Skin anti-aging agent using deep sea water and method for preventing skin aging using said skin anti-aging agent

Abstract

To provide an anti-skin-aging agent comprising deep sea water and specific lactic acid bacteria derived from deep sea water as active ingredients to promote collagen synthesis to effectively prevent wrinkles, stains, etc., and enhance the anti-aging effect of the skin, and to provide an anti-skin-aging method.SOLUTION: An anti-skin-aging agent comprises deep sea water and lactic acid bacteria or a fermented product thereof. The lactic acid bacteria is Lactobacillus plantarum LPAK1-13 strain (accession number: NITE P-03021) derived from deep sea water.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] The present invention relates to a skin anti-aging agent using deep sea water and a skin anti-aging method using the novel skin anti-aging agent, and in particular to a novel skin anti-aging agent containing deep sea water and specific lactic acid bacteria as active ingredients, which can promote collagen synthesis in the skin and further inhibit the decomposition of proteoglycan, thereby improving wrinkles, dark circles under the eyes, dullness, and the like caused by aging and the like, and a skin anti-aging method using the novel skin anti-aging agent. [Background technology]

[0002] It is known that as people age, the production of human dermal matrix components such as collagen and proteoglycan declines, causing wrinkles. Age also reduces the body's defense mechanism, and the ability to defend cells against external stimuli. These factors are said to be deeply involved in the development of skin aging symptoms, particularly wrinkles, dullness, dark circles under the eyes, and sagging skin. It is known that the amount of collagen and proteoglycan in the skin is reduced by such aging and various external stimuli including the effects of exposure to ultraviolet rays and dryness, the moisturizing function and elasticity of the skin are reduced, the skin loses its firmness and luster, and the skin aging symptoms such as wrinkles, dullness, and dark circles are accelerated. In particular, wrinkles and sagging are more prominent in areas exposed to sunlight, and the phenomenon in which wrinkles and sagging occur on skin exposed to sunlight is specifically called photoaging. It is also known that the amount of collagen in the dermis is significantly reduced in photoaged wrinkled areas.

[0003] As described above, preventing and improving wrinkles, dullness, dark circles under the skin, and other skin problems that occur with aging and photoaging is an important cosmetic issue.

[0004] Collagen is a fibrous protein that has a structure in which three polypeptide chains with a molecular weight of 95,000 are intertwined and is present in most tissues such as skin, blood vessels, tendons, teeth, etc., and accounts for approximately 30% of all proteins that make up the body. Collagen also accounts for more than 90% of the dermal matrix, and it is known that its decomposition is accelerated by collagenase activated by ultraviolet light. The dermis also contains proteoglycan, which is one of the components of the dermal matrix and contributes to moisture retention in the skin. Proteoglycan is known to be broken down by aggrecanase (a proteoglycan-degrading enzyme) with aging, causing wrinkles, dark spots, and dark circles under the eyes.

[0005] For these reasons, it is believed that a decrease in dermal matrix components such as collagen and proteoglycan in the skin is the main cause of wrinkles, dullness, dark circles under the eyes, etc. Therefore, if an agent capable of inhibiting the decrease in collagen and proteoglycan could be provided, it would be possible to prevent and improve not only photoaging, but also wrinkles and sagging skin, as well as age-related dark spots and dullness.

[0006] On the other hand, deep ocean water is seawater at a depth of 200m or more, which is the euphotic zone (compensation depth), where organic matter production by photosynthesis is almost nonexistent and decomposition is predominant.In addition, because it contains a large amount of inorganic nutrients, various uses have been attempted.

[0007] As an example of a cosmetic product that uses deep-sea water, JP 2016-94372 A (Patent Document 1) filed by the present applicant describes a high-absorption composition that combines deep-sea water and a carotenoid in order to increase the absorbability of the carotenoid in the body. The carotenoid is preferably derived from citrus fruits, and more preferably the citrus-derived carotenoid is cryptoxanthin. The deep-sea water is raw water or a mixture of raw water and deep-sea water, which is freshwater obtained by desalination using reverse osmosis. The carotenoid high-absorption composition disclosed is characterized in that the carotenoid is preferably derived from citrus fruits, and more preferably the citrus-derived carotenoid is cryptoxanthin, and the deep-sea water is raw water or a mixture of raw water and freshwater obtained by desalination using reverse osmosis.

[0008] In addition, JP 2018-199629 A (Patent Document 2) filed by the present applicant discloses a method for improving skin moisturizing properties in which deep-sea water accounts for 30% or more by weight of the total, and deep-sea water is used in combination with a polyol in a weight ratio of 9:1 to 3:7, wherein the polyol is at least one selected from the group consisting of glycerin, diglycerin, sorbitol, propylene glycol, 1,3-butylene glycol, propanediol, dipropylene glycol, glycereth-26, pentylene glycol, hexanediol, maltitol, xylitol, and caprylyl glycol.

[0009] Furthermore, JP 2008-297205 A (Patent Document 3) discloses a cosmetic product that uses deep sea water together with animal collagen derived from black pigs, specifically describing the use of deep sea water together with purified water to activate dermal connective tissue.

[0010] However, these conventional products do not contain deep sea water, lactic acid bacteria isolated from the deep sea water, or fermentation products thereof. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] JP 2016-94372 A [Patent Document 2] JP 2018-199629 A [Patent Document 3] JP 2008-297205 A Summary of the Invention [Problem to be solved by the invention]

[0012] The object of the present invention is to provide a skin anti-aging agent and a skin anti-aging method which contain deep sea water and specific lactic acid bacteria as active ingredients, thereby promoting collagen synthesis and preventing wrinkles, dullness and dark circles under the eyes, as well as enhancing the anti-aging effect of the skin. In particular, the object of the present invention is to provide a skin anti-aging agent and a skin anti-aging method which, by using lactic acid bacteria derived from deep-sea water, can effectively exhibit, together with deep-sea water, an excellent function of promoting collagen production and a function of inhibiting the decomposition of proteoglycan. [Means for solving the problem]

[0013] As a result of extensive research, the inventors have discovered that by combining specific lactic acid bacteria and / or their fermentation products with deep sea water, when applied to the skin, the collagen synthesis promoting effect is improved compared to deep sea water alone, and skin aging such as wrinkles, dullness and dark circles under the skin is prevented, and further that the function of inhibiting the breakdown of proteoglycan can be exerted, which led to the present invention.

[0014] The skin anti-aging agent according to claim 1 is characterized in that it contains deep sea water and lactic acid bacteria and / or a fermentation product thereof, and the lactic acid bacteria is Lactobacillus plantarum LPAK1-13 strain (accession number NITE P-03021) derived from deep sea water.

