JP4945692B2 - Cell-utilized skin external preparation composition and method for producing the same - Google Patents

Abstract

A fermented product composition and a cosmetic composition comprising a fermented product, which is obtained by fermenting peptides originating in rice bran and beans by using Bacillus subtilis, and at least one member selected from peptides and vitamin A analogs. A production method comprising a mixing step wherein a fermented product, which is obtained by fermenting peptides originating in rice bran and beans by using Bacillus subtilis, is mixed with at least one member selected from peptides and vitamin A analogs.<I/>

Description

  The present invention relates to a cell-utilizing skin external preparation composition and a method for producing the same. More specifically, the present invention relates to a cell-utilizing skin external preparation composition that is highly safe and excellent in collagen synthesis and the like, and a method for producing the same.

  Conventionally, cosmetics such as a skin external preparation containing a fermented product obtained by fermenting rice bran and the like are known. For example, Patent Document 1 discloses a cosmetic composition containing a fermented rice cake and a cosmetic using the same. Patent Document 1 describes that this cosmetic product is excellent in moisture retention, has little irritation when applied to the skin, is less likely to cause inflammation, and is comfortable. Patent Document 2 discloses a method for producing a skin external preparation raw material obtained by fermenting rice bran and soybean peptide with Bacillus subtilis and then purifying it. Patent Document 2 describes that this skin external preparation raw material has high safety and can enhance skin activity.

JP 2004-262829 A JP-A-8-104647

  There are many effects by mix | blending the fermented material of patent document 1 and 2 with cosmetics, such as a skin external preparation. For example, cosmetics such as an external preparation for skin containing this fermented product can suppress wrinkles, stains, sagging, and the like, and can prevent dullness, redness, rough skin, and the like. Moreover, in cosmetics such as an external preparation for skin containing this fermented product, effects such as wound healing and improvement of symptoms such as burns can be obtained.

An object of the present invention is to provide a cell-utilizing skin external preparation composition that is highly safe and excellent in collagen synthesis action and the like, and a method for producing the same.

The present invention is as follows.
[1] A mass ratio of a fermented product obtained by fermenting rice bran and legume-derived peptides after fermentation with Bacillus subtilis, and a peptide obtained by hydrolyzing soy protein and a peptide that is at least one of a silk peptide and a mass ratio of 9 : A cell-applied skin external preparation composition obtained by mixing at a ratio of 1-1: 9.
[2] A mass ratio of a fermented product obtained by fermenting rice bran and legume-derived peptides after fermentation with Bacillus subtilis, and a peptide obtained by hydrolyzing soy protein and a peptide that is at least one of a silk peptide and a mass ratio of 9 The manufacturing method of the cell utilization skin external preparation composition characterized by providing the mixing process mixed in the ratio of: 1-1: 9.

The cell utilization skin external preparation composition of the present invention uses a natural product as a raw material. Therefore, the cell utilization skin external preparation composition of this invention has high safety | security, and has the outstanding collagen synthetic | combination effect | action, cell proliferation effect | action, etc.
According to the manufacturing method of the cell utilization skin external preparation composition of this invention, it can manufacture by fermenting using the Bacillus subtilis easily available. Therefore, the cell utilization skin external preparation composition of this invention which has said outstanding effect | action can be manufactured safely and easily with small manufacturing equipment.

The present invention will be described in detail below.
The cell utilization skin external preparation composition of the present invention is a fermented product obtained by fermenting rice bran and legume-derived peptides with Bacillus subtilis and then refining them, and a peptide obtained by hydrolyzing soy protein and a silk peptide. It is obtained by mixing the peptide as a seed in a mass ratio of 9: 1 to 1: 9. The cell utilization skin external preparation composition of this invention can contain vitamin A further.

  Examples of the “rice bran” include rice bran, defatted rice bran, rice germ, and defatted rice germ. These may be used alone or in combination of two or more. The rice bran is not limited to commercially available rice bran, and various types of rice bran can be used.

  As long as the “bean-derived peptide” is a peptide derived from a peptide contained in a bean or a protein contained in a bean, its specific type and structure are not particularly limited. The “bean-derived peptides” are not limited to commercially available bean-derived peptides, and various kinds of bean-derived peptides can be used. Examples of the beans-derived peptide include soybean peptide, red bean peptide, pea peptide, and broad bean peptide. As the bean-derived peptide, soybean peptide is preferable. The legume-derived peptides may be used alone or in combination of two or more.

