WO2006016582A1

WO2006016582A1 - Liquid culture method for inonotus obliquns

Info

Publication number: WO2006016582A1
Application number: PCT/JP2005/014581
Authority: WO
Inventors: Seihou Shinya; Hiroyuki Kondo; Hideto Uemura; Hiroaki Matsushima
Original assignee: Rohto Pharmaceutical Co., Ltd.; Fujiwara Techno-Art Co., Ltd.; Nishikawa, Tomoko
Priority date: 2004-08-09
Filing date: 2005-08-09
Publication date: 2006-02-16
Also published as: JPWO2006016582A1

Abstract

A method for the liquid culture of Inonotus obliquns; a culture obtained by this liquid culture method and materials relating thereto. Use of the above method can establish an effect of economically and safely obtaining Inonotus obliquns and Inonotus obliquns culture (for example, a liquid culture) on an industrial scale. Because of having a high physiological activity, the culture, etc. obtained by the above culture method can be used for various purposes.

Description

Liquid culture method of birch

Technical field

[0001] The present invention relates to a liquid culture method for birch moth (Scientific name: Fuscoporia obliqua or Inonotus obliquus), a culture obtained by the liquid culture method, and an invention related thereto.

Background art

[0002] The birch is a mushroom that belongs to the genus Coleoptera, and is a carcinogenic fungus that parasitizes mature birch in cold regions such as Hokkaido and Russia. It has been confirmed that it takes more than 10 years to grow to a size of ~ 20cm. This birch is traditionally used to maintain health in Russia as a healthy tea for cold prevention and hangover, and in Japan, the Ainu people decoction and drink as healthy tea. It has been reported that there are effects and antiviral effects.

[0003] Therefore, due to the recent increase in demand for birch oysters due to the health boom, over-exploitation of natural birch moss has progressed, and now the number of white birch parasitized by cyno V anatake is "one per mountain" ”And“ 1 for every 20,000 ”, and in addition to the birch moss that imports mouth sika, its origin is unclear. There is also a problem with this point.

[0004] Therefore, various methods have been studied for artificial culture of cynomolgus (see, for example, Patent Documents 1 to 3).

Patent Document 1: Japanese Patent No. 3008292

Patent Document 2: Japanese Patent Publication No. 52-44613

Patent Document 3: JP-A-10-191783

Disclosure of the invention

Problems to be solved by the invention

[0005] However, the cultivated oyster mushroom obtained by the above known culturing method, The liquid culture was sufficiently high in tumor cell growth inhibitory activity, antiviral activity, superoxide scavenging activity, and the like.

[0006] Therefore, the present inventors conducted extensive research focusing on superoxide-erasing activity among these physiological activities, and identified a weight ratio of potassium and sodium in a medium that has not been focused on conventionally. When cultivated cynomolgus shoots in a liquid range, the superoxide scavenging activity of the culture is remarkably increased, and in particular, the ratio of superoxide scavenging activity to the mycelium weight (specific activity) is remarkably high. I found out for the first time. The present invention has been completed based on such findings.

[0007] That is, an object of the present invention is to provide a birch liquid for obtaining a culture of birch moss having a physiological activity such as superoxide scavenging activity at a low cost, safely, and on an industrial scale. It is an object of the present invention to provide a culture method and a culture of cynomolgus mushroom having high physiological activity obtained by using the liquid culture method.

Means for solving the problem

[0008] The gist of the present invention is as follows.

[1] A fungus which is characterized in that it has a step of culturing mycelium of birch fungus in an aqueous medium, and the weight ratio of potassium to sodium (potassium Z sodium) in the aqueous medium is lZl to 50Zl. Liquid culture method,

[2] a culture obtained by the liquid culture method according to [1],

[3] A composition for eliminating superoxide comprising the culture according to [2],

[4] Use of the culture according to the above [2] for the production of a composition for eliminating superoxide, and

[5] A method for eliminating superoxide using the culture according to [2] above

About.

