Novel Coprinus anatolicus mutant strain and its use

Abstract

To provide Inonotus obliquus that can achieve the mass production of a bioactive substance.SOLUTION: A mutant of Inonotus obliquus disclosed herein is the mutant of Inonotus obliquus with an accession number of NITE AP-03465.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a novel Inonotus obliquus mutant strain, a culture of the mutant strain, a method for culturing the mutant strain, and a method for producing a physiologically active substance using the culture of the mutant strain.

Background Art

[0002] Inonotus obliquus is known to have physiologically active substances such as antitumor activity, antioxidant activity, and blood glucose lowering inhibitory activity (Patent Document 1).

[0003] On the other hand, the abundance of natural Inonotus obliquus is small. In addition, it takes more than 10 years to collect Inonotus obliquus. Therefore, the development of a production technology for physiologically active substances by artificial culture of Inonotus obliquus has been promoted. For example, Patent Document 2 describes a method for culturing Inonotus obliquus using a liquid medium containing a carbon source and adjusted to a specific pH range.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, even with the conventional technologies as described above, mass production of physiologically active substances has not been achieved, and further improvement is required.

[0006] One aspect of the present invention aims to develop Inonotus obliquus capable of achieving mass production of physiologically active substances.

Means for Solving the Problems

[0007] As a result of intensive studies, the inventors of the present invention have found that a mutant strain of Inonotus obliquus prepared by the inventors has a higher growth rate compared to conventional Inonotus obliquus. Then, the inventors have found that it is possible to achieve mass production of physiologically active substances by culturing the mutant strain, and have thus completed the present invention.

[0008] <1>In order to solve the above problems, a mutant strain of Inonotus obliquus according to one aspect of the present invention is a mutant strain of Inonotus obliquus with the accession number NIT-03465. received Entrust number NIT EP -03465.

[0009] <2>A culture of a mutant strain of Inonotus obliquus according to one aspect of the present invention is a culture of a mutant strain of Inonotus obliquus with the accession number NIT-03465. Entrust received EP number NIT

[0010] <3>A method for culturing Inonotus obliquus according to one aspect of the present invention includes a step of aerobically culturing a mutant strain of Inonotus obliquus with the accession number NIT-03465. Entrust received EP number NIT

[0011] <4>In the method for culturing Inonotus obliquus according to one aspect of the present invention, in the above step, the mutant strain of Inonotus obliquus may be cultured in a medium containing yeast extract.

[0012] <5>A production method according to one aspect of the present invention is a production method of at least one selected from an antioxidant component and an antiglycation component, including a step of aerobically culturing a mutant strain of Inonotus obliquus with the accession number NIT-03465. Entrust received EP number NIT

[0013] <6>In the production method according to one aspect of the present invention, the antiglycation component may be dihydroxybenzylacetone.

[0014] <7>An antiglycation agent according to one aspect of the present invention is received Entrust with the number NIT EP -03465, and contains an extract of a culture of a mutant strain of Coprinus comatus as an active ingredient, and the above culture extract contains dihydroxybenzylacetone or a salt thereof.

Effects of the Invention

[0015] According to one aspect of the present invention, it is possible to provide Coprinus comatus capable of realizing mass production of physiologically active substances.

Brief Description of the Drawings

[0016]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0017] One embodiment of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to each configuration described below, and various modifications are possible within the scope shown in the claims. Also, embodiments or examples obtained by appropriately combining the technical means disclosed in different embodiments or examples are also included in the technical scope of the present invention. Furthermore, by combining the technical means disclosed in each embodiment, new technical features can be formed. All academic documents and patent documents described in this specification are incorporated herein by reference. Also, unless otherwise specified in this specification, "A~B" representing a numerical range is intended to mean "A or more (including A and greater than A) and B or less (including B and less than B)".

[0018] 〔Kabanoya anatake mutant strain〕 The mutant strain of Kabanoya anatake (Inonotus obliquus) according to this embodiment (hereinafter sometimes abbreviated as "Kabanoya anatake mutant strain") is the recipient Entrust with the number NIT EP -03465 and is a mutant strain of Kabanoya anatake.

