JP2004147574A - Method for culturing wood-decaying fungus and method for using the fungus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To mass-produce melanin-decomposing matter produced by wood-decaying fungi belonging to the genus Pleurotus ostreatus. <P>SOLUTION: A method comprising culturing the wood-decaying fungi belonging to the genus Pleurotus ostreatus in a medium ≤3 in the ratio C/N( wherein, C is the weight of a carbon source in the medium(g); and N is the weight of a nitrogen source in the medium(g) ) is provided. By this method, the melanin-decomposing matter is mass-produced, and the culture treatment product thereof has high bleaching effect as well. <P>COPYRIGHT: (C)2004,JPO

Description

【００２６】
表１の結果から分かる通り、Ｃ／Ｎ比が３以下である培地で培養した培養液において、メラニン分解性物質であるマンガンパーオキシダーゼが量産される事が分かる。また、培養物、菌糸体の抽出物についても同様の傾向を示した。
【００２７】メラニンの分解
ヒラタケ（ＩＦＯ：Ｎｏ．３０７７６）をＣ／Ｎ比１．０の培地で培養した培養液１００ｍｌに、硫酸アンモニウム５６ｇを加え、溶解させる。４℃で１昼夜放置し、沈殿を生じさせる。３，０００ｒｐｍ、１０分間遠心分離により、沈殿を回収し、乾燥して粗酵素粉末を得る。この粉末を精製水１０ｍｌに溶解し、酵素溶液とする。調製した酵素溶液５ｍｌに０．１ｇ／ｍｌイカスミメラニン（グリコ栄養食品社製）分散水溶液を５０μｌを添加し、室温で３日間放置した。その後、メラニンをろ過した溶液の４０５ｎｍの吸光度を測定し、メラニンの分解度を測定した。
【００２８】
【表２】

【００２９】
表２に示したように、Ｃ／Ｎ比１．０で培養した培養液より調製した酵素溶液はメラニンを分解し、４０５ｎｍにおける吸光度の値が上昇していることがわかった。陰性対象として酵素液の代わりに精製水を用いたものは、４０５ｎｍの吸光度値の増加は全く認められなかった。
【００３０】
次に、本発明の培養処理物を配合した処方例を示すが、本発明はこれに限定されるものでない。
化粧料の処方例
【００３１】
（１）化粧用クリーム
（重量％）
ａ）ミツロウ・・・２．０
ｂ）ステアリルアルコール・・・５．０
ｃ）ステアリン酸・・・８．０
ｄ）スクワラン・・・１０．０
ｅ）自己乳化型グリセリルモノステアレート・・・３．０
ｆ）ポリオキシエチレンセチルエーテル（２０Ｅ．Ｏ．）・・・１．０
ｇ）ヒラタケ培養液（Ｃ／Ｎ比０．２）・・・１０．０
ｈ）１，３−ブチレングリコール・・・５．０
ｉ）水酸化カリウム・・・０．３
ｊ）防腐剤・酸化防止剤・・・適量
ｋ）精製水・・・残部
製法
ａ）〜ｆ）までを加熱溶解し、８０℃に保つ。ｈ）〜ｋ）までを加熱溶解し、８０℃に保ち、ａ）〜ｆ）に加えて乳化し、４０℃まで撹拌しながら冷却する。その後、ｇ）を加え、攪拌し均一に溶解する。
【００３２】
（２）乳液 （重量％）
ａ）ミツロウ・・・０．５
ｂ）ワセリン・・・２．０
ｃ）スクワラン・・・８．０
ｄ）ソルビタンセスキオレエート・・・０．８
ｅ）ポリオキシエチレンオレイルエーテル（２０Ｅ．Ｏ．）・・・１．２
ｆ）タモギタケ菌糸体抽出物（Ｃ／Ｎ比０．０４）・・・１５．０
ｇ）１，３−ブチレングリコール・・・７．０
ｈ）カルボキシビニルポリマー・・・０．２
ｉ）水酸化カリウム・・・０．１
ｊ）精製水・・・残部
ｋ）防腐剤・酸化防止剤・・・適量
ｌ）エタノール・・・７．０
製法
ａ）〜ｅ）までを加熱溶解し、８０℃に保つ。ｇ）〜ｋ）までを加熱溶解し、８０℃に保ち、ａ）〜ｅ）に加えて乳化し、５０℃まで撹拌しながら冷却する。５０℃でｆ）、ｌ）を添加し、４０℃まで攪拌、冷却する。
【００３３】
（３）化粧水
（重量％）
ａ）マンガンパーオキシダーゼ（Ｃ／Ｎ比１．０）・・・０．０１
ｂ）グリセリン・・・５．０
ｃ）ポリオキシエチレンソルビタンモノラウレート（２０Ｅ．Ｏ．）・・・１．０
ｄ）エタノール・・・６．０
ｅ）香料・・・適量
ｆ）防腐剤・酸化防止剤・・・適量
ｇ）精製水・・・残部
製法
