strain was 325mg% and C06S05 × C06S43 strain was 280mg%. Thereafter, strains C06S05 × C06S33 and C06S05 × C06S43 were cultured, respectively, to confirm that the same characteristics were maintained.

The selected strains were filed with the varieties protection application (Application No. 2007-125) as the cultivar name Yedang No. 3 on March 23, 2007, to the National Seed Management Office.

< Example  5> Optimum mycelial culture temperature

C06S05 × C06S33 strains were inoculated into YMA (yeast malt agar) medium and cultured at 15 ° C., 20 ° C., 25 ° C., 30 ° C., and 35 ° C. for 15 days. Growth was good between 20 ~ 25 ^o C and mycelial growth was the best at 25 ℃ (Fig. 3).

1 shows the results of culturing single spores (C06S01, C06S02, C06S03, C06S04, C06S05) isolated from pupa cordyceps.

2 shows the culture results of strains C06S05 × C06S33 and C06S05 × C06S43.

Figure 3 is a graph comparing the mycelial growth according to the culture temperature of strain C06S05 × C06S33.

Claims (4)

(a) separating monospores from the fruiting bodies of Cordyceps sinensis; (b) culturing the isolated single spores to induce fruiting bodies, and selecting strains having excellent fruiting body forming ability, cordycepin content, and combination ability; (c) stably producing a high content of cordycepin from Cordyceps sinensis comprising the step of combining the spores of the strain selected in step (b) with spores of other strains and selecting Cordyceps fungi having high fruiting body forming ability and cordycepin content. How to. The method according to claim 1, wherein the cordyceps is pupa cordyceps. The method of claim 1, wherein the culture temperature of Cordyceps sinensis is maintained at 20-25 ^° C. Chrysalis of the Cordyceps sinensis produced by the method of any one of claims 1 to 3, and applied for the protection of varieties (Application No. 2007-125) to the National Seed Management Office.

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