The invention's effect

[0009] By using the present invention, there is an effect that a culture such as birch bamboo, a culture solution of birch bamboo, etc. can be obtained inexpensively, safely, on an industrial scale. In addition, since the culture obtained by the culture method of the present invention has high physiological activity, it can be used for various applications. BEST MODE FOR CARRYING OUT THE INVENTION

[0010] As described above, the liquid culture method for oyster mushrooms of the present invention has a step of culturing mycelium of birch moss in an aqueous medium, and the weight ratio of potassium and sodium in the aqueous medium (potassium / sodium). ) Is lZl to 50Zl. By having such a feature, there is an effect that a culture such as a culture solution of bonito salmon having high physiological activity can be obtained at an inexpensive and industrial scale. In addition, since the present invention is a liquid culture, there is an advantage that a large-scale culture on an industrial scale is easier than a solid culture, and that the culture can be easily treated.

[0011] In the present invention, the birch (Scientific name: Fuscoporia obliqua or Inonotus obliquus) refers to a mushroom belonging to the order of the genus Cilantroidae. When used in the present invention, the mycelium of Ranunculaceae, parasitic on birch in nature, can be used as is, and the National Institute of Technology and Evaluation (Biotechnology Division, Biogenetic Resources Division ( (NBRC)] (NBRC No. 8681) Strength The cultured mycelium obtained can also be used. In order to prevent the mycelium from being damaged when inoculating the aqueous medium, the mycelium can be pre-cultured and then added to the aqueous medium.

[0012] The aqueous medium used in the present invention is a composition in which the content ratio of potassium and sodium in the aqueous medium is weight ratio (potassium / sodium) 1Zl to 50Zl. The weight ratio here is the ratio of the total weight of potassium ions and non-ionized potassium in the aqueous medium to the total weight of sodium. The presence of potassium and sodium at a strong ratio has the advantage that the superoxide scavenging activity is significantly increased. Among them, the weight ratio (potassium to sodium) is preferably 3Zl to 50Zl, more preferably 6Zl to 45Z1, from a practical viewpoint.

[0013] The weight ratio is a ratio in an aqueous medium used for culturing, and an aqueous medium used for pre-culture and an aqueous medium continuously added during the culture have the same weight ratio. It is preferable. The potassium and sodium contents in the aqueous medium may be calculated by calculating the potassium and sodium strength in the medium raw material, respectively, but preferably each content in the aqueous medium can be calculated by the method described later. .

[0014] The weight ratio of potassium to sodium in the aqueous medium is However, it is preferable that the weight ratio is always achieved during the culture period. In this case, it can be calculated by sampling an aqueous medium and directly measuring the content of potassium or sodium therein. In this case, the content of the sampled aqueous medium can be measured using a known elemental analysis method such as atomic absorption analysis, inductively coupled plasma emission analysis, or ion selective electrode method.

[0015] The content of potassium in the aqueous medium, in view of the involvement in the ^{bioactive, 0. 005 ~1. O 0/} 0 (w / v) force S Preferably, from 0.01 to 0.5 ^_0/0 (w / v) force Ri preferably, 0. 1~0. 5% (w Zv) it is more preferred. In addition, the content of sodium in the aqueous medium is preferably 0.0001 to 0.1% (wZv), taking into account the ratio of bioactivity and potassium. (WZV) is more preferable 0.01 to 0.1% (WZV) is more preferable.

[0016] Furthermore, in consideration of the optimum involvement in physiological activity, one or two selected from the group having minerals such as magnesium, copper and zinc in the aqueous medium and amino acid strengths such as tyrosine and ferrolanine It is preferable to contain more than seeds. The content of magnesium or copper, in the aqueous ^{_{medium, 0. 0001~1. 0 0 / o}} (w / v) force S ^{_{Preferably, 0. 001~1. 0 0 / o}} (w / v) force RiYoshimi More preferred is 0.01 to 0.5% (wZv). The content of zinc in the aqueous medium, from 0.001 to 5.0 ^_0/0 force S Preferably, from 0.001 to 3. More preferably 0 ⁰ / _o (w / v) force S, 0. 01-3 More preferred is 0% (wZv). Moreover, tyrosine or Hue -.. The content of Ruaranin, aqueous ^{_{medium, 0. 0001~5 0 0 / o (}} w / v) force S ^{_{Preferably, 0. 001~1 0 0/0 (}} w / v ) More preferable than force S, and more preferably 0.01 to 0.5% (wZv).

