JPH06219933A - Method for decomposing melanin and decomposable substance of melanin - Google Patents

Abstract

PURPOSE:To decompose and decolor melanin using Basidonycetes having melanin decomposing property. CONSTITUTION:Melanin is decomposed and decolored by bringing melanin into contact with a microbial cell of Basidonycetes having melanin decomposing property, its cultured material and/or its treated material in-vivo or in-vitro. The reaction system is kept in a state where nitrogen is limited (under nitrogen source-free conditions or conditions reduced to as small amount of nitrogen as possible) in contact with melamine and combined use of glucose is further effective in this time. As necessary, the reaction system may be incubated at 10-45 deg.C for a required time. When a microorganism cultured in a culture medium previously containing melanin and/or its precursor under conditions where nitrogen is limited, further good result is obtained. The usable microorganism contains wood-destroying fungus having melanin-decomposability, e.g. NK-1148 strain (FERM BP-1859). This microorganism can be utilized for the production of skin-whitening cosmetics for the treatment of stain and ephelis as well as decoloring of melanin-containing industrial products.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a melanin-decomposing method, a melanin-decomposing substance and a whitening cosmetic composition which utilize microorganisms, especially basidiomycetes having a melanin-decomposing property.

[0002]

BACKGROUND OF THE INVENTION Melanin is biosynthesized not only in the skin but also in the central nerves and retina, and is widely distributed not only in animals but also in plants and microorganisms in nature. Melanin is produced by the conversion of tyrosine into dopa and further dopaquinone by the action of tyrosinase, followed by oxidation to become indole-5,6-dihydroquinone, which is polymerized (reference document 1).

The biosynthesis of melanin is carried out by melanocytes, which are cells located in the basal layer of the epidermis, using ultraviolet light as one of the triggers (reference document 2). In melanocytes, melanin is synthesized and matured in granules called melanosomes, migrates and disperses in epidermal cells, discolors with the metabolism of the skin, and becomes dross and falls off during renewal (Reference 3). Thus, melanin plays an important role in protecting the body from the adverse effects of ultraviolet rays, and is an important factor in medicine. However, when the amount of melanin increases, the skin becomes darker, and the uneven distribution thereof causes spots (melasma) and freckles (mottled freckles), which is a serious cosmetic problem.

The currently known measures against spots and freckles are:
It is roughly divided into two preventive methods and one therapeutic method.

Preventive measures include (1) a method of blocking UV light that triggers melanin biosynthesis with a sunscreen agent or the like (reference document 6), and (2) a drug that inhibits melanin biosynthesis (for example, A method using melanin synthesis inhibitors such as glutathione, vitamin C, cysteine, arbutin, and sodium thiosulfate) (References 7 and 8) is known.

However, the present situation is that a safe method for eliminating the stains and freckles once formed by treatment has no side effect has been developed (References 2 and 4). Previously, hydroquinone, 4-isopropylcatechol, and hydroquinone monobenzyl ether were also used as skin bleaching agents, but they have a strong whitening effect, but they are based on degeneration and lethality of pigment cells. , Continued topical application may cause permanent vitiligo, and other side effects such as pigment abnormalities and rashes are unavoidable, so it is not currently on the market and the development of safe spots and freckles treatment agents in the industry Is strongly desired (reference 5).

[0007] Therefore, at present, as a method for treating spots and freckles, a method of shortening the period during which melanin is removed due to renewal of the epidermis by using keratin softening agents such as resorcin and salicylic acid, and a peeling agent is known. (Reference 4). However, this method originally
It is not a cure for spots and freckles, and it takes a long time of several months to treat, so it cannot be called a realistic cure.

