Nitrated protein degrading agent

Abstract

To provide nitrated proteolytic agents and skin external preparations that have the effect of improving various diseases and symptoms in various tissues in the living body, especially various symptoms caused by protein nitration such as yellowish discoloration in the skin.SOLUTION: Provided are a nitrated proteolytic agent containing a fermented product obtained by fermenting the petal extract of Keihakitami (Ministry of Agriculture, Forestry and Fisheries, Variety Registration No. 10597) with lactic acid bacteria; and a skin external preparation containing the fermented product.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a lactic acid bacteria fermented product of rose petals, and more particularly to a nitrated protease and a topical skin preparation containing a fermented product obtained by fermenting a flower petal extract of Rosa multiflora Thunb. var. carnea Thory with lactic acid bacteria.

Background Art

[0002] Rose is a general term for plants classified in the genus Rosa of the family Rosaceae. In addition to native varieties, there are many horticultural varieties created by crossing and the like. Horticultural varieties are roughly classified into old roses and modern roses. As old roses, there are Rosa Centifolia, Rosa Damask, Rosa Gallica, Rosa Alba, and as modern roses, there are Floribunda Rose, Hybrid Tea Rose, etc. Not limited to classification by scientific name and species, roses are further classified in more detail by the appearance of the plant body and ecological characteristics, etc. The total number of rose varieties including horticultural varieties is said to be 200,000. Also, roses of each variety have diverse component compositions contained in their plant bodies, and it is known that the expected biochemical actions differ depending on the variety. Non-Patent Document 1 describes that the contents of anthocyanins, flavonols, and carotenoids in rose petals differ depending on the variety, and Patent Document 1 describes that the degree of promotion of fat production in adipocytes of rose extracts differs depending on the variety. Therefore, in order to obtain a specific action from the roses to be used, it is necessary to be able to strictly distinguish them from other varieties due to variety registration, trademark registration, etc., and to supply roses of varieties that are stably produced.

[0003] As described above, among roses that are expected to have various biochemical effects depending on the variety, there are some that have been reported to have useful effects on the skin. Patent Document 2 describes that an extract of Rosa Centifolia is effective as a topical skin whitening agent. Patent Document 3 describes that a hybrid tea rose or its extract is effective as an elastase activity inhibitor, an antioxidant, and a collagenase activity inhibitor. In addition to these, although many rose extracts having useful effects on the skin are known, nothing has been known about those having a nitrated protein degradation effect. On the other hand, Patent Document 4 describes that the anti-allergic effect and the whitening effect are enhanced by fermenting roses or their extracts with lactic acid bacteria. However, while no rose extract having a nitrated protein degradation effect has been known, neither an example in which the effect is enhanced nor an example in which the effect is obtained by fermentation with lactic acid bacteria has been known.

[0004] Nitrated proteins are a general term for nitrated derivatives that undergo post-translational modification of proteins called nitration, in which a nitro group is added to the benzene ring in the residues of tyrosine and tryptophan, which are aromatic amino acids constituting the protein, by reactive nitrogen species generated in vivo. The content of tryptophan in many proteins present in vivo is much smaller than that of tyrosine, and it is considered that the nitration reaction of proteins mainly occurs in tyrosine residues (Non-Patent Document 2). Non-Patent Document 3 describes that protein nitration affects cell function by causing a decrease in the function of enzymes and tyrosine kinase-type receptors. Non-Patent Document 4 describes that the accumulation of nitrotyrosine in proteins is involved in various diseases such as arteriosclerosis and ischemic brain diseases. In addition to these, it has been reported that protein nitration is involved in various diseases and symptoms in various tissues in vivo, and in the skin, it is known to be involved in skin dullness. Patent Document 5 states that the cause of skin dullness is not only glycated and carbonylated proteins existing in the dermis, which have been conventionally considered, but also nitrated proteins generated by nitration of amino acids constituting proteins existing in the stratum corneum, and in order to improve various symptoms caused by protein nitration, it is effective to decompose nitrated proteins generated by nitration. Therefore, if nitrated proteins can be decomposed, improvement of various symptoms caused by protein nitration such as dullness is expected.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Literature

[0006]

Non-Patent Literature 1

Non-Patent Literature 2

Non-Patent Literature 3

Non-Patent Literature 4

Summary of the Invention

Problems to be Solved by the Invention

[0007] An object of the present invention is to provide a nitrated protein degrading agent and a topical skin preparation having an effect of improving various diseases and symptoms in various tissues in vivo, particularly in the skin, various symptoms caused by nitration of proteins such as skin dullness.