[0017] Examples of main potassium sources include dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and potassium chloride. Examples of the main sodium source include sodium chloride sodium. Examples of the raw material containing both potassium and sodium include various peptones and yeast extracts.

[0018] The aqueous medium used in the present invention includes glucose, various peptones (soybean peptone, etc.), yeast extract, dipotassium hydrogen phosphate, phosphorus in addition to water from the viewpoint of production cost and handling. Preferred are those containing at least one selected from the group consisting of potassium dihydrogen acid, tyrosine, copper and magnesium, and those containing all of these. It is preferable. Examples of such an aqueous medium include water, glucose, soybean peptone, yeast extract, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and magnesium sulfate.

[0019] The water-based medium may contain components other than those described above as additives. For example, sucrose, mannitol, galactose, trenosose, manoleose, ratatoses, dextrin, corn starch, glycerol and the like can be used. In addition, various peptones (such as casein-made peptone), malt extract, soyton, kashiton, casamino acid, calcium nitrate, ammonium sulfate, and rice bran can be used. Thiamine, piotin, folic acid, calcium chloride and the like can also be included in the aqueous medium.

[0020] Among them, as an aqueous medium, water 90-96% (wZv) is also used, glucose 0.4-4% (wZ v), various peptone 0.01-1% (wZv), yeast extract 0. 01 to 3% (wZv), dipotassium hydrogen phosphate 0.05 to 0.5% (wZv), potassium dihydrogen phosphate 0.01 to l% (wZv), magnesium sulfate 0.01 to 0.5 % (wZv), copper 0.01-0.5% (wZv), and tyrosine 0.01-0.5% (wZv) More preferably, all of these are contained. Examples of such an aqueous medium include water 90-96% (wZv), glucose 0.4-4% (wZv), soybean peptone 0.01-1% (wZv), yeast extract 0.01-3% ( wZv), dipotassium hydrogen phosphate 0.05 to 0.5% (w / v), potassium dihydrogen phosphate 0.01 to l% (wZv), and magnesium sulfate 0.01 to 0.5% (w Zv), water 90-96% (wZv), glucose 0.4-4% (wZv), yeast extract 0.01-3% (wZv), dipotassium hydrogen phosphate 0.05- 90-96% water containing 0.5% (wZv), potassium dihydrogen phosphate 0.01-1% (wZv), and tyrosine 0.01-0.5% (wZv) (wZv), glucose 0.4-4% (wZv), yeast extract 0.01-3% (w Zv), dipotassium hydrogen phosphate 0.05-0.5% (wZv), potassium dihydrogen phosphate Examples include those containing 0.01 to 1% (wZv), tyrosine 0.01 to 0.5% (wZv), and copper 0.01 to 0.5% (w / v).

[0021] The pH of the aqueous medium is preferably 4 to 8 forces, more preferably 5 to 7.5 forces, in consideration of the contribution to physiological activity.

[0022] The aqueous medium is prepared by appropriately sterilizing the above components and then sterilizing them. Media kill There are no particular limitations on the fungus method as long as it is a method that is usually used in liquid culture methods.

[0023] In the liquid culture method of the present invention, it is preferable to inoculate and culture in an aqueous medium characterized in that the weight ratio of potassium to sodium (kalmium Z sodium) is lZl to 50Zl. To start. Here, inoculation refers to the process of planting cynomolgus mushroom mycelium as an inoculum in an aqueous medium. Specifically, the mycelium is punched out together with agar using a 4 mm diameter cork borer from an agar plate medium on which the mycelium has grown. A method of planting in the aqueous medium, a method of pre-culturing mycelium in the aqueous medium, and adding this culture solution to the aqueous medium, or a method of adding natural cynomolgus mycelia directly to the aqueous medium. Etc.

[0024] In addition, in the present invention, when manufacturing on an industrial scale, the aqueous medium obtained as described above can be scaled up and cultured. Among these, it is desirable to culture the inoculated aqueous medium at 15 to 30 ° C, preferably 15 to 27 ° C, more preferably 18 to 27 ° C, from the viewpoint of increasing physiological activity.

[0025] The culture scale is not particularly limited as long as it satisfies the above conditions! /.