[0015] Furthermore, Claim 1 The skin aging inhibitor described in The above deep ocean water but , deep sea water treated by reverse osmosis filtration, deep sea water treated by electrodialysis, or deep sea water treated by ion exchange, which has been subjected to ion selective processing so that the mass ratio of potassium ions, sodium ions, calcium ions, magnesium ions, and chloride ions in the deep sea water is K:Na:Ca:Mg:Cl=1:30-70:10-40:40-150:200-500. Deep sea water, the content of which is 20% by mass or more, preferably 40% by mass or more of the total. The present invention is a skin anti-aging agent characterized by comprising:

[0016] The method for preventing skin aging according to claim 3 comprises: the above The method for preventing skin aging is characterized by applying a cosmetic composition containing a skin aging inhibitor to the skin. Effect of the Invention

[0017] According to the skin anti-aging agent of the present invention, by using deep sea water, preferably deep sea water that has been subjected to ion selective treatment, in combination with a specific lactic acid bacterium derived from deep sea water and / or a fermented product thereof, when applied to the skin, it is possible to effectively prevent skin aging such as wrinkles, dullness, dark circles under the eyes, etc. by increasing collagen in the dermis. Furthermore, by suppressing the decomposition of proteoglycan, which is one of the components of the dermal matrix, it is possible to effectively prevent skin aging such as wrinkles, dullness, dark circles under the eyes, etc. Therefore, by using the skin anti-aging agent of the present invention, which contains deep sea water and specific lactic acid bacteria as active ingredients, it is possible to apply it to cosmetics such as lotions, milky lotions, creams, essences, packs, foundations, and body lotions that are applied directly to the skin, thereby preventing aging such as wrinkles, dullness, and dark circles under the skin. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a diagram showing an example of a method for isolating the lactic acid bacteria of the present invention from deep sea water. [Diagram 2] FIG. 1 shows an example of an evaluation of the collagen synthesis promoting effect of ED mineral water. [Diagram 3] FIG. 1 is a diagram showing an example of evaluation of the collagen synthesis promoting effect of the fermentation liquid of the lactic acid bacteria used in the present invention and that of existing lactic acid bacteria. [Figure 4] FIG. 1 shows an example of an evaluation of the collagen synthesis promoting effect of purified water (DW), ED mineral water, ED mineral water + fermentation liquid of the lactic acid bacteria used in the present invention, and ED mineral water + fermentation liquid of existing lactic acid bacteria. [Diagram 5] FIG. 1 shows an example of evaluation of the degree of inhibition of proteoglycan decomposition. [Figure 6] FIG. 1 shows an example of evaluation of the expression level of the glucuronyltransferase gene. [Figure 7] FIG. 1 shows an example of evaluation of antioxidant activity against hydrogen peroxide. [Figure 8] FIG. 1 is a diagram showing an example of evaluation of the wrinkle improving effect of a lotion containing the skin anti-aging agent of the present invention. [Figure 9] FIG. 2 is a diagram showing an example of evaluation of the effect of a lotion containing the skin anti-aging agent of the present invention on alleviating dark circles under the eyes. [Figure 10] FIG. 2 is a diagram showing an example of evaluation of the dullness-improving effect of a lotion containing the skin aging inhibitor of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention will be described in detail based on the following embodiments. The skin anti-aging agent of the present invention is characterized in that it contains deep-sea water and lactic acid bacteria and / or a fermentation product thereof, and the lactic acid bacteria is Lactobacillus plantarum LPAK1-13 strain (accession number NITE P-03021) derived from deep-sea water. Unless otherwise specified, % indicates % by mass.

[0020] In the present invention, by using deep sea water and the specific lactic acid bacteria and / or fermented products thereof derived from deep sea water in combination, collagen production in the skin can be promoted and skin aging can be alleviated. Also, degradation of proteoglycan, which is a component of the dermal matrix, can be inhibited, thereby alleviating skin aging. In particular, it is desirable for the skin anti-aging agent to contain deep sea water at preferably 20% by mass or more, more preferably 40% by mass or more.

[0021] The skin anti-aging agent of the present invention contains deep sea water as an active ingredient. The "deep-sea water" in the present invention includes the raw deep-sea water itself from a depth of 200 m or more, as well as water obtained by electrodialysis of deep-sea water (ED salt water, ED mineral water), water obtained by reverse osmosis filtration (RO membrane), water obtained by ion exchange membrane treatment, and any treated water that is a liquid containing minerals equivalent to those contained in deep-sea water.

[0022] Deep seawater (DSW) is generally low-temperature, clean, hygienic water found more than 200 m below sea level that is rich in sodium, potassium, calcium, magnesium, and other inorganic nutrients, and is safe and stable. There are currently 15 intake facilities in nine prefectures, including Ito City, Shizuoka Prefecture (Izu Akazawa), for collecting deep sea water (according to the Deep Sea Water Utilization Society website, as of February 2017). Any of the deep sea water collected from these intake locations can be used in the present invention, and it is also possible to use deep sea water collected from locations other than the above mentioned intake locations.

[0023] Particularly preferred is the Izu Akazawa deep sea water, which is pumped up from a depth of about 800 m off the coast of Izu Akazawa and has a microorganism abundance ratio of about one several thousandth of that of surface water. In addition, the Izu Akazawa deep sea water is a place where uncontaminated clean deep sea water can be taken, despite being the closest to the capital and metropolitan areas, because the Kuroshio Current, which flows northeastward in the ocean surface layer near the Izu Peninsula, and the Izu-Ogasawara Arc, which is made up of a group of high underwater volcanoes on the southeast coast of the Izu Peninsula, are formed. Therefore, the Izu Akazawa deep sea water can be effectively applied to the present invention.

[0024] As deep sea water that is preferably used in the present invention, particularly preferred is deep sea water that has been subjected to ion selection processing to set the ions contained in the collected deep sea water to a specific ratio. By setting the ions to such a specific ratio, the above-mentioned effects of the present invention can be particularly effectively exhibited, and further, irritation to the skin caused by salt, etc. can be alleviated.

[0025] The method for processing the ions contained in the raw deep-sea water to a specific ratio is not particularly limited as long as it can achieve the specific ion ratio described below. For example, ion selection processing can be performed using known reverse osmosis membrane filtration, electrodialysis, ion exchange membrane methods, etc. In addition, any other known method for selectively treating raw deep-sea water with ions can be applied as long as it can achieve the specific ion ratios described below.

[0026] For example, the method of selectively treating ions may be a method of desalination using an electrodialysis apparatus "Ashiraiser 25 type 100 pairs x 3 stages continuous type (seawater desalination specification) manufactured by Astom Corporation" to desalinate and concentrate ionic substances in raw deep-sea water by the action of an ion exchange membrane and a direct current using electrodialysis (ion exchange membrane method). Furthermore, as disclosed in JP 2000-159655 A, a method of desalination of deep-sea water using a reverse osmosis membrane apparatus (HR5155 manufactured by Toyobo Co., Ltd.) and then desalination using an electrodialysis apparatus (Selemion CMV, Selemion ASV manufactured by Asahi Glass Co., Ltd.) may be applied to obtain a specific ion ratio as described below. Alternatively, a specific ion ratio may be obtained by using a reverse osmosis membrane desalination method as described in JP 2010-274214 A.

