KR101810854B1 - Cordyceps militaris having improved capability of producing cordycepin - Google Patents

Abstract

The present invention provides a cordyceps militaris strain having an improved cordycepin production ability, and more specifically, to a cordyceps militaris S6xS8 strain having a significantly improved cordycepin production ability. [Consignment number: KCTC 13088BP]

Description

{CORDYCEPS MILITARIS HAVING IMPROVED CAPABILITY OF PRODUCING CORDYCEPIN}

The present invention relates to a Cordyceps sinensis strain having improved cordycepin producing ability, specifically Cordyceps militaris S6xS8 strain.

Some of the fungus of Aspergillus fungus grows in insects with insects as a host. When certain conditions are met, insects penetrate to form fruiting bodies, collectively referred to as caterpillar fungus.

Chinese caterpillar fungus has been used as a traditional medicinal product for a long period of time, as it is said to be the three greatest herbals in China, and there is a legend that the Qin Shi Huang and the poppy have maintained youth by using Cordyceps.

The fungus that forms Cordyceps has host-specificity depending on the species. For example, Cordyceps cicadae hosts cicadas and Ophiocordycepss sinensis , Paecilomyces hepialid , Lecanicillium , and the like are bats The moth larva is used as a host. On the other hand, Cordyceps militaris ) has a wide host specificity and can be used as a host of various insects.

Cordyceps contains various low-molecular substances such as polysaccharides, cordycepin, adenosine, lovastatin, ergosterol and various unsaturated fatty acids, which are known to contribute to the physiological activity of Cordyceps sinensis (Lo et al. A Systematic Review of the Mysterious Caterpillar Fungus Ophiocordyceps sinensis in Dong-ChongXiaCao and Related Bioactive Ingredients J Tradit Complement Med, 2013, 3, 16-32). Among them, nucleic acid derivatives such as cordycepin and adenosine are contained at a high concentration, and they are also used as a main indicator material of Cordyceps.

In particular, codisepin is known to be an active ingredient that exhibits physiological activity such as anticancer, antioxidant and anti-inflammation (Tuli et al., Pharmacological and therapeutic potential of Cordyceps with Cordycepin, 3 Biotech, 2014, , And as a major pharmacological component of Cordyceps in Korea.

Cordycepsin is the most common species of Cordyceps sinensis) and cicada fungus (Cordyceps cicadae) or not in production, whereas only present in trace amounts, Cody sepseu Millie other leases (Cordyceps militaris ) is found in high concentration in the infected Cordyceps.

Cordyceps The production of cordycepin of militaris may vary depending on the isolating region of the strain and the type of the medium. Cordyceps (mildaris) may be added to the rice, silkworm, militaris ) were found to produce 3.41, 1.85 and 2.72 mg / g of cordycepin, respectively. (Determination and analysis of cordycepin and adenosine in the products of Cordyceps spp. Afr. , 957-961).

Accordingly, the present invention relates to a method for producing Cordyceps sp. militaris ) strains.

The present invention has been made in view of the above problems, and it is an object of the present invention to provide a method of manufacturing the same.

In order to solve the above-mentioned problems, the present invention provides a Cordyceps militaris S6xS8 strain (accession number: KCTC 13088BP) according to one embodiment.

In addition, the strain may have cordycepin-producing ability.

In addition, the strain may be cultured in a pupae culture medium.

Further, the strain can be identified from the 18S rDNA of SEQ ID NO: 1.

The details of other embodiments are included in the detailed description and drawings.

The embodiments of the present invention have at least the following effects.

(About 10.15 mg / g) of cordycepsin compared to the wild-type strain, Cordyceps militaris ) S6xS8 strain can be obtained.

In addition, it is possible to produce cordisepin as an active ingredient such as anti-cancer, antioxidant and anti-inflammation in a high concentration and in a large amount.

The effects according to the embodiments of the present invention are not limited by the contents exemplified above, and more various effects are included in the specification.

FIG. 1 is a graph showing the results of experiments on Cordyceps militaris ) hyphae cultured on a silkworm pupa culture medium.
FIG. 2 is a photograph showing growth patterns of some of mycelium according to Experimental Example of the present invention. FIG.
FIG. 3 is a graph summarizing growth characteristics of spore-derived haploid mycelium according to Experimental Example of the present invention.
FIG. 4 is a photograph showing the diploid strain according to the hybridization of haploid mycelium according to the experimental example of the present invention.
FIG. 5 is a graph of absorbance at 214 nm of cordycepin and adenosine content of S6xS8 strain and wild type strain (IUM4188) according to the experimental example of the present invention.
FIG. 6 is a graph showing the results of analyzing the content of cordycepin in each sample according to Experimental Example of the present invention. FIG.

