A topical skin preparation containing a lactic acid fermented product of aloe vera leaves and a method for producing the same.

Abstract

This invention provides a novel topical skin preparation and a method for producing the same, using a new strain of Lactiplantibacillus plantarum, strain FL-781, which contains a lactic acid fermented product of aloe vera leaf juice as the active ingredient. [Solution] The present invention relates to a topical skin preparation containing a lactic acid fermented product of aloe vera leaves, This invention relates to a topical skin preparation and a method for producing the same, wherein the lactic acid bacterium is Lactiplantibacillus plantarum FL-781 strain (accession number: NITE ABP-04146).

Description

Technical Field

[0001] The present invention relates to a skin external preparation containing a lactic acid bacteria fermented product of aloe vera leaves, a method for producing the same, and the like.

Background Art

[0002] Since lactic acid bacteria fermented products exhibit an effect of improving skin wrinkles and / or sagging, they are generally formulated in lotions, basic cosmetics, and the like. Among them, as those using aloe vera of plants, a moisturizer using aloe fermentation products by various lactic acid bacteria (Patent Document 1) and a cleansing cosmetic containing an aloe lactic acid bacteria fermented product (Patent Document 2) have been reported. However, for Lactiplantibacillus plantarum, existing strains such as standard strains have been used.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] As a result of intensive studies, the present inventors have found a novel strain, FL-781 strain, of Lactiplantibacillus plantarum. Therefore, an object is to provide a novel skin external preparation containing, as an active ingredient, a lactic acid bacteria fermented product of aloe vera leaf juice using the strain, a method for producing the same, and the like.

Means for Solving the Problems

[0005] (1) A skin external preparation according to an embodiment for achieving the above object is It contains lactic acid fermented aloe vera leaves, The lactic acid bacterium in question is Lactiplantibacillus plantarum strain FL-781 (accession number: NITE ABP-04146). (2) In another embodiment of the topical skin preparation, preferably, it has hyaluronic acid production promoting activity. (3) In another embodiment of a topical skin preparation, preferably, the preparation prevents and / or improves skin dryness. (4) In another embodiment of a topical skin preparation, it is preferable to promote collagen production. (5) In another embodiment of a topical skin preparation, for example, there are cosmetics for skin application, hair cosmetics, and so on. (6) A method for producing a topical skin preparation containing a lactic acid ferment product produced from the juice of aloe vera leaves according to one embodiment for achieving the above objective is: In the process of producing lactic acid fermented products, The process includes a fermentation step in which the aforementioned liquid is fermented using Lactiplantibacillus plantarum FL-781 strain (accession number: NITE ABP-04146). The process for producing the lactic acid fermented product is preferably: A step of removing aloin from the aforementioned liquid, The process includes a fermentation step in which the liquid after the aloin removal step is fermented with Lactiplantibacillus plantarum FL-781 (accession number: NITE ABP-04146). [Effects of the Invention]

[0006] According to the present invention, for example, by using a novel strain of Lactiplantibacillus plantarum, strain FL-781, it is possible to provide a novel topical skin preparation and a method for producing the same, which uses a lactic acid fermented product of aloe vera leaf juice as an active ingredient. [Brief explanation of the drawing]

[0007] [Figure 1] This is a schematic diagram of the test methods for Test 2 and Test 3 as described in the examples. [Figure 2] The test piece (a 5cm x 5cm copy paper) used in Test 5, described in the examples, is shown. Artificial sebum (five circles) was placed on this piece as shown in the figure. [Figure 3] This is the calculation formula used in the measurement of hair tensile strength in Test Example 7. [Modes for carrying out the invention]

[0008] Next, we will describe each embodiment of the present invention. It should be noted that the embodiments described below are not intended to limit the invention as defined in the claims, and not all of the elements and combinations thereof described in each embodiment are necessarily essential to the solution of the present invention.

[0009] (Aloe vera leaves) The aloe vera leaves used in this invention consist of an outer layer composed of a thick cuticle and a leaf pulp located inside the outer layer. In this invention, "aloe vera leaves" may be used fresh or dried, or they may be crushed. The amount of aloe vera leaves used as a raw material in this invention is not particularly limited, but it is preferably 0.0001% or more, preferably 0.001% or more, preferably 0.01% or more, more preferably 0.1% or more, and particularly preferably 1% or more, of the total weight of the raw materials used in the fermentation process. The aloe vera leaf juice produced from these aloe vera leaves includes not only the form of juice (liquid), but also powders produced by a predetermined drying method (such as spray drying, freeze-drying, vacuum drying, shelf drying, belt drying, drum drying, etc.).

[0010] (FL-781 stock) In this invention, the lactic acid fermentation of aloe vera leaves is performed using Lactiplantibacillus plantarum strain FL-781 (hereinafter sometimes simply referred to as strain FL-781). Strain FL-781 is a novel strain of Lactiplantibacillus plantarum isolated from Shimizu white peaches from Okayama Prefecture. Strain FL-781 was deposited with the Patent Microbial Depositary Center (NPMD) of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, Room 122) on August 23, 2024, under accession number NITE ABP-04146.