[0027] As the ion selective treatment, it is desirable to subject deep sea water to ion selective treatment so that the ratio of potassium ions, sodium ions, calcium ions, magnesium ions, and chlorine ions contained therein is about K:Na:Ca:Mg:Cl=1:30-70:10-40:40-150:200-500. Specifically, it is desirable to use ED mineral water, which is deep sea water that has been treated so that, for example, in 100 g of deep sea water, K is about 0-0.1 g, Na is about 0.01-0.50 g, Ca is about 0.01-0.04 g, Mg is about 0.05-0.5 g, and Cl is about 0.1-1.0 g. By using ion-selectively treated deep sea water (ED mineral water) having the above-mentioned mineral composition in combination with the above-mentioned specific lactic acid bacteria (accession number NITE P-03021), it is possible to particularly effectively enhance the moisture retention ability of the stratum corneum of the skin and improve moisturizing properties. A comparison of the composition of surface seawater from Akazawa, Izu, deep ocean water (raw water) from Akazawa, and an example of ED mineral water is shown in Table 1 below.

[0028] [Table 1]

[0029] Generally, the ratio of potassium ions, sodium ions, calcium ions, magnesium ions, and chloride ions in natural bittern is about K:Na:Ca:Mg:Cl=1:2-25:0-1:1-5:10-50, and the mineral composition of an aqueous solution using natural bittern is significantly different from that of deep seawater that has been subjected to ion-selective treatment. In addition, it is not desirable to apply an aqueous solution using bittern to the present invention. For example, deep sea water (ED mineral water) obtained by selectively treating ions of deep sea water from Akazawa, Izu using the above-mentioned electrodialysis method (electrodialysis device "Ashiraizer 25 type, manufactured by Astom Corporation"), deep sea water (ED mineral water) from Namerikawa, Toyama Prefecture, and various bitterns have the composition of ion components shown in Table 2 below.

[0030] [Table 2]

[0031] Furthermore, the electrical conductivity of the deep sea water used in the present invention is desirably 0 to 30 mS / cm, and more preferably 5 to 15 mS / cm, in order to more effectively exert the effects of the present invention.

[0032] The lactic acid bacteria contained in the skin anti-aging agent of the present invention are Lactobacillus plantarum, and in particular, are lactic acid bacteria belonging to Lactobacillus plantarum and derived from deep sea water, and Lactobacillus plantarum LPAK1-13 strain (accession number NITE P-03021) is preferably used. Here, the "LPAK1-13" strain is a number given to the novel strain by the present applicant.

[0033] The Lactobacillus plantarum LPAK1-13 strain used in the present invention was received at the Patent Microorganism Deposit Center of the National Institute of Technology and Innovation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) on September 19, 2019 under the accession number NITE AP-03021 and has also been deposited under NITE P-03021 (Deposit Certificate Notification No. 2019-0373).

[0034] The above-mentioned strain is derived from deep sea water, is separated from deep sea water, and is contained in the skin anti-aging agent of the present invention. The method for isolating the above-mentioned lactic acid bacteria from deep sea water is not particularly limited as long as it is possible to isolate the lactic acid bacteria. However, the following method can be exemplified, and an overview of the method is shown in FIG. 1. As an example shown in Figure 1, the bottom of a bag filter (pore size 0.5 μm) used to filter suspended matter in the taken deep seawater is aseptically cut into 3 cm squares, which are then placed in sterilized deep seawater and stirred to prepare a suspension. The prepared suspension is then smeared onto MRS seawater agar medium (containing 0.005% cycloheximide: Table 3), which is a selective medium for lactic acid bacteria, and cultured at 27°C in the dark under stationary conditions. After culture, the colonies that appear on the agar medium are isolated, and the 16S rDNA is analyzed to identify and isolate them as lactic acid bacteria (Lactobacillus plantarum). The isolated lactic acid bacteria was named Lactobacillus plantarum LPAK1-13 strain (LPAK1-13 lactic acid bacteria).

[0035] [Table 3]

[0036] The mycological properties of the Lactobacillus plantarum LPAK1-13 strain used in the present invention are shown in Tables 4 and 5 below. Table 4 shows the properties of this strain, and Table 5 shows the results of testing its sugar assimilation ability. In Table 4, "+" indicates fermentable and "-" indicates non-fermentable. In addition, the LPAK1-13 strain in Table 4 is the Lactobacillus plantarum LPAK1-13 strain (accession number NITE P-03021), and the type culture strain means the type strain, specifically the Lactobacillus plantarum JCM11125 strain (type culture strain).

[0037] [Table 4]

[0038] [Table 5]

[0039] The Lactobacillus plantarum LPAK1-13 strain (accession number NITE P-03021), isolated from deep seawater, can promote collagen production in the skin and reduce skin aging. It also inhibits the decomposition of proteoglycan, a component of the dermal matrix, making it possible to reduce skin aging and has an antioxidant effect.

[0040] It is also possible to prepare a fermentation liquid using Lactobacillus plantarum LPAK1-13 strain (accession number NITE P-03021) isolated from deep sea water, and such a fermentation liquid can be used together with deep sea water as an active ingredient in the skin anti-aging agent of the present invention in place of the above-mentioned LPAK1-13 lactic acid bacteria itself, or in combination with it. For example, a fermentation liquid can be obtained by preparing a solution of glucose, yeast extract, and purified water, sterilizing it, inoculating it with Lactobacillus plantarum LPAK1-13 strain (accession number NITE P-03021), and allowing it to stand at 30°C for 3 days. In addition to the glucose and yeast extract, soy milk, vegetables, etc. may also be used to prepare the fermentation liquid.

[0041] Furthermore, in the present invention, deep sea water is combined with LPAK1-13 lactic acid bacteria and / or a lactic acid bacteria fermentation liquid (calculated as lactic acid bacteria), and it is desirable to set the final concentration of the LPAK1-13 lactic acid bacteria or the lactic acid bacteria fermentation liquid to 0.01 to 50.00%, preferably 0.10 to 5.00%, in order to particularly effectively express the effects of the present invention.

[0042] As described above, by combining deep sea water with Lactobacillus plantarum LPAK1-13 strain, which is a lactic acid bacterium of the Lactobacillus genus, it is possible to promote collagen production in the skin and prevent skin aging such as wrinkles, dark spots, dark circles under the eyes, etc. In addition, it is possible to inhibit the decomposition of proteoglycan, which is one of the matrix components in the dermis of the skin, which also makes it possible to inhibit skin aging.

[0043] The dermis contains proteoglycan, a component that contributes to the skin's moisture retention. As the skin ages, proteoglycan is broken down by aggrecanase (a proteoglycan-degrading enzyme), causing wrinkles, dark spots, and dark circles under the eyes. The skin anti-aging agent of the present invention suppresses the expression of the aggrecanase gene, thereby inhibiting the breakdown of proteoglycan. Furthermore, the skin anti-aging agent of the present invention can increase the expression level of glucuronyltransferase I, an enzyme that contributes to the biosynthesis of glycosaminoglycans such as hyaluronic acid, thereby making it possible to inhibit skin aging.