[0019] The Kabanoana mutans strain can be prepared, for example, by irradiating the mycelium of Kabanoana with an ion beam to induce mutations. The ion beam used for irradiating the mycelium of Kabanoana is not particularly limited as long as it can induce mutations. Examples of the ion beam include carbon ion beam, hydrogen ion beam, as well as nitrogen, neon, argon, iron, and helium ion beams, with carbon ion beam or hydrogen ion beam being preferred. The irradiation dose of the ion beam may be determined according to the type of ion beam used, the strain of Kabanoana, etc. In the case of a carbon ion beam, it may be 100 - 1000 Gy, preferably 200 - 750 Gy, more preferably 300 - 600 Gy. In the case of a hydrogen ion beam, it is 200 - 2000 Gy, preferably 300 - 1500 Gy, more preferably 400 - 1500 Gy. If it is less than 100 Gy, the effect of ion beam irradiation will not appear, and if it is 2500 Gy or more, all the mycelia of Kabanoana will die, so neither case is preferable from the perspective of obtaining various mutant strains. As the high-energy accelerator for generating the ion beam, a synchrotron or a cyclotron, which is characterized by having a high linear energy transfer (LET) compared to gamma rays, etc., and a long range in water compared to low-energy accelerators, is preferred. After ion beam irradiation, for the mycelium of Kabanoana being cultured on a solid medium, by measuring the diameter of the grown Kabanoana over time and comparing the growth rate (cm / day) with that of the non-irradiated ones, it can be used as an index for mutant strain selection. The growth rate in this specification refers to the average value (cm / day) of the growth rate for 14 days after the start of culture.

[0020] The Kabanoana mutans strain was deposited at the Patent Microorganisms Depositary Center (NPMD) of the National Institute of Technology and Evaluation (hereinafter abbreviated as "NITE"), 2-5-8 Kazusa Kamashima, Kisarazu City, Chiba Prefecture, Room 122 (Received Entrust Accession No.: NIT EP -03465).

[0021] NIT EP The strain with the strain number -03465 has naturally mutated, and strains having properties equivalent to those of NIT EP are also within the scope of the mutant strains of the present case.

[0022] 〔Culturing method of Coprinus comatus mutant strain〕 The culturing method of the Coprinus comatus mutant strain according to the present embodiment includes a step of aerobically culturing the Coprinus comatus mutant strain. The aerobic culture may be carried out according to the general aerobic culture performed for Coprinus comatus. As the culture medium, a culture medium containing necessary nutrient sources such as a carbon source, a nitrogen source, or inorganic salts may be used. In terms of increasing the production amount of physiologically active substances, etc., it is preferable that the culture medium contains yeast extract. The culture form may be a liquid culture medium or a solid culture medium. In terms of ease of growth, etc., the culture form is preferably a liquid surface culture. Also, the initial culture pH is preferably 5.0 to 7.0, the culture temperature is preferably 20°C to 35°C, and the culture time is preferably 20 to 35 days.

[0023] The culture obtained by the above culturing method of the Coprinus comatus mutant strain is also included in the present embodiment.

[0024] 〔Production method of physiologically active substance〕 The production method of the physiologically active substance according to the present embodiment includes a step of aerobically culturing the Coprinus comatus mutant strain. Examples of the physiologically active substance include antioxidant components and anti-glycation components, etc. Examples of the anti-glycation component include dihydroxybenzylacetone and its salts, etc.

[0025] Dihydroxybenzylacetone is also referred to as 4-dihydroxyphenyl-but-3-en-2-one or dihydroxybenzylideneacetone.

[0026] Dihydroxybenzylacetone or its salt may be a cis form, a trans form, or a mixture of a cis form and a trans form. Since it has excellent anti-glycation activity, it is more preferably a trans form.

[0027] In a preferred embodiment, dihydroxybenzylacetone is 3,4-dihydroxybenzylacetone represented by the following formula (2). The IUPAC name of 3,4-dihydroxybenzylacetone is 4-(3,4-dihydroxyphenyl)-but-3-en-2-one.

Chemical formula

[0028] In a particularly preferred embodiment, dihydroxybenzylacetone is (E)-3,4-dihydroxybenzylacetone. The IUPAC name of (E)-3,4-dihydroxybenzylacetone is (E)-4-(3,4-dihydroxyphenyl)-but-3-en-2-one (CAS number: 123694-03-1).

[0029] In one embodiment, dihydroxybenzylacetone may be a derivative of the above-described substances. As used herein, the term "derivative" refers to a group of compounds formed by substituting a part of a specific compound molecule with another functional group or another atom.

[0030] Examples of the above other functional groups include alkyl group, alkoxy group, alkylthio group, aryl group, aryloxy group, arylthio group, arylalkyl group, arylalkoxy group, arylalkylthio group, arylalkenyl group, arylalkynyl group, allyl group, amino group, substituted amino group, silyl group, substituted silyl group, silyloxy group, substituted silyloxy group, arylsulfonyloxy group, alkylsulfonyloxy group, nitro group, etc. Examples of the above other atoms include carbon atom, hydrogen atom, oxygen atom, nitrogen atom, sulfur atom, phosphorus atom, halogen atom, etc.