BRIEF DESCRIPTION OF THE DRAWINGS The advantages and features of the present invention and the manner of achieving them will become apparent with reference to the embodiments described in detail below with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

It is to be understood that the term "comprising " herein is intended to encompass a stated step or material, but not to the exclusion of any other step or material, unless the context clearly indicates otherwise.

Although the first, second, etc. are used to describe various components, it goes without saying that these components are not limited by these terms. These terms are used only to distinguish one component from another. Therefore, it goes without saying that the first component mentioned below may be the second component within the technical scope of the present invention.

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. Also, preferred methods or samples are described in this specification, but similar or equivalent ones are also included in the scope of the present invention. The contents of all publications referred to herein are incorporated herein by reference.

Hereinafter, embodiments of the present invention will be described.

Cordyceps sinensis strain in accordance with an embodiment of the present invention to coordinate sepseu ms other less sepseu coordination mm in a strain belonging to the (Cordyceps militaris), Microbial Resource Center, Korea, May 26 to 08, 2016 Date of other less (Cordyceps militaris ) S6xS8 and deposited under accession number KCTC 13088BP.

The Cordyceps The militaris S6xS8 strain can be identified from the 18S rDNA of SEQ ID NO: 1 and has a significantly improved ability to produce cordycepin.

The Cordyceps militaris ) S6xS8 strain may have about 10.15 mg / g of codysepine producing ability when cultured in a pupae culture medium. However, the present invention is not limited to the kind of the medium, but it can be cultured in rice medium, wheat medium and the like.

The Cordyceps militaris ) S6xS8 strain specifically includes (a) Cordyceps militaris ) wild type strain to a host to obtain spore-derived haploid mycelium; (b) randomly crossing (crossing) the grown haploid hyphae; And (c) measuring the content of codistin in the hybridized heterozygous strains, thereby selecting a strain having a remarkably improved ability to produce codistin. However, this is not the case with the Cordyceps militaris ) S6xS8 strains.

Codisepin is a substance having a structural formula as shown below or derivatives thereof, and is known to exhibit antibiotics, anticancer, anti-inflammation, immunity and metabolism activity.

[Chemical Formula 1]

Cancers that are the subject of treatment, prevention or amelioration of codedin include, for example, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, pituitary adenocarcinoma, adenocarcinoma, papillary adenocarcinoma, papillary adenocarcinoma, papillary adenocarcinoma, endometrioid adenocarcinoma, Cancer of the central nervous system (CNS), primary central nervous system (CNS) neoplasia, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, prostate cancer, prostate cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, Lymphoma, spinal cord tumor, brainstem glioma and pituitary adenoma, and the like.

The Cordyceps The militaris S6xS8 strain may also be capable of producing components such as adenosine, amino acid, vitamin precursor and / or mannitol.

Adenosine is effective in improving immunity, preventing aging and improving heart / liver. Amino acids and vitamin precursors can be used for the synthesis of physiologically active substances. Mannitol is used for prevention and treatment of vasoconstriction, cerebral thrombosis and kidney weakness Which is known to exhibit its efficacy.

The present invention provides a Chinese cabbage extract according to another embodiment. The Cordyceps sinensis extract was obtained from Cordyceps militaris ) S6xS8 strain, which may contain codisepin as an active ingredient, and may further contain adenosine, an amino acid, a vitamin precursor, and / or mannitol.

The Cordyceps sinensis extract was obtained from Cordyceps militaris ) S6xS8, as well as the purified or processed Cordyceps militaris S6xS8 itself or a part thereof.

Conventional methods in the art can be used to extract the extract from natural products in order to obtain the Chinese cabbage extract. Specifically, an extract of the Cordyceps militaris S6xS8 strain can be obtained by using a conventional extraction solvent under ordinary temperature and pressure conditions. As the extraction solvent, water, an anhydrous Or a solvent commonly used in an extraction process such as a functional lower alcohol, acetone, ethyl acetate, butyl acetate, dichloromethane and 1,3-butylene glycol can be used. However, the present invention is not limited thereto.

According to still another embodiment of the present invention, there is provided a health functional food, a health supplement food, a health food, a cosmetic product, a medicine, and a quasi-drug, which comprise a Chinese cabbage extract.

The health functional food, the health supplement food, the health food, the cosmetic product, the medicine and the quasi-drug including the Cordyceps monocotyl extract may further include foodstuffs, cosmetics or pharmaceutically acceptable ingredients.