[0044] In addition, other ingredients may be contained as long as they do not inhibit the effects of the present invention. Examples of such other ingredients include known ultraviolet absorbers and ultraviolet blockers, whitening agents, anti-inflammatory agents, animal and vegetable oils, waxes, polyhydric alcohols, fluorinated oils, higher alcohols, higher fatty acids, other surfactants such as nonionic surfactants, anionic surfactants, and amphoteric surfactants, antioxidants, fragrances, preservatives, pH adjusters, etc.

[0045] The method for preventing skin aging of the present invention is a method for preventing skin aging by incorporating the above-mentioned skin aging inhibitor of the present invention into a conventional cosmetic composition and bringing the composition into contact with the skin, and can be applied to skin in any state, whether it is aged skin or skin that has not yet shown signs of aging such as wrinkles, sagging, dullness, or blemishes.

[0046] The method for bringing the cosmetic preparation containing the skin aging inhibitor of the present invention into contact with the skin is not particularly limited, and can be carried out, for example, by applying the cosmetic preparation containing the skin aging inhibitor of the present invention directly to the skin or by diluting and applying the cosmetic preparation.

[0047] For example, the skin anti-aging agent of the present invention can be applied in the form of cosmetics such as lotion, milky lotion, cream, essence, pack, foundation, and body lotion that are applied directly to the skin. Furthermore, it is expected that it can be used in foods (especially health foods) by combining it with ingredients that are not harmful to humans. EXAMPLES

[0048] The present invention will be described with reference to the following examples, comparative examples and test examples, but is not limited thereto. (1) Preparation of deep sea water (ED mineral water) The deep sea water contained in the skin anti-aging agent is deep sea water pumped up from a depth of about 800m off the coast of Akazawa, Izu (Izu Akazawa origin, latitude 34°50'19" north, longitude 139°08'11" east, depth 800m, stored at 4℃ after intake), which has been subjected to ion selective treatment using the following method to obtain electrodialysis water (deep sea water (ED mineral water)) (electrical conductivity 12.5mS / cm). Specifically, using an electrodialysis device "Astrom Corporation's Acilyser 25 type 100 pairs x 3 stages continuous type (seawater desalination specifications)", deep seawater from Akazawa, Izu was subjected to electrodialysis (ion exchange membrane method) using an ion exchange membrane to apply a direct current to desalt and concentrate the ionic substances present in the raw deep seawater, and after the desalting process, ion-treated deep seawater (ED mineral water) was obtained. The obtained deep sea water (ED mineral water) has the component composition shown in Table 2 above (produced in Akazawa, Izu).

[0049] In addition, the K, Na, Ca, and Mg ion contents in the obtained deep sea water (ED mineral water) were measured using a multi-type ICP optical emission spectrometer (manufactured by Shimadzu Corporation), and the Cl content was measured using a high-performance liquid chromatograph (manufactured by Shimadzu Corporation) (Table 2). Next, 20% (w / w) ED mineral water was prepared by blending ED mineral water:purified water at a ratio of 1:4, 40% (w / w) ED mineral water:purified water at a ratio of 2:3, 60% (w / w) ED mineral water at a ratio of 3:2, and 80% (w / w) ED mineral water at a ratio of 4:1.

[0050] (2) Isolation of lactic acid bacteria from deep sea water 1) The bottom of a bag filter (pore size 0.5 μm) used to filter deep ocean water taken from a depth of 800 m off the coast of Akazawa, Izu, was aseptically cut into 3 cm squares, which were then placed in the sterilized deep ocean water and stirred to prepare a suspension. 2) Next, the suspension was smeared onto MRS seawater agar medium (containing 0.005% cycloheximide: Table 3 above), which is a selective medium for lactic acid bacteria, and left to stand in the dark at a temperature of 27°C for 3 days. 3) After cultivation, colonies that appeared on the agar medium were collected using a platinum loop (sterilized rod), and 16S rDNA was analyzed to confirm that they were lactic acid bacteria (Lactobacillus plantarum). In this manner, the lactic acid bacterium Lactobacillus plantarum LPAK1-13 strain (accession number NITE P-03021) was obtained.

[0051] (3) Preparation of lactic acid bacteria fermentation liquid In the present invention, the fermentation liquid of Lactobacillus plantarum LPAK1-13 strain lactic acid bacteria refers to one prepared by the following steps. A) LPAK1-13 lactic acid bacteria fermentation liquid The lactic acid bacteria Lactobacillus plantarum LPAK1-13 strain isolated from deep seawater in (2) above was cultured in the same MRS seawater liquid medium (Table 3 above) as described in (2) 2) above, and the resulting culture solution was centrifuged, the supernatant was removed, and the cells were collected by sedimentation. The obtained cells were washed with sterile physiological saline, and the washed cells were suspended in sterile physiological saline to obtain a cell suspension. Next, yeast extract (Kokubase, manufactured by Dai-Nippon Meiji Sugar Co., Ltd.) was added to 1% (w / w), glucose (D-glucose, manufactured by Wako Pure Chemical Industries, Ltd.) to 1% (w / w), phosphate buffer to 25 μM, GP-1 (manufactured by Nisshin Pharma Co., Ltd.) to 0.08 (w / w)%, and hydrogenated lysolecithin (Resinol LL20, manufactured by Nikko Chemicals Co., Ltd.) to 0.05 (w / w)% in purified water. The mixture was then sterilized by heating at 121° C. for 20 minutes, and the Lactobacillus plantarum LPAK1-13 strain was cultured at 1×10 5The bacteria were inoculated at a concentration of 1.03 g / mL, fermented at 27° C. for 3 days, and the bacterial cells were removed using a φ0.2 μm filter to prepare a fermentation liquid of Lactobacillus plantarum LPAK1-13 strain lactic acid bacteria.

[0052] Type culture fermentation liquid Yeast extract (Kokubase, Dai-Nippon Meiji Sugar Co., Ltd.) was added to the purified water to make 1% (w / w), glucose (D-glucose, Wako Pure Chemical Industries, Ltd.) to make 1% (w / w), phosphate buffer to make 25 μM, GP-1 (Nisshin Pharma Co., Ltd.) to make 0.08% (w / w), and hydrogenated lysolecithin (Resinol LL20, Nikko Chemicals Co., Ltd.) to make 0.05% (w / w). After sterilization by heating at 121°C for 20 minutes, 1 × 10 type culture strain (Lactobacillus plantarum JCM11125) was added to the purified water to make 1% (w / w), glucose (D-glucose, Wako Pure Chemical Industries, Ltd.) to make 1% (w / w), phosphate buffer to make 25 μM, GP-1 (Nisshin Pharma Co., Ltd.) to make 0.08% (w / w), and hydrogenated lysolecithin (Resinol LL20, Nikko Chemicals Co., Ltd.) to make 0.05% (w / w). 5 The bacteria were inoculated at a concentration of 1000 cells / mL, fermented at 27°C for 3 days, and the bacterial cells were removed using a φ0.2 μm filter to prepare a fermentation liquid of Type culture strain lactic acid bacteria.