ｃ）〜ｇ）までを混合し、均一に溶解する。使用時にａ）、ｂ）を混合して使用する。
【００３４】
（４）洗顔料 （重量％）
ａ）ステアリン酸・・・１２．０
ｂ）ミリスチン酸・・・１４．０
ｃ）ラウリン酸・・・５．０
ｄ）ホホバ油・・・３．０
ｅ）グリセリン・・・１０．０
ｆ）ソルビトール・・・１５．０
ｇ）１，３−ブチレングリコール・・・１０．０
ｈ）ＰＯＥ（２０）グリセロールモノステアリン酸・・・２．０
ｉ）水酸化カリウム・・・５．０
ｊ）水・・・残部
ｋ）キレート剤・・・適量
ｌ）香料・・・適量
ｍ）ウスヒラタケ培養液乾燥物（Ｃ／Ｎ比３．０）・・・０．１
製法 ａ）〜ｈ）までを加熱溶解し７０℃に保つ。ｊ）にｉ）を溶解後ａ）〜ｈ）に加えケン化する。その後ｋ）、ｌ）を入れ攪拌しながら冷却する。５０℃でｍ）を添加し、４０℃まで攪拌、冷却する。
【００３５】
（５）ドリンク剤 （重量％）
ａ）トキイロヒラタケ菌糸体抽出物（Ｃ／Ｎ比０．２）・・・２０．０
ｂ）防腐剤・・・適量
ｃ）砂糖・・・１０．０
ｄ）酸化防止剤・・適量
ｅ）精製水・・・残部
製法 ａ）〜ｅ）までを混合攪拌し、よく分散させ均一にする。
【００３６】
（６）錠剤 （重量％）か
ａ）ヒラタケ培養物乾燥物（Ｃ／Ｎ比２．０）・・・２０．０
ｂ）ショ糖・・・８０．０
製法 ａ）ｂ）を混合し、よく攪拌分散させ均一にする。その後適量毎に打錠する。
【００３７】
【効果確認試験】
（１）塗布によるヒトでの効果確認試験
被験者として、２０〜５０歳の女性１５名に１日２回（朝、夜）連続２ヵ月間、本発明品と比較品のそれぞれを使用させ、塗布部位の状態を試験前後で比較し、改善効果を調べた。本試験には、実施例１として処方例中の（１）化粧用クリームで示した化粧料を用い、比較例 １ には実施例１からヒラタケ培養液を除き、代わりにＣ／Ｎ比２５．７で培養したヒラタケ培養液を配合した化粧料、比較例 ２には実施例１からヒラタケ培養液を除いた化粧料を作成し、その使用による効果について調べた。本発明の有効成分を配合した化粧料を毎日使用しながら肌の美白効果及び使用後の肌の感触について塗布開始前及び２ヶ月塗布後におけるアンケートで集計し、効果の確認を行った。
【００３８】評価基準
＜美白効果＞
著効：しみ・ソバカスが消えた部分がある。
有効：しみ・ソバカスが目立たなくなった。
やや有効：しみ・ソバカスがやや目立たなくなった。
変化なし：なんら変化なし。
【００３９】
【表３】

【００４０】
表３より、比較例１にもやや美白効果が見られるが、Ｃ／Ｎ比０．２の培地で培養した培養液を配合した実施例１は、非常に高い美白効果があることがわかる。また、使用後の肌感触についてはしっとり・つるつるになった人が半数以上にのぼり、使用後の肌感触が良くなっていることがわかる。実施例１と比較例１を比較すると、実施例１の方が比較例１よりはるかにこれらの効果が高いことから、これらの効果は培地のＣ／Ｎ比を３．０以下に設定したことに起因していることが明らかになった。
尚、本試験において実施例１を使用して何らかの刺激があったと答えた人はいなかった。
【００４１】
（２）服用によるヒトでの効果確認試験
被験者として、２０〜５０歳の女性１５名に１日１回（夜）連続２ヵ月間、本発明品と比較品のそれぞれを服用させ、肌の状態を試験前後で比較し、改善効果を調べた。本試験には、実施例２として処方例で示した（６）錠剤を用い、比較例３には実施例２からヒラタケ培養物乾燥物を除き、代わりにＣ／Ｎ比４．０で培養したヒラタケ培養物乾燥物を配合した錠剤、比較例４には実施例２からヒラタケ培養物乾燥物を除きショ糖のみとした錠剤を作成し、その使用による効果について調べた。本発明の有効成分を配合した錠剤を毎日服用しながら肌の美白効果の有無を服用開始前及び２ヶ月服用後におけるアンケートで集計し、効果の確認を行った。尚、評価基準は連続塗布による美白効果で行なった基準と同様である。
【００４２】
【表４】

【００４３】
表４より、比較例３にもやや美白効果が見られるが、Ｃ／Ｎ比２の培地で培養した培養物乾燥物を配合した実施例２は、非常に高い美白効果があることがわかる。美白効果が培地のＣ／Ｎ比を３．０以下設定したことに起因していることが明らかになった。
尚、本試験において実施例２を使用して何らかの体調不良があったと答えた人はいなかった。
【発明の効果】
以上詳述したごとく、本発明の培養法によれば、安定的にメラニン分解性物質を量産でき、それを利用したものは、角質中のメラニンを分解し、高い美白効果を有するとともに安全性の高いものである。[0001]
BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a culture method for effectively mass-producing melanin-decomposing substances produced by microorganisms, particularly basidiomycetes, and a method of using the same.