Food acceptable ingredients include, for example, proteins, carbohydrates, fats, nutrients and seasonings. Specific examples of the carbohydrate include carbohydrate such as monosaccharide (glucose, fructose, etc.), disaccharide (maltose, sucrose, etc.), oligosaccharide, polysaccharide (dextrin, cyclodextrin) and sugar alcohols (xylitol, sorbitol, Threitol, etc.), natural flavoring agents (tau martin, stevia extract, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) as a flavoring agent.

Cosmetically or pharmaceutically acceptable ingredients include, for example, carriers, binders, lubricants, disintegrants, excipients, solubilizers, stabilizers, bases, lubricants, preservatives, wetting agents, emulsifiers, suspending agents, , A surfactant, an antioxidant, a moisturizing agent, a perfume and a coloring agent. Specific examples of the carrier include cellulose derivatives such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, Syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

The medicines and the quasi-drugs containing the Cordyceps sinensis extract may be formulated in a unit dosage form by using pharmaceutically acceptable carriers and / or excipients according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs. Or may be manufactured by penetrating into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

Medicines and quasi-drugs containing the Cordyceps sinensis extract are generally administered orally, but they may be administered parenterally by topical application to the skin, intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection or transdermal administration.

Hereinafter, the above-mentioned Cordyceps militaris ) S6xS8 strain and the ability to produce cocidepine will be described in detail.

< Manufacturing example  1: On the puppy badge Cody's Millitaris ( Cordyceps militaris ) Culture and spore collection>

First, Cordyceps &lt; RTI ID = 0.0 &gt; militaris ) wild type strain, IUM4188 strain, was cultured in PDB medium at 20 DEG C for 1 week with shaking.

Next, the above-mentioned Cordyceps militaris) silkworm pupa 5ml sterile culture medium for (traditional markets supplied) to 50g are inoculated in either polypropylene culture plates (103 x 78.6 mm, SPL Life Sciences, Pocheon, Republic of Korea), and cultured at 20 ℃ 40 ilgan solid.

As a result of solid culture, Cordyceps militaris mycelium completely penetrated into the silkworm pupa as shown in Fig. 1, and small fruiting body analogues were formed on the surface.

The silkworm cultivated pupa cultured with mycelium was collected and suspended in sterilized water, the mycelium was settled and the supernatant liquid was taken. The spores contained in the supernatant were separated through centrifugation (2,500 x g, 10 min).

< Manufacturing example  2: Spore origin Haploid  Securing hyphae>

The suspension of spores isolated in Preparation Example 1 was plated on a PDA medium containing 100 g / ml of ampicillin and then cultured at 20 ° C for 3 days.

Then, the mycelia growing independently on the solid medium (PDA medium) were inoculated into a new PDA medium and cultured at 20 ° C for 1 week.

FIG. 2 is a photograph showing growth patterns of some of the mycelium. As shown in FIG. 2, each spore-derived haploid mycelium exhibited various growth characteristics and colors. For example, strains S1 and S8 showed a slow growth rate and a relatively close white color, while strains S5 to S7 showed fast growth rates and showed a color close to yellow.

FIG. 3 is a graph summarizing the growth characteristics of the spore-derived haploid mycelium. As shown in FIG. 3, most of the haploid mycelia grew into a mycelial body having a diameter of about 2.5 cm when cultured in a solid medium for one week, but hyphae such as S3 to S7, S9 and S24 were grown to a size of 3.5 cm or more .

< Manufacturing example  3: Haploid  Through hybridization of hyphae Cody's Millitaris ( Cordyceps  militaris ) Breeding>

Cordyceps The above haploid hyphae were randomly crossed for breeding new varieties of militaris .

As a result, mating hybridization which does not cause mycelial fusion in most mating combinations such as S1xS33, S1xS37, S9xS37, S22xS33, S22xS37, and S33xS37 was shown to be caused by the mating type difference of each haploid hyphae.

On the other hand, as shown in Fig. 4, in the crossing combination with the S6 haploid strain, many haploid strains were crossed. In particular, the hybrid between S6 strain and S2, S3, S8, S20, S22, S36, S50, S51 and S53 strains was proved to be a diploid combination having excellent mycelial growth characteristics. Respectively.

< Experimental Example : Codispecin of diploid strains Production capacity  Research>

In order to investigate the ability of the nine hybrids hybridized with excellent growth characteristics to produce coryneformin, each hybridoma was first cultured in a PDB medium, and 5 ml of the culture broth was inoculated in a 50 g pupae culture medium and cultured at 25 DEG C for 30 days Respectively.

