JP2016113386A - Whitening agent and whitening cosmetic - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a whitening agent and whitening cosmetic that can be ingested daily, easily, and safely and has excellent tyrosinase inhibitory activity or melanin generation inhibitory activity.SOLUTION: The whitening agent contains, as an active ingredient, a lactic acid fermented material of Vitis obtained by fermenting Vitis seeds and peels with a lactic acid bacteria comprising at least Lactobacillus plantarum and Lactobacillus brevis, and/or extract thereof. And the whitening cosmetic also contains, as an active ingredient, the lactic acid fermented material of Vitis obtained by fermenting the Vitis seeds and peels with the lactic acid bacteria comprising at least the Lactobacillus plantarum and the Lactobacillus brevis, and/or extract thereof.SELECTED DRAWING: None

Description

  The present invention relates to a whitening agent and a whitening cosmetic useful for preventing and recovering sunburn and suppressing pigmentation such as spots and freckles.

  Prevention and recovery of sunburn and suppression of pigmentation such as spots and freckles are important elements of beauty, and there is a great interest in technology for suppressing the production of melanin that causes pigmentation of the skin. In addition, since tyrosinase of pigment cells is involved in melanin production, there is a great interest in a technology that inhibits the activity of tyrosinase. There is a demand for the development of an active ingredient that has no side effects, can be used easily and safely on a daily basis, and has excellent effects.

  For such a problem, for example, the following Patent Document 1 describes that a fermented product of grape skin and seed has tyrosinase inhibitory activity and melanin production inhibitory activity, and is useful for skin whitening. .

International Publication No. 2008/093670

  However, the grape skin and seed lactic acid bacteria fermented product of Patent Document 1 is a fermented product using a single lactic acid bacterium, and its tyrosinase inhibitory activity and melanin production inhibitory activity were not sufficient.

  An object of the present invention is to provide a whitening agent and a whitening cosmetic that can be easily and safely ingested on a daily basis and are excellent in tyrosinase inhibitory activity and melanin production inhibitory activity.

  As a result of various studies, the present inventor has conducted lactic acid fermentation of grape seeds and fruit skins in combination with Lactobacillus plantarum and Lactobacillus brevis, each having superior tyrosinase inhibitory activity than lactic acid fermentation alone. And it discovered that melanin production inhibitory activity was show | played and came to complete this invention.

That is, this invention provides the whitening agent and cosmetics for whitening which have the following structures.
[1] A lactic acid fermentation product obtained by fermenting grape seeds and skins with a lactic acid bacterium containing at least Lactobacillus plantarum and Lactobacillus brevis and / or an extract thereof as an active ingredient Whitening agent.
[2] The whitening agent according to [1], wherein the lactic acid fermented product of grape and / or the extract thereof has tyrosinase inhibitory activity.
[3] The whitening agent according to [1] or [2], wherein the lactic acid fermented product of grape and / or an extract thereof has melanin production inhibitory activity.
[4] A lactic acid fermentation product obtained by fermenting grape seeds and pericarp with lactic acid bacteria containing at least Lactobacillus plantarum and Lactobacillus brevis and / or an extract thereof as an active ingredient Whitening cosmetics.
[5] The whitening cosmetic according to [4], wherein the lactic acid fermented product of grape and / or the extract thereof has tyrosinase inhibitory activity.
[6] The whitening cosmetic according to [4] or [5], wherein the lactic acid fermented product of grape and / or an extract thereof has a melanin production inhibitory activity.

  The whitening agent and whitening cosmetic composition of the present invention comprises a grape lactic acid fermented product obtained by fermenting grape seeds and skins with a lactic acid bacterium containing at least Lactobacillus plantarum and Lactobacillus brevis and / or an extract thereof. Since it contains as an active ingredient, it is excellent in tyrosinase inhibitory activity and melanin production inhibitory activity. In addition, since the active ingredient is derived from natural materials, it is highly safe and can be taken easily and safely on a daily basis, thereby preventing and / or recovering from sunburn and suppressing pigmentation such as spots and freckles. be able to.