[0002]
2. Description of the Related Art Melanin is biosynthesized not only in skin but also in the central nervous system and retina, and is widely distributed not only in animals but also in nature from plants to microorganisms. Melanin is produced by converting tyrosine into dopa and further to dopaquinone by the action of tyrosinase, and then oxidizing to indole-5,6-dihydroquinone, which polymerizes.
[0003] Biosynthesis of melanin is carried out in cells, melanocytes, which are present in the basal layer of the epidermis, using ultraviolet light as a trigger. In melanocytes, melanin is synthesized and matured in granules called melanosomes, migrates and disperses to epidermal cells, discolors as the skin metabolizes, and falls off as dirt when renewed. Thus, melanin plays an important role in protecting the body from the adverse effects of ultraviolet light, and is a medically important factor. However, when the amount of melanin increases, the skin becomes dark and dark, and its uneven distribution becomes spots (hepatic spots) and freckles (sparrow eggs), which is a serious cosmetic problem.
[0004] Currently known measures against spots and freckles are roughly classified into two preventive methods and one therapeutic method.
[0005] Prevention methods include a method of blocking UV light that triggers melanin biosynthesis with a sunscreen or the like, and a method of inhibiting melanin biosynthesis (eg, glutathione, vitamin C, cysteine, arbutin, thiosulfate). And a method using a melanin synthesis inhibitor such as soda).
[0006] However, at present, a safe method for eliminating treatment of spots and freckles once formed without side effects has not yet been developed. In the past, hydroquinone, 4-isopropylcatechol, and hydroquinone monobenzyl ether have been used as skin bleaching agents, but they have a strong whitening effect, but they are based on pigment cell degeneration and death. If topical use is continued, permanent vitiligo may occur, and other side effects such as abnormal pigmentation and rash are inevitable. Therefore, it is not currently marketed as a cosmetic.
