Melanin production inhibitor and whitening composition

Abstract

To provide a melanin production inhibitor that exhibits high safety and superior melanin production inhibitory activity, and a whitening composition that exhibits high safety and superior whitening activity.SOLUTION: A melanin production inhibitor comprising, as an active ingredient, particles secreted in fermented milk whey resulting from fermentation of milk by lactic acid bacteria.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a melanin production inhibitor and a skin-whitening composition. [Background technology]

[0002] Melanin protects the body from ultraviolet rays and is produced in the skin when exposed to ultraviolet rays. However, excessive production or uneven accumulation can cause skin darkening, age spots, and freckles. Generally, melanin is produced by the enzyme tyrosinase, which is biosynthesized in pigment cells, converting tyrosine to dopa and dopa to dopaquinone, followed by intermediates such as 5,6-dihydroxyindophenol. Therefore, inhibiting melanin production is considered effective in preventing and improving skin darkening (cutaneous pigmentation), i.e., for skin whitening.

[0003] On the other hand, fermented milk whey obtained by fermenting milk with lactic acid bacteria (hereinafter sometimes referred to as "fermented milk whey") is known to have various excellent effects. For example, fermented milk whey obtained by fermenting milk with Lactobacillus helveticus has been reported to have moisturizing effects (see, for example, Patent Document 1) and epidermal cell differentiation and keratinization promoting effects (see, for example, Patent Document 2).

[0004] In recent years, extracellular vesicles (EVs) secreted by cells have attracted attention as a tool for intercellular communication. EVs are defined as particles released from cells that are surrounded by a lipid bilayer membrane and lack a nucleus. Based on differences in their production mechanisms, EVs are classified into exosomes, microvesicles, and apoptotic vesicles. EVs are found in various body fluids, and their functions vary depending on the cells from which they originate and the conditions under which they are produced.

[0005] To date, for example, compositions for preventing hair loss or promoting hair growth (see, for example, Patent Document 3) and compositions for immunoregulation (see, for example, Patent Document 4) have been proposed that contain extracellular vesicles derived from lactic acid bacteria as active ingredients. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2005-206578 [Patent Document 2] International Publication No. 2006 / 137513 [Patent Document 3] Japanese Patent Application Publication No. 2018-529720 [Patent Document 4] Patent Publication No. 2021-501136 Summary of the Invention [Problem to be solved by the invention]

[0007] Although materials that inhibit melanin production have been developed to date, there is still a need for new materials that are highly safe, can inhibit melanin production, and have excellent whitening properties.

[0008] The present invention aims to solve the above-mentioned conventional problems and achieve the following objectives: Namely, the present invention aims to provide a melanin production inhibitor that is highly safe and has an excellent melanin production inhibitory effect, and a whitening composition that is highly safe and has an excellent whitening effect. [Means for solving the problem]

[0009] As a result of extensive research conducted by the present inventors to solve the above problems, they discovered that particles secreted into fermented milk whey obtained by fermenting milk with lactic acid bacteria have excellent melanin production inhibitory effects and are useful as an active ingredient in melanin production inhibitors and skin whitening compositions.

[0010] The present invention is based on the above findings of the present inventors, and the means for solving the above problems are as follows: <1> The melanin production inhibitor is characterized by containing, as an active ingredient, particles secreted in fermented milk whey obtained by fermenting milk with lactic acid bacteria. <2> The particle diameter of the particles is 50 to 150 nm. <1> The melanin production inhibitor is described in the above. <3> The particles are precipitated by ultracentrifugation of the fermented milk whey at 210,000×g or more. <1> or <2> The melanin production inhibitor is described in the above. <4> The lactic acid bacterium is at least one selected from the group consisting of Lactobacillus delbrueckii bulgaricus, Lactobacillus helveticus, Lactobacillus acidophilus, and Lactobacillus casei. <1> from <3> The melanin production inhibitor according to any one of the above items. <5> The lactic acid bacterium is Lactobacillus helveticus. <1> from <4> The melanin production inhibitor according to any one of the above items. <6> The aforementioned <1> from <5> 1. A whitening composition comprising the melanin production inhibitor according to any one of claims 1 to 9. [Effects of the Invention]

[0011] According to the present invention, it is possible to solve the above-mentioned problems in the prior art, achieve the above-mentioned object, and provide a melanin production inhibitor that is highly safe and has an excellent melanin production inhibitory effect, and a whitening composition that is highly safe and has an excellent whitening effect. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 shows the results of the melanin production inhibitory effect test in Test Example 1. [Figure 2]FIG. 2 shows the results of the melanin production inhibitory effect test in Test Example 2. [Figure 3] FIG. 3 is a diagram showing the measurement results of Test Example 3 using a particle size distribution analyzer. [Figure 4] FIG. 4 shows the results of the melanin production inhibitory effect test in Test Example 4. [Figure 5] FIG. 5 shows the results of measuring the expression level of Tyr mRNA in Test Example 5. [Figure 6] FIG. 6 shows the results of measuring the expression level of Trp1 mRNA in Test Example 5. [Figure 7] FIG. 7 shows the results of measuring the expression level of Dct mRNA in Test Example 5. [Figure 8] FIG. 8 shows the results of the melanin production inhibitory effect test in Test Example 6. DETAILED DESCRIPTION OF THE INVENTION

[0013] (Melanin production inhibitor) The melanin production inhibitor of the present invention contains at least particles secreted in fermented milk whey obtained by fermenting milk with lactic acid bacteria as an active ingredient, and may further contain other ingredients as necessary.