Means for Solving the Problems

[0008] As a result of intensive studies to solve the above problems, the present inventors solved the above problems by using a fermented product obtained by fermenting a petal extract of Plumeria rubra with lactic acid bacteria.

Effects of the Invention

[0009] The fermented product obtained by fermenting the petal extract of *Rosa hybrida* `Keihakitami` with lactic acid bacteria has a nitrated protein-degrading effect that has not been known to be possessed by conventional rose petals, their extracts, or fermented products obtained by fermenting those extracts. Moreover, because this effect is extremely high, it has a high effect of improving various symptoms caused by nitration of proteins such as skin dullness. In addition, the nitrated protein-degrading agent and external skin preparation of the present invention can prevent the accumulation of nitrated proteins by continuously or continuously using them to repeat nitrated protein degradation. As a result, it suppresses an increase in the nitration ratio of proteins in various tissues in vivo including the skin, and thereby can also prevent various symptoms.

Mode for Carrying Out the Invention

[0010] Hereinafter, the present invention of the application will be described in detail.

[0011] *Rosa hybrida* `Keihakitami` used in the present invention of the application is classified as a modern rose of the genus *Rosa* in the family Rosaceae and is a horticultural variety also known by the alias "Moonlight". It is registered under the Ministry of Agriculture, Forestry and Fisheries Variety Registration No. 10597 (Keisei Rose Horticulture, 1999). The fermented product of the present invention is a fermented product (hereinafter referred to as the *Rosa hybrida* `Keihakitami` fermented product) obtained by fermenting the petal extract of this *Rosa hybrida* `Keihakitami` (hereinafter referred to as the *Rosa hybrida* `Keihakitami` extract) with lactic acid bacteria. However, for reasons such as industrial production and work, it does not matter if parts other than the petals (for example, sepals, filaments, anthers, stigmas, styles, ovaries, ovules, flower stalks, flower branches) are slightly included in the preparation process.

[0012] The method for preparing the extracts of *Rhododendron mucronulatum* for fermentation by lactic acid bacteria is not particularly limited. For example, it can be prepared by extracting the petals of *Rhododendron mucronulatum* with a solvent. The solvent for obtaining the extracts of *Rhododendron mucronulatum* is not particularly limited. For example, water (such as purified water), aqueous solutions of inorganic salts (such as sodium chloride, potassium chloride, magnesium chloride, ammonium carbonate, etc.), buffer solutions (such as phosphate buffer, acetate buffer, Tris-hydrochloride buffer, carbonate buffer, borate buffer, etc.), aqueous solutions of inorganic acids (such as hydrochloric acid, carbonic acid, sulfuric acid, nitric acid, phosphoric acid, etc.), aqueous solutions of organic acids (such as acetic acid, citric acid, lactic acid, succinic acid, ascorbic acid, fumaric acid, malic acid, etc.), aqueous solutions of surfactants (such as saponin, lecithin, etc.), lower alcohols (such as methanol, ethanol, propanol, butanol, etc.), ketones (such as acetone, ethyl methyl ketone, etc.), hydrocarbons (such as propane, butane, hexane, cyclohexane, etc.), ethers (such as diethyl ether, etc.), glycols (such as ethylene glycol, propylene glycol, 1,3-butylene glycol, glycerin, etc.), halogenated hydrocarbons (such as dichloromethane, 1,1,1,2-tetrafluoroethane, 1,1,2-trichloroethene, etc.), acetate esters (such as ethyl acetate, methyl acetate, etc.), saccharides (such as glucose, fructose, sucrose, maltose, oligosaccharides, etc.) or a mixture thereof can be used. The mixing ratio (weight ratio) of the petals of *Rhododendron mucronulatum* to the solvent is generally in the range of 1:1 to 1:1000, preferably 1:5 to 1:100, more preferably 1:10 to 1:50 in terms of the dry weight conversion of the petals.