As a method for decomposing melanin, a method using a melanin decomposed product extracted from seaweed (Japanese Patent Laid-Open No. 3-251514) and a method using a melanin decomposed product collected from cultured fungal cells ( JP-A-7-213294), a method using a culture of a basidiomycete or a processed product thereof (JP-A-6-219933) and the like, but the degree of melanin decomposition is not sufficient, or mass production cannot be performed stably, There were problems such as irritation.
[0008]
SUMMARY OF THE INVENTION The present invention has been made in view of the state of the art, and does not prevent the generation of spots and freckles, but removes the stains and freckles that have already been formed. The purpose of the present invention is to provide a method for stably mass-producing a substance that degrades melanin using basidiomycete.
[0009]
Means for Solving the Problems The inventors of the present invention screened melanin-degrading mushrooms from wood-rotting fungi and cultured them in a specific medium to obtain a melanin-degrading substance produced by the wood-rotting fungi. Was found to be mass-produced, and the present invention was finally completed. In other words, the present invention uses a culture-treated product of wood rot fungi belonging to the genus Pleurotus cultivated, which is cultured in a medium having a C / N ratio represented by Formula 1 and having a C / N ratio of 3 or less.
[0010]
As the microorganisms usable in the present invention, any wood-rotting fungi having a melanin-degrading property can be used regardless of whether they are white (lignin-decomposing bacteria) or brown, but Pleurotus. Is particularly preferably used.
[0011]
Wood rot fungi belonging to the genus Pleurotus (Pleurotus) include oyster mushrooms (Pleurotus ostreatus (Jacq .: Fr.) Kummer), syrup mushrooms (Pleurotus cornucopiae (Paulet) Rolland var. L.
Vass. ), Pleurotus pulmonarius (Fr.) Quel, and the like.
[0012]
Basidiomycetes can induce hyphae from natural fruiting bodies and soil, and can also be obtained from a strain distribution agency. By culturing these hyphae, a melanin-decomposable substance such as manganese peroxidase is produced in the culture solution. Manganese peroxidase is known as an enzyme that degrades wood lignin, and is used for pretreatment of wood saccharification, decolorization of dyes, decolorization of colored wastewater, and decomposition of hardly decomposable substances.
[0013]
The medium in the present invention may have a C / N ratio represented by Formula 1 of 3 or less.
As a carbon source component of the medium, for example, glucose, maltose, trehalose, sucrose, malt extract, potato extract, oatmeal, wheat malt or a mixture thereof can be used. Examples of the nitrogen source component that can be used in the medium include amino acids, ammonium salts, nitrates, peptone, skim milk, yeast extract, and mixtures thereof. In addition, as the inorganic salt component that can be used in the medium, for example, sodium chloride, magnesium sulfate, phosphate, ferrous sulfate, potassium chloride, or a mixture thereof can be used.
[0014]
The culturing conditions are a temperature of 15 to 37 ° C., a pH of 2.5 to 10.0, and an illumination of 0 to 2000 Lux. The culturing is performed under aerobic conditions by a method such as shaking culture or aeration stirring culture for 7 to 30 days. Is preferred. Although the main culture can be carried out from the beginning, it is preferable to carry out a small-scale preculture in advance and to inoculate the main culture.
[0015]
The culture-treated product referred to in the present invention is a culture after culturing, a culture solution in which mycelium is separated by a method such as filtration, a mycelium extract obtained by extracting a mycelium of a cultured wood rot fungus by a usual method, And those obtained by separating and purifying a melanin-decomposable substance such as manganese peroxidase from a common method.
[0016]
The preparation of the extract of mycelium is not particularly limited, but for example, it is extracted from a low temperature to a heated state using various appropriate organic solvents. Examples of the extraction solvent include water; lower alcohols such as methyl alcohol and ethyl alcohol; liquid polyhydric alcohols such as glycerin, propylene glycol and 1,3-butylene glycol; ketones such as acetone and methyl ethyl ketone; One or more kinds of alkyl esters; hydrocarbons such as benzene and hexane; ethers such as diethyl ether; halogenated alkanes such as dichloromethane and chloroform can be used. In particular, water, ethyl alcohol, and a mixed solvent of one or more of 1,3-butylene glycol are particularly preferable.