2 is a chart showing tyrosinase inhibitory activity examined in Test Example 1. 6 is a chart showing melanin production inhibitory activity examined in Test Example 2.

  The whitening agent and whitening cosmetic composition of the present invention comprises a lactic acid fermented product of grape and / or an extract thereof as an active ingredient.

  In the present invention, the grape as a raw material is not particularly limited, and may be white grape, red grape or the like. Examples of white grapes include Chardonnay, Riesling, and Muscat. As red grapes, Koshu, Kyoho, Pione, Cabernet Franc, Cabernet Sauvignon, Merlot, Pinot Noir, Pinot Meunier, Muscat Berry A, Schiller, Gamay, Examples include Grenache, Mourvedre, Sunsaw and Gloro. Among them, red grapes are preferably used because the skin of red grapes contains a relatively large amount of anthocyanins and flavonoids. The use site of the raw grapes is high in the content of polyphenols and is also an inexpensive raw material, so seeds and skins are used.

  In the present invention, a lactic acid bacterium used for lactic acid fermentation of a raw grape is a lactic acid bacterium containing at least Lactobacillus plantarum and Lactobacillus brevis. Fermentation with lactic acid bacteria containing at least Lactobacillus plantarum and Lactobacillus brevis may increase the amount of polyphenols and other components contained in the raw grapes, and increase the absorption of these components into the body. Thus, tyrosinase inhibitory activity and melanin production inhibitory activity are improved. In addition, the grape-derived component, lactic acid bacteria containing at least Lactobacillus plantarum and Lactobacillus brevis act synergistically to further improve tyrosinase inhibitory activity and melanin production inhibitory activity. Furthermore, astringency, bitterness and blue odor are reduced, and flavor is improved. As a result, it is suitably used as an active ingredient in whitening agents and whitening cosmetics that prevent and recover from sunburn and suppress pigmentation such as spots and freckles.

  As the lactic acid bacteria, other lactic acid bacteria may be added as long as they do not interfere with the growth of Lactobacillus plantarum and Lactobacillus brevis.For example, enterococcus faecalis, enterococcus Fesium, Enterococcus abium, Enterococcus durans, Enterococcus maladolats, Enterococcus caselliflavus, Enterococcus galinar, Leuconostoc cremoris, Leuconostok citrobolum, Leuconostok mezenteroides, Pediococcus cervus Halophilus, Streptococcus acetinicus, Streptococcus ebium, Streptococcus cremoris, Streptococcus thermo Streptococcus sangius, Streptococcus soye, Streptococcus dulans, Streptococcus paracitrobolus, Streptococcus lactis, etc. And Bifidobacterium such as Bifidobacterium bifidum, Bifidobacterium longum and Bifidobacterium breve.

  There is no limitation in particular as a solvent used for extraction of the lactic acid fermented product of grape. Examples include water, alcohol solvents such as methanol and ethanol. Preferably, ethanol or hydrous ethanol is suitable because of its high safety and extraction efficiency.

  The grape lactic acid fermented product and / or extract thereof, which is an active ingredient of the present invention, can be obtained, for example, as follows.

  First, raw grapes are pulverized to obtain a pulverized grape product. Preferably, it grind | pulverizes until a particle size will be 100 micrometers or less. When the grain size of the pulverized grape is 100 μm or less, the graininess is lost, the texture is improved, and the fermentation efficiency by lactic acid bacteria is improved. As the raw grapes, grapes containing grape seeds and pericarp are used. In that case, the fruit of the grape itself containing a seed and a fruit skin may be used, and the residue containing the seed and the fruit skin remaining after recovering the pulp and juice from the fruit of the grape may be used.

  The method for pulverizing the raw grapes is preferably wet pulverization. The wet pulverization can be performed using, for example, a grinder, a cutter or the like.

  Next, an appropriate amount of water is added to the obtained grape pulverized product, and heat-sterilized to prepare a medium.