[0011] (Lactic acid fermented product) In the present invention, "lactic acid bacteria fermented product" refers to a product obtained by fermenting aloe vera leaves with the above-mentioned FL-781 strain. The form of the lactic acid bacteria fermented product is not particularly limited and may be a liquid or a solid such as a powder, and can be arbitrarily selected depending on the form of use of the present invention. Furthermore, the lactic acid bacteria fermented product according to the present invention may contain additives such as preservatives, antibacterial agents, and chelating agents. As preservatives, antibacterial agents, and chelating agents, hydroxybenzoic acids and their salts or esters such as 1,3-propanediol, methylparaben, ethylparaben, propylparaben, and butylparaben; salicylic acid; sodium benzoate; phenoxyethanol; 1,2-diols such as 1,2-pentanediol and 1,2-hexanediol; isothiazolinone derivatives such as methylchloroisothiazolinone and methylisothiazolinone; imidazolinium urea; dehydroacetic acid and its salts; phenols; halogenated bisphenols such as triclosan, acid amides, quaternary ammonium salts; trichlorocarbanide, zinc pyrithione, benzalkonium chloride, benzethonium chloride, sorbic acid, chlorhexidine, chlorhexidine gluconate, halocarban, hexachlorophene, hinokitiol; phenol, iso Examples of additives include propylphenol, cresol, thymol, parachlorophenol, phenylphenol, sodium phenylphenol and other phenols; phenylethyl alcohol, photosensitizers, antibacterial zeolites, silver ions; EDTA, EDTA2Na, EDTA3Na, EDTA4Na and other EDTA salts (ethylenediaminetetraacetate); HEDTA3Na and other hydroxyethylethylenediaminetriacetate; pentetates (diethylenetriaminepentaacetate); phytic acid; phosphonic acids such as etidronic acid and their sodium salts; sodium oxalate; polypolyamino acids such as polyaspartic acid and polyglutamic acid; sodium polyphosphate, sodium metaphosphate, phosphoric acid; sodium citrate, citric acid, alanine, dihydroxyethylglycine, gluconic acid, ascorbic acid, succinic acid, tartaric acid, etc. The content of additives in the lactic acid bacteria fermented product is not particularly limited, but is preferably 1% or less, more preferably 0.5% or less, and particularly preferably 0.1% or less, relative to the total weight of the lactic acid bacteria fermented product.

[0012] (Nicotinamide) In order to exert more effects, the agent of the present invention may preferably further contain nicotinamide. It is an amide compound of nicotinic acid (vitamin B3, niacin), a derivative of nicotinic acid, also called nicotinamide or niacinamide. Nicotinamide is a water-soluble vitamin and a substance belonging to vitamin B group. For example, it can be extracted from natural products (such as rice bran) or synthesized by known methods. Specifically, those listed in the 15th revised Japanese Pharmacopoeia 2008 can be used.

[0013] (Form of external preparation for skin) The external preparation for skin according to the present invention can be in a suitable form for use such as ampoule, capsule, powder, granule, liquid, gel, foam, emulsion, sheet, mist, spray agent, etc. including 1) pharmaceuticals, 2) quasi-drugs, 3) external preparations for skin for local or systemic use (for example, basic cosmetics such as lotion, emulsion, cream, ointment, lotion, oil, pack, etc., cleansing agents for face and skin such as solid soap, liquid soap, hand wash, etc., massage agents, cleansing agents, depilatory agents, hair removal agents, beard shaving treatment agents, after shave lotion, pre-shave lotion, shaving cream, foundation, lipstick, blush, eyeshadow, eyeliner, mascara, etc., makeup cosmetics, perfumes, nail cosmetics, nail enamel, nail enamel remover, patch agents, plaster agents, tape agents, sheet agents, adhesive agents, aerosol agents, etc.), 4) pharmaceutical or / and cosmetic preparations for scalp and hair (for example, shampoo, rinse, hair treatment agent, pre-hair treatment agent, permanent solution, hair dyeing agent, hair styling agent, hair tonic, hair growth and nourishing agent, patch agent, plaster agent, tape agent, sheet agent, aerosol agent, etc.), 5) bath agents to be added to bath water, 6) others such as axillary odor preventives, deodorants, antiperspirants, sanitary products, sanitary napkins, wet tissues, etc.

[0014] (Form of external preparation for skin, for example, hair cosmetics) The topical skin preparation according to the present invention includes, for example, hair cosmetics. Hair cosmetics, for example, repair the cuticle CMC (lipid-cell membrane complex) of hair, shorten the drying time of washed hair, improve the tensile strength of hair, and the like. Hair is damaged by living environment (ultraviolet rays and heat from sunlight), daily hair care actions (friction by shampooing and brushing), and chemical treatments (coloring, perming, etc.). The cuticle is the outermost tissue of hair and originally serves to protect hair and prevent the outflow of nutrients and moisture from the inside of hair. However, in damaged hair, the cuticle is damaged, so the original function of the cuticle deteriorates. The damaged cuticle has innumerable small cavities and absorbs a large amount of moisture like a sponge, and it takes time to evaporate the moisture and dry it. Hair is known to consist of three structures: the medulla (hair medulla) at the center, the cortex (hair cortex) in the intermediate part surrounding it, and the outermost cuticle (hair cuticle). It is said that the damage is caused by the cuticle CMC (lipid-cell membrane complex), which acts like an adhesive between the outermost cuticles, becoming spongy. If the components for repair can stay, it is expected that the damage can be suppressed, and further effects can be expected by repeated use. Although various hair cosmetics targeting the cuticle have been reported, there are hair protection cosmetics intended to recover the cuticle of hair damaged by chlorine in pool water and tap water and prevent the hair from becoming limp and rough (Japanese Patent Laid-Open No. 2024-034405).

[0015] "Applying a hair cosmetic to hair" in the present invention includes applying the hair cosmetic to the hair by taking it with the hand or a brush, directly applying the hair cosmetic to the hair, and the like. That is, the hair cosmetic comes into contact with the hair.

[0016] (Components of the topical skin preparation) Furthermore, in the manufacture of such agents, the following components and additives may be optionally selected and used in combination, as necessary, within the limits that do not impair the effects of the present invention. There are no specific requirements for the amount of these components to be included in the formulation system, but generally, amounts of approximately 0.0001 to 100% are considered preferable.