  There is no limitation in particular in the method of obtaining the said beans origin peptide. For example, the above-mentioned bean-derived peptide is obtained by using beans (for example, one or more of soybeans, red beans, peas, broad beans, etc.) as a solvent (water, hot water, alcohol (ethanol etc.) organic solvent or water-organic solvent mixture. It can be obtained by extraction with a solvent. In this process, fractionation and purification can be performed as necessary. Furthermore, the obtained legume-derived peptide or legume protein can be further fragmented by subjecting it to a hydrolysis treatment with protease, acid or alkali. Further, the bean-derived peptide may be a peptide artificially synthesized by chemically bonding amino acids to each other or by a known genetic engineering technique.

  There is no particular limitation on the form of the legume-derived peptide. The legume-derived peptide can be a solid (for example, a powdery product or a granular product) solidified by drying or the like after the extraction. Moreover, the said extract itself can be used as said bean origin peptide. That is, in the present invention, an extract containing a bean-derived peptide (bean extract, particularly soybean extract) can be used as the bean-derived peptide. For example, in the present invention, beans (for example, one or more of soybeans, red beans, peas, broad beans, etc.) are used as solvents (water, hot water, organic solvents such as alcohol (ethanol etc.) or water as the beans-derived peptides. An extract obtained by extraction with (organic solvent mixed solvent) can be used. In the present invention, an extract (soybean extract) containing soybean peptide is particularly preferably used.

  There is no limitation in particular about the extraction method and extraction conditions for obtaining the extract containing the said beans origin peptide. For example, beans that are extraction raw materials may be unground or pulverized. Further, as long as the quality of the extract can be maintained, pretreatment such as impurity removal may be performed. Examples of the extraction solvent include water and hot water, alcohols such as methanol and ethanol, esters such as ethyl acetate, organic solvents such as n-hexane, and mixed solvents of these organic solvents and water or hot water. Can be used. As the extraction solvent, water or hot water, alcohol (such as methanol and ethanol), and a mixed solvent of water or hot water and alcohol are preferable. The temperature of the hot water is usually 40 to 100 ° C, preferably 50 to 80 ° C, more preferably 50 to 70 ° C. In addition, the pH of the extraction solvent during extraction is usually 3 to 7, preferably 4 to 6, and more preferably 4 to 5. By setting the pH within the above range, it is preferable because the stability of various components contained in the extraction raw material can be maintained. The extraction temperature is not particularly limited, but normal temperature or heat extraction is preferable. In the case of heat extraction, the heating temperature is usually 40 to 100 ° C, preferably 50 to 80 ° C, more preferably 50 to 70 ° C. By setting the heating temperature to such a range, extraction can be efficiently performed, which is preferable.

  There is no particular limitation on the ratio of the rice bran and the beans-derived peptide. Usually, when the said beans origin peptide is 100 mass parts, the ratio of the said rice bran is 1-20 mass parts, Preferably it is 5-20 mass parts, More preferably, it is 8-18 mass parts. If the rice bran is within the above range, a fermented product having a sufficient collagen synthesis action and the like can be obtained.

  The above-mentioned “Bacillus subtilis” [Bacillus subtilis] is a typical type of aerobic spore-forming bacteria. The type of Bacillus subtilis is not particularly limited as long as safety is guaranteed. As the Bacillus subtilis, for example, Bacillus natto [Bacillus natto] or the like can be used. As the Bacillus subtilis, Bacillus natto which is easily available and inexpensive is preferable. In addition, as said Bacillus subtilis, the common Bacillus subtilis marketed can be used. In addition, as long as a fermented product having a collagen synthesizing action or the like is obtained, the Bacillus subtilis may be produced naturally or by an artificial mutation means such as a chemical substance such as nitrosoguanidine, X-rays, ultraviolet rays, etc. Mutant strains with altered morphological properties can also be used.

The “fermented product” is obtained by purifying the rice bran and the legume-derived peptide after fermentation with Bacillus subtilis. The fermented product is usually obtained by ingesting Bacillus subtilis into a medium containing the rice bran and the bean-derived peptide and fermenting and culturing under appropriate conditions. The fermentation culture conditions for obtaining the above “fermented product” are not particularly limited. Fermentation culture is usually performed by aeration and agitation. The medium is not particularly limited as long as the Bacillus subtilis can grow. The medium is usually a liquid medium, but may be a solid medium. Moreover, pH (especially at the time of fermentation) of the said culture medium is 5.5-8.5 normally, Preferably it is 6.0-8.0, More preferably, it is 6.5-7.5. If this pH is within the above range, it is preferable because the stability of various components contained in the extraction raw material can be maintained. Further, the culture temperature is not particularly limited as long as fermentation is performed. The culture temperature is usually 15 to 50 ° C, preferably 20 to 45 ° C, more preferably 30 to 45 ° C, more preferably 35 to 43 ° C , and particularly preferably 35 to 37 ° C. In addition, various nutrients such as sugar and acid and alkali for pH adjustment may be added to the medium. Furthermore, the fermentation may be mixed fermentation or continuous fermentation.