[0031] As used herein, the term "salt" is not limited as long as it is a physiologically acceptable salt for administration to a subject as a pharmaceutical. Examples of salts include alkali metal salts (such as sodium salts and potassium salts), alkaline earth metal salts (such as calcium salts and magnesium salts), ammonium salts, organic base salts (such as trimethylamine salts, triethylamine salts, pyridine salts, picoline salts, dicyclohexylamine salts, and N,N'-dibenzylethylenediamine salts), organic acid salts (such as acetate salts, maleate salts, tartrate salts, methanesulfonate salts, benzenesulfonate salts, formate salts, toluenesulfonate salts, and trifluoroacetate salts), and inorganic acid salts (such as hydrochloride salts, hydrobromide salts, sulfate salts, and phosphate salts). "Salts" have advantages such as being easily soluble in water and easy to administer.

[0032] The recovery of the bioactive substance from the culture of the Cabanoana taka mutant strain may be carried out according to the general extraction methods and extraction conditions performed on the Cabanoana taka culture. The culture includes culture supernatant, cultured mycelia, disrupted cultured mycelia, etc. Examples of extraction methods include extraction using water and / or an organic solvent, supercritical extraction, etc. For the organic solvent, alcohols such as methanol, ketones such as methyl ethyl ketone, alkanes such as hexane, esters such as ethyl acetate, ethers such as diethyl ether, and halogenated alkyls such as chloroform can be used. Also, for the extraction temperature, it is sufficient if the solvent used is in a liquid state. The bioactive substance may be recovered using one type of extraction method, or two or more types of extraction methods may be used for the recovery of the bioactive substance.

[0033] Examples of the uses of the bioactive substance include food and drink products, feeds, cosmetics, pharmaceuticals, supplements, and pet foods, etc.

[0034] 〔Antiglycation agent〕 The anti-glycation agent according to this embodiment (hereinafter sometimes referred to as "this anti-glycation agent") contains an extract of a culture of the Coprinus ananiceps mutant strain (culture extract) as an active ingredient. The culture extract contains dihydroxybenzylacetone or a salt thereof. The culture of the Coprinus ananiceps mutant strain and the extract of the culture can be obtained by the methods described in the above [Method for culturing Coprinus ananiceps mutant strain] and [Method for producing physiologically active substance]. Further, the anti-glycation agent according to this embodiment is subjected to Entrust a Coprinus ananiceps mutant strain with the number NIT EP -03465-produced dihydroxybenzylacetone or a salt thereof, or an anti-glycation agent containing, as an active ingredient, a structure equivalent thereto (for example, chemically synthesized dihydroxybenzylacetone or a salt thereof) may also be used.

[0035] In this specification, the "anti-glycation agent" is intended to mean an agent having anti-glycation activity. Further, "anti-glycation" includes an action of inhibiting glycation and suppressing the production of advanced glycation end products (AGEs) (hereinafter also referred to as "glycation inhibition action" or "AGEs production inhibition action"), an action of suppressing the accumulation of AGEs in vivo (hereinafter also referred to as "AGEs accumulation inhibition action"), and an action of promoting the decomposition of AGEs (hereinafter also referred to as "AGEs decomposition promotion action"). Therefore, in this specification, the anti-glycation agent can also be referred to as a "glycation inhibitor", an "AGEs production inhibitor", an "AGEs accumulation inhibitor", or an "AGEs decomposition promoter".

[0036] As a method for examining anti-glycation activity, there can be mentioned a method of measuring the fluorescence spectrum from fluorescent AGEs in a reaction solution containing a sample, a protein, and a reducing sugar. Since fluorescent AGEs have the property of emitting fluorescence when excited by a wavelength of about 370 nm or in the vicinity thereof, by measuring the fluorescence intensity of the reaction solution, the fluorescent AGEs in the reaction solution can be quantified, and the anti-glycation activity of the sample can be examined.

[0037] "Advanced glycation end products (AGEs)" is a general term for products generated by glycation (also referred to as the "Maillard reaction"). AGEs are known to be related to various diseases (for example, conditions and diseases related to aging (hereinafter also referred to as "age-related diseases"), diabetic complications, and inflammation, etc.).