[0014] The melanin production inhibitor has the effect of inhibiting the production of melanin. As used herein, inhibiting melanin production means that when the melanin production inhibitor is used, the amount of melanin produced is reduced compared to when the melanin production inhibitor is not used.

[0015] The melanin production inhibitor is preferably based on at least one action selected from the group consisting of an action of inhibiting tyrosinase mRNA expression, an action of inhibiting tyrosinase-related protein 1 mRNA expression, and an action of inhibiting tyrosinase-related protein 2 mRNA expression, but is not limited to these actions.

[0016] [particle] The particles are those secreted into fermented milk whey obtained by fermenting milk with lactic acid bacteria. It was not previously known that the particles have an excellent melanin production inhibitory effect and are useful as an active ingredient in melanin production inhibitors and skin-whitening compositions, and this is a new discovery made by the present inventors.

[0017] <Fermented milk whey> The fermented milk whey is whey obtained by fermenting milk with lactic acid bacteria.

[0018] The lactic acid bacteria are not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include lactic acid bacteria belonging to the genera Streptococcus, Lactococcus, Lactobacillus, Bifidobacterium, etc. Among these, lactic acid bacteria belonging to the genus Lactobacillus are preferred. The lactic acid bacteria may be used alone or in combination of two or more kinds.

[0019] The lactic acid bacteria belonging to the genus Lactobacillus are not particularly limited and can be appropriately selected depending on the purpose. Examples include Lactobacillus delbrueckii bulgaricus, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus casei, and Lactobacillus fermentum.

[0020] Among the lactic acid bacteria, at least one selected from the group consisting of Lactobacillus delbrueckii bulgaricus, Lactobacillus helveticus, Lactobacillus acidophilus, and Lactobacillus casei is preferred, with Lactobacillus helveticus being more preferred.

[0021] The strain of each of the lactic acid bacteria is not particularly limited and can be appropriately selected depending on the purpose.

[0022] The Lactobacillus helveticus strain is preferably one with high extracellular proteinase activity. For example, a strain with a U / OD590 value of 400 or more measured according to the method of Yamamoto et al. (Yamamoto, N. et al., J. Biochem. (1993) 114, 740), which is based on the method of Twining et al. (Twining, S., Anal. Biochem. 143, 3410 (1984)). Furthermore, Lactobacillus helveticus strains having the following bacteriological properties can be used.

[0023] -Mycological properties- 1.Morphological properties: 1) Cell shape: Bacillus 2) Motility: None 3) Presence or absence of spores: None 4) Gram staining: positive 2.Physiological properties: 1) Catalase: negative 2) Indole production; negative 3) Nitrate reduction; negative 4) Attitude towards oxygen; facultative anaerobes 5) DL(-) lactic acid is produced from glucose by homolactic fermentation, and no gas is produced.

[0024] Among the strains of Lactobacillus helveticus, the Lactobacillus helveticus JCM1120 strain and the Lactobacillus helveticus CM4 strain are preferred. The Lactobacillus helveticus JCM1120 strain is a type strain and is available from the RIKEN BioResource Research Center (Riken BRC). The Lactobacillus helveticus CM4 strain was deposited on August 15, 1997, at the then Ministry of International Trade and Industry's Agency of Industrial Science and Technology's Microbial Research Institute (now the National Institute of Technology and Evaluation's Patent Organism Deposit Center: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) under accession number FERM BP-6060. This Lactobacillus helveticus CM4 strain has been registered under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure under the above accession number, and this strain has already been patented.

[0025] The method for producing the fermented milk whey is not particularly limited and can be selected appropriately depending on the purpose. For example, the fermented milk whey can be obtained by adding the lactic acid bacteria directly or a fermented milk starter containing the lactic acid bacteria to milk and fermenting the milk under appropriately selected fermentation conditions such as fermentation temperature.

[0026] The fermented milk whey can be obtained by separating whey from the obtained fermented milk by a conventional separation procedure such as centrifugation or filtration.

[0027] The lactic acid bacteria are preferably used as a starter that has been pre-cultured in advance and has a sufficiently high activity. 5 ~10 9 It is about 1 / mL.

[0028] The raw milk material is not particularly limited and can be appropriately selected depending on the purpose. Examples include animal milks such as cow's milk, horse's milk, sheep's milk, and goat's milk, plant milks such as soy milk, and processed milks such as skim milk, reconstituted milk, milk powder, and condensed milk. Cow's milk, soy milk, and processed milks thereof are preferred, and cow's milk or processed milks thereof are particularly preferred. The solids concentration of the milk is not particularly limited and can be selected appropriately depending on the purpose. For example, when skim milk is used, the non-fat milk solids concentration is usually about 3 to 15% by mass, and from the viewpoint of productivity, 6 to 15% by mass is preferable.

[0029] The fermentation can usually be carried out by static or agitated culture, for example, at a fermentation temperature of 25 to 45°C, preferably 30 to 45°C, for a fermentation time of 3 to 72 hours, preferably 12 to 36 hours, and by stopping the fermentation when the lactic acid acidity reaches 1.5 or more.

[0030] <Particle preparation> The method for preparing the particles from the fermented milk whey is not particularly limited, and any known method for preparing extracellular vesicles can be appropriately selected, including, for example, ultracentrifugation, ultrafiltration, density gradient centrifugation, polymer precipitation, immunoprecipitation, etc. These methods may be used alone or in combination of two or more.