There is no limitation in particular in the form of the said fermented material. The said fermented material is refine | purified and the liquid which filtered the fermentation liquid obtained by fermentation culture may be sufficient. In addition, the fermented product is subjected to sterilization treatment or pH adjustment, if necessary, or post-treatment such as deodorization / decolorization using an ion exchange resin, an activated carbon column or a dialysis membrane. Fermented liquid may be used. Further, the fermented product may be a concentrated solution or a paste-like product obtained by concentrating the fermented solution. In addition, the fermented product may be a solid product and a powdered product obtained by removing the solvent from the fermented solution by a known method such as freeze drying. Further, the fermented product is a solution or dispersion in which the fermented liquid or the solid and powder are added to water or an organic solvent such as ethanol, propylene glycol and 1,3-butylene glycol, or a mixed solvent thereof. Also good.

  The “peptide” is a polypeptide in which amino acids are bound by peptide bonds. The amino acid is usually an L-amino acid, but may be a D-amino acid or a mixture of both. That is, the amino acid constituting the peptide may be any of L-amino acids, D-amino acids, and a mixture of both.

There is no particular limitation on the structure of the peptide. The peptide structure may contain not only a linear structure but also a disulfide bond. Examples of the peptides include peptides obtained by hydrolyzing soy protein (proteins contained in soybean) and silk peptides such as silk fibroin (peptides obtained by hydrolyzing peptides contained in silk and proteins contained in silk) ) Is used. Since use of these peptides, it is possible to obtain an excellent collagen synthesis activity and cell proliferation effect.

  The method for obtaining the peptide is not particularly limited. The peptide can be usually obtained by hydrolyzing various proteins with protease, acid, alkali or the like. Of course, the specific hydrolysis method and conditions are not particularly limited.

  The number of amino acids and molecular weight of the peptide are not particularly limited. The lower limit of the number of amino acids of the peptide can be set to 2, 3, or 4, for example. Moreover, the upper limit of the amino acid number of the said peptide can be set to 100, 80, 60, or 40, for example. The lower limit of the molecular weight of the peptide can be, for example, 50, 100, or 200. Moreover, as an upper limit of the molecular weight of the said peptide, it can be set as 300000, 200000, 100,000, 50000, or 30000, for example. In particular, for the purpose of cell proliferation, it is preferable to use a high molecular weight peptide, for example, a peptide having a molecular weight of 40,000 or more as the peptide. The molecular weight of the peptide can be measured by various known methods. The molecular weight of the peptide can be measured, for example, by electrophoresis using SDS-PAGE and gel filtration chromatography using HPLC and a column. The number of amino acids and the molecular weight of the peptide can be appropriately adjusted by hydrolyzing the protein or peptide with a protease, acid, alkali, or the like.

  The peptide is preferably (1) a peptide having a molecular weight of 200 to 200,000, (2) a peptide having 4 to 40 amino acids and a molecular weight of 220 to 8000, and (3) having 2 to 3 amino acids. And a mixture of peptides having different molecular weights of 100 or less, 100 to 250, and 250 to 500. Further, as these peptides (1) to (3), silk fibroin is more preferable. By containing these peptides and the fermented product, a more excellent collagen synthesizing action and cell proliferating action can be obtained than when only the fermented product is contained.

Moreover, when the said peptide is what hydrolyzed soybean protein, the peptide whose number of amino acids is 2-6 and whose molecular weight is 100-1200 is preferable. By this soy protein to contain a hydrolyzed peptide and fermented product, than when containing only fermented product can Rukoto obtain better collagen synthesis activity and cell proliferation effect.

  The said peptide may be used individually by 1 type, and may use 2 or more types. For example, as said peptide, only 1 type in said various peptides may be used, and 2 or more types may be used together. Moreover, the said peptide may contain 2 or more types of peptides from which an amino acid number and / or molecular weight differ.