[0017] (1) Various oils and fats Avocado oil, almond oil, fennel oil, perilla oil, olive oil, orange oil, orange rafur oil, sesame oil, cocoa butter, chamomile oil, carrot oil, cucumber oil, beef tallow fatty acid, kukui nut oil, safflower oil, shea butter, liquid shea butter, soybean oil, camellia oil, corn oil, rapeseed oil, peach oil, castor oil, cottonseed oil, peanut oil, turtle oil, mink oil, egg yolk oil, palm oil, palm kernel oil, Japanese wax, coconut oil, beef tallow, lard, squalene, squalane, pristane, or hydrogenated versions of these oils (hydrogenated oils, etc.).

[0018] (2) Waxes Beeswax, carnauba wax, whale wax, lanolin, liquid lanolin, reduced lanolin, hard lanolin, candelilla wax, montane wax, shellac wax, rice wax, etc.

[0019] (3) Mineral oil Liquid paraffin, petrolatum, paraffin, ozokeride, ceresin, microcrystalline wax, etc.

[0020] (4) Fatty acids Natural fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, linolenic acid, docosahexaenoic acid, eicosapentaenoic acid, 12-hydroxystearic acid, undecylenic acid, tall oil, and lanolin fatty acids; and synthetic fatty acids such as isononanoic acid, caproic acid, 2-ethylbutanoic acid, isopentanoic acid, 2-methylpentanoic acid, 2-ethylhexanoic acid, and isopentanoic acid.

[0021] (5) Alcohols Natural alcohols such as ethanol, isopropanol, lauryl alcohol, cetanol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, phytosterol, and phenoxyethanol, and synthetic alcohols such as 2-hexyldecanol, isostearyl alcohol, and 2-octyldodecanol.

[0022] (6) Polyhydric alcohols Ethylene oxide, ethylene glycol, diethylene glycol, triethylene glycol, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, polyethylene glycol, propylene oxide, propylene glycol, polypropylene glycol, 1,3-butylene glycol, pentyl glycol, glycerin, pentaerythritol, treitol, arabitol, xylitol, ribitol, galactitol, sorbitol, mannitol, lactitol, maltitol, etc.

[0023] (7) Esters Isopropyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, oleyl oleate, decyl oleate, octyldodecyl myristate, hexyldecyl dimethyloctanoate, cetyl lactate, myristyl lactate, diethyl phthalate, dibutyl phthalate, lanolin acetate, ethylene glycol monostearate, propylene glycol monostearate, propylene glycol dioleate, etc.

[0024] (8) Metal soaps Aluminum stearate, magnesium stearate, zinc stearate, calcium stearate, zinc palmitate, magnesium myristate, zinc laurate, zinc undecylenate, etc.

[0025] (9) Gum, sugars or water-soluble polymer compounds Acacia gum, benzoin gum, dammar gum, guaiac butter, Irish moss, karaya gum, tragacanth gum, carob gum, quince seed, agar, casein, lactose, fructose, sucrose or its esters, trehalose or its derivatives, dextrin, gelatin, pectin, starch, carrageenan, carboxymethyl chitin or chitosan, hydroxyalkyl (C2-C4) chitin or chitosan to which alkylene (C2-C4) oxides such as ethylene oxide have been added, low molecular weight chitin or chitosan, chitosan salts, sulfated chitin or chitosan, phosphorylated chitin or chitosan, alginic acid or its salts, hya Rulonic acid or its salts, chondroitin sulfate or its salts, heparin, ethylcellulose, methylcellulose, carboxymethylcellulose, carboxyethylcellulose, sodium carboxyethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, nitrocellulose, crystalline cellulose, polyvinyl alcohol, polyvinyl methyl ether, polyvinylpyrrolidone, polyvinyl methacrylate, polyacrylates, polyalkylene oxides such as polyethylene oxide and polypropylene oxide or their crosslinked polymers, carboxyvinyl polymers, polyethyleneimines, etc.

[0026] (10) Surfactants Anionic surfactants (alkyl carboxylates, alkyl sulfonates, alkyl sulfates, alkyl phosphates), cationic surfactants (alkylamines, alkyl quaternary ammonium salts), amphoteric surfactants: carboxylic acid type amphoteric surfactants (amino type, betaine type), sulfate type amphoteric surfactants, sulfonic acid type amphoteric surfactants, phosphate type amphoteric surfactants, nonionic surfactants (ether type nonionic surfactants, ether ester type nonionic surfactants, ester type nonionic surfactants, block polymer type nonionic surfactants, nitrogen-containing nonionic surfactants), other surfactants (natural surfactants, derivatives of protein hydrolysates, polymer surfactants, surfactants containing titanium and silicon, fluorinated carbon surfactants), etc.

[0027] (11) Various vitamins Vitamin A group: retinol, retinal (vitamin A1), dehydroretinal (vitamin A2), carotene, lycopene (provitamin A), Vitamin B group: thiamine hydrochloride, thiamine sulfate (vitamin B1), riboflavin (vitamin B2), pyridoxine (vitamin B6), cyanocobalamin (vitamin B12), folic acids, nicotinic acids, pantothenic acids, biotins, choline, inositols, Vitamin C group: vitamin C acid or its derivatives, Vitamin D group: ergocalcifer Vitamin D2 (Cholecalciferol), Vitamin D3 (Cholecalciferol), Dihydrotachisterol, Vitamin E group: Vitamin E or its derivatives, Ubiquinones, Vitamin K group: Phytonadione (Vitamin K1), Menaquinone (Vitamin K2), Menadione (Vitamin K3), Menadiol (Vitamin K4), Others, Essential fatty acids (Vitamin F), Carnitine, Ferulic acid, γ-Oryzanol, Orotic acid, Vitamin P group (Rutin, Eriocitrin, Hesperidin), Vitamin U, etc.