[References] 1. Protein / nucleic acid / enzyme, 15, p. 550 (197
0) 2. Ryoichi Fukushiro, "Dermatological Diagnosis and Treatment Type 4" Kodansha, p.
38 3. Cells, 4, (9), p. 16 (1972) 4. Junichi Miyazaki, “External preparation for skin, its preparation and application”, Nanzandou, p. 273-275, 353-355 5. Lin, 44, (6), p. 629 (1990) 6. Cosmetic Science Study Group "Latest Cosmetic Science"
(Sho 61-4-15), p. 391-395 7. JP-A-53-142515 JP-A-48-44442

[0009]

SUMMARY OF THE INVENTION The present invention has been made in view of the current state of the art, and has not been successful in the conventional development, rather than preventing the generation of spots and freckles. It was made for the purpose of developing a new system for safely treating the generated spots and freckles.

Since the main cause of spots and freckles is melanin, melanin is also contained in in vitro substances such as various colored products in addition to the skin, and decolorization thereof is also strongly demanded in the industry. Taking into account
The present invention has been made for the purpose of developing a new system capable of degrading melanin in vo and in vitro.

[0011]

[Means for Solving the Problems] In order to achieve the above-mentioned object, as a result of examinations from various aspects, attention was paid to biological treatment from the viewpoint of safety, and intensive screening was conducted for microorganisms, but it did not succeed in the end, and there For the first time, I changed my mind and focused on wood-destroying fungi that had nothing to do with the degradation of melanin.

The present invention succeeded in screening a microorganism capable of degrading melanin from wood-destroying fungi, and finally completed the present invention. The present invention provides a basidiomycete having melanin degradability. It is a new technical idea to decompose melanin using a culture or a treated product thereof as a basic technical idea.

Microorganisms that can be used in the present invention include all wood-decaying fungi, regardless of whether they are white (lignin-decomposing fungi) or brown, as long as they are wood-decaying fungi having melanin decomposing property. -1148 strain (FERM B
P-1859) or Polo discus (Por
Microorganisms belonging to the genus Odisculus) can be advantageously used.

The NK-1148 strain is a microorganism isolated by the present inventors and is an excellent melanin-degrading bacterium, and its mycological properties are described in JP-B-3-32996.
The details are as follows, but the main ones are as follows.

[(1) Growth state in culture medium] As shown in Table 1 below.

[0016]

[Table 1]

[(2) Physiological and ecological properties] pH range of growth (potato-glucose agar medium, 2
Cultivated at 8 ° C. for 4 days) Grows at around pH 3 to 9, but not at pH 2 and 10. The optimum pH is around 4-6. Growth temperature range (potato-glucose agar, p
H5, 4 days culture) Grows at around 10 to 45 ° C, but does not grow at 50 ° C. The optimum temperature is around 28 to 37 ° C. Phenol oxidase reaction (28 ° C, 4 days culture) Shows weak or negative. Characteristics of mycobacterium (potato, glucose agar, pH
5, 28 ° C., 4 days culture) White and felt-like.

The physiological and ecological properties and the mycological properties such as the growth state in each medium were examined in detail, but it could not be identified as a known bacterium. Therefore, this strain was identified as a new strain, named as NK-1148 strain, and deposited at Micro Engineering Laboratories (FERM BP-1859).

Examples of the bacterium belonging to the genus Porodiscus include, for example, a strain NK-729W (FERM BP-1860) belonging to Nurudetake (Porodiscus pendulus) which the present inventors succeeded in newly separating.
Is exemplified. This strain is extremely excellent in that it can highly decompose melanin, and its mycological properties are described in detail in JP-B-3-32997, the main ones of which are as follows. is there.

[(1) Growth state in culture medium] As shown in Table 2 below.