[0017]
As for the extract of mycelium, each mycelium was dried and then finely pulverized, and the solvent was used in a weight ratio of 1 to 1000 times, especially 10 to 100 times, and in the case of room temperature extraction, 0 to 100 times. It is preferable to carry out at a temperature of 20 ° C. or more, particularly 20 ° C. to 30 ° C. for 1 hour or more, particularly 3 to 4 hours.
[0018]
Cultures, culture solutions, and mycelium extracts obtained under the above conditions may be used as they are, but if necessary, concentrated and powdered by filtration or the like may be used as appropriate. Can be done. When used as it is, the compounding amount can be used in the range of 0.01 to 100% by weight, and preferably in the range of 10.0 to 50.0% by weight.
[0019]
When the melanin-decomposable substance is used by separating peroxidases present in the culture solution, salting out is performed using a salt such as ammonium sulfate. The substance precipitated by salting out may be dried and used as it is. However, if necessary, it can be used after further purification treatment. The amount of the purified peroxidases used in the present invention is preferably 0.00001 to 10.0% by weight, and most preferably 0.001 to 0.1% by weight.
[0020]
In addition to the cultured product according to the present invention, aqueous components, oil components, plant extracts, animal extracts, powders, excipients, surfactants, oils, alcohols, pH adjusters, preservatives, antioxidants, It is prepared by mixing a thickener, a sweetener, a coloring matter, a flavor and the like as necessary and appropriately blending them. The dosage form of the cosmetic of the present invention is not particularly limited, and may be various dosage forms such as lotion, emulsion, cream, pack, powder, spray, ointment, dispersion, and detergent. When used as a food or drink, the dosage form may be tablet, capsule, beverage, gum, candy and the like.
[0021]
Hereinafter, examples of the present invention will be described, and examples of application of the material to skin external preparations and foods and drinks will be described. However, it goes without saying that the present invention is not limited to the examples described herein.
Culture of Wood-rot Fungi As a wood-rot fungus of the genus Pleurotus, a strain purchased from the Institute for Fermentation: Hirate (IFO: No. 30776) was used. Glucose was used as a carbon source and polypeptone was used as a nitrogen source. Using a medium with a different C / N ratio, screening of a medium in which a melanin-decomposable substance was mass-produced was performed. In order to avoid the influence of other components, screening was performed without adding inorganic components and the like.
For culture, 100 ml of the culture solution was added to a 200 ml Erlenmeyer flask, sterilized, inoculated with mycelia, and subjected to rotary culture at 24 ° C. and 150 rpm for 10 days.
Preparation of culture solution The culture cultured by the above-mentioned method was filtered to remove mycelia to obtain a culture solution.
Measurement of manganese peroxidase titer 2 ml of a culture solution was added to 1 ml of a 1% aqueous solution of 2,6-dimethyphenol (2,6-DMP), and a buffer adjusted to pH 4.5 with 0.2 M citric acid-NaOH. 0.3 ml of the liquid and 0.7 ml of purified water were added, and the change in absorbance at 475 nm after 10 minutes was measured.
[0025]
[Table 1]

[0026]
As can be seen from the results in Table 1, it is understood that manganese peroxidase, which is a melanin decomposable substance, is mass-produced in a culture solution cultured in a medium having a C / N ratio of 3 or less. In addition, cultures and extracts of mycelium showed the same tendency.
Melanin Decomposition Oyster mushroom (IFO: No. 30776) is dissolved in 100 g of a culture obtained by culturing the medium in a medium having a C / N ratio of 1.0 by adding 56 g of ammonium sulfate. Leave at 4 ° C. for one day to form a precipitate. The precipitate is collected by centrifugation at 3,000 rpm for 10 minutes, and dried to obtain a crude enzyme powder. This powder is dissolved in 10 ml of purified water to obtain an enzyme solution. To 5 ml of the prepared enzyme solution, 50 μl of an aqueous solution of 0.1 g / ml squid mimelanin (manufactured by Glyco Nutrition Foods) was added, and the mixture was allowed to stand at room temperature for 3 days. Thereafter, the absorbance at 405 nm of the solution obtained by filtering the melanin was measured, and the degree of decomposition of the melanin was measured.