[0053] (4) Collagen synthesis test (1) (ED mineral water) Normal human skin fibroblasts were used, and the effect of adding the ED mineral water obtained in (1) above to the cells on collagen synthesis was tested by ELISA (human type I collagen). Specifically, Eagle's MEM medium (Nissui Pharmaceutical) was dissolved in purified water to a concentration of 0.94% (w / v), sterilized by heating at 121°C for 20 minutes, and then sterilized with a φ0.2μm filter. Sodium bicarbonate (Wako Pure Chemical Industries) was added to a concentration of 0.2%, L-glutamine (Wako Pure Chemical Industries) was added to a concentration of 4mM, and FBS (Corning) was added to a concentration of 10% (v / v) FBS to prepare a medium (Eagle's MEM medium containing 10% (v / v) FBS). Normal human skin fibroblasts (NHDF, Riken BioResource Research Center) were seeded in a 96-well microplate (AGC Technoglass Co., Ltd.) at 20,000 cells / well, and pre-cultured for 2 days at 37°C and 5% CO2.

[0054] After the preliminary culture, the Eagle MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) was dissolved in 20% (w / w), 40% (w / w), 60% (w / w), and 80% (w / w) ED mineral water prepared in (1) above so that the Eagle MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) became 0.94% (w / v), and the medium was sterilized by heating at 121°C for 20 minutes. After that, sodium bicarbonate (manufactured by Wako Pure Chemical Industries, Ltd.) sterilized with a φ0.2 μm filter was added to a concentration of 0.2%, L-glutamine (manufactured by Wako Pure Chemical Industries, Ltd.) to a concentration of 4 mM, and FBS (manufactured by Corning) to a concentration of 0.5%. In this medium (Eagle MEM medium containing 0.5% (v / v) FBS), evaluation culture was carried out for 1 day at 37°C and 5% CO2. After the evaluation culture, the culture supernatant was collected and the amount of collagen synthesis was evaluated by ELISA (human type I collagen). For comparison, purified water (DW) was used instead of ED mineral water, and the same evaluation culture was carried out for one day as above. The results are shown in Figure 2. The results in FIG. 2 when purified water (DW) was used are the values ​​showing no increase in collagen.

[0055] It can be seen from Figure 2 that ED mineral water has a collagen synthesis promoting effect. It can also be seen that the higher the ED mineral water content, the better the collagen synthesis promoting effect in cultured fibroblasts.

[0056] (5) Collagen synthesis test (2) (Lactic acid bacteria fermentation liquid) The following tests were carried out using the lactic acid bacteria fermentation liquid prepared in (3) above, LPAK1-13 lactic acid bacteria fermentation liquid and Type culture fermentation liquid, as lactic acid bacteria fermentation liquid. Normal human skin fibroblasts (NHDF, Riken BioResource Research Center) were seeded into a 24-well microplate at 115,000 cells / well and pre-cultured for 2 days at 37°C and 5% CO2 in Eagle's MEM medium containing 10% (v / v) FBS as in (4) above.

[0057] After the preliminary culture, 1) Eagle's MEM medium containing 0.5% (v / v) FBS prepared with purified water (DW) instead of the ED mineral water in (4) above 2) Eagle's MEM medium containing 0.5% (v / v) FBS, prepared using purified water (DW) instead of the ED mineral water in (4) above, and further containing the LPAK1-13 lactic acid bacteria fermentation liquid prepared in (3) above at a final concentration of 2% (w / w); and 3) Eagle's MEM medium containing 0.5% (v / v) FBS, prepared using purified water (DW) instead of the ED mineral water in (4) above, and further supplemented with the lactic acid bacteria fermentation liquid of Type culture (Lactobacillus plantarum JCM11125) prepared in (3) above to a final concentration of 2% (w / w). Evaluation culture was carried out for one day using each of the above. After the evaluation culture, the culture supernatant was collected and the amount of collagen synthesis was evaluated by ELISA (human type I collagen). These results of collagen synthesis promotion are shown in FIG. 3, and the results are expressed as relative values ​​(%) to the results of the comparative example (DW). The results in FIG. 3 when purified water (DW) was used are the values ​​showing no increase in collagen.

[0058] As can be seen from FIG. 3, the LPAK1-13 lactic acid bacteria fermentation liquid, which is a skin aging inhibitor of the present invention, significantly increased collagen synthesis promotion more than the Type culture lactic acid bacteria fermentation liquid.

[0059] (6) Collagen synthesis test (3) (ED mineral water + lactic acid bacteria fermentation liquid) As a representative example of ED mineral water, 40% ED mineral water was used, and as the lactic acid bacteria fermentation liquid, the LPAK1-13 lactic acid bacteria fermentation liquid and Type culture fermentation liquid prepared in (3) above were used to carry out the following tests. Normal human dermal fibroblasts (NHDF, Riken BioResource Research Center) cells were seeded into a 24-well microplate at 115,000 cells / well and pre-cultured for 2 days at 37°C and 5% CO2 in Eagle's MEM medium containing 10% (v / v) FBS, as in (4) above.

[0060] After the preliminary culture, 1) Eagle's MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 0.5% FBS, prepared using purified water (DW) instead of the ED mineral water described above in (4), 2) Eagle's MEM medium containing 0.5% (v / v) FBS prepared with 40% (w / w) ED mineral water in the same manner as in (4) above; 3) Eagle's MEM medium containing 0.5% (v / v) FBS, prepared with 40% (w / w) ED mineral water in the same manner as in (4) above, and further containing the LPAK1-13 lactic acid bacteria fermentation liquid prepared in (2) above at a final concentration of 2% (w / w), and 4) Eagle's MEM medium containing 0.5% (v / v) FBS, prepared with 40% (w / w) ED mineral water in the same manner as in (4) above, and further containing the lactic acid bacteria fermentation liquid of Type culture (Lactobacillus plantarum JCM11125) prepared in (2) above at a final concentration of 2% (w / w). Evaluation culture was carried out for one day using each of the above. After the evaluation culture, the culture supernatant was collected and the amount of collagen synthesis was evaluated by ELISA (human type I collagen). These results of collagen synthesis promotion are shown in FIG. 4, which shows the results of collagen synthesis promotion as relative values ​​(%) to the results of the comparative example (DW). The results in FIG. 4 when purified water (DW) was used are the values ​​showing no increase in collagen.