[0038] Examples of AGEs include Nε-carboxymethyllysine (CML), Nε-carboxyethyllysine (CEL), argpyrimidine, pentosidine, pyrraline, crossline, GA-pyridine, Nω-carboxymethylarginine (CMA), furoylfuranyl imidazole, and glucosepane, etc.

[0039] The mechanism of action by which this anti-glycation agent exerts anti-glycation activity is presumed to be any one or a combination of the following (i) to (iii), but the present invention is not limited to such presumption: (i) By inhibiting the glycation of the target protein, the production of AGEs is suppressed; (ii) The accumulation in vivo of AGEs generated by the glycation of the target protein is suppressed; and, (iii) By promoting the degradation of AGEs generated by the glycation of the target protein, the amount of AGEs is reduced.

[0040] This anti-glycation agent has anti-glycation activity against the glycation of various substances such as proteins. Among these, this anti-glycation agent exhibits high anti-glycation activity against the glycation of collagen or elastin. In particular, by appropriately adjusting the dosage of the active ingredient (dihydroxybenzylacetone or its salt) in this anti-glycation agent, it exhibits excellent anti-glycation activity significantly superior to aminoguanidine, which is typically used as a positive control having anti-glycation activity. Since the glycation of collagen and elastin has been reported to have a high correlation with skin aging phenomena, arteriosclerosis, osteoporosis, etc. associated with aging, this effect is very important.

[0041] Therefore, the present anti-glycation agent can be suitably used as a collagen glycation inhibitor for suppressing the glycation of collagen, or an elastin glycation inhibitor for suppressing the glycation of elastin. In other words, the present invention provides a collagen glycation inhibitor and an elastin glycation inhibitor containing dihydroxybenzylacetone or a salt thereof as an active ingredient. The collagen glycation inhibitor and the elastin glycation inhibitor according to one embodiment of the present invention can be suitably used, for example, in cosmetics for improving the skin condition related to AGEs.

[0042] The present anti-glycation agent has anti-glycation activity against glycation caused by various reducing sugars. Examples of reducing sugars include all monosaccharides (e.g., glucose, fructose, xylose, arabinose, and galactose), lactose, and glyceraldehyde. Among these reducing sugars, glucose and fructose are present in large amounts in the living body. In addition, AGEs generated from the glycation by glyceraldehyde have been reported to have a high correlation with the onset of diseases.

[0043] Among these reducing sugars, the present anti-glycation agent exhibits excellent anti-glycation activity against glycation by glucose, fructose, xylose, arabinose, galactose, lactose, and glyceraldehyde. In other words, the present anti-glycation agent can be suitably used to suppress glycation by glucose, fructose, xylose, arabinose, galactose, lactose, and glyceraldehyde.

[0044] The amount of dihydroxybenzylacetone or its salt contained in the present anti-glycation agent is not particularly limited as long as the desired effect can be obtained. The suitable amount of the active ingredient varies depending on the administration subject, administration form, etc. of the present anti-glycation agent. For example, it may be 0.00005% by mass to 100% by mass, preferably 0.0001% by mass to 100% by mass, more preferably 0.001% by mass to 100% by mass, still more preferably 0.01% by mass to 100% by mass, even more preferably 0.1% by mass to 100% by mass, even more preferably 0.1% by mass to 95% by mass, even more preferably 0.1% by mass to 90% by mass, even more preferably 0.1% by mass to 80% by mass, even more preferably 0.1% by mass to 70% by mass, even more preferably 0.1% by mass to 60% by mass, even more preferably 0.1% by mass to 50% by mass, even more preferably 0.1% by mass to 40% by mass, even more preferably 0.1% by mass to 30% by mass, even more preferably 0.1% by mass to 20% by mass, still more preferably 0.1% by mass to 10% by mass, and particularly preferably 0.1% by mass to 5% by mass, based on the total mass of the present anti-glycation agent.

[0045] The administration subject of the present anti-glycation agent is not particularly limited and may be a human or a non-human animal (e.g., livestock, pet animals, and experimental animals). Examples of non-human animals include monkeys, chimpanzees, cows, pigs, sheep, goats, horses, dogs, cats, rabbits, mice, rats, chickens, and fish and shellfish.

[0046] The dosage of the present anti-glycation agent is not particularly limited as long as the desired effect can be obtained. The suitable dosage varies depending on the administration subject, administration form, etc.