[0028] (12) Various amino acids Valine, leucine, isoleucine, threonine, methionine, phenylalanine, tryptophan, lysine, glycine, alanine, asparagine, glutamine, serine, cysteine, cystine, tyrosine, proline, hydroxyproline, aspartic acid, glutamic acid, hydroxylysine, arginine, ornithine, histidine, etc., or their sulfates, phosphates, nitrates, citrates, or amino acid derivatives such as pyrrolidone carboxylic acid.

[0029] (13) Various additives derived from plant or animal raw materials These can be supplied by performing processing according to the type and form of product to be added (for example, crushing, milling, washing, hydrolysis, fermentation, purification, pressing, extraction, fractionation, filtration, drying, powdering, granulation, dissolution, sterilization, pH adjustment, deodorization, decolorization, etc., which can be arbitrarily selected and combined), and then arbitrarily selected from various materials.

[0030] The upper limit of the lactic acid bacteria fermented product content in the topical skin preparation is not particularly limited, and may be, for example, 100% by mass, 50% by mass, 10% by mass, 5% by mass, or 1% by mass of the total weight of the topical skin preparation. Similarly, the lower limit of the lactic acid bacteria fermented product content in the topical skin preparation is not particularly limited, but may be, for example, 0.001% by mass, 0.01% by mass, 0.05% by mass, or 0.1% by mass of the total weight of the topical skin preparation.

[0031] (Dry skin) "Dry skin" is a condition in which moisture is lost from the stratum corneum of the skin. When moisture is lost from the stratum corneum, the skin's barrier function weakens, making it easier for external stimuli to penetrate. When inflammatory cytokines are produced in response to the penetration of external stimuli, inflammation is triggered, causing itching and redness. Scratching the affected area can lead to eczema, and the skin's turnover period shortens. Consequently, the number of stratum corneum cells with immature moisturizing functions increases, making the skin even more prone to dryness.

[0032] Another aspect of the present invention provides a method for producing a topical skin preparation.

[0033] (Aloin removal process) Aloin is a medicinal component found in the outer layer of aloe vera leaves, possessing bitterness and laxative activity. Aloin is usually removed during the manufacturing process of cosmetics. In the present invention, the method for producing a topical skin preparation preferably includes an aloin removal step. This aloin removal step is not limited as long as it can remove aloin, and any known method such as adsorption to activated carbon can be used.

[0034] (Fermentation process) The fermentation process in this invention will be described in detail below.

[0035] In the present invention, the fermentation process is carried out by mixing the sap component of aloe vera leaves after the aloin removal process with the FL-781 strain. The sap component of aloe vera leaves after the aloin removal process is in powder form in the examples described later, but is not limited to this, and may be in liquid form, for example. The sap component is suspended or added to a fermentation medium before being subjected to the fermentation process. The fermentation medium is water or a mixture of water and lower alcohols (methanol, ethanol, propanol, etc.) or glycols (ethylene glycol, propylene glycol, 1,3-butylene glycol, glycerin, etc.) in an appropriate amount relative to the sap component. Sugars such as glucose, fructose, sucrose, sugar, granulated sugar, and refined sugar may be added to the fermentation medium. Yeast extract may also be added to the fermentation medium.

[0036] Before being subjected to the fermentation process, the suspension of the liquid components is sterilized to remove any bacteria that could hinder fermentation. In this case, the sterilization method may involve washing and sterilizing the fermentation material with sterilizing ethanol or the like beforehand and then suspending it in a sterile medium such as sterile water, or suspending the fermentation material in a medium and then heating the suspension. Common heat sterilization methods include autoclave sterilization, in which the suspension is heated at 120-130°C for 10-20 minutes, and intermittent sterilization, in which the suspension is kept at 80-90°C for 60-120 minutes, once a day for 2-3 days.

[0037] Fermentation is carried out by adding a culture medium containing the FL-781 strain to the fermentation medium after heat sterilization. The time required for fermentation is not particularly limited, but 12 to 36 hours is preferred, 15 to 30 hours is more preferred, and 18 to 24 hours is particularly preferred. The temperature conditions during fermentation are preferably 15 to 37°C, more preferably 20 to 35°C, and particularly preferred at 25 to 32°C. After fermentation is complete, the FL-781 strain cells are removed, but the method of removing the cells is not particularly limited, and known methods such as centrifugation and filtration can be used. The sugar content (Brix) of the lactic acid bacteria fermented product is not particularly limited, but 0.1 to 20° is preferred, 0.5 to 15° is more preferred, and 1 to 10° is particularly preferred.

[0038] A preservative may be added to the lactic acid bacteria fermented product after the removal of bacterial cells. Here, the preservative is not particularly limited as long as it is a preservative added to the topical skin preparation described above. Preferred preservatives with antiseptic properties include, for example, aromatic alcohols, glycols, parabens, or mixtures thereof. Since preservatives can cause skin irritation or stickiness when added to, for example, topical skin preparations, it is desirable to reduce the amount used as much as possible. Therefore, there is no limit to the lower limit of the concentration when adding a preservative, as long as it is above 0% by mass. The upper limit of the concentration is 1% by mass, preferably 0.5% by mass, and more preferably 0.1% by mass. Preferred preservatives with antiseptic properties at these low concentrations include, for example, parabens, benzoic acid and its salts, and more preferably sodium benzoate.

[0039] The pH of the mixture containing the suspension and the culture medium containing strain FL-781 (hereinafter simply referred to as the mixture) before fermentation is preferably about 4 to 6, more preferably about 4.5 to 5.5, and particularly preferably about 5.0. The pH after fermentation is preferably about 3 to 6, more preferably about 3.5 to 5.0, and particularly preferably about 4.5. Furthermore, the acidity of the mixture before fermentation is preferably about 0.1 to 0.3 in lactic acid equivalent, and more preferably about 0.1. The acidity of the mixture after fermentation is preferably about 0.1 to 0.3 in lactic acid equivalent, and more preferably about 0.25.