  10-40 mass% is preferable at solid content conversion content, and the density | concentration of the grape ground material in a culture medium has more preferable 25-35 mass%. If the concentration of the pulverized grape is less than 10% by mass, the drying cost is increased and the bulk specific gravity is decreased, which tends to cause inconvenience in formulation. Further, when the concentration of the pulverized grape product exceeds 40% by mass, the viscosity increases and it becomes difficult to stir, and lactic acid fermentation tends to be performed unevenly. Tends to decrease.

  The heat sterilization is preferably performed at 110 to 125 ° C. for 10 to 30 minutes, more preferably at 115 to 120 ° C. for 10 to 20 minutes. By performing heat sterilization under the above-described conditions, propagation of miscellaneous bacteria and the like can be suppressed without destroying components responsible for tyrosinase inhibitory activity and melanin production inhibitory activity.

Next, lactic acid fermentation is performed by adding lactic acid bacteria to the grape-derived composition prepared as described above. As described above, lactic acid bacteria containing at least Lactobacillus plantarum and Lactobacillus brevis are used. Although the addition amount of lactic acid bacteria is not particularly limited, it is preferable to add them so that the total number of cells is 1 × 10 ⁹ to 1 × 10 ¹⁰ per liter of the medium, and the bacteria of Lactobacillus plantarum It is more preferable to add so that the number of cells is 5 × 10 ^{8 to} 5 × 10 ^{9 and the} number of Lactobacillus brevis cells is 5 × 10 ^{8 to} 5 × 10 ⁹ .

  Lactic acid fermentation is preferably performed under conditions of a temperature of 27 to 37 ° C., a pH of 3 to 7, and a culture time of 24 to 72 hours, and more preferably a temperature of 30 to 33 ° C., a pH of 4.5 to 6 and a culture time of 40 to 50 hours. Perform under the conditions of

Thus, the lactic acid fermented product of grape obtained by lactic acid fermentation has an increased polyphenol content as compared with the state before lactic acid fermentation, and the absorbability of polyphenols into the body is enhanced. The reason for this is thought to be that glycosides of polyphenols are cleaved or bound with organic acids by lactic acid fermentation. Further, the lactic acid fermentation product, in solid 100mg of lactic acid fermentation product, it is preferable that lactic acid bacteria are contained ^{10 10} 1 × ¹⁰ 8 ~1 ×. Lactobacillus plantarum and Lactobacillus brevis have 5 × 10 ^{8 to} 5 × 10 ⁹ cells of Lactobacillus plantarum and 100 cells of Lactobacillus brevis in a solid content of 100 mg of the same fermented product. It is preferable that 5 * 10 < ⁸ > -5 * 10 < ⁹ > pieces are contained.

  And the said lactic acid fermented product is heat-sterilized at 60-80 degreeC for 5 to 10 minutes, and saccharides, such as galactooligosaccharide and sucrose, fruit juice, etc. are added as needed, and a liquid lactic acid fermented product becomes. can get.

  Moreover, a powdery lactic acid fermented product is obtained by drying and pulverizing the said lactic acid fermented product after heat sterilization. Examples of the drying method of the lactic acid fermented product after heat sterilization include hot air drying, spray drying, drum drying, vacuum drying, etc., but there is little destruction of components responsible for tyrosinase inhibitory activity and melanin production inhibitory activity, and excellent physiological Lyophilization is preferred because it is easy to maintain activity. The freeze-drying is preferably performed, for example, by freezing the fermented product at −60 to −40 ° C. for 1 to 3 hours and under a pressure of 0.1 mbr or less, a time of 24 to 48 hours, and a temperature of 20 to 30 ° C.

  Moreover, the extract of a lactic acid fermented product is obtained by extracting from the said lactic acid fermented product using an extraction solvent. As described above, the extraction solvent is not particularly limited. Examples include water, alcohol solvents such as methanol and ethanol. Preferably, ethanol or hydrous ethanol is suitable because of its high safety and extraction efficiency. Examples of the extraction method include a method in which the lactic acid fermented product is suspended in an extraction solvent and shaken at 20 to 40 ° C. for 2 to 48 hours. If necessary, the extract may be used after removing solids by filtration, and may further be concentrated or dried powder.