[0047] When the administration target is an adult human and the anti-glycation agent is administered orally, a suitable dosage is, for example, 50 to 3,000 mg / kg (body weight) as the mass of the active ingredient (dihydroxybenzylacetone or its salt), and more preferably 100 to 2,000 mg / kg (body weight). Also, when the anti-glycation agent is administered orally on a daily basis over a long period, a suitable dosage is, for example, 1 to 500 mg / kg (body weight) as the mass of the active ingredient (dihydroxybenzylacetone or its salt).

[0048] When the administration target is a non-human animal and the anti-glycation agent is administered orally, a suitable dosage is, for example, 50 to 3,000 mg / kg (body weight) as the mass of the active ingredient (dihydroxybenzylacetone or its salt), and more preferably 100 to 2,000 mg / kg (body weight). Also, when the anti-glycation agent is administered orally on a daily basis over a long period, a suitable dosage is, for example, 1 to 500 mg / kg (body weight) as the mass of the active ingredient (dihydroxybenzylacetone or its salt).

[0049] The anti-glycation agent can be administered to the administration target by any administration route. Examples of the administration route include oral administration and parenteral administration (for example, transdermal administration, transmucosal administration, nasal administration, intravenous administration).

[0050] The anti-glycation agent may be in any form such as solid (powder, granule, etc.), liquid (solution, suspension, etc.), and paste, etc. Also, the dosage form of the anti-glycation agent can be any dosage form. Examples of the dosage form include powder, pill, granule, tablet, coated tablet, capsule, troche, solution, suspension, emulsion, syrup, infusion, decoction, tincture, injection, ointment, cream, gel, patch, external solution, external powder, suppository, inhalant, eye drop, etc.

[0051] The anti-glycation agent can be used as a component of products such as, for example, food and drink (food and drink compositions, food and drink additives, etc.), feed (feed compositions, feed additives, etc.), cosmetics, and pharmaceuticals.

[0052] According to the Kabanoanatake mutant strain and the like according to this embodiment, since it is possible to realize mass production of physiologically active substances, the time and energy required for culturing can be reduced. As a result, a sustainable production and consumption pattern can be ensured, contributing to the achievement of sustainable development goals (SDGs).

[0053] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0054] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples only.

Examples

[0055] In the following examples, unless otherwise specified, % represents mass %.

[0056] 〔Example 1〕Preparation of Kabanoanatake mutant strain As the strain, Inonotus obliquus NY-1 stored at 5°C on a potato dextrose agar slant was used. To prepare a plate for ion beam irradiation, one platinum loopful was scraped from the stored strain and inoculated into the center of a potato dextrose agar plate with a diameter of 3.5 cm, and cultured at 25°C for 10 days. The mycelial membrane on the plate was irradiated with a 660 MeV carbon beam generated by the multi-purpose synchrotron tandem accelerator (custom-made product of Hitachi, Ltd., no model number) at the Wakasa Bay Energy Research Center at a dose of 500 Gy. From the central and peripheral parts of the mycelial membrane after carbon beam irradiation, one platinum loopful of mycelium was scraped and inoculated into the center of a potato dextrose agar plate. The diameter of the mycelial membrane that grew over time was measured, and those with a growth rate (cm / day) increased to 120% or more of that of the non-irradiated ones were selected.

[0057] The selected mutant strains were cultured on the liquid surface, and the mycelial weight was measured over time to evaluate the growth rate. The parent strain Inonotus obliquus NY-1 and the selected mutant strains were each precultured on a potato dextrose agar plate at 30 °C for 2 weeks to obtain inoculum. 150 mL of the main culture medium with the composition shown in Table 1 was placed in a container for static culture (capacity 500 mL). After autoclaving, the caps of the inoculum that had grown to 3.5 - 4.5 cm by preculture were punched out with a sterilized cylindrical cutter (1 cm) and floated on the main culture medium for static culture.

[0058]

Table 1

[0059] Every 5 days, the entire contents of the culture container were suction filtered using filter paper (5A made by Advantec) to recover the mycelium. After washing this with distilled water, it was dried at 100 °C for 24 hours to measure the weight of the mycelium.

[0060] The mutant strain C500-1, which reached the weight (growth amount) that the parent strain reaches in 30 days in 25 days of culture, was selected. An application for deposit of the mutant strain C500-1 was made to the Patent Biological Deposit Center of the National Institute of Technology and Evaluation (accession number NIT - 03465). As the morphological and physiological characteristics of this mutant, although no change was observed in the form of the mycelium compared to the parent strain, the colony changed slightly to brown, etc. Entrust Accession number NIT EP -03465). As the morphological and physiological characteristics of this mutant, although no change was observed in the form of the mycelium compared to the parent strain, the colony changed slightly to brown, etc.