For example, any apparatus that is used for normal liquid culture, such as a small-scale apparatus that is cultured in a flask or a large-scale apparatus that uses a silo, can be used. Further, depending on the culture scale, methods such as shaking culture and stirring culture can be selected as appropriate. Examples of the shaking condition include 100 to 150 rpm.

[0026] Although the culture period is not particularly limited, it is, for example, 7 to 60 days, and preferably 7 to 50 days from the viewpoint of increasing the physiological activity in the culture and reducing the production cost. 15 to 50 days is more preferred. 30 to 45 days is more preferred. Other culture conditions are not particularly limited.

[0027] By virtue of the method, a culture having a high physiological activity of the bamboo shoot can be obtained. For example, a culture filtrate having a high superoxide scavenging activity such as a superoxide scavenging activity value of 0% or more in the culture filtrate can be obtained. In addition, for example, a culture having a high superoxide elimination specific activity such that the superoxide elimination specific activity (the magnitude of the superoxide elimination activity relative to the dry weight of the mycelium) is 35% Zg or more can be obtained. Incidentally, the method for measuring the superoxide scavenging activity can be exemplified by the method described in Examples below. It is done.

[0028] The obtained culture exhibits high physiological activity such as superoxide scavenging activity, and natural birch salmon itself has historically had very little side effects on the human body, so it eliminates superoxide. Therefore, it is possible to take a strong culture directly and it is considered safe as a food additive. For example, the above-mentioned culture is anticancer, immunity enhancement, blood sugar level lowering, blood pressure lowering, cholesterol lowering, antithrombosis, antiviral activity, active oxygen elimination, energy recovery, atopic dermatitis, hay fever, It can also be suitably used as various therapeutic agents such as rhinitis, infection of pathogenic bacteria, skin spots, cosmetics such as prophylactic agents, pharmaceutical compositions, foods or their raw materials.

[0029] The culture is a generic term for a medium obtained by culturing, and includes a culture solution, a culture filtrate (separated culture fluid cells), and cells (viable and dead bacteria). ), Cell extracts, purified products thereof, active fractions, dilutions, concentrates or dried products. The method for producing the purified product, active fraction, dilution, concentrate and dried product is not particularly limited as long as it is a known method.

[0030] The present invention also provides a superoxide scavenging composition comprising the culture. Due to the strong characteristics, the composition has a physiological activity such as a high superoxide scavenging activity, and therefore can be suitably used when superoxide scavenging is required. For example, the composition can be suitably used as cosmetics, pharmaceutical compositions, foods, research reagents and the like. Therefore, the present invention also provides a cosmetic, a pharmaceutical composition, and a food characterized by including the culture.

[0031] The culture content in the composition for eliminating superoxide of the present invention may be the culture itself without particular limitation.

[0032] The method for producing the superoxide scavenging composition of the present invention is not particularly limited as long as the superoxide scavenging activity is maintained. Ingredient strength It can manufacture using a well-known manufacturing method.

[0033] The dosage form of the cosmetic of the present invention is not particularly limited, and examples thereof include basic cosmetics such as lotion, milky lotion, cream, makeup cosmetics, and hair cosmetics. These cosmetics can be obtained by formulating the above culture and optional ingredients for cosmetics by a conventional method. Examples of the optional component that can be used include hydrocarbons, tridalylides, fatty acids, surfactants, polyhydric alcohols, thickeners, ultraviolet absorbers, and the like.

[0034] The cosmetic of the present invention can be used for prevention and improvement of hair stains, prevention and improvement of hair loss, prevention of generation of body odor, etc. by eliminating superoxide. The content of the culture in the cosmetic of the present invention is preferably 0.001 to 80% by weight, more preferably 0.001 to 50% by weight, and still more preferably 0.01 to 30% by weight. . The cosmetic of the present invention can be used such that the culture is applied, for example, 5 mg to 50 g per day per adult.

[0035] The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and examples thereof include injections, tablets, and liquids. These pharmaceutical compositions can be obtained by formulating the above culture and optional ingredients for the pharmaceutical composition by conventional methods. Illustrative optional ingredients include excipients, binders, disintegrants, coating agents and the like.