After the incubation was completed, the sample was dried at 65 DEG C for 36 hours, and the dried sample was crushed into a mortar. 10 ml of distilled water was added to 1 g of the pulverized sample, and the cordycepin in the sample was extracted with an ultrasonic extraction apparatus for 1 hour.

After extraction, 1 ml of the supernatant was filtered through a 0.22 m filter, and the filtrate was analyzed by HPLC. The HPLC column for the analysis was a Waters Symmetry Shield RP18 (4.6 x 150 mm, 5 m) and eluted at a flow rate of 0.4 ml / min using mobile phase water-methanol (87.5: 12.5, v / v). The content of codistin and adenosine in the sample was quantified by absorbance at 214 nm.

As a control, Cordyceps militaris) were analyzed in the same manner as above and the coordination sepin adenosine content of the wild type strain (IUM4188) and the wild type strain (IUM1091) of Cordyceps sp..

FIG. 5 is a graph of cytotoxin content and adenosine content of S6xS8 strain and wild type strain (IUM4188) measured by absorbance at 214 nm, and FIG. 6 is a graph showing the results of analysis of content of cordycepin in each sample.

As shown in FIG. 5 and FIG. 6, the codistin content of the other hybrid strains was 2.27 to 3.13 mg / g, the content of the cordissepin of the control strain IUM4188 was 2.87 (± 0.22) mg / g and the control strain IUM1091 Showed almost no production of codistepin, whereas the codistin content of the S6xS8 hybrid was 10.15 (+/- 0.39) mg / g, which was significantly higher than the other hybrid and wild-type strains.

Thus, the present Cordyceps &lt; RTI ID = 0.0 &gt; militaris ) S6xS8 hybrids have remarkably superior corysepine production ability than other hybrids and wild type strains.

While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, You will understand. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> LEE, won-jae <120> CORDYCEPS MILITARIS HAVING IMPROVED CAPABILITY OF PRODUCING          CORDYCEPIN <130> GP170023KRAA <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1079 <212> DNA <213> Artificial Sequence <220> 18S rDNA (Cordyceps militaris S6XS8) <400> 1 ttgtctcaaa gattaagcca tgcatgtcta agtataagca attatacagc gaaactgcga 60 atggctcatt atataagtta tcgtttattt gatagtacct tactacttgg ataaccgtgg 120 taattctaga gctaatacat gctaaaaatc ccgacttcgg aagggatgta tttattagat 180 taaaaaccaa tgccctctgg gctccttggt gattcataat aacttttcga atcgcacggc 240 cttgcgccgg cgatggttca ttcaaatttc ttccctatca actttcgatg tttgggtctt 300 ggccaaacat ggtcgcaacg ggtaacggag ggttagggct cgaccccgga gaaggagcct 360 gagaaacggc tactacatcc aaggaaggca gcaggcgcgc aaattaccca atcccgattc 420 ggggaggtag tgacaataaa tactgataca gggctctttt gggtcttgta attggaatga 480 gtacaattta aatctcttaa cgaggaacaa ttggagggca agtctggtgc cagcagccgc 540 ggtaattcca gctccaatag cgtatattaa agttgttgtg gttaaaaagc tcgtagttga 600 accttgggcc tggctggccg gtccgcctca ccgcgtgcac tggtccggcc gggcctttcc 660 ctctgtggaa ccccatgctc ttcactgagt gtggcggtga aacaggactt ttactttgaa 720 aaaattagag tgctccaggc aggcctatgc tcgaatacat tagcatggaa taataaaata 780 ggacgcgtgg ttctattttg ttggtttcta ggaccgccgt aatgattaat agggacagtc 840 gggggcatca gtattcaatt gtcagaggtg aaattcttgg atttattgaa gactaactac 900 tgcgaaagca tttgccaagg atgttttcat taatcaggaa cgaaagttag gggatcgaag 960 acgatcagat accgtcgtag tcttaaccat aaactatgcc gactagggat cggacgatgt 1020 tattttttga cgcgttcggc accttacgag aaatcaaagt gcttgggctc cagggggag 1079

Claims (4)

Cordyceps militaris S6xS8 strain [cord number: KCTC 13088BP] having ability to produce cordycepin. delete The method according to claim 1,
The strain is a Codisps militaris S6xS8 strain cultivated in a pupae culture. The method according to claim 1,
Wherein said strain is identified from the 18S rDNA of SEQ. ID. NO. 1, the Coryseps. Millilitaris S6xS8 strain.

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