[0061] FIG. 4 shows that the skin aging inhibitor of the present invention, ED mineral water + LPAK1-13 lactic acid bacteria fermentation liquid, promoted collagen synthesis to an almost equal or greater extent (n=3, p<0.01) than ED mineral water + Type culture lactic acid bacteria fermentation liquid. Furthermore, when 20% (w / w) ED mineral water, 60% (w / w) ED mineral water, or 80% (w / w) ED mineral water was used instead of the 40% ED mineral water, results showing a similar tendency to those described above were observed.

[0062] (7) Inhibitory effect on proteoglycan degradation As a representative example of ED mineral water, 40% ED mineral water was used, and as the lactic acid bacteria fermentation liquid, the LPAK1-13 lactic acid bacteria fermentation liquid and Type culture fermentation liquid prepared in (3) above were used to carry out the following tests. Normal human dermal fibroblasts (NHDF, Riken BioResource Research Center) cells were seeded into a 96-well microplate at 20,000 cells / well and pre-cultured for 2 days at 37°C and 5% CO2 in Eagle's MEM medium containing 10% (v / v) FBS, as in (4) above.

[0063] After the preliminary culture, 1) Eagle's MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 0.5% FBS, prepared using purified water (DW) instead of the ED mineral water described above in (4), 2) Eagle's MEM medium containing 0.5% (v / v) FBS prepared with 40% (w / w) ED mineral water in the same manner as in (4) above, 3) Eagle's MEM medium containing 0.5% (v / v) FBS, prepared with 40% (w / w) ED mineral water in the same manner as in (4) above, and further containing the lactic acid bacteria fermentation liquid prepared in (2) so that the final concentration is 2% (v / v). Evaluation culture was carried out for one day using each of the above.

[0064] After the evaluation culture, the medium components in each well were removed by suction, and the wells were washed once with 0.1 mL / well of PBS (-) (manufactured by Nissui Pharmaceutical Co., Ltd.). RNA was extracted and reverse transcribed using SuperPrep Cell Lysis & RT Kit for qPCR (manufactured by Toyobo Co., Ltd.) according to the kit's instructions, and a cDNA solution was prepared. Specifically, 24.85μL of lysis solution and 0.15μL of gDNA Remover were prepared per well, and 25.0μL was added to each well and left at room temperature for 5 minutes. Then, 4.75μL of Stop solution and 0.25μL of RNase inhibitor were prepared per well, and 5.0μL was added to each well. After leaving at room temperature for 2 minutes, 30.0μL of the cell lysate prepared in a 0.2mL tube was transferred. 4.0μL of 5×RT Master Mix and 12.0μL of Nuclease-free water were prepared per reaction, and 16.0μL was dispensed into 0.2mL tubes, and 4μL of the cell lysate was dispensed into each 0.2mL tube to make a total of 20μL. The 0.2 mL tube was placed in a thermal cycler (MyCycler, BIO-RAD) and incubated at 37° C. for 15 minutes, 50° C. for 5 minutes, and 98° C. for 5 minutes, and then cooled to 4° C. to prepare a cDNA solution.

[0065] Quantitative polymerase chain reaction (qPCR) was performed using THUNDERBIRD qPCR Mix (manufactured by Toyobo Co., Ltd.) according to the kit's instructions, and cDNA quantification of each gene was performed. Specifically, 5.0 μL of THUNDERBIRD SYBR qPCR Mix, 0.3 μL of Forward Primer (10 μM), 0.3 μL of Reverse Primer (10 μM), 0.2 μL of 50×ROX reference dye, and 2.2 μL of sterile water were prepared per reaction, and then 8.0 μL was poured into 0.1 mL tubes, and 2.0 μL of 10-fold diluted cDNA solution was added. Triplicates were used to reduce variation between tubes.

[0066] The PCR conditions were as follows: using a real-time PCR device (StepOnePlus, Applied Biosystems), heating at 95°C for 1 minute, followed by 40 cycles of 95°C for 30 seconds and 60°C for 30 seconds. In addition, melting curve analysis was performed after PCR to confirm the absence of nonspecific amplification. The base sequences of the analyzed genes and the primers used in the chain reaction are as follows:

[0067] glyceraldehyde-3-phosphate dehydrogenase (GAPDH); 5'-CATTGATGGCAACAATATCCACTT-3' 3'-GGTTATGCTGGTTTAGGCAAC-5'

[0068] ADAM metallopeptidase with thrombospondin type 1 motif 5(ADAMTS5); 5'-CCCGAAGAACTCCCAGGACA-3' 3'-GACACCACACGACACCATGC-5'

[0069] For quantitative analysis, the expression level of ADAMTS5 relative to the expression level of GAPDH was calculated, and then the expression level in the purified water group was divided by the expression level in each test group to relatively evaluate the effect of inhibiting proteoglycan degradation compared to the purified water group.

[0070] The results are shown in Figure 5. As shown in Figure 5, the addition of 40% (w / w) ED mineral water significantly (n=3, p<0.05) inhibited proteoglycan degradation compared to purified water (DW), and it was found to inhibit the expression of the aggrecanase synthesis gene (ADAMTS-5). In addition, the addition of LPAK1-13 lactic acid bacteria fermentation liquid obtained in (3) above (n=3, p<0.05) to 40% (v / v) ED mineral water maintained an effect almost equivalent to that of the addition of the conventional Type culture lactic acid bacteria fermentation liquid. Therefore, it is possible to inhibit skin aging. Furthermore, when 20% (w / w) ED mineral water, 60% (w / w) ED mineral water, or 80% (w / w) ED mineral water was used instead of the 40% ED mineral water, results showing a similar tendency to those described above were observed.

[0071] (8) Enhancement of glucuronyltransferase I expression Normal human skin fibroblasts (NHDF, Riken BioResource Research Center) were seeded into a 24-well microplate (AGC Technoglass) at 118,750 cells / well, and pre-cultured for 2 days at 37°C and 5% CO2 using Eagle's MEM medium (Nissui Pharmaceutical Co., Ltd.) containing 10% (v / v) FBS, as in (4) above.

[0072] After the preliminary culture, 1) Eagle's MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 0.5% FBS, prepared using purified water (DW) instead of the ED mineral water described above in (4), 2) Eagle's MEM medium containing 0.5% FBS prepared with 40% ED mineral water in the same manner as in (4) above; 3) Eagle's MEM medium containing 0.5% FBS, prepared with 40% ED mineral water in the same manner as in (4) above, and further supplemented with 1% (v / v) of the fermentation liquid prepared by Type culture according to the method in (3) above. 4) Eagle's MEM medium containing 0.5% FBS, prepared with 40% ED mineral water in the same manner as in (4) above, and further supplemented with 1% (v / v) of the LPAK1-13 lactic acid bacteria fermentation liquid prepared in (3) above. The evaluation culture was carried out for one day at 37°C and 5% CO2.