[0013] The extraction process for obtaining the camellia extract is not particularly limited, but for example, it can be carried out by the following method. First, immerse or disperse the petals of the camellia to be extracted in the solvent. In this case, the petals may be used as raw, or may be used after being dried or semi-dried in advance. Also, the shape is not particularly limited, and the collected ones can be used as they are, but those that have been shredded or pulverized and made finer can also be used. The extraction process is sufficient just by immersing or dispersing the petals of the camellia in the solvent, and there is no particular need to set an extraction time, but an appropriate extraction time for a certain period may be set to the extent that the petals do not rot. Before the obtained camellia extract is subjected to the fermentation process, it is preferable to separate the liquid phase by solid-liquid separation means such as filtration or centrifugation and remove the immersed or dispersed petals, etc., but if it does not cause an obstacle in the fermentation process, this solid-liquid separation process may be omitted. After the camellia extract is prepared, it may be used directly for fermentation, or may be used after performing solvent replacement or purification treatment according to a conventional method, or may be used after appropriately adjusting the concentration by dilution or concentration if necessary. In some cases, the liquid phase after solid-liquid separation may be solidified according to a conventional method such as the spray drying method or the freeze drying method, and further pulverized into a powder form if necessary before use.

[0014] Before the camellia extract prepared by the above method is subjected to the fermentation process, it is preferable to perform sterilization as necessary to remove contaminants that may cause obstacles to fermentation. In this case, as a method for removing contaminants, a method of washing and sterilizing the petals to be used in advance with sterilizing ethanol, etc. and then extracting with a sterile solvent, or a method of extracting the petals with a solvent, obtaining an extract, and then performing heat sterilization may be used. As the heat sterilization method, an autoclave sterilization method of heating at 105 to 121 °C for 10 to 20 minutes, or an intermittent sterilization method of maintaining at 80 to 90 °C for 60 to 120 minutes once a day for 2 to 3 days is generally used. However, this sterilization process is not an essential process. If it does not cause an obstacle in the next fermentation process, this sterilization process may be omitted.

[0015] The lactic acid bacteria used in fermentation in the present invention are not particularly limited, but from the perspective of versatility, it is preferable to use Lactobacillus plantarum. It is preferable to perform preculture in a basic medium before subjecting it to the fermentation process. The culture at this stage can be carried out in the usual manner as long as the lactic acid bacteria can grow. As the composition of the basic medium for lactic acid bacteria, it is sufficient to contain a minimum carbon source, nitrogen source, and phosphorus source. For example, it is preferable to use a medium developed as a medium showing good growth of the whole lactic acid bacteria, such as MRS medium (MRS medium composition: peptone 10 g, beef extract 10 g, yeast extract 5 g, glucose 20 g, Tween 80 1 g, K 2 HPO 4 2 g, sodium acetate 5 g, diammonium citrate 2 g, MgSO 4 ·7H 2 O 0.2 g, MnSO 4 ·nH 2O 0.05 g, purified water 1 L). Even if it is a medium composition other than the above, as long as it is a substance that lactic acid bacteria can assimilate and grow, it goes without saying that it is applicable to the present invention. Furthermore, various additives can be added to the basic medium, for example, an aqueous solution of inorganic salts (sodium chloride, potassium chloride, magnesium chloride, ammonium carbonate, etc.) or a buffer solution (phosphate buffer, acetate buffer, Tris-hydrochloride buffer, carbonate buffer, borate buffer, etc.), an aqueous solution of inorganic acids (hydrochloric acid, carbonic acid, sulfuric acid, nitric acid, phosphoric acid, etc.), an aqueous solution of organic acids (acetic acid, citric acid, lactic acid, succinic acid, ascorbic acid, fumaric acid, malic acid, etc.), an aqueous solution of surfactants (saponin, lecithin, etc.), lower alcohols (methanol, ethanol, propanol, butanol, etc.), ketones (acetone, ethyl methyl ketone, etc.), hydrocarbons (propane, butane, hexane, cyclohexane, etc.), ethers (diethyl ether, etc.), glycols (ethylene glycol, propylene glycol, 1,3-butylene glycol, glycerin, etc.), halogenated hydrocarbons (dichloromethane, 1,1,1,2-tetrafluoroethane, 1,1,2-trichloroethene, etc.), acetate esters (ethyl acetate, methyl acetate, etc.), saccharides (glucose, fructose, sucrose, maltose, oligosaccharides, etc.) or a mixture thereof, etc. can be added, but when adding, it is preferably added within a range that does not affect the growth of lactic acid bacteria. Furthermore, in the subsequent fermentation process, the above various components may be contained in the solvent at a concentration within a range that does not affect the fermentation of lactic acid bacteria.