[0021]

[Table 2]

[(2) Physiological and ecological properties] Growth pH range (potato-glucose agar medium, 2
8 ° C, 4 days of culture) Grows at around pH 3 to 7, but not at pH 2 and 8. The optimum pH is around 4-5. Growth temperature range (potato-glucose agar, p
H5, 4 days of culture) Grows at around 10 to 32 ° C, not at 37 ° C. The optimum temperature is around 20 to 30 ° C. Phenol oxidase reaction (28 ° C, 4 days of culture) shows positive. Bacterial shape (potato, glucose agar, pH
5, 28 ° C, 4 days of culture) White and hairy. Shape of fruiting body ・ Size: Diameter 2-5mm ・ Shape: Inverted bowl shape (shape of nose) ・ Rim and surface: Edge curls inward, surface is yellowish brown with brown hair on the entire surface.・ Pipe hole surface: Light grayish white, dented like a dish, and the hole is small. -Meat quality: It is a soft leather and almost white. Spore shape Sausage shape of about 3 to 4 × 1 μm, which is colorless and smooth.

Based on these mycological characteristics, the present bacterium is recognized as belonging to the genus Porodiscus, and taxonomically, especially, P.
S. pendulus) is considered appropriate.

In the present invention, a basidiomycete having a melanin degrading property is used, but in addition to the bacterium itself, a culture product and / or a processed product thereof can also be used. The culture broadly refers to a mixture of cells and a culture solution obtained by culturing a bacterium, but in the present invention, cells such as wet cake separated from the cell culture, its residue, and solids. It is also possible to use the culture medium after removing all the substances.
In addition, the processed product refers to all of the above-mentioned products that have been concentrated, dried, or diluted.

In order to decompose melanin according to the present invention, the microbial cells, cultures and / or processed products of basidiomycetes having melanin-degrading property are treated with melanin in vivo or in v.
It may be contacted in vitro, and in that case, it may be incubated at about 10 to 45 ° C. for the required time. During the contact with melanin, the reaction system is preferably kept in a nitrogen-restricted state, and it is preferable that the nitrogen source is not present or the amount is as small as possible. At this time, it is more effective to use glucose together.

Therefore, the melanin-degrading substance according to the present invention comprises a basidiomycete fungus having melanin-decomposing property, a culture and / or a treated product as an active ingredient, and an inorganic or organic carrier commonly used therein, In solid, semi-solid or liquid form, it may be formulated into an oral administration agent as well as a parenteral administration agent such as an external preparation or a bath agent, but in the case of industrial use, it may contain only the active ingredient.

The preparations for oral administration include tablets, pills, granules, soft and hard capsules, powders, fine granules, powders,
Emulsifiers, suspensions, syrups, elixirs and the like can be mentioned. Formulations for parenteral administration include injections, ointments, lotions, tonics, sprays, suspensions, oils,
Examples thereof include emulsions and suppositories. In order to formulate the active ingredient of the present invention, conventional methods may be followed, including surfactants, excipients, coloring agents, flavoring agents, preservatives, stabilizers, buffers, suspending agents, isotonic agents. Other commonly used substances are used as appropriate.

The dose of the substance according to the present invention varies depending on the degree of spots, freckles, administration method, age and the like, but in the case of oral or enteral administration, the active ingredient is 0.1% per adult per day. It is administered within the range of 1 to 100 mg / kg. When administered as an external preparation for skin, an appropriate amount may be applied to the affected area several times. In the case of industrial application, there is no particular limitation as described above, and the substance and the melanin removal target may be brought into contact with each other as appropriate. The substance was orally administered to rats at a dose of 100 mg / kg of body weight, but no toxicity was observed and it was safe.

The present invention also relates to a whitening cosmetic composition containing a melanin-degrading basidiomycete fungus body, a culture and / or a treated product as an active ingredient. The whitening cosmetic composition of the present invention is mainly a lotion. , Creams, emulsions, packs, bath agents, hair depigmenting agents and the like, and the above-mentioned active ingredient is contained in 0.001 to 15% in the external bases and auxiliaries usually used for each of these external preparations. Preferably, 0.01 to 10% is blended to obtain an external preparation.

For example, in the case of lotion, moisturizers such as glycerin and propylene glycol, skin nutrients, etc. are dissolved in purified water, preservatives, fragrances, etc. are dissolved in alcohol, and both are mixed at room temperature. In the production of a general lotion to be solubilized, the active ingredient of the present invention is added in an amount of 0.01 to
Add to 10% to make up lotion.