  The whitening agent of the present invention contains the lactic acid fermented product and / or the extract thereof, and exhibits an excellent effect in terms of tyrosinase inhibitory activity and melanin production inhibitory activity. Sunburn can be prevented and / or recovered, and pigmentation such as spots and freckles can be suppressed. The dosage form is not particularly limited, but is preferably taken orally or by skin application. And it is used in various fields, such as a pharmaceutical, cosmetics, and a foodstuff, and can be used as an active ingredient of the pharmaceutical, a raw material of cosmetics, a foodstuff, etc.

  For example, in the case of a pharmaceutical product, it can be formulated with a pharmaceutically acceptable substrate or carrier and provided as a pharmaceutical composition. Examples of the form of the pharmaceutical composition include pills, powders, tablets, granules, capsules, syrups, liquids, jellies, lozenges and the like.

  Moreover, when it is set as cosmetics, it can formulate with the base material and carrier accept | permitted in cosmetics, and can mix | blend as a raw material of cosmetics. Examples of cosmetic forms include creams, gels, ointments, cosmetic solutions, spray solutions, foam preparations, packs, face masks and the like.

  Moreover, when it is set as a foodstuff, it can be mix | blended and used for a functional display food, a nutritional supplement, a health food etc. especially in addition to a general food.

The effective intake of the whitening agent of the present invention is preferably 100 mg to 3 g in terms of solid content of the above active ingredient per day for an adult when taken orally. Also, when applied to the skin, adult per day, applied area 1 cm ² per, 50Mg～250g preferably in terms of solid content of the active ingredient.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but these examples do not limit the scope of the present invention.

Example 1
1 L of water was added to 1 kg of Koshu grape seeds and pericarp, and wet pulverized using a fine grinder to obtain a crushed grape having a particle size of 100 μm or less. 1 L of water was added to 2 kg of the pulverized grapes to adjust the pH to 6, and sterilized by heating at 120 ° C for 20 minutes. To this heat-sterilized product, 5 mL of Lactobacillus plantarum preculture solution (5 × 10 ⁹ cells) and 5 mL of Lactobacillus brevis preculture solution (5 × 10 ⁹ cells) are added. The temperature was maintained at 33 ± 1 ° C. and fermented for 48 hours.

After completion of fermentation, heat sterilized at 70 ° C. for 10 minutes, frozen at −40 ° C., freeze-dried under conditions of pressure 0.1 mbr or less, 24 hours, 30 ° C., and powdered lactic acid fermentation product of grape Of 250 g was obtained. The grapes during lactic acid fermentation product 100mg of killed microbial cells of Lactobacillus plantarum is 5 × 10 ⁸ cells, dead cells of Lactobacillus brevis was included 5 × 10 ⁸ pieces. The total polyphenol content was 23 mg in terms of gallic acid, and the DPPH radical scavenging activity was 242 μmol in terms of Trolox.

(Comparative Example 1)
As a lactic acid bacterium, a lactic acid fermented product of grapes was used in the same manner as in Example 1 except that Lactobacillus plantarum was used in addition to Lactobacillus plantarum with 10 × 10 ⁹ bacterial cells and added to the pulverized grape. Got.

(Comparative Example 2)
A lactic acid fermentation product of grapes in the same manner as in Example 1 except that Lactobacillus brevis was used as a lactic acid bacterium without adding Lactobacillus plantarum to the pulverized product of 10 × 10 ⁹ cells. Got.

(Comparative Example 3)
Example 1 except that, instead of Lactobacillus plantarum and Lactobacillus brevis, Lactobacillus casei and Streptococcus lactis were used as lactic acid bacteria in addition to 5 × 10 ⁹ cells in addition to the crushed grapes, respectively. In the same manner, a lactic acid fermentation product of grape was obtained.

(Comparative Example 4)
Example except that, instead of Lactobacillus plantarum and Lactobacillus brevis, Lactobacillus thermophilus and Enterococcus faecalis were used as lactic acid bacteria in addition to 5 × 10 ⁹ cells in addition to the pulverized grapes, respectively. In the same manner as in No. 1, a lactic acid fermentation product of grape was obtained.