[0061] 〔Example 2〕Antioxidant components of the Kabanatana ke mutant Mutant strain C500-1 was cultured on the liquid surface for 25 days. The obtained mycelia were washed with distilled water and then freeze-dried. The freeze-dried product was ground in a mortar. 1 g of the ground freeze-dried product was transferred to a beaker, 20 mL of hexane was added, and the mixture was stirred at room temperature for 2 hours. Then, it was centrifuged (4 °C, 10,000 rpm, 10 minutes) to collect the supernatant. The precipitate remaining in the centrifuge tube was completely recovered in the beaker using 20 mL of ethyl acetate. After stirring again at room temperature for 2 hours, it was centrifuged (4 °C, 10,000 rpm, 10 minutes) to collect the supernatant. Next, the precipitate in the centrifuge tube was completely recovered in the beaker using 20 mL of 99% (v / v) ethanol. After stirring at room temperature for 1 hour, it was centrifuged (4 °C, 10,000 rpm, 10 minutes) to collect the supernatant. Subsequently, the precipitate in the centrifuge tube was completely recovered in the beaker using 30 mL of distilled water and subjected to hot water extraction at 100 °C for 4 hours while stirring using a water bath with a stirrer. The suspension after hot water extraction was suction filtered and further washed with 10 mL of distilled water. The collected filtrate and wash solution were put into a separatory funnel, an equal volume of methyl isobutyl ketone (MIBK) was added for liquid-liquid extraction, and the MIBK layer and the aqueous layer were collected. The MIBK layer was concentrated and freeze-dried. This was designated as the MIBK fraction.

[0062] The antioxidant activities of the MIBK fraction fractionated from the cultured mycelia of mutant strain C500-1 and the MIBK fraction fractionated from natural Wolfiporia extensa sclerotia by the same procedure were measured by five methods: the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method, the superoxide dismutase (SOD) method, the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) method, the ferric reducing antioxidant power (FRAP) method, and the oxygen radical absorbance capacity (ORAC) method. The details of each measurement method are shown below.

[0063] (DPPH method) Into each well of a 96-well plate (Costar, for UV use, transparent, #3635), 50 μL of the sample solution was injected, and 300 μL of a 100 μM DPPH methanol solution was added and mixed. Then, the reaction was carried out at room temperature in the dark for 10 minutes. As a control, 50 μL of methanol was used instead of the sample solution. After the reaction for 10 minutes at room temperature, the absorbance was measured at 517 nm, and the antioxidant activity was determined from the following formula (1). Also, the solution concentration EC 50 value was determined. Antioxidant activity (%) = (1 - absorbance of sample solution / absorbance of control) × 100 ··· (1)

[0064] (SOD method) Into each well of a 96-well plate (Costar, for UV use, transparent, #3635), 125 μL of 50 mM phosphate buffer (pH 7.4), 20 μL of 15 mM disodium ethylenediaminetetraacetate dihydrate (Na 2 EDTA·H 2 O), 30 μL of 3 mM hypoxanthine, 50 μL of 0.6 mM nitroblue tetrazolium (NBT), 25 μL of the sample solution, and 50 μL of 0.1 U / mL xanthine oxidase were added, and the reaction was carried out at 25 °C. After 2.5 minutes, the absorbance was measured at 540 nm, and the scavenging activity of superoxide anion radical was determined from formula (1). As a control, distilled water was used instead of the sample solution. Also, EC 50 was calculated in the same manner as the DPPH method.

[0065] (ABTS method) To 88 μL of an aqueous solution of 140 mM potassium peroxydisulfate, 5 mL of a 7 mM ABTS solution was added and mixed. Then, the reaction was carried out at room temperature in the dark for 12 - 14 hours to prepare an ABTS radical solution. 2 mL of the ABTS radical solution and 20 μL of the sample solution were injected into a quartz cell, and after reacting for 6 minutes in a constant temperature bath at 30 °C, the absorbance was measured at 734 nm. As a control, 20 μL of distilled water was used instead of the sample solution. The antioxidant activity was determined from formula (1). Also, EC 50 was calculated in the same manner as the DPPH method.