[0028]
[Table 2]

[0029]
As shown in Table 2, it was found that the enzyme solution prepared from the culture solution cultured at a C / N ratio of 1.0 decomposed melanin and the absorbance at 405 nm was increased. When purified water was used instead of the enzyme solution as a negative control, no increase in the absorbance at 405 nm was observed.
[0030]
Next, a formulation example in which the cultured product of the present invention is blended is shown, but the present invention is not limited to this.
Example of formulation of cosmetics
(1) Cosmetic cream (% by weight)
a) Beeswax 2.0
b) Stearyl alcohol ... 5.0
c) Stearic acid ... 8.0
d) Squalane ... 10.0
e) Self-emulsifying glyceryl monostearate: 3.0
f) Polyoxyethylene cetyl ether (20EO) 1.0
g) Oyster mushroom culture solution (C / N ratio 0.2) ... 10.0
h) 1,3-butylene glycol 5.0
i) Potassium hydroxide 0.3
j) Preservatives / antioxidants: appropriate amount k) Purified water: The remaining processes a) to f) are dissolved by heating and maintained at 80 ° C. h) to k) are dissolved by heating, kept at 80 ° C., added to a) to f), emulsified, and cooled to 40 ° C. with stirring. Then, add g) and stir to dissolve uniformly.
[0032]
(2) Emulsion (% by weight)
a) Beeswax 0.5
b) Vaseline 2.0
c) Squalane ... 8.0
d) Sorbitan sesquioleate 0.8
e) Polyoxyethylene oleyl ether (20EO) 1.2
f) Mycelium extract of C. versicolor (C / N ratio 0.04) ... 15.0
g) 1,3-butylene glycol 7.0
h) Carboxyvinyl polymer 0.2
i) Potassium hydroxide 0.1
j) Purified water: balance k) Preservative / antioxidant: appropriate amount l) Ethanol: 7.0
Heating and dissolving processes a) to e), and keeping at 80 ° C. g) to k) are dissolved by heating, kept at 80 ° C, added to a) to e), emulsified, and cooled to 50 ° C with stirring. At 50 ° C add f), l), stir to 40 ° C and cool.
[0033]
(3) Lotion (weight%)
a) Manganese peroxidase (C / N ratio: 1.0) ... 0.01
b) Glycerin 5.0
c) Polyoxyethylene sorbitan monolaurate (20EO) 1.0
d) Ethanol 6.0
e) Perfume: proper amount f) Preservative / antioxidant: proper amount g) Purified water: balance The remaining processes c) to g) are mixed and uniformly dissolved. At the time of use, a) and b) are mixed and used.
[0034]
(4) Face wash (% by weight)
a) Stearic acid 12.0
b) Myristic acid 14.0
c) Lauric acid ... 5.0
d) Jojoba oil 3.0
e) Glycerin 10.0
f) Sorbitol 15.0
g) 1,3-butylene glycol 10.0
h) POE (20) glycerol monostearic acid 2.0
i) Potassium hydroxide 5.0
j) water ... balance k) chelating agent ... appropriate amount l) flavoring agent ... appropriate amount m) dried Mushroom mushroom culture (C / N ratio 3.0) ... 0.1
Preparation method a) to h) are dissolved by heating and kept at 70 ° C. After dissolving i) in j), add it to a) to h) and saponify. Then, k) and l) are added and cooled while stirring. At 50 ° C., add m), stir to 40 ° C. and cool.
[0035]
(5) Drinks (% by weight)
a) Mycelium extract of Japanese oyster mushroom (C / N ratio: 0.2) 20.0
b) Preservative: appropriate amount c) Sugar: 10.0
d) Antioxidant: proper amount e) Purified water: The rest of the production method a) to e) are mixed and stirred, well dispersed, and made uniform.