<Test Example 1> (Tyrosinase inhibitory activity)
Tyrosinase inhibitory activity was measured for the lactic acid fermented products of grapes of Example 1 and Comparative Examples 1 to 4. The inhibitory activity was determined by comparing with the tyrosinase activity when water was added instead of the lactic acid fermented product of grape. In addition, as a reference example, tyrosinase inhibitory activity was also measured for a non-fermented product obtained by freeze-drying the heat-sterilized processed product of the pulverized grape used in the preparation of the lactic acid fermented product. The test samples are summarized below. Moreover, the measuring method of tyrosinase inhibitory activity is shown.

[Test sample]
A: Control (water)
B: Reference example (non-fermented product of grape seeds and pericarp)
C: Example 1 (Lactobacillus plantarum + Lactobacillus brevis)
D: Comparative example 1 (Lactobacillus plantarum)
E: Comparative Example 2 (Lactobacillus brevis)
F: Comparative Example 3 (Lactobacillus casei + Streptococcus lactis)
G: Comparative example 4 (Lactobacillus thermophilus + Enterococcus faecalis)

[Measuring method]
-Preparation of sample 100 mg of fermented or non-fermented product was suspended in 10 mL of 80% methanol and extracted with shaking for 3 hours. Methanol was removed from the supernatant obtained by centrifugation, and the mixture was prepared again to 10 mL to prepare a sample.

Analytical method Take 0.3 mL of the above sample, add 3 mL of citrate buffer (20 mM, pH 6.8) to this, and further tyrosinase solution (prepared with citrate buffer so that mushroom-derived tyrosinase is 400 U / mL) 0.3 mL was added and incubated at 37 ° C. for 10 minutes. Then, 3 mL of tyrosine solution (1.11 μmol / mL) was added and reacted at 37 ° C. for 20 minutes. Absorbance at 490 nm was measured, and the relative activity was determined when the tyrosinase activity was set to 100 when 0.3 mL of water was used instead of 0.3 mL of the sample.

  The results are shown in FIG.

  As shown in FIG. 1, in the reference example using the non-fermented product, the tyrosinase activity was about 60% of the control. In Comparative Examples 1 and 2 in which lactic acid fermentation was performed using Lactobacillus plantarum or Lactobacillus brevis alone, the inhibitory activity was improved by the lactic acid fermentation, and Lactobacillus plantarum and Lactobacillus brevis were used in combination. In Example 1 subjected to lactic acid fermentation, the inhibitory activity was further improved. On the other hand, as a lactic acid bacterium, Lactobacillus casei and Streptococcus lactis were used in combination with Comparative Example 3, and Lactobacillus thermophilus and Enterococcus faecalis were used in combination with lactic acid fermentation. In No. 4, only inhibitory activity comparable to the reference example using a non-fermented material was obtained.

<Test Example 2> (Melanin production inhibitory activity)
About the lactic acid fermented product of the grape of Example 1 and Comparative Examples 1-2, the activity which suppresses the melanin production | generation of B16 melanoma cell was tested. The inhibitory activity was determined by comparing with the amount of melanin produced when water was added instead of the lactic acid fermented product of grape. Moreover, as a reference example, the melanin production inhibitory activity was tested also about the non-fermented material which freeze-dried the heat-sterilized processed product of the grape ground material used for preparation of a lactic acid fermented product as it is. The test samples are summarized below. Moreover, the test method of melanin production inhibitory activity is shown.

[Test sample]
A: Control (water)
B: Reference example (non-fermented product of grape seeds and pericarp)
C: Example 1 (Lactobacillus plantarum + Lactobacillus brevis)
D: Comparative example 1 (Lactobacillus plantarum)
E: Comparative Example 2 (Lactobacillus brevis)

[Test method]
-Preparation of sample 100 mg of fermented products and non-fermented products were suspended in 10 mL of distilled water and extracted with shaking for 3 hours. The supernatant obtained by centrifugation was freeze-dried to prepare a sample.