[0073] After the evaluation culture, the medium components in each well were aspirated and removed, 1.0 mL of lysis buffer was added with a micropipette, and the cultured NHDF was dissolved by pipetting several times. The entire amount of the NHDF dissolution solution was transferred to a 1.5 mL tube, 0.2 mL of chloroform was added, and the mixture was stirred with a vortex mixer for 10 seconds. After centrifugation at 12,000 × g for 10 minutes at 4 ° C, 500 μL of the upper layer (aqueous phase) was collected and transferred to a new 1.5 mL tube. An equal amount (500 μL) of 2-propanol was added, mixed by inversion, and then allowed to stand at 4 ° C for 10 minutes. After centrifugation at 12,000 × g for 10 minutes at 4 ° C, the supernatant was discarded, and the gel-like RNA precipitate was dissolved in 200 μL of nuclease-free water. 20 μL of 3 M sodium acetate (pH 5.2) and 200 μL of 2-propanol were added and mixed, and then allowed to stand at 4 ° C for 10 minutes. After centrifugation at 12,000×g for 10 minutes at 4°C, 250 μL of 75% ethanol was added to the RNA precipitate to wash off any guanidine salts that had been carried over. After completely removing the supernatant, the mixture was air-dried, and 50 μL of nuclease-free water was added to completely dissolve the RNA precipitate, preparing an RNA solution.

[0074] A reverse transcription reaction was performed using the ReverTra Ace qPCR RT Kit (Toyobo) according to the kit's instructions to prepare a cDNA solution. Specifically, 2.0 μL of 5×RT Buffer, 0.5 μL of RT Enzyme Mix, and 0.5 μL of Primer Mix were prepared per reaction, and 3.0 μL was dispensed into 0.2 mL tubes. The RNA solution was then mixed with nuclease-free water and dispensed into each 0.2 mL tube to make a total of 7.0 μL. The 0.2 mL tubes were placed in a thermal cycler (MyCycler, BIO-RAD), incubated at 37°C for 15 minutes and 98°C for 5 minutes, then cooled to 4°C to prepare a cDNA solution.

[0075] Quantitative polymerase chain reaction (qPCR) was performed using THUNDERBIRD qPCR Mix (Toyobo) according to the kit's instructions, and cDNA quantification of each gene was performed. Specifically, the required amount of THUNDERBIRD SYBR qPCR Mix 5.0 μL, forward primer (10 μM) 0.3 μL, reverse primer (10 μM) 0.3 μL, 50×ROX reference dye 0.2 μL, and sterile water 2.2 μL were prepared for each reaction, and then 8.0 μL was dispensed into 0.1 mL tubes, and 2.0 μL of 10-fold diluted cDNA solution was added to each reaction. Triplicates were used to reduce variation between tubes.

[0076] The PCR conditions were as follows: using a real-time PCR device (StepOnePlus, Applied Biosystems), heating at 95°C for 1 minute, followed by 40 cycles of 95°C for 30 seconds and 60°C for 30 seconds. In addition, melting curve analysis was performed after PCR to confirm the absence of nonspecific amplification. The base sequences of the analyzed genes and the primers used in the chain reaction are as follows:

[0077] glyceraldehyde-3-phosphate dehydrogenase (GAPDH); 5'-CATTGATGGCAACAATATCCACTT-3' 3'-GGTTATGCTGGTTTAGGCAAC-5'

[0078] Glucuronyltransferase I; 5'-TGCCTCTGGCCTCCTCTTCA-3' 3'-TGCCTCTGGCCTCCTCTTCA-3'

[0079] For quantitative analysis, the expression level of glucuronyltransferase relative to the expression level of GAPDH was calculated, and then the expression level of glucuronyltransferase I relative to the expression level of purified water was evaluated by dividing the expression level of each test area by the expression level of purified water area.

[0080] The results are shown in Figure 6. As shown in Figure 6, compared with purified water (DW), 40% (w / w) ED mineral water, and 40% (w / w) ED mineral water + Type culture, ED mineral water + Lactobacillus plantarum LPAK1-13 strain (Accession No. NITE P-03021) was able to increase the expression level of glucuronyltransferase I, an enzyme that contributes to the biosynthesis of glycosaminoglycans such as hyaluronic acid. Therefore, it is possible to suppress skin aging. Furthermore, when 20% (w / w) ED mineral water, 60% (w / w) ED mineral water, or 80% (w / w) ED mineral water was used instead of the 40% ED mineral water, results showing a similar tendency to those described above were observed.

[0081] (9) Antioxidant effect against hydrogen peroxide The antioxidant (anti-aging) effect of the lactic acid bacteria fermentation liquid obtained in (3) above was evaluated in the following respects regarding the carbonylation (oxidation) of stratum corneum proteins by treatment with hydrogen peroxide on the exfoliated stratum corneum from the inner part of the human arm. Specifically, the inside of the left forearm of a Japanese male in his 60s was washed with tap water and soap. Thereafter, cellophane tape (registered trademark, manufactured by Nichiban) was applied to three locations on the cleansed inner side of the left forearm and then peeled off to exfoliate the stratum corneum on the inner side of the left anterior arm.

[0082] The cellophane tape (registered trademark) with the peeled stratum corneum attached was attached to a slide glass previously coated with adhesive, and the slide glass with the cellophane tape (registered trademark) attached was immersed in xylene overnight, after which only the cellophane tape (registered trademark) was peeled off and the stratum corneum was transferred to the slide glass. The peeled stratum corneum was immersed in ethanol and purified water, and then 100 μL of a solution prepared with 50 mM hydrogen peroxide (Wako Pure Chemical Industries, special grade) and purified water to make 5% LPAK1-13 lactic acid bacteria fermentation liquid or 5% Type culture lactic acid bacteria fermentation liquid was dropped onto the peeled stratum corneum and allowed to stand at room temperature for 20 minutes.

[0083] The peeled stratum corneum was then washed with purified water and treated with 50 μM FTCZ (AnaSpec, Inc.). A MES buffer solution containing (Fluorescence-5-thiosemicarbazide) was dropped onto the sample and allowed to stand at room temperature for 10 minutes to detect carbonylated proteins in the stratum corneum. Fluorescence images were taken and the fluorescence intensity per unit area was converted into numerical values ​​using ImageJ to evaluate the carbonylated proteins. For comparison, the case where purified water (Blank) was used instead of the 5% lactic acid bacteria fermentation liquid is also shown.

[0084] Next, the fluorescence intensity of each test area per unit area was quantified, and the antioxidant effect was calculated as the fluorescence intensity of each test area / fluorescence intensity of blank. The antioxidant effect of blank was set at 100 and expressed as a relative value. The results are shown in Figure 7. When hydrogen peroxide was brought into contact with exfoliated stratum corneum, oxidation (carbonylation) of the stratum corneum was observed. It was found that while Type culture lactic acid bacteria fermentation liquid had no inhibitory effect on this oxidation of the stratum corneum, LPAK1-13 lactic acid bacteria fermentation liquid had a significantly high inhibitory effect.

[0085] (10) Anti-aging effect of ED mineral water and lactic acid bacteria fermentation liquid blend Several cosmetic compositions were prepared by combining 40% ED mineral water and the lactic acid bacteria fermentation liquid obtained in (3) above, with the compositions shown below.