[0031] The ultracentrifugation method is a method in which lactic acid bacteria residues are removed by low-speed centrifugation, followed by ultracentrifugation to separate extracellular vesicles. The conditions for the low-speed centrifugation are not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include 8,000 to 15,000×g. The conditions for the ultracentrifugation are not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include 100,000 to 250,000×g. Between the low-speed centrifugation and the ultracentrifugation, the supernatant may be filtered as needed. After the ultracentrifugation, the resulting precipitate may be suspended in phosphate buffered saline (PBS) or the like, and then subjected to low-speed centrifugation.

[0032] The ultrafiltration method is a method for separating extracellular vesicles using a filter according to the target size.

[0033] The density gradient centrifugation is a method in which a crude fraction obtained by ultracentrifugation is further purified by passing it through a density gradient.

[0034] The polymer precipitation method is a method for separating extracellular vesicles by low-speed centrifugal sedimentation using a polymer.

[0035] The immunoprecipitation method is a method for separating extracellular vesicles by an affinity method for marker proteins or lipids of extracellular vesicles.

[0036] Extracellular vesicles are defined as particles without a nucleus surrounded by a lipid bilayer membrane that are released from cells. Extracellular vesicles are further classified into exosomes, microvesicles, and apoptotic vesicles based on differences in their production mechanisms. The exosomes are small vesicles of about 50 to 150 nm in size derived from the endosomal membrane and formed during the process of endocytosis. The microvesicles are small vesicles with a size of about 100 to 1,000 nm that bud directly from the cell membrane and are secreted outside the cell. The apoptotic vesicles are micrometer-sized vesicles that are budded from the membrane of apoptotic cells and can be easily distinguished from other vesicles by low-speed centrifugation at about 2,000 × g.

[0037] The particles of the present invention are thought to be exosome-like extracellular vesicles because they are secreted into fermented milk whey obtained by fermenting milk with lactic acid bacteria, they sediment by ultracentrifugation, and they have a particle size of 50 to 150 nm.

[0038] The particles are preferably precipitated by ultracentrifugation of the fermented milk whey at 210,000×g or more.

[0039] The average particle size of the particles is not particularly limited and can be appropriately selected depending on the purpose, and may be, for example, 80 to 150 nm.

[0040] Furthermore, the particles of the present invention are contained in a fraction of 3 KDa or more when the fermented milk whey is fractionated based on molecular weight.

[0041] The content of the particles in the melanin production inhibitor is not particularly limited and can be selected appropriately depending on the purpose, but is preferably 0.00005 (w / w or w / v)% or more, more preferably 0.0001 (w / w or w / v)% or more, even more preferably 0.0005 (w / w or w / v)% or more, and particularly preferably 0.001 (w / w or w / v)% or more.

[0042] The melanin production inhibitor may consist of the particles alone, or may be a formulation of the particles in any dosage form.

[0043] [Other ingredients] The other components in the melanin production inhibitor are not particularly limited as long as they do not impair the effects of the present invention, and can be appropriately selected depending on the purpose. Examples of such components include auxiliary raw materials or additives that are commonly used in producing cosmetics or foods and beverages, and known components that have a melanin production inhibitory effect. The auxiliary raw materials and additives that are typically used in producing the cosmetics are not particularly limited and can be appropriately selected depending on the purpose. Examples include oils and fats, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, metal soaps, pH adjusters, preservatives, fragrances, moisturizers, powders, ultraviolet absorbers, thickeners, pigments, and antioxidants. Examples of auxiliary raw materials or additives commonly used in producing the above-mentioned foods and beverages include carriers, excipients, fillers, preservatives, stabilizers, binders, pH adjusters, buffers, thickeners, gelling agents, preservatives, and antioxidants. The other components in the melanin production inhibitor may be used alone or in combination of two or more.

[0044] The content of the other components in the melanin production inhibitor is not particularly limited and can be appropriately selected depending on the purpose.

[0045] The melanin production inhibitor preferably does not contain lactic acid bacteria-fermented milk whey, i.e., the particles contained in the melanin production inhibitor are preferably isolated from lactic acid bacteria-fermented milk whey.

[0046] <Application> The use of the melanin production inhibitor is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include a wide range of uses such as pharmaceuticals, quasi-drugs, cosmetics, and foods and beverages. The melanin production inhibitor is highly safe and has an excellent melanin production inhibitory effect, and therefore can be suitably used, for example, as an active ingredient in a whitening composition described below.

[0047] The melanin production inhibitor may be administered orally or parenterally.

[0048] The dosage form of the melanin production inhibitor is not particularly limited, and any known dosage form can be appropriately selected depending on the purpose. Examples include oral preparations such as tablets, pills, hard capsules, soft capsules, microcapsules, powders, granules, and liquid preparations; and parenteral preparations such as injections, suppositories, and liniments. The method for producing the melanin production inhibitor in various dosage forms is not particularly limited, and any known method can be appropriately selected.

[0049] The melanin production inhibitor is highly safe and can be used on a daily basis, and the particles, which are the active ingredient, can exert a highly effective melanin production inhibitory effect.

[0050] The method of use, amount used, area to be used, period of use, interval of use, etc. of the melanin production inhibitor are not particularly limited, and can be appropriately selected taking into consideration the age and health condition of the subject, etc.

[0051] The amount of the melanin production inhibitor used (sometimes referred to as the dosage or intake amount) is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include a daily amount for an adult human in which the amount of the particles is in the range of 0.001 to 0.005 (w / w or w / v)%. The daily amount may be used in one dose or in divided doses.

[0052] The timing of use of the melanin production inhibitor is not particularly limited and can be appropriately selected depending on the purpose. For example, the melanin production inhibitor may be used continuously or intermittently before exposure to an environment in which inhibition of melanin production is required, or may be used continuously or intermittently after exposure to such an environment.