[0016] The fermentation process for obtaining the fermented product of keihakitami is not particularly limited. For example, lactic acid bacteria can be inoculated into the keihakitami extract prepared by the above method and fermented and cultured. The inoculation amount of lactic acid bacteria is not particularly limited, but preferably 10 5 ~10 10It is in the range of / mL. The fermentation temperature is not particularly limited, but generally it is in the range of 5 to 50 °C, preferably in the range of 25 to 40 °C which is the optimum growth temperature of lactic acid bacteria. The fermentation days are not particularly limited, but for example, at the optimum temperature, it is generally in the range of 1 to 15 days, preferably 3 to 10 days. Static fermentation culture is sufficient, but for shortening the fermentation time, etc., shaking culture or aeration culture can also be carried out. If the above fermentation treatment is completed, in order to stop the fermentation, it is preferable to perform a heat sterilization treatment at 80 to 120 °C for about 15 to 120 minutes on the fermentation culture solution that has undergone the fermentation process under the above conditions. The fermentation culture solution after the sterilization treatment can be used as it is, or the liquid phase can be separated preferably by common solid-liquid separation means such as filtration or centrifugation to obtain a fermented product.

[0017] The fermented product of *Caulerpa lentillifera* may be used as it is after preparation, or may be used after purification treatment according to a conventional method, or may be used after being adjusted to an appropriate concentration by dilution or concentration if necessary. In some cases, the liquid phase after solid-liquid separation may be solidified according to a conventional method such as spray drying method or freeze drying method, and further pulverized into a powder form if necessary before use. When using the fermented product of *Caulerpa lentillifera* as each agent of the present invention, the fermented product of *Caulerpa lentillifera* may be used as an agent as it is, or it may be mixed with a general base and used. As the final form, it can be in any form such as liquid, emulsion, gel, solid, powder, granule, etc., and optional compounding components used in external skin preparations can be appropriately compounded as needed within the range that does not impair the effect. Examples of the optional compounding components include, for example, oils, surfactants, powders, colorants, water, alcohols, thickeners, chelating agents, silicones, antioxidants, ultraviolet absorbers, moisturizers, preservatives, fragrances, various medicinal components, pH adjusters, neutralizing agents, and the like. Further, as the final form, for example, in the case of an external skin preparation, basic cosmetics such as emulsions, creams, lotions, essences, gels, packs, facial washes, makeup cosmetics such as lipsticks, foundations, liquid foundations, makeup pressed powders, cleansing cosmetics such as facial washes, body shampoos, soaps, and furthermore bath agents, etc. can be mentioned, but of course it is not limited to these. The blending amount of the fermented product of *Caulerpa lentillifera* in each agent may be appropriately adjusted according to the desired effect, but the range of 0.0001 to 10% by mass, preferably 0.001 to 1% by mass, in terms of evaporation residue is optimal.