[0036] The dosage of the pharmaceutical composition varies depending on the type of disease, symptoms, patient age, body weight, and the like. As for the dosage, the above culture can be administered orally at 1 mg to 20 g Zkg body weight once a day or several times a day, for example, 5 mg to 50 g per adult. Once a day or in several divided doses, it should be administered by transdermal absorption.

[0037] The form of the food of the present invention is not particularly limited. Examples of such forms include candies, gummies, jellies, drinks, breads, and the like. Examples of dosage forms include tablets, capsules, jellies, liquids, and the like. These foods can be obtained by blending the above-mentioned culture and optional ingredients for foods by a conventional method and calcining.

[0038] The content of the cultures in these foods, different forces good Mashiku the type of food from 0.001 to 60 weight ^_0/0, more preferably 0.001 to 40 weight ^_0/0 More preferably, the content is 0.01 to 20% by weight. The food of the present invention can be ingested so that the culture is ingested, for example, 1 mg to 20 gZkg body weight per day per adult.

[0039] In general, the influence of superoxide on a living body includes tissue damage due to generation of radicals. As a result, skin aging symptoms such as blemishes, tarmi, blemishes, hair loss, body odor, allergic reactions such as hay fever, rhinitis, bronchial asthma, atopic dermatitis, myocardial infarction, arteriosclerosis, Arrhythmia, pneumonia, gastric ulcer, cirrhosis, splenitis, dementia, It is known that leukemia, hyperlipidemia, diabetes, autonomic neuropathy, cataract, collagen disease, rheumatoid arthritis, etc. are caused. Here, natural cynomolgus mushrooms actually have excellent effects such as anti-cancer action and immune enhancement action, and can exert a superoxide elimination action in a subject requiring superoxide elimination. There is power. Therefore, the superoxide erasing composition of the present invention (cosmetics, pharmaceutical composition, food, etc.) containing the above-mentioned culture derived from natural cynomolgus moss generally has an excellent superoxide erasing action. It can be effectively used for the improvement and prevention of the above-mentioned diseases, which are considered to be related to superoxide, skin aging symptoms, and the generation of body odor, and delay.

[0040] The present invention further provides a method for eliminating superoxide using the culture.

[0041] In the method of the present invention, the culture may be used in an effective amount or more that provides a superoxide scavenging action.

Example

[0042] Hereinafter, the liquid culture method for birch moss according to the present invention will be specifically described by way of examples. However, the present invention is not intended to be limited to the scope of the powerful examples.

[0043] (Examples 1 to 5, Comparative Examples 1 to 4)

Place 200 mL of aqueous medium having the composition shown in Table 1 into a culture container (container with a cotton cap on an Erlenmeyer flask), then heat sterilize at 121 ° C for 15 minutes, isolate Hokkaido natural cynomolgus moss, and agar plate culture The obtained mycelium was aseptically inoculated into each culture container. In Table 1, the weight ratio of potassium and sodium was calculated from the potassium and sodium contents in the raw material of the initial culture medium.

[0044] Next, shaking culture (condition: 120 rpm) was performed at 23 ° C for 30 days, and then the culture was taken out from each culture vessel, and the mycelium was filtered off to obtain a culture filtrate.

[0045] Table 1 shows the dry weight of the obtained birch mycelia, and the super 1-year-old oxidase scavenging activity value of the culture filtrate (3-fold diluted solution). Moreover, these measuring methods are shown below.

[0046] (Dry weight measurement of birch mycelium)

The mycelium is filtered, washed with distilled water, and 30 to 80 ° C, preferably 30 to 60 ° C for 24 hours. [0047] (Measurement of superoxide erasing activity and superoxide erasing specific activity value)

Using “SOD Assay kit-WST” (Dojin Institute of Science), the superoxide scavenging activity of a solution obtained by diluting the culture filtrate obtained above three times was measured. For calibration, superoxide dismutase (manufactured by Wako Pure Chemical Industries, Ltd.) derived from red blood cells was used as a standard sample. The superoxide elimination specific activity (SOD specific activity value) was calculated by dividing the superoxide elimination activity value (SOD activity value) of the measured 3-fold diluted culture filtrate by the dry mycelium weight. The results are shown in Table 1.