-Culture of B16 melanoma cells B16 melanoma cells were prepared to a concentration of 1.0 × 10 ⁴ cells / well using Doulbecco's modified Eagle's Medium (5% FBS-containing DMEM) containing 5% fetal bovine serum (FBS). The cells were seeded on a plate and cultured at 37 ° C. in a 5% CO ² gas phase. After culturing for 24 hours, the medium was changed using a medium in which 0.05% of the sample was added to DMEM containing 5% FBS. The cells were cultured for 6 days while changing the medium every 72 hours. As a control, ultrapure water was used instead of the sample.

-Analysis method After culture, the cells were detached with a 0.25% trypsin solution, and the solution was centrifuged to prepare a cell pellet. The pellet was suspended in 1.5 mL of PBS (−) buffer, 0.3 mL was used for protein measurement, and 1.2 mL was used for melanin determination.

  Protein measurement was performed as follows. That is, 0.3 mL of the cell suspension was centrifuged to pellet the cells, and the pellet was washed 3 times with PBS (−) buffer. 1 mL of PBS (-) buffer containing 0.5% Triton X-100 was added to the pellet and vortexed for 20 minutes to obtain a cell protein lysate. About the obtained cell protein solution, protein amount was quantified by the Lowry method according to a conventional method.

  Melanin quantification was performed as follows. That is, 1.2 mL of the cell suspension was centrifuged to pellet the cells, and the pellets were each three times with PBS (−) buffer, 5% trichloroacetic acid solution, and ethanol / diethyl ether (1: 3) solution. After washing, it was washed once with diethyl ether. The pellets were heated to 50 ° C. for 1 hour and sufficiently dried, and then 150 μL of 1M sodium hydroxide solution was added and heated to 100 ° C. for 10 minutes to solubilize melanin. The absorbance at 405 nm of melanin was measured using a microplate reader, and the amount of melanin was compared with the absorbance per protein amount.

  The results are shown in FIG.

  As shown in FIG. 2, in the reference example using the non-fermented product, the melanogenesis activity was about 80% of the control. In Comparative Examples 1 and 2 in which lactic acid fermentation was performed using Lactobacillus plantarum or Lactobacillus brevis alone, the melanin production inhibitory activity was improved by the lactic acid fermentation, but Lactobacillus plantarum and Lactobacillus brevis were used in combination. In Example 1, which was used for lactic acid fermentation, the melanin production inhibitory activity was further improved.

<Production Example 1>
Tablets containing the lactic acid fermentation product of grape obtained in Example 1 were produced according to a conventional method with the formulation shown in Table 1 below.

<Production Example 2>
A drink containing the lactic acid fermented product of grape obtained in Example 1 was produced according to a conventional method with the formulation shown in Table 2 below.

<Production Example 3>
A cream containing the lactic acid fermentation product of grape obtained in Example 1 was prepared according to a conventional method with the formulation shown in Table 3 below.

Claims (6)

  A whitening agent comprising, as an active ingredient, a lactic acid fermented product of grape obtained by fermenting grape seeds and skins with a lactic acid bacterium containing at least Lactobacillus plantarum and Lactobacillus brevis and / or an extract thereof. .   The whitening agent according to claim 1, wherein the lactic acid fermented product of grape and / or an extract thereof has tyrosinase inhibitory activity.   The whitening agent according to claim 1 or 2, wherein the lactic acid fermented product of grape and / or an extract thereof has a melanin production inhibitory activity.   For whitening, comprising as an active ingredient a lactic acid fermented product and / or an extract thereof obtained by fermenting grape seeds and skins with lactic acid bacteria containing at least Lactobacillus plantarum and Lactobacillus brevis Cosmetics.   The whitening cosmetic according to claim 4, wherein the lactic acid fermented product of grape and / or an extract thereof has tyrosinase inhibitory activity. The whitening cosmetic according to claim 4 or 5, wherein the grape lactic acid fermented product and / or an extract thereof has a melanin production inhibitory activity.