[0018] The nitrated protein degradation of the present invention includes not only the decomposition and digestion of nitrated proteins and their constituent amino acids, but also the disappearance of nitro groups by reduction, oxidation, and other chemical reactions.

Examples

[0019] Hereinafter, examples of the effect test of the fermented product of *Caulerpa lentillifera* in the present invention will be shown. Furthermore, application formulation examples and the like of external skin preparations using the fermented product of *Caulerpa lentillifera* will be described, but it is not limited to the examples described herein.

[0020] <Preparation of lactic acid bacteria inoculated into the extract> Lactic acid bacteria were used, specifically Lactobacillus plantarum. One platinum loopful of lactic acid bacteria strain was inoculated into 30 mL of MRS medium and statically cultured at 30 °C for 3 days to obtain a lactic acid bacteria culture solution. (MRS medium composition: peptone 10 g, beef extract 10 g, yeast extract 5 g, glucose 20 g, Tween 80 1 g, K 2 HPO 4 2g, sodium acetate 5 g, ammonium dihydrogen citrate 2 g, MgSO 4 ·7H 2 O 0.2 g, MnSO 4 ·nH 2 O 0.05 g, purified water 1 L)

[0021] <Preparation of extracts and fermented products> 100 mL of purified water was added to 10 g of pre-dried petals of Hydrangea macrophylla, and after extraction at room temperature, the petals were removed by filtration to obtain a Hydrangea macrophylla extract. 5 mL of the above lactic acid bacteria culture solution was added to the obtained extract and stirred well. The extract inoculated with lactic acid bacteria was statically cultured at 30 °C for 3 days and then sterilized at 105 °C for 15 minutes. After sterilization, the solid content was removed by filtration to obtain a Hydrangea macrophylla fermented product. As a comparative control, a double delight extract and a double delight fermented product were prepared from the dried petals of double delight, another variety of rose classified as a hybrid tea rose, in the same manner as the above Hydrangea macrophylla extract and fermented product.

[0022] <Measurement of nitrated protein degradation activity> Nitrotyrosine (3-nitrotyrosine), a constituent amino acid of nitrated protein, was dissolved in PBS(-) to prepare a nitrotyrosine solution with a final concentration of 0.2 mM. 250 μL of PBS(-) or the nitrotyrosine solution was dispensed into each well of a 96-well plate, and each sample was added to each well so that the final concentration of the extract and fermented product was 100 ppm and the final concentration of sodium dithionite was 1 mM (174 ppm). After incubation at 37 °C for 72 hours, the absorbance at 450 nm was measured using a plate reader. The value calculated by the following formula was used as the nitrated protein degradation degree for analysis.

[0023] [Number]

[0024] [Table 1]

[0025] Table 1 shows the results of the nitrated protein degradation effect of the fermented product of . In Comparative Example 1 where purified water as a negative control was added, the degree of nitrated protein degradation was 0.0%. In contrast, in Comparative Example 2 where sodium dithionite, which is used in various organic synthesis reactions etc. as a nitrated protein reducing agent, was added in an amount greatly exceeding an equivalent amount to nitrotyrosine, it was 53.5% under these conditions. On the other hand, in Comparative Example 3 where a double delight extract was added, Comparative Example 4 where a double delight fermented product was added, and Comparative Example 5 where a extract was added, all were far lower than Comparative Example 2 and did not exhibit an effect to the extent that substantial usefulness as a nitrated protein degrading agent was recognized. In contrast, surprisingly, only in Example 1 where the fermented product of was added, a nitrated protein degradation effect was found at a lower concentration than Comparative Example 2 and exceeding Comparative Example 2.

[0026] Next, formulation examples of an external preparation for skin containing the fermented product of of the present invention are shown, but the present invention is not limited thereto. In the following, the numerical values described after the component names indicate the blending amounts, and each example is expressed as a total of 100% by mass. All parts are parts by mass, and all % are % by mass. The fermented products of shown in the following examples are fermented products obtained by fermenting the extracts extracted with the following various solvents in the extraction step by lactic acid bacteria according to the method according to

[0021] , and the blending amounts are shown as mass % in terms of the evaporation residue. However, the extracts extracted with 100% EtOH or 50% EtOH aqueous solution were subjected to solvent substitution with purified water before being used for fermentation. In addition, in each example, the effects of the present application were confirmed.