[0048] [Table 1]

In Table 1, glucose is manufactured by -Sushi Co., Ltd. (trade name: Glu'Final), Polypeptone (registered trademark), and powdered yeast extract s is manufactured by Nippon Pharmaceutical Co., Ltd. Umu 'potassium dihydrogen phosphate, magnesium sulfate heptahydrate is manufactured by Wako Pure Chemical Industries, Ltd. (food additive). [0050] (Examples 6 and 7)

Place 200 mL of aqueous medium having the composition shown in Table 2 into a culture vessel (container with a cotton cap on an Erlenmeyer flask), then heat sterilize at 121 ° C for 15 minutes, isolate Hokkaido natural cynomolgus mosquito, and agar plate culture The obtained mycelium was aseptically inoculated into each culture container. In Table 2, the weight ratio of potassium and sodium was calculated from the contents of potassium and sodium in the raw material of the initial culture medium.

[0051] Next, shaking culture (condition: 120 rpm) was performed at 28 ° C for 30 days, and then the culture was taken out from each culture vessel, and the mycelium was filtered to obtain a culture filtrate.

[0052] Table 2 shows the superoxide scavenging activity values of the obtained culture filtrate (3-fold diluted solution). The method for measuring the superoxide erasing activity value is as described above.

[0053] [Table 2]

In Table 2, glucose is manufactured by Nisshi Co., Ltd. (trade name: Guru's final), yeast extract is manufactured by Nippon Paper Chemicals Co., Ltd. (trade name: SK yeast extract S-2), dipotassium hydrogen phosphate 'phosphoric acid Potassium dihydrogen is manufactured by Yoneyama Chemical Co., Ltd., L-tyrosine is manufactured by Kyowa Hakko Co., Ltd., and the mineral yeast is manufactured by Oriental Yeast Co., Ltd. (trade name: mineral yeast Cu). Since mineral yeast contains abundant copper, the aqueous medium containing 0.1% by weight of mineral yeast as in Example 7 contains about 0.03% by weight of copper. .

[0055] From the results of Tables 1 and 2, it can be seen that in Examples 1 to 7, the superoxide scavenging activity value in the culture filtrate is extremely high as compared with Comparative Examples 1 to 4. In particular, it can be seen that in Examples 1 to 5, superoxide erasing specific activity is extremely high as compared with Comparative Examples 1 to 4.

Industrial applicability

[0056] By using the liquid culture method for cynomolgus of the present invention, it is possible to stably cultivate cynomolgus mushroom in large quantities. In addition, the cultivated cynomolgus mushroom obtained by the present invention and the superoxide erasing composition containing the culture have high physiological activity and can be suitably used when superoxide erasing is required. The culture and composition are, for example, anticancer, immunity enhancement, blood glucose level lowering, blood pressure lowering, cholesterol lowering, antithrombosis, antiviral action, active oxygen elimination, energy recovery, atopic dermatitis, pollen It can also be suitably used as cosmetics such as various therapeutic agents such as infectious diseases, rhinitis, infection of pathogenic bacteria, skin spots, and preventive agents, pharmaceuticals, foods, and raw materials thereof.

Claims

The scope of the claims

[1] A birch salmon liquid characterized in that it has a step of culturing mycelium of birch salmon in an aqueous medium, and the weight ratio of potassium to sodium (potassium Z sodium) in the aqueous medium is lZl to 50Zl. Culture method.

[2] The potassium content in the aqueous medium is 0.005 to 1.0% (wZv), and the sodium content is 0.0001-1.0% (wZv). Liquid culture method.

[3] The liquid culture method according to claim 1 or 2, wherein the content of magnesium in the aqueous medium is 0.0001-1.0% (wZv).

[4] The liquid culture method according to any one of claims 1 to 3, wherein the aqueous medium further contains at least one selected from the group consisting of copper, zinc, tyrosine and phenylalanin.

[5] A culture obtained by the liquid culture method according to any one of claims 1 to 4.

[6] A composition for eliminating superoxide, comprising the culture according to claim 5.

[7] The superoxide eliminating composition according to claim 6, which is a cosmetic, a pharmaceutical composition, or a food.

[8] Use of the culture according to claim 5 for the production of a composition for eliminating superoxide.

[9] A method for eliminating superoxide using the culture according to claim 5.