[0086] Toner: Ingredients Amount (mass%) ED Mineral Water 40.0 Lactic acid bacteria fermentation liquid 0.1 Glycerin 5.0 Ethanol 10.0 1,3 Butylene Glycol 3.0 Phenoxyethanol 0.5 Purified water Remainder (mix so that the total amount is 100)

[0087] Cream 1: Ingredients Amount (mass%) ED Mineral Water 20.0 Lactic acid bacteria fermentation liquid 0.1 POE(20) Cetyl Ether 2.0 Lipophilic glyceryl monostearate 4.0 Cetanol 2.0 Cetyl Palmitate 2.0 White Vaseline 6.0 Squalane 30.0 Glyceryl triethylhexanoate 8.0 Glycerin 15.0 Preservatives 0.2 Carbomer 0.2 Arginine 0.1 Purified water Remainder (mix so that the total amount is 100)

[0088] Cream 2: Ingredients Amount (mass%) ED Mineral Water 20.0 Lactic acid bacteria fermentation liquid 0.1 POE(20)POP(4) Cetyl Ether 1.0 POE(50) hydrogenated castor oil 1.0 Self-emulsifying glyceryl monostearate 1.0 Stearic Acid 3.0 Behenyl Alcohol 3.0 Microcrystalline Wax 3.0 Squalane 13.0 Cetyl ethylhexanoate 7.0 Jojoba oil 5.0 Polyethylene glycol 1500 5.0 Preservatives 0.2 Xanthan gum 0.1 1,3 Butylene Glycol 4.0 Glycerin 2.0 Purified water Remainder (mix so that the total amount is 100)

[0089] Emulsion: Ingredients Amount (mass%) ED Mineral Water 30.0 Lactic acid bacteria fermentation liquid 0.5 Caprylic / Capric Triglyceride 5.0 Shea Butter 2.0 Glyceryl triethylhexanoate 2.0 Tocopherol 0.6 Ethylhexylglycerin 0.4 1,3 Butylene Glycol 5.0 Pentylene glycol 3.0 Dipropylene glycol 2.0 Glycerin 2.0 Hydrogenated lysolecithin 0.5 (Acrylic acid / alkyl acrylate (C10-30)) Copolymer 0.4 Xanthan gum 0.1 Arginine 0.4 Purified water Remainder (mix so that the total amount is 100)

[0090] Beauty serum: Ingredients Amount (mass%) ED Mineral Water 40.0 Lactic acid bacteria fermentation liquid 0.5 Carboxyvinyl polymer (2% aqueous solution) 10.00 Carboxyethyl cellulose 3.00 Glycerin 10.00 Triethanolamine 0.15 Methylparaben 0.10 Quince seed solution (1% aqueous solution) 10.00 Sodium hyaluronate solution (1% aqueous solution) 4.00 Pyruvate 1.00 Levulinic acid 1.00 L-Arginine 2.00 Purified water Remainder (mix so that the total amount is 100.00)

[0091] pack: Ingredients Amount (mass%) ED Mineral Water 20.0 Lactic acid bacteria fermentation liquid 0.1 Hydroxyethoxycellulose 3.0 Carboxyvinyl polymer (2% aqueous solution) 10.0 Polyoxyethylene oleyl ether (15EO) 1.0 Glyoxylic Acid 1.0 Levulinic acid 1.0 L-Arginine 2.0 Ethanol 5.0 Preservatives 0.2 Purified water Remainder (mix so that the total amount is 100.0)

[0092] Seventeen healthy Japanese women applied each of the above cosmetic preparations to half of their faces, three times a day (morning, noon, and night) for eight consecutive weeks. The lotion, cream, milky lotion, and serum were applied three times a day, going from the inner corner of the eye to the outer corner twice, and then from the inner corner of the eye to the temples twice more, tapping the skin lightly to blend in. The pack was also applied from the inner corner of the eye to the outer corner. Before application (W0), after 4 weeks of use (W4), and after 8 weeks of use (W8), the effects of improving wrinkles, dark circles under the eyes, and dullness were evaluated and compared with the side where no cosmetic was applied.

[0093] As a representative example, the average results of 17 subjects who used lotion are shown in Figures 8 to 10. FIG. 8 is a graph showing the effect of improving wrinkles, FIG. 9 is a graph showing the effect of improving dark circles, and FIG. 10 is a graph showing the effect of improving dark circles.

[0094] The wrinkle improvement effect in FIG. 8 was determined by visually assigning a score based on standard wrinkle grade photographs at each measurement point in accordance with the Anti-Wrinkle Product Evaluation Guidelines (Japan Cosmetic Science Society). The effect of improving dark circles in Figure 9 was evaluated by measuring the change in melamine and hemoglobin values ​​using a Megxameter (product number: MX18, manufacturers: Courage and Khazaka), while the effect of improving dullness in Figure 10 was evaluated by measuring the L value using a color difference meter (product number: CR-400, manufactured by Konica Minolta Japan, Inc.).

[0095] As a result of continuous application of a cosmetic composition containing the skin improvement and prevention agent of the present invention, it can be seen that, as shown in Figure 8, a significant (p<0.01) wrinkle improvement effect was obtained from 4 weeks after application, and as shown in Figure 9, a significant (p<0.05) improvement effect on dark circles was obtained from 4 weeks after application, and as shown in Figure 10, a significant (p<0.05) increase in brightness was obtained from 4 weeks after application, indicating that an improvement effect on dullness was obtained. The creams (Cream 1, Cream 2), milky lotion, beauty essence, and pack showed results similar to those shown in Figs. 8 to 10 for the lotion. [Industrial Applicability]

[0096] The skin anti-aging agent of the present invention not only has the effect of promoting collagen production in dermal matrix components, but also inhibits the decomposition of proteoglycan, and is therefore capable of preventing skin aging such as wrinkles, dullness, and dark circles under the eyes, and can be applied by being incorporated into cosmetics.

Claims

1. A skin anti-aging agent comprising deep-sea water and a lactic acid bacteria fermentation liquid, the deep-sea water being deep-sea water that has been treated by reverse osmosis filtration, deep-sea water that has been treated by electrodialysis, or deep-sea water that has been treated by ion selection such that the mass ratio of potassium ions, sodium ions, calcium ions, magnesium ions, and chloride ions in the deep-sea water is K:Na:Ca:Mg:Cl=1:30-70:10-40:40-150:200-500, the lactic acid bacteria being contained in an amount of 20% by mass or more of the total, and the lactic acid bacteria being Lactobacillus plantarum LPAK1-13 strain (accession number NITE P-03021) derived from the deep-sea water.

2. 2. The skin anti-aging agent according to claim 1, wherein the deep sea water is contained in an amount of 40 mass % or more of the total.

3. A method for preventing skin aging, comprising applying a cosmetic composition containing the skin aging inhibitor according to claim 1 or 2 to the skin.

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