The “vitamins A” are compounds having isoprenoid polyene alcohol as a basic skeleton and derivatives of the compounds. Specific examples of the vitamin A include, for example, vitamin A1 (retinol), vitamin A2, vitamin A3, and 3,4-dihydroretinol, and aldehydes (retinal and the like), carboxylic acids (retinoic acid) and the like in which these are oxidized. Can be mentioned. In addition, the “vitamin A” includes pharmaceutically acceptable salts and pharmaceutically acceptable derivatives. Examples of the derivatives include carboxylic acid esters such as the above retinol. More specifically, examples of the derivative include C1-C30 esters such as retinol acetate and retinol palmitate. In the present invention, the vitamin A may be used alone or in combination of two or more.

In the present invention, the form of vitamin A is not particularly limited. The vitamin A itself may be contained in the cell-utilizing skin external preparation composition of the present invention, or may be contained in combination with other substances. For example, the vitamin A can be contained in the cell-utilizing skin external preparation composition of the present invention as an inclusion compound that is included in a host compound such as cyclodextrin. Moreover, the said vitamin A can be contained in the cell utilization skin external preparation composition of this invention as a composition or mixture containing the said vitamin A. For example, the vitamin A can be contained in the cell-utilizing skin external preparation composition of the present invention as a composition containing the vitamin A such as liver oil. Furthermore, the said vitamin A can be contained in the cell utilization skin external preparation composition of this invention as a solution or dispersion liquid in which the said vitamin A melt | dissolved or disperse | distributed. For example, the vitamin A can be dissolved in animal oil or vegetable oil, and the solution can be contained in the cell-utilizing skin external preparation composition of the present invention.

When the cell utilization skin external preparation composition of this invention contains vitamin A, there is no limitation in particular in the ratio of the said fermented material, the said peptide, and the said vitamin A. When the total of the fermented product, the peptide and the vitamin A is 100% by mass, the ratio of the fermented product is usually 0.1 to 99.9% by mass, preferably 10 to 80% by mass, Preferably it is 40-60 mass%. In this invention, a collagen synthetic | combination effect | action etc. improve by containing the said fermented material, the said peptide, and 1 type, or 2 or more types of the said vitamin A. In the present invention, when there is no significant difference in the content of one or more of the fermented product, the peptide and the vitamin A, that is, when the mass ratio of the fermented product is 40 to 60% by mass. Is preferable because collagen synthesis and the like are further improved.

The cell utilization skin external preparation composition of this invention can be used for various uses. There is no particular limitation on this application. The cell utilization skin external preparation composition of this invention can be used for cosmetics, cosmetic raw materials, bath preparations, etc., such as skin external preparations, for example.

The cell utilization skin external preparation composition of this invention can be mix | blended with cosmetics. When blended in cosmetics , the blending amount of the cell utilization skin external preparation composition of the present invention is not particularly limited. The blending amount can be blended in an appropriate amount depending on the type of cosmetic. The compounding quantity of the cell utilization skin external preparation composition of this invention can be 0.001-100 mass% when a cosmetics is 100 mass%, and can be 0.001-50 mass% especially. If content of the cell utilization skin external preparation composition of this invention is in the said range, it can be set as the cosmetics which have the outstanding collagen synthetic effect etc.

There is no particular limitation on the type of cosmetics that can contain the cell- utilized skin external preparation composition of the present invention. Examples of the cosmetics include freeze-drying, packs, pack sheets, peeling, powders, skin lotions, white powders, solid white powders, lipsticks, solid blushers, foundation creams, emulsions, lotions, body lotions, cleansing lotions, facial creams, skin care creams. , Hair rinse, hair liquid, eye shadow and eyebrows. In addition, depending on the type of these cosmetics, if necessary, other ingredients such as plant extracts, vitamins, vitamin-like substances, minerals, lower alcohols, polyhydric alcohols, fats and oils, surfactants, Antioxidants, ultraviolet absorbers, thickeners, pigments, preservatives, fragrances and the like can also be added.