[0053] The melanin production inhibitor is preferably applied to humans, but can also be applied to animals other than humans as long as its action and effect are exhibited.

[0054] The melanin production inhibitor can also be used as a reagent for research into melanin production inhibitory activity.

[0055] As shown in the test examples described below, the particles can reduce the expression levels of mRNA for tyrosinase, tyrosinase-related protein 1, and tyrosinase-related protein 2, which are involved in melanin production. Therefore, the present invention also relates to a tyrosinase mRNA expression inhibitor, a tyrosinase-related protein 1 mRNA expression inhibitor, or a tyrosinase-related protein 2 mRNA expression inhibitor, characterized in that it contains, as an active ingredient, particles secreted into fermented milk whey obtained by fermenting milk with lactic acid bacteria.

[0056] As used herein, suppressing mRNA expression means reducing the expression level of the mRNA when an mRNA expression inhibitor is used compared to when the mRNA expression inhibitor is not used.

[0057] (Whitening composition) The whitening composition of the present invention contains at least the melanin production inhibitor of the present invention, and further contains other ingredients as needed.

[0058] [Melanin production inhibitor] The melanin production inhibitor is the melanin production inhibitor of the present invention described above.

[0059] The content of the melanin production inhibitor in the whitening composition is not particularly limited and can be selected appropriately depending on the purpose, but the amount of the particles contained in the melanin production inhibitor is preferably 0.00005 (w / w or w / v)% or more, more preferably 0.0001 (w / w or w / v)% or more, even more preferably 0.0005 (w / w or w / v)% or more, and particularly preferably 0.001 (w / w or w / v)% or more. The whitening composition may consist solely of the melanin production inhibitor, but the upper limit of the amount of the melanin production inhibitor contained in the whitening composition is preferably 0.005 (w / w or w / v)% or less in terms of the amount of the particles contained in the melanin production inhibitor.

[0060] [Other ingredients] The other components in the whitening composition are not particularly limited as long as they do not impair the effects of the present invention, and can be appropriately selected depending on the form of use of the whitening composition. Examples of the other components include those similar to the other components in the melanin production inhibitors described above, and known components having a whitening effect. The other components in the whitening composition may be used alone or in combination of two or more.

[0061] The content of the other components in the whitening composition is not particularly limited and can be appropriately selected depending on the purpose.

[0062] The manner of use of the whitening composition is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include parenteral compositions such as topical skin preparations, oral compositions such as foods, etc. Among these, parenteral compositions such as topical skin preparations are preferred.

[0063] The topical skin preparation is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include cosmetics, quasi-drugs, pharmaceuticals, etc. Among these, cosmetics are preferably used.

[0064] The dosage form of the topical skin preparation is not particularly limited and can be appropriately selected depending on the purpose. Examples include lotion, cream, emulsion, serum, lotion, gel, aerosol, ointment, cataplasm, essence, pack, cleanser, bath agent, and foundation.

[0065] The foods include pharmaceuticals, quasi-drugs, foods (including beverages, the same applies below), supplements, etc. The foods and supplements are sometimes referred to as functional foods. The foods and supplements also include foods for specified health uses, foods with nutrient functions, and foods with functional claims.

[0066] The food is not particularly limited and can be appropriately selected depending on the purpose. Examples include soft drinks (carbonated drinks, non-alcoholic drinks, juices, coffee drinks, tea drinks, mineral water, sports drinks, etc.), dairy drinks, soy milk drinks, fermented milk, lactic acid bacteria drinks, cocoa, alcoholic drinks (beer, happoshu, other brewed alcoholic drinks, liqueurs, sake, amazake, wine, shochu, etc.), drinks prepared from instant powders, other drinks; bread, cereal, confectioneries (biscuits, cookies, chocolate, etc.), processed foods (bonito flakes, salted fish, kusaya, etc.), pickles (pickled vegetables, kimchi, pickles), fermented foods (natto, cheese, yogurt, etc.), seasonings (soy sauce, miso, vinegar, etc.), and liquid foods.

[0067] The method for producing the whitening composition is not particularly limited, and any known method can be appropriately selected depending on the embodiment.

[0068] The whitening composition is highly safe and can be used daily, and the particles contained in the melanin production inhibitor, which is an active ingredient, can exert a very effective whitening effect.

[0069] The method of use, amount used, area to be used, period of use, interval of use, etc. of the whitening composition are not particularly limited and can be appropriately selected taking into consideration the age and health condition of the subject.

[0070] The amount of the whitening composition to be used is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include an amount of the particles contained in the melanin production inhibitor in the range of 0.001 to 0.005 (w / w or w / v)% as the amount to be used per day for a human adult. The amount to be used per day may be used once or divided into multiple doses.

[0071] The timing of use of the whitening composition is not particularly limited and can be appropriately selected depending on the purpose. For example, the composition may be used continuously or intermittently before exposure to an environment requiring skin whitening, or may be used continuously or intermittently after exposure to such an environment.

[0072] The whitening composition is preferably applied to humans, but can also be applied to animals other than humans as long as the composition exhibits its functional effects.

[0073] As described above, the melanin production inhibitor of the present invention has an excellent melanin production inhibitory effect. Therefore, the present invention also relates to a method for inhibiting melanin production, which comprises administering the melanin production inhibitor to a subject.