[0027] (Example 2) Cosmetic Cream (mass%) 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 (20 E.O.) ··· 1.0 g) Fermented Product of Pittosporum tobira Fruit (Extraction Solvent: Purified Water) ··· 0.001 h) 1,3-Butylene Glycol ··· 5.0 i) Potassium Hydroxide ··· 0.3 j) Preservative · Antioxidant ··· Appropriate amount k) Purified Water ··· The balance Production Method a) to f) are heated and dissolved, and maintained at 80°C. h) to k) are heated and dissolved, maintained at 80°C, added to a) to f) and emulsified, and cooled with stirring to 40°C. Then, g) is added and stirred until uniformly dissolved.

[0028] (Example 3) Skin Lotion (mass%) a) Fermented Product of Pittosporum tobira Fruit (Extraction Solvent: Purified Water) ··· 1.0 b) Glycerin ··· 5.0 c) Polyoxyethylene Sorbitan Monolaurate (20 E.O.) ··· 1.0 d) Ethanol ··· 6.0 e) Fragrance ··· Appropriate amount f) Preservative · Antioxidant ··· Appropriate amount g) Purified Water ··· The balance Production Method a) to g) are mixed and uniformly dissolved.

[0029] (Example 4) Emulsion (mass%) a) Beeswax ··· 0.5 b) Vaseline ··· 2.0 c) Squalane ··· 8.0 d) Sorbitan Sesquioleate ··· 0.8 e) Polyoxyethylene oleyl ether (20 E.O.) ··· 1.2 f) Fermented product of Prunella vulgaris (extraction solvent: 1% aqueous citric acid solution) ··· 0.05 g) 1,3 - Butylene glycol ··· 7.0 h) Carboxyvinyl polymer ··· 0.2 i) Potassium hydroxide ··· 0.1 j) Purified water ··· The balance k) Preservative · Antioxidant ··· Appropriate amount l) Ethanol ··· 7.0 Production method a) to e) are heated and dissolved, and maintained at 80°C. g) to k) are heated and dissolved, maintained at 80°C, added to a) to e) and emulsified, and cooled with stirring to 50°C. At 50°C, f) and l) are added, and stirred and cooled to 40°C.

[0030] (Example 5) Facial wash (mass%) 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 monostearate ··· 2.0 i) Potassium hydroxide ··· 5.0 j) Water ··· The balance k) Chelating agent ··· Appropriate amount l) Fragrance ··· Appropriate amount m) Fermented product of Prunella vulgaris (extraction solvent: 0.2% aqueous citric acid solution) ··· 0.1 Production method a) to h) are heated and dissolved and maintained at 70°C. i) is dissolved in j) and then added to a) to h) for saponification. Then k) and l) are added and cooled with stirring. At 50°C, m) is added, and stirred and cooled to 40°C.

[0031] (Example 6) Essence (mass%) a) Keihakita fermented product (extraction solvent: 100% EtOH) ··· 0.01 b) Carboxyvinyl polymer ··· 0.05 c) L-Arginine ··· appropriate amount d) Glycerin ··· 5.0 e) Polyoxyethylene sorbitan monolaurate (20 E.O.) ··· 1.0 f) Ethanol ··· 6.0 g) Fragrance ··· appropriate amount h) Preservative · antioxidant ··· appropriate amount i) Purified water ··· the balance Production method Disperse b) in part of i), then add c) and adjust the pH to 6.5. Then mix a) to i) and dissolve uniformly.