Examples of cosmetics containing the cell-utilizing skin external preparation composition of the present invention include skin external preparations. The cell-applied skin external preparation composition of the present invention has an excellent collagen synthesis action and the like. Therefore, the cell utilization skin external preparation composition of this invention is especially useful for the cosmetics used by apply | coating to skin. A skin external preparation containing the cell rejuvenating skin external preparation composition of the present invention as an active ingredient is prepared using the cell rejuvenating skin external preparation composition of the present invention and other compounding agents used for the skin external preparation. can do. Examples of the other compounding agents include liquid oil (such as squalane and jojoba oil), solid oil (such as beeswax and cetyl alcohol), various activators, and humectants (such as glycerin and 1,3-butylene glycol). It is done. There is no particular limitation on the dosage form of the external preparation for skin. This external preparation for skin can be made into various dosage forms according to the purpose, such as lotion, cream and emulsion. In this external preparation for skin, the blending amount of the cell-use skin external preparation composition of the present invention is not particularly limited. The blending amount of the fermented product composition of the present invention can be 0.001 to 100% by mass, particularly 0.001 to 50% by mass, when the skin external preparation is 100% by mass. If content of the cell utilization skin external preparation composition of this invention is in the said range, it can be set as the skin external preparation which has the outstanding collagen synthetic effect etc.

In addition, the formulation which mix | blended the cell utilization skin external preparation composition of this invention can also be used as a cosmetic raw material, a bath agent, etc.

The method for producing a cell-utilizing skin external preparation composition of the present invention comprises a fermented product obtained by fermenting rice bran and a bean-derived peptide with Bacillus subtilis and then purifying it, a peptide obtained by hydrolyzing soy protein, and a silk peptide. A mixing step of mixing at least one peptide at a mass ratio of 9: 1 to 1: 9 .

The contents described in the fermented product composition of the present invention are applied to the “rice bran”, the “bean-derived peptide”, the “Bacillus subtilis”, and the “peptide”.

The “fermented product” is usually obtained by a fermentation process in which Bacillus subtilis is inoculated and fermented in a medium containing rice bran and legume-derived peptides , and a purification process described later . The above-mentioned “medium” in the “fermentation step” may be any medium that contains rice bran and a bean-derived peptide and is capable of fermenting culture by Bacillus subtilis growth. There is no limitation in particular in the kind and composition of the said culture medium. The medium is usually a liquid medium, but may be a solid medium. Moreover, pH (especially at the time of fermentation) of the said culture medium is 5.5-8.5 normally, Preferably it is 6.0-8.0, More preferably, it is 6.5-7.5. Moreover, in order to adjust pH, nutrient conditions, etc. to a preferable range, saccharides, such as glucose, enzymes, such as protease, water, such as purified water, etc. can be added to the said culture medium.

The culture temperature during the fermentation is not particularly limited as long as fermentation is performed. The culture temperature is usually 15 to 50 ° C, preferably 20 to 45 ° C, more preferably 30 to 45 ° C, more preferably 35 to 43 ° C , and particularly preferably 35 to 37 ° C. Fermentation culture is usually performed by aeration and agitation.

The fermented material obtained by the fermentation process is purified and then mixed with the peptide. The method for this purification treatment is not particularly limited. For example, the fermented product can be purified by squeezing and filtering the fermentation broth. Moreover, the said fermented liquor can also be refine | purified by squeezing and filtering a fermentation liquid, and then performing the deodorizing process by activated carbon etc., a decoloring process, and / or the removal process of a deposit, and filtering again after that.
The form of the fermented product obtained by the fermentation process and the purification process is not particularly limited. The content described in the fermented product composition of the present invention is applied to the fermented product.

In the “mixing step”, the method for mixing the fermented material with the peptide and at least the peptide of vitamin A is not particularly limited. A fermented material and a peptide can be mixed using appropriate apparatuses, such as mixers, such as a homomixer, according to each form and each mass ratio. Moreover, the temperature at the time of mixing is not specifically limited, It is preferable to set with each kind etc. of fermented material and a peptide. The fermented product and the peptide can be mixed while cooling as necessary in the range of 5 to 20 ° C., and can be mixed while heating as necessary in the range of 35 to 85 ° C.

  The manufacturing method of the present invention may further include other steps.

Hereinafter, the present invention will be specifically described by way of examples.
[1] Production of fermented product composition 3 kg of rice bran, 0.4 kg of soybean peptide, 0.5 kg of glucose, 0.01 kg of alkaline protease and 50 kg of purified water were mixed to prepare a medium. Thereafter, the pH of the medium was adjusted to 6.0 to 8.0, then inoculated with Bacillus natto, and cultured at 37 ° C. for 38 hours for fermentation. Thereafter, the fermentation broth was squeezed and filtered, and then decolorized and deodorized with activated carbon. Then, the fermented material was manufactured by filtering and refine | purifying a fermentation liquid again.