[0074] Furthermore, the tyrosinase mRNA expression inhibitor of the present invention has an excellent effect of inhibiting tyrosinase mRNA expression, the tyrosinase-related protein 1 mRNA expression inhibitor has an excellent effect of inhibiting tyrosinase-related protein 1 mRNA expression, and the tyrosinase-related protein 2 mRNA expression inhibitor has an excellent effect of inhibiting tyrosinase-related protein 2 mRNA expression. Therefore, the present invention also relates to a method for suppressing the expression of tyrosinase mRNA, characterized by using the tyrosinase mRNA expression inhibitor in a subject; a method for suppressing the expression of tyrosinase-related protein 1 mRNA, characterized by using the tyrosinase-related protein 1 mRNA expression inhibitor in a subject; or a method for suppressing the expression of tyrosinase-related protein 2 mRNA, characterized by using the tyrosinase-related protein 2 mRNA expression inhibitor in a subject.

[0075] Furthermore, as described above, the whitening composition of the present invention has an excellent whitening effect. Therefore, the present invention also relates to a method for whitening skin, which comprises applying the whitening composition to a subject. [Example]

[0076] Test examples of the present invention will be explained below, but the present invention is not limited to these test examples.

[0077] (Test Example 1) <Preparation of fermented milk whey> A milk medium consisting of skim milk powder with a solid content of 9% by mass was inoculated with one of the following lactic acid bacteria (final inoculation amount: 1 to 2 × 10 6 The resulting fermented milk was subjected to ultrafiltration (MW<5 kDa) to obtain each fermented milk whey. [Lactic acid bacteria] The following lactic acid bacteria were used. Note that for lactic acid bacteria other than Lactobacillus helveticus CM4 strain, the respective type strains were used. Lactobacillus delbrueckii bulgaricus type strain (JCM1002 strain (obtained from Riken BRC)) Lactobacillus helveticus type strain (JCM1120 strain (obtained from Riken BRC)) Lactobacillus acidophilus type strain (JCM1132 strain (obtained from Riken BRC)) Lactobacillus casei type strain (JCM1134 strain (obtained from Riken BRC)) Lactobacillus helveticus strain CM4 (Accession number: FERM BP-6060)

[0078] <Melanin production inhibitory effect test> Melanoma B16 cells were seeded onto plates (seeding density: 3,000 cells / cm). 2 ), and 24 hours later, a melanin production inducer (α-melanocyte stimulating hormone (α-MSH)) was added (amount added: 50 nM) to induce melanin production. Furthermore, 48 hours after the addition of the α-MSH, the fermented milk whey obtained above (addition amount: 3 (v / v)%) was added simultaneously with α-MSH (addition amount: 50 nM), and the cells were cultured for 4 days. The amount of melanin after the incubation was measured as follows. The cells on the plate were washed with phosphate-buffered saline (PBS) and then lysed with 1 M NaOH. The cell lysate was collected in a tube and incubated at 80°C for 60 minutes. After cooling, the absorbance at 415 nm was measured using a plate reader to calculate the amount of melanin.

[0079] The results are shown in Figure 1. 1, the horizontal axis represents the samples tested, as described below, and the vertical axis represents the relative amount of melanin produced (%) when the amount of melanin produced in Control I, as described below, is taken as 100%. I (Control I) Melanin production inducer: No addition, fermented milk whey: No addition. II (Control II) Melanin production inducer: added, fermented milk whey: not added. 1-1 Melanin production inducer: added, Fermented milk whey: fermented milk whey prepared using the standard strain of Lactobacillus delbrueckii bulgaricus was added. 1-2 Melanin production inducer: added, Fermented milk whey: fermented milk whey prepared using the standard strain of Lactobacillus helveticus was added. 1-3 Melanin production inducer: added, Fermented milk whey: fermented milk whey prepared using the standard strain of Lactobacillus acidophilus was added. 1-4 Melanin production inducer: added, Fermented milk whey: fermented milk whey prepared using a standard strain of Lactobacillus casei was added. 1-5 Melanin production inducer: added, fermented milk whey: fermented milk whey prepared using Lactobacillus helveticus CM4 strain was added.

[0080] In Control II, melanin production was significantly increased compared to Control I (p<0.001). In Samples 1-1 to 1-5, to which fermented milk whey was added, melanin production was significantly decreased compared to Control II (Samples 1-1 and 1-4: p<0.01, Samples 1-2, 1-3, and 1-5: p<0.001).

[0081] (Test Example 2) Fermented milk whey prepared using the standard strain of Lactobacillus helveticus and fermented milk whey prepared using the CM4 strain of Lactobacillus helveticus, which had a strong inhibitory effect on melanin production in Test Example 1, were each fractionated by molecular weight to estimate the active fractions with inhibitory effect on melanin production.

[0082] <Fractionation by molecular weight> Using Amicon-Ultra4 (Merck Millipore), the fermented milk whey was separated into a fraction of 3 KDa or more (hereinafter sometimes referred to as "fraction A") and a fraction of 3 KDa or less. The fraction of 3 kDa or less was then fractionated into a water-soluble fraction (hereinafter sometimes referred to as "fraction B") and a fat-soluble fraction (hereinafter sometimes referred to as "fraction C") using Sep-pak tC18 (Waters).

[0083] <Melanin production inhibitory effect test> The melanin production inhibitory effect test was conducted in the same manner as in Test Example 1, except that instead of adding 3 (v / v)% fermented milk whey in the melanin production inhibitory effect test in Test Example 1, the fractions A to C fractionated above were added at 1 (v / v)%. In addition, in the same manner as in Test Example 1, a test was also carried out in the case where fermented milk whey was added.