[0066] (FRAP method) As the FRAP solution, a solution prepared by mixing 25 mL of 300 mM acetate buffer, 2.5 mL of 10 mM TPTZ, and 2.5 mL of 20 mM ferric chloride was used. 15 μL of the sample solution was injected into each well of a 96-well plate (Costar, for UV, transparent, #3635), and 285 μL of the FRAP solution was added and mixed. Then, the reaction was carried out at 37 °C for 30 minutes in the dark, and the absorbance was measured at 593 nm. As a control, 15 μL of distilled water was used instead of the sample solution. Using the Trolox solution as a reference, the antioxidant activity was determined as the Trolox equivalent.

[0067] (ORAC method) 20 μL of the sample solution, 200 μL of 96 nM fluorescein prepared with 75 mM phosphate buffer, and 80 μL of 80 mM AAPH (2,2’-azobis(2-methylpropionamidine) dihydrochloride) solution prepared with 75 mM phosphate buffer were added to a 96-well plate (Greiner bio-one, 96 well microplate, black, 655076). The reaction was started at 37 °C, and the fluorescence (Ex: 485 nm, Em: 520 nm) was measured over time. As a control, 75 mM phosphate buffer was used instead of the sample solution. The ORAC value was determined as the integrated value of the fluorescence intensity of the mixture, and the antioxidant activity was calculated using the following formula (2). The fluorescence intensity of the well without the added mixture was used as the blank. Antioxidant activity = {(Integrated value of the fluorescence intensity of the sample solution - Integrated value of the fluorescence intensity of the blank) / (Integrated value of the fluorescence intensity of the control - Integrated value of the fluorescence intensity of the blank)} × 100 ··· (2)

[0068] Based on the reciprocal EU of the EC value of each obtained antioxidant activity 50 the antioxidant activities of the MIBK fraction fractionated from the cultured mycelium of mutant strain C500-1 and the MIBK fraction fractionated from natural Grifola frondosa sclerotia by the same procedure were compared. The comparison results are shown in Fig. 1. 50

[0069] ​"MIBK extract (Cultured)" in Fig. 1 shows the results of mutant strain C500-1. "MIBK extract (Natural)" shows the results of natural Flammulina velutipes sclerotia. The closer the plot is to the circumference (the farther it is from the center of the circle), the higher the antioxidant activity.

[0070] As shown in Fig. 1, it was found that the SOD activity of the MIBK fraction of mutant strain C500-1 cultured by the liquid surface culture method was higher than that of the MIBK fraction of natural Flammulina velutipes sclerotia. Also, from the comparison of antioxidant activities by five methods, it was found that the MIBK fraction of mutant strain C500-1 had higher antioxidant activity than the MIBK fraction of natural Flammulina velutipes sclerotia.

[0071] Also, Fig. 2 is a graph comparing the growth rates of mutant strain C500-1 (Mutant) and Flammulina velutipes wild strain NY-1 strain (Wild type) in the liquid surface culture method. The vertical axis of Fig. 2 indicates the growth amount (g / L), and the horizontal axis indicates the culture time (days). As shown in Fig. 2, it was found that the growth rate of mutant strain C500-1 was higher compared to the Flammulina velutipes wild strain NY-1 strain.

[0072] [Example 3] Analysis of MIBK Fraction When the MIBK fraction obtained in Example 2 was analyzed by LC / MS, a peak was detected at m / z = 177. Also, 1 from the measurement results of 1H-NMR and 13 13C-NMR, it was revealed that 3,4-dihydroxybenzylacetone (hereinafter referred to as "DBL") was contained as the main component. The measurement conditions of LC / MS are shown below. Column: COSMOSIL PBr φ4.6mm×250mm Column temperature: 40°C Eluent: water / methanol = 2 / 8 Flow rate: 0.8 mL / min Ionization: ESI, negative mode

[0073] [Example 4] Anti-glycation Activity against Glycation of Bovine Serum Albumin The anti-glycation activity (AGEs production inhibitory activity) of this anti-glycation agent against the glycation of bovine serum albumin (BSA) by sugar (glucose or fructose) was evaluated according to the following method. As this anti-glycation agent, (E)-3,4-dihydroxybenzylacetone (manufactured by Fujifilm Wako Pure Chemical Corporation, hereinafter also referred to as "DBL") was used.

[0074] First, 300 μL of a 40 g / L aqueous solution of bovine serum albumin, 150 μL of a 2 M aqueous solution of glucose or 2 M aqueous solution of fructose, 50 μL of an aqueous solution of DBL, and 900 μL of phosphate buffered saline (pH 7.4) were added to a 2 mL screw-cap tube and stirred to obtain a mixture. The above aqueous solution of DBL was prepared by diluting the stock solution (high-concentration aqueous solution of DBL) with water so that the final concentration of DBL in the mixture was 0.0004 to 0.0500 g / L. As a positive control, an aqueous solution of aminoguanidine (final concentration of aminoguanidine in the mixture 0.060 to 0.500 g / L) was used instead of the aqueous solution of DBL.