[2] Evaluation of Fermented Product Composition Containing Silk Fibroin The fermented product produced in [1] above, vitamin C (manufactured by Wako Pure Chemical Industries, Ltd.), and silk fibroin (manufactured by Audleman, trade name “Fibroin Sheet”) Each was dissolved in a buffer solution (PBS) so that the solid concentration was 2% (mass / volume). The solution was then filtered through a 0.22 μm filter. Then, the sample solution of Experimental Examples 2-15 was prepared by diluting and mixing this solution so that each component might become the density | concentration of Table 1 and Table 2. FIG. In Experimental Example 1, the buffer solution (PBS) was used as a sample solution.

  Each of two 96-well plates (referred to as “P1” and “P2” in the table) was distributed with 5000 human skin fibroblasts per well. Subsequently, the cells were cultured in a MEM medium supplemented with 5% FBS for 3 days. The sample addition medium was prepared by adding the samples of Experimental Examples 1 to 15 to the MEM medium containing 5% FBS. The concentration of the sample solutions of Experimental Examples 1 to 15 in the sample-added medium is 1% (mass / volume, excluding the mass of PBS) (therefore, the final concentration of each component in the fermentation composition in the medium). The concentration is 1/100 of the concentration in the sample solution.) Then, the 5% FBS-added MEM medium in the well was replaced with the sample-added medium, and further cultured for 3 days.

  Next, using a “collagen stain kit” (manufactured by Collagen Technology Research Group), the amount of collagen synthesis (μg) was measured according to the manual attached to the kit. Tables 1 and 2 show the average values of the amount of collagen synthesis in Plate 1 (P1) and Plate 2 (P2). Moreover, the collagen synthesis amount of Experimental Example 1 was set to 1, and the relative value of the collagen synthesis amount of Experimental Examples 2 to 8 was obtained. Similarly, the collagen synthesis amount of Experimental Example 9 was set to 1, and the relative value of the collagen synthesis amount of Experimental Examples 10 to 15 was obtained. The results are also shown in Tables 1 and 2. In Tables 1 and 2, “VC” means vitamin C, and “SF” means silk fibroin.

  From Tables 1 and 2, in Experimental Examples 5 to 8 and 13 to 15 in which silk fibroin and a fermented product are used in combination, Experimental Examples 3 and 11 using only the fermented product and an experiment using only silk fibroin It can be seen that the collagen synthesizing action is superior to Examples 4 and 12.

[3] Evaluation of fermented product composition containing peptides derived from other silk raw materials Fermented product manufactured in [1] above, vitamin C (manufactured by Wako Pure Chemical Industries, Ltd.), silk fibroin, and the following silk peptide (a) and (B) was dissolved in a buffer solution (PBS) so that the solid concentration was 2% (mass / volume). The solution was then filtered through a 0.22 μm filter. Then, the sample solution of Experimental Examples 17-19, 24-28, and 33-35 was prepared by diluting and mixing this solution so that each component might become the density | concentration of Table 3-Table 5. Moreover, the sample solutions of Experimental Examples 20 to 22, 29 to 31, and 36 were prepared by mixing the sample solutions of Experimental Examples 17 to 19, 24 to 28, and 33 to 35 in equal amounts by volume. In Experimental Examples 16, 23 and 32, the buffer solution (PBS) was used as the sample solution.

Vitamin C and silk fibroin are the same as those used in [2] above. Moreover, the following were used as silk peptides (a) and (b). Silk peptides (a) and (b) were both liquids, and were diluted with a buffer solution so that the final concentration as a solid content concentration was 1% (mass / volume).
Silk peptide (a); manufactured by Cosmo Foods, trade name “Silk Peptide M-500”
Silk peptide (b); manufactured by Cosmo Foods, trade name “Silk Peptide M-500 # 60”

  By the same method as [2] above, the collagen synthesis amount (μg) of Experimental Examples 16 to 36 was measured. Tables 3 to 5 show average values of the amount of collagen synthesis in Plate 1 (P1) and Plate 2 (P2). Further, assuming that each collagen synthesis amount of Experimental Examples 16, 23 and 32 was 1, the relative value of the collagen synthesis amount of other experimental examples was obtained. The results are also shown in Tables 3-5. In Tables 3 to 5, “VC” means vitamin C, “SF” means silk fibroin, and “SP” means silk peptide.