[0084] The results are shown in Figure 2. The horizontal axis of Figure 2 represents the samples tested, as described below, and the vertical axis represents the relative amount of melanin produced (%) when the amount of melanin produced in Control I, as described below, is taken as 100%. I (Control I) Melanin production inducer: No addition, fermented milk whey and fractions A to C: No addition. II (Control II) Melanin production inducer: added; fermented milk whey and fractions A to C: not added. 2-1 Melanin production inducer: added, fermented milk whey and fractions A to C: fermented milk whey prepared using the standard strain of Lactobacillus helveticus was added. 2-1-A Melanin production inducer: added, fermented milk whey and fractions A to C: fraction A, which was fractionated from fermented milk whey prepared using a standard strain of Lactobacillus helveticus, was added. 2-1-B Melanin production inducer: added, fermented milk whey and fractions A to C: fraction B, which was fractionated from fermented milk whey prepared using a standard strain of Lactobacillus helveticus, was added. 2-1-C Melanin production inducer: added, fermented milk whey and fractions A to C: fraction C, which was fractionated from fermented milk whey prepared using a standard strain of Lactobacillus helveticus, was added. 2-2 Melanin production inducer: added, fermented milk whey and fractions A to C: fermented milk whey prepared using Lactobacillus helveticus CM4 strain was added. 2-2-A Melanin production inducer: added, fermented milk whey and fractions A to C: fraction A obtained by fractionating fermented milk whey prepared using Lactobacillus helveticus CM4 strain was added. 2-2-B Melanin production inducer: added, fermented milk whey and fractions A to C: fraction B obtained by fractionating fermented milk whey prepared using Lactobacillus helveticus CM4 strain was added. 2-2-C Melanin production inducer: added, fermented milk whey and fractions A to C: fraction C obtained by fractionating fermented milk whey prepared using Lactobacillus helveticus CM4 strain was added.

[0085] In Control II, the amount of melanin produced was significantly increased compared to Control I (p<0.001). As in Test Example 1, in Samples 2-1 and 2-2 to which fermented milk whey was added, the amount of melanin produced was significantly decreased compared to Control II (p<0.001). Furthermore, samples 2-1-A and 2-2-A, which contained fraction A, a fraction of fermented milk whey with a molecular weight of 3 kDa or more, also showed a significant decrease in melanin production compared to control II (p<0.01). Sample 2-1-B, which contained fraction B, also showed a significant decrease in melanin production compared to control II (p<0.05).

[0086] (Test Example 3) The presence of exosome-like extracellular vesicles in fermented milk whey prepared using the standard strain of Lactobacillus helveticus, which had a strong inhibitory effect on melanin production in Test Example 1, was investigated as follows.

[0087] <Preparation of exosome-like extracellular vesicles> 9 mL of fermented milk whey prepared using the type strain of Lactobacillus helveticus prepared in Test Example 1 was centrifuged at 10,000 × g, 4°C, and for 10 minutes, and the supernatant was filtered using a 0.22 μm filter. The entire amount of the obtained filtrate was ultracentrifuged at 210,000×g, 4° C., and 70 minutes, and the obtained precipitate was suspended in PBS. The suspension was centrifuged at 10,000×g, 4° C., and 5 minutes, and the resulting precipitate was suspended in 0.2 mL of PBS to prepare a PBS suspension.

[0088] The particle size of the particles contained in the PBS suspension was confirmed using a particle size distribution analyzer (ZETASIZER NANO, manufactured by Malvern Instruments). It was found that particles with a size of 50 to 150 nm were present (see Figure 3), confirming the presence of exosome-like extracellular vesicles in the PBS suspension.

[0089] (Test Example 4) <Melanin production inhibitory effect test> The melanin production inhibitory effect test was conducted in the same manner as in Test Example 1, except that 3 (v / v)% fermented milk whey was added in Test Example 1, and the PBS suspension containing exosome-like extracellular vesicles prepared in Test Example 3 was added to the following concentrations. In addition, in the same manner as in Test Example 1, a test was also carried out in the case where fermented milk whey was added. [Amount of PBS suspension added] The protein concentration of the PBS suspension was measured, and based on that concentration, the particle concentrations in the medium were adjusted to one of the following concentrations and added. 0.5μg (protein amount) / mL 1μg (protein amount) / mL 5μg (protein) / mL 10μg (protein amount) / mL

[0090] The results are shown in Figure 4. 4, the horizontal axis represents the samples tested, as described below, and the vertical axis represents the relative amount of melanin produced (%) when the amount of melanin produced in Control I, as described below, is taken as 100%. I (Control I) Melanin production inducer: None added; fermented milk whey and PBS suspension: None added. II (Control II) Melanin production inducer: added; fermented milk whey and PBS suspension: not added. 4-1 Melanin production inducer: added, fermented milk whey and PBS suspension: fermented milk whey prepared using the standard strain of Lactobacillus helveticus was added. 4-2 Melanin production inducer: added, fermented milk whey and PBS suspension: PBS suspension was added (amount to give a particle concentration of 0.5 μg (protein amount) / mL). 4-3 Melanin production inducer: added, fermented milk whey and PBS suspension: PBS suspension was added (amount to give a particle concentration of 1 μg (protein amount) / mL). 4-4 Melanin production inducer: added, fermented milk whey and PBS suspension: PBS suspension was added (amount to give a particle concentration of 5 μg (protein amount) / mL). 4-5 Melanin production inducer: added, fermented milk whey and PBS suspension: PBS suspension was added (amount to give a particle concentration of 10 μg (protein amount) / mL).