[0040] When providing the lactic acid bacteria fermented product produced in the above fermentation process in the form of a topical skin preparation, it can be provided, for example, together with a pharmaceutically or cosmetically acceptable carrier (water, oily component, etc.) in addition to the lactic acid bacteria fermented product. Furthermore, if necessary, the above-mentioned commonly used components and additives can be added, and the product can be prepared in the desired form by conventional methods.

[0041] The present invention will now be described in more detail with reference to examples, but the present invention is not limited in any way to these examples. In the following examples, the unit % in the numerical values ​​indicating the amount of each component added means mass %. [Examples]

[0042] The experimental materials used in the experiments described in the following example tests are as follows: Human epidermal keratinocytes: Normal human epidermal keratinocytes, Kurabo, KK-4009, derived from frozen NHEK(NB) neonatal cells, KURABO • Culture medium for keratinocytes (1): HuMedia-KG2 custom GC supplement medium, Kurabo, KK-2170S. This is the custom GC supplement medium to which calcium (final concentration 0.06 mM) has been added. • Culture medium for keratinocytes (2): Medium obtained by removing BPE (Bovine Pituitary Extract) and EGF (Epidermal Growth Factor) from medium (1). • Staphylococcus epidermidis: Standard strain of Staphylococcus epidermidis (ATCC12228) • NB medium: Nutrient Broth No. 2 liquid medium (Oxoid Limited, CM0067B)

[0043] (Example 1: Preparation of lactic acid bacteria fermented product) The following process is used to produce the lactic acid bacteria fermented product (aloe vera liquid that has undergone the fermentation process) used in the following test example. A juice component (aloe powder) was prepared.

[0044] The unfermented material used in the following test examples is aloe powder (organic SQ freeze-dried aloe vera leaf sap component after the aloin removal process, manufactured by Aroe Laboratories, Inc.).

[0045] 1. Pre-culture of FL-781 strain FL-781 strain was placed in a culture medium with the composition shown in Table 1 below, in a quantity of 30 × 10⁶ units. 8 The cells were inoculated to a concentration of cfu / mL and incubated at 30°C for 18 hours.

[0046] [Table 1]

[0047] 2. Preparation of the sap component (aloe powder) of aloe vera leaves after the aloin removal process. Organic SQ Freeze Dried Aloe Vera Leaf Powder 100X (manufactured by Aroe Laboratories, Inc.), an aloe powder, was used as the sap component of aloe vera leaves that had undergone the aloin removal process. The unfermented material shown in the following test examples is this aloe powder.

[0048] 3. Fermentation process of aloe powder 6.0 kg of the above aloe powder (approximately 1% of the total weight of the mixture) was suspended in a fermentation medium with the composition shown in Table 2 below, yielding a total suspension of 594.0 kg.

[0049] [Table 2]

[0050] The suspension had a pH of 6.542 and an acidity (lactic acid equivalent) of 0.042%. The suspension was heat-sterilized at 90°C for 1 minute. The heat-sterilized suspension was cooled to approximately 30°C. 6.0 kg of pre-culture medium containing strain FL-781 was added to the cooled suspension.

[0051] The suspension after the addition was subjected to a fermentation process at 30°C for 18 hours. After the fermentation process, the pH of the suspension was 4.39, the acidity (lactic acid equivalent) was 0.310%, and the sugar content (Brix) was 4.50.

[0052] 4. Removal of FL-781 strain As described below, the FL-781 strain was removed from the suspension after the fermentation process. The lactic acid bacteria fermented product after the fermentation process was centrifuged (8,200×g) using a continuous centrifuge, and the supernatant was collected. The supernatant was filtered by precoating to obtain the filtrate (Example 1: lactic acid bacteria fermented product).

[0053] (Test Example 1) Method for confirming the moisture retention capacity of the stratum corneum In this study, purified water or a lactic acid bacteria ferment (Example 1) was added to the flexor side of the forearm of a healthy human (51-year-old female, N=1), and the stratum corneum water content (capacitance) was measured.

[0054] (Test Procedure) Approximately 0.3 ml of each sample (purified water or lactic acid bacteria fermented product) was applied to the flexor side of the forearm, and filter paper was placed on top. After 30 seconds, the excess liquid was removed. Immediately afterward, the electrical conductivity of the stratum corneum was measured using a Corneometer. (TM) Capacitance was measured using a CM825MP (Courage+Khazak). Measurements were continued every 30 seconds for 5 minutes. The final measurement was performed 180 minutes after application. The capacitance of purified water and the blank (purified water) were measured before application in Example 1.

[0055] (Measurement results) The measurement results are shown in Table 3. The results in Table 3 show relative values ​​with the measurement result of the blank (measurement result before sample application) set to 100. As shown in Table 3, it was confirmed that capacitance (stratum corneum moisture content) increased with the application of Example 1 compared to the group applied with purified water, particularly from immediately after application to 5 minutes later. [Table 3]

[0056] (Test Example 2) Test using a three-dimensional skin model (Permeability test of niacinamide) A three-dimensional skin model (KURABO:EFT-400) including the epidermis and dermis was subjected to a penetration test of niacinamide (nicotinamide) and measurement of procollagen production when a predetermined sample was added.