  According to the results of Tables 3 to 5, in Experimental Examples 22, 29 and 36 in which silk fibroin and fermented material are used in combination, Experimental Examples 18, 25 and 34 using only fermented material, and only silk fibroin are used. Compared with the experimental examples 19, 26 and 35 used, the collagen synthesizing action is more excellent as described above. Furthermore, it turns out that Experimental example 30 and 31 which uses a silk peptide and fermented material together also has the outstanding collagen synthetic | combination effect | action similarly to the case of silk fibroin. From Experimental Examples 17, 24 and 33, it can be seen that vitamin C also has a collagen synthesizing action. However, it can be seen that in Example 20 in which fermented material and vitamin C are used in combination and in Example 21 in which vitamin C and silk fibroin are used in combination, the synergistic effect due to the combination is not observed or very slight. Therefore, it turns out that the synergistic effect by combined use of a fermented material and a peptide is large.

[4] Evaluation of fermented product composition containing soybean peptide Fermented product produced in [1] above, vitamin C (manufactured by Wako Pure Chemical Industries, Ltd.), silk fibroin, and the following soybean peptides (a) to (c) Each was dissolved in a buffer solution (PBS) so that the solid concentration was 2% (mass / volume). The solution was then filtered through a 0.22 μm filter. Then, the sample solution of Experimental Examples 38-43 and 49-54 was prepared by diluting and mixing this solution so that each component might become the density | concentration of Table 6 and Table 7. Moreover, the sample solutions of Experimental Examples 44 to 47 and 55 to 56 were prepared by mixing equal amounts of the sample solutions of Experimental Examples 38 to 43 and 49 to 54 by volume. In Experimental Examples 37 and 48, the buffer solution (PBS) was used as the sample solution.

Vitamin C and silk fibroin are the same as those used in [2] above. Moreover, the following were used as soybean peptides (a) to (c). The soybean peptides (a), (b) and (c) were all liquids, and were diluted with a buffer solution so that the final concentration as a solid content concentration was 1% (mass / volume).
Soy peptide (a): Fuji Oil Co., Ltd., trade name “High New SMP”
Soy peptide (b): Fuji Oil Co., Ltd., trade name “High New R”
Soybean peptide (c): Fuji Oil Co., Ltd., trade name “High New DC5”

  By the same method as [2] above, the collagen synthesis amount (μg) of Experimental Examples 37 to 47 was measured. Table 6 shows the average value of the amount of collagen synthesis in plate 1 (P1) and plate 2 (P2). Further, assuming that the collagen synthesis amount of Experimental Example 37 was 1, the relative value of the collagen synthesis amount of the other Experimental Examples was determined. The results are also shown in Table 6. In Table 6, “VC” means vitamin C, “SF” means silk fibroin, and “SBP (a) to (c)” means soybean peptides (a) to (c).

  Each of two 96-well plates was spread with 2000 human skin fibroblasts per well. Subsequently, the cells were cultured in a MEM medium supplemented with 5% FBS for 3 days. Thereafter, the medium was replaced with a serum-free MEM medium and further cultured for 1 day. Samples of Experimental Examples 48 to 56 were added to a MEM medium supplemented with 0.5% serum to prepare a sample-added medium. The concentration of the sample solutions of Experimental Examples 48 to 56 in the sample-added medium is 1% (mass / volume, excluding the mass of PBS) (therefore, the concentration of each component in the fermentation composition in the medium). The final concentration is 1/100 of the concentration in the sample solution.) Then, the serum-free MEM medium in the well was replaced with the sample-added medium, and further cultured for 4 days.

  Then, based on Dojindo “Cell Counting Kit 8”, the absorbance was measured by measuring the water-soluble formazan produced by reduction by a dehydrogenase in living cells at a wavelength of 450 nm using WST-8 tetrazolium salt as a chromogenic substrate. It was measured (the number of living cells is proportional to the absorbance value). Further, assuming that the number of living cells in Experimental Example 48 was 1, the relative value of the amount of living cells in other Experimental Examples was calculated, and the cell proliferation action was evaluated. The results are also shown in Table 7. In Table 7, “VC” means vitamin C, “SF” means silk fibroin, and “SBP (a) to (c)” means soybean peptides (a) to (c). Moreover, the average value of the term of cell growth rate in Table 7 is the average value of the absorbance values.

  From Table 6, Experimental Example 44 in which silk fibroin and fermented material are used in combination is superior in collagen synthesis to Experimental Example 39 using only fermented material and Experimental Example 40 using only silk fibroin. It turns out that it has an effect | action. Furthermore, it turns out that Experimental Example 45-47 which uses fermented material and a soybean peptide together also has the collagen synthesis effect | action excellent in the same way as the case of silk fibroin.