[0091] In Control II, the amount of melanin produced was significantly increased compared to Control I (p<0.001). As in Test Example 1, in Sample 4-1 to which fermented milk whey was added, the amount of melanin produced was significantly decreased compared to Control II (p<0.001). Furthermore, in samples 4-2 to 4-5, which were supplemented with a PBS suspension containing exosome-like extracellular vesicles from lactic acid bacteria-fermented milk whey, melanin production decreased in a concentration-dependent manner, and was significantly reduced compared to control II (samples 4-2 and 4-3: p<0.05, samples 4-4 and 4-5: p<0.001).

[0092] In addition, a WST-1 assay to measure cell viability was performed simultaneously with the above test. Even when a PBS suspension containing exosome-like extracellular vesicles was added to a particle concentration of 50 μg (protein amount) / mL, no decrease in cell number was observed.

[0093] (Test Example 5) <Expression levels of genes related to melanin production> The expression levels of genes related to melanin production were examined as follows. Melanoma B16 cells were seeded onto plates (seeding density: 3,000 cells / cm). 2 ), and 24 hours later, a melanin production inducer (α-MSH) was added (amount added: 50 nM) to induce melanin production. Furthermore, 48 hours after the addition of the α-MSH, fermented milk whey prepared using the standard strain of Lactobacillus helveticus prepared in Test Example 1 above (addition amount: 3 (v / v)%) or a PBS suspension containing exosome-like extracellular vesicles prepared in Test Example 3 above (addition amount: an amount to give a particle concentration of 1 μg (protein amount) / mL or 10 μg (protein amount) / mL) was added together with the α-MSH (addition amount: 50 nM), and the cells were cultured for 4 days. After culturing, RNA was extracted from the cells, and the mRNA expression levels of tyrosinase (Tyr), tyrosinase-related protein 1 (Trp1), and tyrosinase-related protein 2 (Dct), which are related to melanin production, were confirmed by real-time PCR.

[0094] The results are shown in Figures 5 to 7. FIG. 5 shows the results of measuring the expression level of Tyr mRNA, FIG. 6 shows the results of measuring the expression level of Trp1 mRNA, and FIG. 7 shows the results of measuring the expression level of Dct mRNA. 5 to 7, the horizontal axis represents the samples tested, as described below, and the vertical axis represents the relative expression level (%) of Tyr, Trp1, or Dct mRNA when the expression level of Tyr, Trp1, or Dct mRNA in Control I, as described below, is set to 100%. I (Control I) Melanin production inducer: None added; fermented milk whey and PBS suspension: None added. II (Control II) Melanin production inducer: added; fermented milk whey and PBS suspension: not added. 5-1 Melanin production inducer: Added. Fermented milk whey prepared using the standard strain of Lactobacillus helveticus was added. 5-2 Melanin production inducer: added, PBS suspension was added (amount to give particle concentration of 1 μg (protein amount) / mL). 5-3 Melanin production inducer: added, PBS suspension was added (amount to give a particle concentration of 10 μg (protein amount) / mL).

[0095] As shown in Figure 5, the expression level of Tyr mRNA was significantly increased in Control II compared to Control I (p<0.001). The expression level of Tyr mRNA was significantly decreased in Sample 5-1, which contained fermented milk whey, compared to Control II (p<0.001). Furthermore, Samples 5-2 to 5-3, which contained a PBS suspension containing exosome-like extracellular vesicles from lactobacillus-fermented milk whey, decreased the expression level of Tyr mRNA in a concentration-dependent manner, and in particular, Sample 5-3 showed a significantly decreased expression level of Tyr mRNA compared to Control II (p<0.001).

[0096] As shown in Figure 6, the expression level of Trp1 mRNA was significantly increased in Control II compared to Control I (p<0.05). Sample 5-1, to which fermented milk whey was added, had a significantly decreased expression level of Trp1 mRNA compared to Control II (p<0.05). Furthermore, Samples 5-2 to 5-3, to which a PBS suspension containing exosome-like extracellular vesicles from lactic acid bacteria-fermented milk whey was added, decreased the expression level of Trp1 mRNA in a concentration-dependent manner. In particular, Sample 5-3 had a significantly decreased expression level of Trp1 mRNA compared to Control II (p<0.05).

[0097] As shown in Figure 7, Dct mRNA expression levels were significantly increased in Control II compared to Control I (p<0.001). Sample 5-1, to which fermented milk whey was added, showed significantly decreased Dct mRNA expression levels compared to Control II (p<0.001). Furthermore, Samples 5-2 to 5-3, to which a PBS suspension containing exosome-like extracellular vesicles from lactobacillus-fermented milk whey was added, showed a concentration-dependent decrease in Dct mRNA expression levels. In particular, Sample 5-3 showed significantly decreased Dct mRNA expression levels compared to Control II (p<0.001).

[0098] From the above, it was confirmed that the addition of exosome-like extracellular vesicles contained in lactobacillus-fermented milk whey significantly reduced the expression level of genes related to melanin production.

[0099] (Test Example 6) The samples used in this test were prepared as follows.

[0100] [Suspension containing exosome-like extracellular vesicles from lactobacillus-fermented milk whey] The PBS suspension containing exosome-like extracellular vesicles prepared in Test Example 3 above was used in this test.

[0101] [Suspension containing exosome-like extracellular vesicles contained in lactic acid bacteria culture medium] Lactobacillus helveticus JCM1120 strain was inoculated into MRS liquid medium (final inoculation volume: 5.0 × 10 7 The cells were cultured at 37°C for 24 hours at a concentration of 10 ... The obtained lactic acid bacteria culture solution was treated in the same manner as in <Preparation of exosome-like extracellular vesicles> in Test Example 3 above to prepare a PBS suspension containing exosome-like extracellular vesicles.