[0036]
(6) Tablet (% by weight) or a) Dried oyster mushroom culture (C / N ratio: 2.0) 20.0
b) Sucrose 80.0
Production method a) b) are mixed, well stirred and dispersed, and made uniform. Thereafter, tableting is performed at appropriate doses.
[0037]
[Effect confirmation test]
(1) Test for confirming the effect of human application by application As subjects, 15 women aged 20 to 50 were allowed to use each of the product of the present invention and the comparative product twice a day (morning and night) for two consecutive months. The state of the site was compared before and after the test, and the improvement effect was examined. In this test, the cosmetic shown in (1) Cosmetic Cream in the Formulation Example as Example 1 was used. In Comparative Example 1, the oyster mushroom culture solution was removed from Example 1 and the C / N ratio was changed to 25. A cosmetic composition containing the oyster mushroom culture solution cultivated in Example 7, and a cosmetic material in Comparative Example 2 from which the oyster mushroom culture solution was removed from Example 1 were prepared, and the effect of use thereof was examined. While using the cosmetics containing the active ingredient of the present invention every day, the whitening effect of the skin and the feel of the skin after use were tabulated by questionnaires before the start of application and after two months of application, and the effect was confirmed.
Evaluation criteria <Whitening effect>
Significant effect: Some spots and freckles disappeared.
Effective: spots and freckles are less noticeable.
Slightly effective: spots and freckles are slightly less noticeable.
No change: No change.
[0039]
[Table 3]

[0040]
Table 3 shows that Comparative Example 1 has a slight whitening effect, but Example 1 in which a culture solution cultured in a medium having a C / N ratio of 0.2 is blended has a very high whitening effect. In addition, as for the skin feel after use, more than half of moist and slippery people climbed, and it can be seen that the skin feel after use is improved. Comparing Example 1 and Comparative Example 1, Example 1 has much higher effects than Comparative Example 1. Therefore, these effects are obtained by setting the C / N ratio of the medium to 3.0 or less. It became clear that it was caused by.
In this test, no one answered that there was any irritation using Example 1.
[0041]
(2) Test for confirming the effect of human taking on humans As subjects, fifteen women aged 20 to 50 were allowed to take each of the present invention product and the comparative product once a day (night) for two consecutive months, and the condition of the skin was examined. Were compared before and after the test to examine the improvement effect. In this test, the tablet (6) shown in the prescription example as Example 2 was used, and in Comparative Example 3, the oyster mushroom culture dried product was removed from Example 2 and the cells were cultured at a C / N ratio of 4.0 instead. Tablets containing dried oyster mushroom cultures, and Comparative Example 4 were prepared from Example 2 using only sucrose except for dried oyster mushroom cultures, and the effects of their use were examined. While taking the tablet containing the active ingredient of the present invention daily, the presence or absence of skin whitening effect was tabulated by questionnaires before the start of administration and after two months of administration, and the effect was confirmed. The evaluation criteria are the same as those used for the whitening effect by continuous application.
[0042]
[Table 4]

[0043]
From Table 4, it can be seen that Comparative Example 3 has a slight whitening effect, but Example 2 in which a dried culture obtained by culturing in a medium having a C / N ratio of 2 is blended has a very high whitening effect. It became clear that the whitening effect was caused by setting the C / N ratio of the medium to 3.0 or less.
In this test, no one answered that there was any unhealthy condition using Example 2.
【The invention's effect】
As described in detail above, according to the culture method of the present invention, melanin-decomposable substances can be stably mass-produced, and those utilizing the same can degrade melanin in the keratin, have a high whitening effect and have safety. It is expensive.

Claims (4)

A method for cultivating wood rot fungi belonging to the genus Pleurotus, characterized by culturing in a medium having a C / N ratio represented by Formula 1 of 3 or less.

A wood rot fungus culture belonging to the genus Pleurotus, characterized by being cultured in a medium having a C / N ratio represented by Formula 1 of 3 or less. An external preparation for skin, comprising a wood rot fungus culture belonging to the genus Pleurotus according to claim 2. A food or drink comprising the culture of wood rot fungi belonging to the genus Pleurotus according to claim 2.