[0057] (Sample preparation, etc.) I prepared the following: • Three-dimensional skin model: KURABO (EFT-400) • Example 1 • EFT-400 assay medium: KURABO, EFT-400ASY Nicotinamide: DSM, Product Code: 0487848340 • Sample 1: Purified water • Sample 3: Example 1

[0058] (Test method) Figure 1 shows a schematic diagram of the test method. A three-dimensional skin model was transferred to a 24-well culture plate, 2.5 mL of EFT-400 assay medium was added, and the mixture was cultured for 24 hours (preliminary culture). After this culture, an insert-type lid was attached to a 12-well culture plate, and 5.5 mL of fresh EFT-400 assay medium was added to the bottom of the 12-well culture plate. 275 μL of the sample (Sample 1 or Sample 3) and nicotinamide (final concentration 10%) were added to the insert surface, and the mixture was cultured in a CO2 incubator at 37°C. At 15 minutes, 30 minutes, 4 hours, and 24 hours after the start of culture, 1 mL of supernatant was collected at each time point, and after being frozen and stored at -80°C, the concentration of niacinamide contained in the supernatant collected at all time courses was measured by HPLC under the following conditions.

[0059] HPLC conditions: ·Instrument: Shimadzu LC-20ADxr - SPD-M20A ·Column:Mightysil RP-18 GP 250X4.6mm (5µm) ·Mobile phase:MeOH:0.1% 1-Heptanesulfonic Acid Sodium Salt1 = 30:70 • Flow rate: 0.5 mL / min Column Temp.: 25℃ Detector: UV250nm

[0060] (Test results) The test results are shown in Table 4. In Table 4, "Sample 1 (μg / mL)" and "Sample 3 (μg / mL)" show the measured values ​​of niacinamide concentration measured by the HPLC, and "Sample 1 (%)" and "Sample 3 (%)" show the measurement results at each time as relative values, with the value before sample addition for each group set to 100 using these measured values. From the results in Table 4, it was found that Sample 3 (Example 1: Lactic acid bacteria fermented product according to the present invention) showed improved permeability of niacinamide (nicotinamide) compared to Sample 1. [Table 4]

[0061] (Example of test 3) Test using a three-dimensional skin model (changes in collagen production) The amount of procollagen produced was measured when a predetermined sample was added to a three-dimensional skin model (KURABO:EFT-400) including the epidermis and dermis.

[0062] (Sample preparation, etc.) I prepared the following: • Three-dimensional skin model: KURABO (EFT-400) • Example 1 • EFT-400 assay medium: KURABO, EFT-400ASY · Nicotinamide: DSM, product code: 0487848340 · Sample 1: Purified water · Sample 2: A composition containing 0.1% of the nicotinamide in purified water at the final concentration · Sample 3: Example 1 · Sample 4: A composition containing 0.1% of the nicotinamide in purified water added to Example 1

[0063] (Test method) Figure 1 shows a schematic diagram of the test method. The three-dimensional skin model was transferred to a 24-well culture plate, 2.5 mL of EFT-400 assay medium was added, and cultured for 24 hours (pre-culture). After this culture, an insert hanging-type lid was attached to a 12-well culture plate, 5.0 mL of new EFT-400 assay medium was added to the bottom surface of the 12-well culture plate, 250 μL of the sample (Sample 1, Sample 2, Sample 3 or Sample 4) was added to the insert surface, and cultured in a CO2 incubator at 37°C for 72 hours. After this culture, the insert was taken out, the supernatant was recovered, and the amount of collagen production in the supernatant was measured using Procollagen type I C-peptide (PIP) EIA Kit (TAKARA, MK101).

[0064] (Measurement results) First, the results of comparing the group of Sample 1 and the group of Sample 2 are described. When the measurement result of Sample 1 is shown as 100.0, it was 90.3 for Sample 2. Next, the results of comparing the group of Sample 3 and the group of Sample 4 are described. When the measurement result of Sample 3 is shown as 100.0, it was 188.2 for Sample 4 (p < 0.05, Student's t-test compared with the value of the group of Sample 3). From the comparison results between Sample 3 and Sample 4, when Example 1 (the lactic acid bacteria fermented product according to the present invention) was used instead of purified water, promotion of collagen production (enhancing the addition effect of nicotinamide) was confirmed.

[0065] (Test Example 4) Human monitor test using an iontophoresis beauty device (measurement of fine lines) An ion introduction beauty device (e.g., LINKA multi facial treatment RM-5 (manufactured by Ivibet)) is known as a beauty device that can penetrate beauty components by passing a weak electric current through the skin. In this test, the change in nasolabial lines was confirmed by combining the treatment with an ion introduction beauty device and the application of a composition containing Example 1 (lactic acid bacteria fermentate) to the skin of 10 humans. The 10 subjects were 10 healthy women who answered in a prior questionnaire that they had wrinkles and signs of aging.

[0066] (Preparation of samples, etc.) The following (Sample 5 and placebo, aqueous composition for application to human skin) were prepared. · Sample 5: An aqueous composition containing the following · An aqueous solution (using water as a solvent, containing 99.0% of Example 1 and 1.0% of phenoxyethanol) at a final concentration of 99.4% · Nicotinamide (DSM, product code: 0487848340) at a final concentration of 0.1% · Caesalpinia spinosa gum (SOLAGUM TARA, SEPPIC S.A.) at a final concentration of 0.5% · Placebo: An aqueous composition containing the following · An aqueous solution containing 1% of phenoxyethanol with purified water as a solvent at a final concentration of 99.4% · Nicotinamide (DSM, product code: 0487848340) at a final concentration of 0.1% · Caesalpinia spinosa gum (SOLAGUM TARA, SEPPIC S.A.) at a final concentration of 0.5%

[0067] (Test method) For one week (7 days), once a day (at night), the composition of Sample 5 was applied to the left half of the face, and the composition of the placebo was applied to the right half of the face. After the application, a self-massage treatment was performed for 90 seconds on each half of the face using an ion introduction beauty device (LINKA multi facial treatment RM-5 (manufactured by Ivibet)). This application and this massage treatment were repeated for 7 days (7 times). Antera3D was applied to the face before the start of the test (day 0) and after the treatment on day 7 (day 7). TM The measurement (nasolabial fold measurement) was performed using Miravex, Dublin, and Ireland.