  From Table 7, it can be seen that Experimental Example 56 using both the fermented product and soybean peptide (c) has a more improved cell proliferation effect than Experimental Example 50 using only the fermented product. Furthermore, it can be seen that Experimental Example 55 using both fermented material and silk fibroin also improves the cell proliferation action.

[5] Evaluation of fermented product composition containing vitamins A fermented product produced in the above [1], vitamin C (manufactured by Wako Pure Chemical Industries, Ltd.), and cyclodextrin inclusion retinol (trade name “CAVAMAX W8 / Retinol Complex” Cyclochem Co., Ltd.) was dissolved in a buffer solution (PBS) so that the solid concentration was 2% (mass / volume). The solution was then filtered through a 0.22 μm filter. Then, the sample solution of Experimental Examples 58-66 was prepared by diluting and mixing this solution so that each component might become the density | concentration of Table 8. In Experimental Example 57, the buffer solution (PBS) was used as the sample solution.

  The collagen synthesis amount (μg) and cell proliferation rate of Experimental Examples 57 to 66 were measured by the same method as described in [2] and [4] above. The average value is shown in Table 8. Further, assuming that the amount of collagen synthesis and the amount of living cells in Experimental Example 57 was 1, relative values of the amount of collagen synthesis and the amount of living cells in other Experimental Examples were obtained. The results are also shown in Table 8. In Table 8, “VC” means vitamin C, and “CDR” means cyclodextrin inclusion retinol. Moreover, the average value of the term of cell growth rate in Table 8 is the average value of absorbance values.

  From Table 8, Experimental Examples 64 to 66 using both retinol and fermented product are superior to those of Experimental Example 59 using only fermented product and Experimental Examples 60 to 63 using only retinol. It can be seen that it has a synthetic action and a cell proliferation action.

[6] Evaluation of fermented product composition containing peptide and vitamin A The fermented product produced in the above [1], the silk fibroin, the soy peptide (a), and the cyclodextrin inclusion retinol are each in a solid content concentration. To 2% (mass / volume) in a buffer solution (PBS). The solution was then filtered through a 0.22 μm filter. Then, the sample solutions of Experimental Examples 68, 69, 71, and 72 were prepared by diluting and mixing the solutions so that each component had a concentration described in Table 9 and Table 10. In Experimental Examples 67 and 70, the buffer solution (PBS) was used as the sample solution.

  By the same method as in the above [2] and [4], the collagen synthesis amount (μg) and cell proliferation rate of Experimental Examples 67 to 72 were measured. The average values are shown in Tables 9 and 10. Moreover, the collagen synthesis amount and the amount of living cells in Experimental Examples 67 and 70 were taken as 1, and the relative values of the amount of collagen synthesis and the amount of living cells in other Experimental Examples were determined. The results are also shown in Table 9 and Table 10. In Tables 9 and 10, “SF” means silk fibroin, “SBP” means soybean peptide, and “CDR” means cyclodextrin inclusion retinol. Moreover, the average value of the term of cell growth rate in Table 9 and Table 10 is the average value of the absorbance value.

  From Table 10, it can be seen that Experimental Examples 69 and 72 in which retinol and a peptide are used in combination with a fermented product have excellent collagen synthesis action and cell proliferation action.

  In addition, this invention is not limited to the said specific Example. The present invention can be variously modified within the scope of the present invention depending on the purpose and application.

  The fermented product composition of the present invention is excellent in safety and has a collagen synthesis action and the like. The fermented product composition of the present invention can be used by blending it with various cosmetics such as external preparations for skin and various foods such as health foods and functional foods. That is, the present invention can be used in the fields of cosmetics and foods and drinks.

Claims (2)

A fermented product obtained by fermenting rice bran and legume-derived peptides after fermentation with Bacillus subtilis and a peptide obtained by hydrolyzing soy protein and a peptide that is at least one of a silk peptide and a mass ratio of 9: 1 to 1 A cell-utilizing skin external preparation composition characterized by being obtained by mixing at a ratio of 1: 9. A fermented product obtained by fermenting rice bran and legume-derived peptides after fermentation with Bacillus subtilis and a peptide obtained by hydrolyzing soy protein and a peptide that is at least one of a silk peptide and a mass ratio of 9: 1 to 1 The manufacturing method of the cell utilization skin external preparation composition characterized by including the mixing process mixed in the ratio of 1: 9.