In the cream, a hydrophilic component such as glycerin or sorbit is added to purified water to form a water phase part, and an oil phase part is beeswax, paraffin, microcrystalline wax, ceresin, higher fatty acid, hardened oil, etc. Solid oil, plus squalane, liquid paraffin,
It is prepared by adding oil components such as preservatives and surfactants to liquid oil components such as various ester oils. The water phase obtained in this manner is heated, and in the production of a general cream to be an emulsified cream by gradually adding the oil phase heated to the same temperature while gently stirring, The active ingredient in 0.
Add to make it 10 to 10% to make a cream.

In the emulsion, a moisturizing agent such as glycerin, a pH adjusting agent for an acid or an alkali is added to purified water, and the mixture is heated and mixed to add ethanol to form a water phase portion. Solid oil components such as beeswax and paraffin, petrolatum, lanolin, etc. Of semi-solid oil, liquid oil such as squalane, liquid paraffin, and various ester oils, and oil ingredients such as preservatives and surfactants are added and prepared, mixed and heated to form the oil phase, and the oil phase is converted to the water phase. In addition, preliminary emulsification is performed, and a protective colloid agent such as carboxyvinyl polymer and carboxymethyl cellulose is added to this, and in the production of a general emulsion to be uniformly emulsified with a homomixer, the active ingredient of the present invention in the aqueous phase part. Is added to the aqueous phase so as to be 0.01 to 10% to obtain an emulsion.

In the pack, a moisturizing agent such as glycerin, a filming agent such as polyvinyl alcohol or bee gum is added to purified water to swell, and powders of kaolin, talc, zinc oxide and the like are added thereto if necessary, and a fragrance is added. In the production of a general pack in which ethanol containing a preservative and the like is added and kneaded until it becomes a paste, the active ingredient of the present invention is added to 0.01 to 10% to form a pack. There is no problem in preparing a bath agent and a hair decolorizing agent according to a conventional method.

In carrying out the present invention, it has already been described that the contact with melanin is carried out under the condition that nitrogen is restricted, and at this time, as a basidiomycete capable of degrading melanin, Even better results are obtained with microorganisms which have been cultivated in a medium in the presence of melanin and / or its precursors under pre-determined nitrogen-limited conditions. As the precursor for melanin, various low-molecular and high-molecular compounds which have already been mentioned can be used alone or in combination.

Examples of the present invention will be described below.

[0036]

Example 1 0% glucose and 0.1% glucose were added to a potato dextrose agar medium (containing 2% glucose) and a nitrogen-restricted synthetic medium (L-N medium shown in Table 3 below), respectively.
%, 1% added medium, a total of 4 types of medium were prepared in Petri dishes. As a melanin sample, sasaika ink (about 50% of which is melanin) was dispersed.

[0037]

[Table 3]

On the other hand, basidiomycete NK having melanin degrading property
Strain -1148 (FERM BP-1859) was inoculated on the edge of the Petri dish and incubated at 28 ° C.

As a result, it was confirmed that the strain NK-1148 was capable of decolorizing or degrading melanin, and particularly when 1% of glucose was added to the nitrogen-restricted medium (Petri dish on the right side of FIG. 1: N50G1, that is, Squid solution 50 μl).
/ Medium 20 ml, glucose 1% added in the medium of Table 3),
The phenomenon appeared fastest, demonstrating the effectiveness of the present invention.
The growth of hypha was most remarkable in the potato dextrose medium, but decolorization or decomposition of melanin was not observed at all. From these, it was found that nitrogen limitation is effective for the decomposition of melanin. Further, when glucose was not added or when it was limited to 0.1%, decolorization or decomposition of melanin was slight even though it was observed although the growth of mycelium was not strong. It was
From these, it was found that sugar plays some role in the degradation of melanin.