[0032] (Example 7) Cosmetic gel (mass%) a) Keihakita fermented product (extraction solvent: 50% EtOH aqueous solution) ··· 0.1 b) Carboxyvinyl polymer ··· 0.5 c) Sodium hydroxide ··· 0.05 d) Methyl paraben ··· 0.1 e) Bis(ethoxydiglycol) cyclohexane-1,4-dicarboxylate ··· 0.5 f) (Eicosanedioic acid / tetradecanedioic acid) polyglyceryl-10 ··· 0.5 g) PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerin ··· 0.5 h) Polyoxyethylene (60 E.O.) hydrogenated castor oil ··· 0.1 i) Fragrance ··· appropriate amount j) Preservative · antioxidant ··· appropriate amount k) Purified water ··· the balance Production method Disperse b) in part of k), then add c). Then mix a) to k) and dissolve uniformly.

[0033] (Example 8) Lipstick (mass%) a) Aspergillus oryzae fermented product (extraction solvent: 0.5% BG aqueous solution) ··· 0.01 b) Candelilla wax ··· 9.0 c) Paraffin wax ··· 8.0 d) Beeswax ··· 5.0 e) Carnauba wax ··· 5.0 f) Cochineal ··· 11.0 g) Castor oil ··· remainder h) Cetyl 2-ethylhexanoate ··· 0.5 i) Isopropyl myristate ··· 10.0 j) Titanium dioxide ··· 5.0 k) Red No. 201 ··· 0.6 l) Red No. 202 ··· 1.0 m) Red No. 223 ··· 0.2 n) Perfume ··· appropriate amount o) Preservative · antioxidant ··· appropriate amount Manufacturing method Disperse j) to m) in a part of g) and process with a roller. Then mix a) to o), heat and melt, and disperse uniformly. After dispersion, pour into a mold and rapidly cool to make it stick-shaped.

[0034] (Example 9) Powder foundation (mass%) a) Aspergillus oryzae fermented product (extraction solvent: 5% BG aqueous solution) ··· 0.05 b) Squalane ··· 10.0 c) Sorbitan sesquioleate ··· 3.5 d) Preservative · antioxidant ··· appropriate amount e) Titanium oxide ··· 13.0 f) Sericite ··· 25.0 g) Talc ··· remainder h) Cinnabar ··· 0.8 i) Yellow iron oxide ··· 2.5 j) Black iron oxide ··· 0.1 Manufacturing method Mix e) to j), grind with a grinder. Then add a) to d) which are mixed and dissolved, stir and mix, grind again, and press into a gold dish container.

[0035] (Example 10) Bath agent (mass%) a) Fermented product of Hibiscus sabdariffa flower (extraction solvent: 2% glucose aqueous solution) ··· 0.5 b) Bis(ethoxydiglycol) cyclohexane-1,4-dicarboxylate ··· 0.2 c) (Eicosanedioic acid / Tetradecanedioic acid) polyglyceryl-10 ··· 0.2 d) PEG / PPG / Polybutylene glycol-8 / 5 / 3 glycerin ··· 0.2 e) Sodium hydrogen carbonate ··· 50.0 f) Sodium sulfate ··· remainder g) Perfume ··· appropriate amount h) Preservative · Antioxidant ··· appropriate amount Production method a) to h) are uniformly mixed.

Industrial applicability

[0036] As described in detail above, it was confirmed that the fermented product obtained by fermenting the petal extract of Hibiscus sabdariffa with lactic acid bacteria has a remarkable effect of promoting the decomposition of nitrated proteins. In the skin, nitrated proteins are known to be the causative substances of skin yellowing. Therefore, by applying the external preparation for skin of the present invention to the skin with prominent skin yellowing, improvement of various symptoms caused by protein nitration is expected.

Claims

1. A nitrated protein degrading agent containing a fermented product obtained by fermenting a petal extract of Keihakikami (Ministry of Agriculture, Forestry and Fisheries Variety Registration No. 10597) with lactic acid bacteria.

2. A topical skin preparation for improving skin yellowing caused by nitration, containing the fermented product according to Claim 1.

3. The topical skin preparation according to Claim 2, wherein the lactic acid bacteria is Lactobacillus plantarum.

4. Use of the fermented product according to Claim 1 or the topical preparation according to Claim 2 for nitrated protein degradation (however, excluding use for treating humans).

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