[0068] (Measurement results) The results shown below are relative values ​​calculated by taking the average value of the 10 subjects (average value of the measurement) and setting the value on day 0 after the calculation to 100. On day 7, the value for the group treated with sample 5 was 95.469, and for the placebo group it was 99.069. The measurement result indicated that "the nasolabial folds tended to be smaller in the group treated with sample 5 compared to the placebo group."

[0069] (Test Example 5) Sebum Shine Inhibition Test Shine caused by foundation is triggered by external factors such as sebum secretion, humidity, and temperature, and is a major cause of makeup breakdown. In particular, when sebum mixes with foundation, it is more likely to crease and become uneven, worsening the longevity of the makeup. Therefore, we investigated whether Example 1 (lactic acid fermented aloe vera leaf) could suppress shine caused by sebum.

[0070] (Sample preparation, etc.) • Artificial sebum model: Artificial sebum model described in paragraphs 0049 and 0050 of Japanese Patent No. 5923409 • Test piece: The test piece shown in Figure 2 consists of five circular pieces placed on a flat surface of copy paper, each measuring 5cm x 5cm. • Gloss meter: Gloss Checker IG-340 (Horiba, Ltd.) • Example 1 aqueous solution: An aqueous solution containing 99.0% of Example 1 and 1.0% phenoxyethanol, with water as the solvent. Placebo water: An aqueous solution containing 1.0% phenoxyethanol with water as the solvent. • Liquid foundation: W / O emulsion, Double Wear Stay-in-Place (Estée Lauder). BB cream; O / W emulsion, Uno Face Color Creator (Fine Today).

[0071] (Preparation of samples, etc., artificial sebum model) The artificial sebum model used was the artificial sebum model described in paragraphs 0049 and 0050 of Japanese Patent 5923409 (a model that faithfully reproduces the cleansing and removal of sebum components unique to middle-aged and elderly people). Liquid paraffin, squalane, wax ester, triglycerides, fatty acids, cholesterol, and ceramide were blended to the composition shown in Table 5, resulting in an artificial sebum model with a triglyceride / fatty acid (mass ratio) of 0.81 / 1.

[0072] [Table 5]

[0073] As the liquid paraffin, the product name: KAYDOL (manufactured by Sonneborn Inc.) was used. As the squalane, the product name: Squalane (manufactured by Wako Pure Chemical Industries, Ltd.) was used. As the wax ester, a mixture of hexadecyl palmitate (product name: hexadecyl palmitate, manufactured by Wako Pure Chemical Industries, Ltd.), myristyl myristate (product name: Excepal MY-M, manufactured by Kao Corporation), and oleyl oleate (product name: CETIOL, manufactured by Cognis Japan Inc.) (mass ratio: hexadecyl palmitate: myristyl myristate: oleyl oleate = 7:2:5) was used. As the aforementioned triglycerides, a mixture of tristearin (trade name: Tristearin, manufactured by Nacalai Tesque), tripalmitin (trade name: Tripalmitin, manufactured by Wako Pure Chemical Industries, Ltd.), trimiristin (trade name: Triiristin, manufactured by Wako Pure Chemical Industries, Ltd.), and triolein (trade name: Triolein, manufactured by Wako Pure Chemical Industries, Ltd.) (mass ratio: Tristearin: Tripalmitin: Triiristin: Triolein = 1:8:3:9) was used. As the fatty acid, a mixture of myristic acid (trade name: myristic acid, manufactured by Nacalai Tesque), palmitic acid (trade name: palmitic acid, manufactured by Nacalai Tesque), stearic acid (trade name: stearic acid, manufactured by Wako Pure Chemical Industries, Ltd.), and oleic acid (trade name: oleic acid, manufactured by Wako Pure Chemical Industries, Ltd.) (myristic acid:palmitic acid:stearic acid:oleic acid (mass ratio) = 4:10:2:10) was used. As the cholesterol, the trade name: Cholesterol (manufactured by Wako Pure Chemical Industries, Ltd.) was used. As the ceramide, ceramide 2 (trade name: ceramide TIC-001, manufactured by Takasago International Corporation) was used.

[0074] (Preparation of samples, test pieces, etc.) Test pieces (5cm x 5cm copy paper) as shown in Figure 2 were prepared in a room temperature environment (approximately 25°C). Four types of test pieces were prepared, as shown in Table 6 below. Using a micropipette, a base coat (Aqueous solution of Example 1 or placebo water) was dropped into five locations on the copy paper (120 μL each). After dropping, it was left to stand for about 10 minutes and then air-dried. Foundation (liquid foundation or BB cream) was applied to the dried copy paper (40 times in total from each direction). Another 5cm x 5cm copy paper was prepared, and artificial sebum (0.3g) was dropped into five locations (60 μL each) as shown in Figure 2. After dropping, it was left to stand at room temperature for 3 minutes. After standing, the test piece with foundation applied was placed on top of the test piece with artificial sebum applied to complete the test piece (artificial skin film) to be used in the following measurements.

[0075] (measurement) The gloss level was measured using a gloss meter at the time of completion of the test piece (0 minutes, initial value), 15 minutes after completion (15 minutes), 30 minutes after completion (30 minutes), and 60 minutes after completion (60 minutes). The measurement results are shown in Table 6. The measurement results shown are expressed as a percentage change compared to the initial value (100%). As shown in Table 6, in both the liquid foundation case and the BB cream case, the gloss level (shininess) was lower in the aqueous solution of Example 1 compared to the placebo water treatment.

[0076] [Table 6]

[0077] (Test Example 6) Measurement of hair gloss In Example 1 (lactic acid fermented aloe vera leaf), we confirmed whether the glossiness (shininess) of hair could be improved.