[0102] [Suspension containing exosome-like extracellular vesicles in skim milk solution] An aqueous solution containing skim milk powder at a concentration of 9% by mass was prepared. The obtained skim milk solution was treated in the same manner as in <Preparation of exosome-like extracellular vesicles> in Test Example 3 above to prepare a PBS suspension containing exosome-like extracellular vesicles.

[0103] <Average particle size> The size of the particles (exosome-like extracellular vesicles) contained in each of the obtained PBS suspensions was confirmed using a particle size distribution analyzer (NanoSight NS300, Malvern Panalytical). The volume-average particle diameter of each exosome-like extracellular vesicle was as follows: Exosome-like extracellular vesicles contained in lactobacillus-fermented milk whey: 132nm Exosome-like extracellular vesicles contained in lactic acid bacteria culture medium: 130nm Exosome-like extracellular vesicles contained in skim milk solution: 113nm

[0104] <Melanin production inhibitory effect test> The melanin production inhibitory effect test was conducted in the same manner as in Test Example 1, except that 3 (v / v)% fermented milk whey was added in the test, and the PBS suspension containing exosome-like extracellular vesicles prepared above was added so that the particle concentration was 1 μg (protein amount) / mL or 10 μg (protein amount) / mL. In addition, in the same manner as in Test Example 1, a test was also carried out in the case where fermented milk whey was added.

[0105] The results are shown in Figure 8. 8, the horizontal axis represents the samples tested, as described below, and the vertical axis represents the relative amount of melanin produced (%) when the amount of melanin produced in Control I, as described below, is taken as 100%. I (Control I) Melanin production inducer: None added; fermented milk whey and PBS suspension: None added. II (Control II) Melanin production inducer: added; fermented milk whey and PBS suspension: not added. 6-1 Melanin production inducer: added, fermented milk whey and PBS suspension: fermented milk whey prepared using the standard strain of Lactobacillus helveticus was added. 6-2-1 Melanin production inducer: added, fermented milk whey and PBS suspension: a suspension containing exosome-like extracellular vesicles contained in lactic acid bacteria-fermented milk whey was added (amount to make the particle concentration 1 μg (protein amount) / mL). 6-2-2 Melanin production inducer: added; fermented milk whey and PBS suspension: a suspension containing exosome-like extracellular vesicles contained in lactic acid bacteria-fermented milk whey was added (amount to make the particle concentration 10 μg (protein amount) / mL). 6-3-1 Melanin production inducer: added; fermented milk whey and PBS suspension: added a suspension containing exosome-like extracellular vesicles contained in lactic acid bacteria culture medium (amount to make particle concentration 1 μg (protein amount) / mL). 6-3-2 Melanin production inducer: added; fermented milk whey and PBS suspension: added a suspension containing exosome-like extracellular vesicles contained in lactic acid bacteria culture medium (amount to make particle concentration 10 μg (protein amount) / mL). 6-4-1 Melanin production inducer: added, fermented milk whey and PBS suspension: a suspension containing exosome-like extracellular vesicles contained in skim milk solution was added (amount to make particle concentration 1 μg (protein amount) / mL). 6-4-2 Melanin production inducer: added, fermented milk whey and PBS suspension: a suspension containing exosome-like extracellular vesicles contained in skim milk solution was added (amount to make particle concentration 10 μg (protein amount) / mL).

[0106] In Control II, the amount of melanin produced was significantly increased compared to Control I (p<0.001). As in Test Example 1, in Sample 6-1 to which fermented milk whey was added, the amount of melanin produced was significantly decreased compared to Control II (p<0.001). Furthermore, in samples 6-2-1 and 6-2-2, which contained a PBS suspension containing exosome-like extracellular vesicles from lactobacillus-fermented milk whey, melanin production was significantly reduced compared to control II (sample 6-2-1: p<0.01, sample 6-2-2: p<0.001). On the other hand, the addition of a suspension containing exosome-like extracellular vesicles contained in a lactic acid bacteria culture medium (samples 6-3-1 and 6-3-2) and a suspension containing exosome-like extracellular vesicles contained in a skim milk solution (samples 6-4-1 and 6-4-2) had no effect on melanin production.

[0107] Based on the above results, the component that is presumed to have an inhibitory effect on melanin production is thought to be a particle with a diameter of 50 to 150 nm derived from lactic acid bacteria, secreted into fermented milk whey obtained when milk is fermented with lactic acid bacteria. Furthermore, the mechanism of action is presumed to be a decrease in the expression of genes related to melanin production. [Accession number]

[0108] FERM BP-6060

Claims

1. A melanin production inhibitor characterized by containing particles secreted in fermented milk whey obtained by fermenting milk with lactic acid bacteria as an active ingredient.

2. 2. The melanin production inhibitor according to claim 1, wherein the particle diameter is 50 to 150 nm.

3. 2. The melanin production inhibitor according to claim 1, wherein the particles are precipitated by ultracentrifugation of the fermented milk whey at 210,000 x g or more.

4. 2. The melanin production inhibitor according to claim 1, wherein the lactic acid bacterium is at least one selected from the group consisting of Lactobacillus delbrueckii bulgaricus, Lactobacillus helveticus, Lactobacillus acidophilus, and Lactobacillus casei.

5. 2. The melanin production inhibitor according to claim 1, wherein the lactic acid bacterium is Lactobacillus helveticus.

6. A whitening composition comprising the melanin production inhibitor according to any one of claims 1 to 5.

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