As is clear from these results, NK-
It has been scientifically proven that strain 1148 exhibits excellent melanin degrading property.

[0041]

Example 2 NK-729W in place of NK-1148 strain
Example 1 except using (FERM BP-1860)
By repeating the same operation as above, the degradability of melanin under nitrogen limitation was similarly confirmed.

[0042]

[Example 3: Whitening skin cream] Glucose 10 g, squid solution 2 were obtained by removing agar from the LN medium shown in Table 3.
Using a culture of the NK-1148 strain cultured in a liquid medium containing ml, an O / W emulsion was prepared according to a conventional method according to the formulation shown in Table 4 below to produce a skin cream for treating spots and freckles. .

[0043]

[Table 4]

[0044]

[Example 4: Whitening pack] Using the culture of NK-729W strain, the agar was removed from the L-N medium shown in Table 3 and cultured in a liquid medium containing 10 g of glucose and 2 ml of Squid solution, and the following Table 5 was used. In the prescription of, according to the usual method,
A is mixed and dissolved to form a gel, and then B and C are sequentially A
To prepare a pack for treating spots and freckles.

[0045]

[Table 5]

[0046]

As a whitening cosmetic for spots and freckles, only a product that prevents the formation of spots and freckles has been developed so far, and a product that eliminates or reduces the spots and freckles that have been once formed, that is, safety. It has not been successful in developing a therapeutic agent for freckles and freckles, which has a high level of stains.

On the other hand, the present invention, as described above, is a highly safe stain which has never been developed by the conventional method.
It was the first successful development of a freckle remedy.
Moreover, the present invention has a remarkable effect that it is effective not only for external use but also for internal use.

The action of the present invention is based on the excellent melanin degrading action, which is as described above.
It is played not only in vivo but also in vitro, and can be used in industrial aspects. For example, in vitro substances such as various colored products contain melanin, but by treating it with the substance of the present invention, it becomes possible to decompose and decolorize melanin, and therefore, according to the present invention, The remarkable effect that the colored industrial products derived from melanin can be efficiently decolorized.

[Brief description of drawings]

FIG. 1 is a photograph showing melanin-degrading ability of basidiomycetes having melanin-degrading properties. (The left side is a control without inoculation of basidiomycetes having melanin-degrading property. As is clear from the Petri dish on the right side, the melanin is decomposed by the present invention and the black part of melanin disappears, and whitening occurs. Recognize.)

Claims (9)

[Claims] 1. A method for degrading melanin, which comprises degrading melanin with a basidiomycete fungus having melanin degrading property, a culture product thereof, or a treated product thereof. 2. The method for degrading melanin according to claim 1, wherein the melanin is brought into contact with nitrogen in a state where nitrogen is limited. 3. A method for decomposing melanin, characterized in that the basidiomycete fungus body having melanin degrading property according to claim 1, and the culture thereof are cultured in a medium to which melanin or a precursor thereof is added. 4. A melanin-degrading substance containing a basidiomycete culture having melanin-degrading property or a treated product thereof as an active ingredient. 5. A melanin-degrading substance containing a culture obtained by culturing a basidiomycete having melanin-degrading property in a medium restricted with nitrogen or a treated product thereof as an active ingredient. 6. A melanin-degrading substance containing a culture product obtained by culturing a basidiomycete having melanin-degrading property in a medium to which melanin or a precursor thereof is added, or a treated product thereof as an active ingredient. 7. A whitening cosmetic comprising a basidiomycete culture having a melanin degrading property or a treated product thereof as an active ingredient. 8. A whitening cosmetic comprising as an active ingredient a culture obtained by culturing a basidiomycete having melanin degrading ability in a medium in which nitrogen is restricted, or a treated product thereof. 9. A whitening cosmetic comprising as an active ingredient a culture obtained by culturing a basidiomycete having melanin degrading ability in a medium containing melanin or a precursor thereof, or a treated product thereof.