[0075] Next, the fluorescence intensity of the obtained mixture at 0 hours of reaction was measured. Then, the above mixture was incubated at 60 °C for 48 hours under light-shielded conditions, and the fluorescence intensity of the obtained reaction solution was measured. The measurement of the fluorescence intensity was performed using a microplate reader at an excitation wavelength of 370 nm and an emission wavelength of 440 nm.

[0076] From the measured fluorescence intensity, the AGEs production inhibitory rates of DBL and aminoguanidine were calculated according to the following formula. The results are shown in Figure 3 (glucose) and Figure 4 (fructose).

Equation

[0077] From the calculated AGEs production inhibition rate, the EC 50 values of DBL and aminoguanidine were calculated. The results are shown in Table 2 below.

[0078]

Table 2

[0079] As shown in Table 2, for both glucose and fructose, the EC 50 value of DBL was about 1 / 13 of that of aminoguanidine. From this result, it became clear that DBL exhibits a high anti-glycation activity significantly exceeding that of aminoguanidine, which is the positive control.

[0080] 〔Example 5〕Anti-glycation activity against collagen glycation The anti-glycation activity (AGEs production inhibitory activity) of this anti-glycation agent against the glycation of collagen by glyceraldehyde was evaluated using the "Collagen Anti-Glycation Assay Kit Glyceraldehyde" manufactured by Cosmo Bio Co., Ltd. DBL was used as this anti-glycation agent. The specific test method followed the protocol attached to the above kit. The test method will be briefly described below.

[0081] First, an aqueous DBL solution was added to the collagen solution, glyceraldehyde solution, and buffer solution of the above kit, and stirred to obtain a mixture. The above aqueous DBL solution was prepared by diluting the stock solution (high-concentration aqueous DBL solution) with water so that the final concentration of DBL in the mixture was 0.001 - 0.250 g / L. As a positive control, an aqueous aminoguanidine solution (final concentration of aminoguanidine in the mixture 0.001 - 0.250 g / L) was used instead of the aqueous DBL solution.

[0082] Next, the fluorescence intensity of the obtained mixture at 0 hours of reaction was measured. Next, the above mixture was incubated at 37°C for 24 hours, and the fluorescence intensity of the obtained reaction solution was measured. The measurement of the fluorescence intensity was carried out in the same manner as in Example 4.

[0083] From the measured fluorescence intensity, the AGEs production inhibitory rates of DBL and aminoguanidine were calculated in the same manner as in Example 4. The results are shown in Fig. 5.

[0084] As shown in Fig. 5, DBL showed a high anti-glycation activity that was significantly higher than that of aminoguanidine, which is the positive control, especially in the high-concentration range, against the glycation of collagen by glyceraldehyde. Also, in the low-concentration range, it showed a good anti-glycation activity equivalent to that of aminoguanidine.

[0085] In Examples 3 to 5, the anti-glycation activity was evaluated using commercially available DBL. It is naturally expected that the MIBK fraction containing DBL as the main component also has anti-glycation activity.

Industrial Applicability

[0086] The mutant strain of Ganoderma tsugae of the present invention has a high growth rate, and it is possible to realize the mass production of bioactive substances. The present invention can be used in the fields of health and beauty, etc.

Deposit Number

[0087] NIT EP -03465

Claims

1. A mutant strain of Inonotus obliquus with the deposit number NITE P-03465.

2. A culture containing a mutant strain of Inonotus obliquus with the deposit number NITE P-03465.

3. A method for culturing Inonotus obliquus, comprising the step of aerobically culturing a mutant strain of Inonotus obliquus with the deposit number NITE P-03465.

4. The culturing method according to claim 3, wherein, in the step, the mutant strain of Inonotus obliquus is cultured in a medium containing yeast extract.

5. A method for producing at least one selected from the group consisting of an antioxidant component and an anti-glycation component, comprising the step of aerobically culturing a mutant strain of Inonotus obliquus with the deposit number NITE P-03465.

6. The production method according to claim 5, wherein the anti-glycation component is dihydroxybenzylacetone or a salt thereof.

7. An anti-glycation agent comprising, as an active ingredient, a culture extract containing a mutant strain of Inonotus obliquus with the deposit number NITE P-03465, wherein the culture extract contains dihydroxybenzylacetone or a salt thereof.

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