[0078] (Sample preparation, etc.) • Black hair: Hair length 10cm, 100% human black hair (BS-BA) (Beaulux) • Brown hair: Hair length 10cm, human hair medium brown (14LV, BR-2-A) (Beaulux) • Example 1 aqueous solution: An aqueous solution containing 99.0% of Example 1 and 1.0% phenoxyethanol, with water as the solvent. Placebo water: An aqueous solution containing 1.0% phenoxyethanol with water as the solvent. • Ultrasonic treatment beauty device: LINKA Shiny Glow Hair Treatment Iron (iVivid) • Gloss meter: Gloss Checker IG-340 (Horiba, Ltd.)

[0079] (Preparation of hair samples to be used in the test) The extracted black or brown hair was immersed in a 1% SDS solution at 40°C in a 25°C environment for 1 hour. After immersion, the black or brown hair was removed and washed. The washed black or brown hair was dried with 25°C air (cold air) for 1 hour. This process is referred to as "SDS treatment".

[0080] After this cold-air drying, the groups were set up as shown in Table 7 below. The following processes were performed according to the set groups. In Table 7, "○" indicates that the process was performed, and "×" indicates that the process was not performed. • Example 1 Aqueous Solution Treatment: Black or brown hair was immersed in the Example 1 aqueous solution at 25°C for 1 minute. After immersion, the black or brown hair was removed. After removal, the black or brown hair was washed. The washed black or brown hair was then air-dried with cold air for 1 hour. This treatment was performed up to the point of air-drying. • Placebo water treatment: Black or brown hair was immersed in placebo water at 25°C for 1 minute. After immersion, the black or brown hair was removed. After removal, the black or brown hair was washed. The washed black or brown hair was then air-dried with cold air for 1 hour. This treatment was performed up to the point of air-drying. • Heat treatment: This involves heating black or brown hair at 180°C for 10 minutes. • Ultrasonic treatment: This treatment involved using an ultrasonic treatment beauty device on black or brown hair for 3 minutes in a room temperature environment of approximately 25°C. To dry the sample after this treatment, it was dried using a drying oven (AS ONE, PCL-101).

[0081] Group 1 (Control 1) consists of black hair (after SDS treatment) that has not been treated with the aqueous solution of Example 1 or placebo water, nor with heat treatment or ultrasonic treatment. Group 2 consists of black hair (after SDS treatment) that was treated with the "Aqueous Solution of Example 1". Group 3 consists of black hair (after SDS treatment) that underwent "treatment with aqueous solution from Example 1" and "ultrasonic treatment after treatment with aqueous solution from Example 1". Group 4 (control group 2) consists of black hair (after SDS treatment) that underwent "heat treatment". Group 5 consists of black hair (after SDS treatment) that has been subjected to "heat treatment" followed by "aqueous solution treatment of Example 1". Group 6 consists of black hair (after SDS treatment) that underwent "heat treatment" followed by "placebo water treatment," and then "ultrasonic treatment after placebo water treatment." Group 7 consists of black hair (after SDS treatment) that has undergone "heat treatment" followed by "treatment with aqueous solution from Example 1" and "ultrasonic treatment after treatment with aqueous solution from Example 1". Group 8 (Control Group 3) consists of brown hair (after SDS treatment) that has not been treated with the aqueous solution of Example 1 or placebo water, nor with heat treatment or ultrasonic treatment. Group 9 consists of brown hair (after SDS treatment) that was treated with the "Aqueous Solution of Example 1". Group 10 consists of brown hair (after SDS treatment) that underwent "treatment with aqueous solution of Example 1" and "ultrasonic treatment after treatment with aqueous solution of Example 1". Group 11 (control group 4) consisted of brown hair (after SDS treatment) that underwent "heat treatment". Group 12 consists of brown hair (after SDS treatment) that has been subjected to "heat treatment" followed by "aqueous solution treatment of Example 1". Group 13 consists of brown hair (after SDS treatment) that underwent "heat treatment" followed by "placebo water treatment," and then "ultrasonic treatment after placebo water treatment." Group 14 consists of brown hair (after SDS treatment) that has undergone "heat treatment" followed by "treatment with aqueous solution from Example 1" and "ultrasonic treatment after treatment with aqueous solution from Example 1".

[0082] (Measurement of gloss level) After the treatment, the gloss level was measured in each group using a gloss meter. The measurement results are shown in Table 7. Compared to the placebo water treatment group, the group treated with the Example 1 aqueous solution showed higher gloss levels (improved gloss).

[0083] [Table 7]

[0084] (Test Example 7) Measurement of hair tensile strength We confirmed whether Example 1 (lactic acid fermented aloe vera leaf) increased the tensile strength of hair.

[0085] (Sample preparation, etc.) The hair samples (groups 1 to 14) listed in Table 7, which were prepared in Test Example 6, were prepared. Then, hair chips were made using the prepared hair as follows.

[0086] (Preparation of samples, etc., and creation of hair chips) Hair from each group was cut one strand at a length of 4 to 5 cm. The cut hair from each group was then sandwiched between 1.5 x 1.5 cm square pieces of copy paper and double-sided tape to create hair chips. These hair chips were then acclimatized using a predetermined method.

[0087] (Hair strength measurement) Using the acclimatized hair chips, hair diameter and hair tensile strength were measured for n=5 samples per group using a hair strength meter (Rheometer Sun Science) and a hair diameter measuring instrument (Laser Microgauge, Tokyo Optoelectronics Co., Ltd.).

[0088] Hair strength, hair cross-sectional area, and work done in the elastic deformation region (mJ) were calculated using the formula shown in Figure 3.

[0089] The measurement results of hair strength (tensile strength) are shown in Table 8 (case using black hair) and Table 9 (case using brown hair). The average value for each group (n=5) was calculated, and in Table 8, the value (average) for group 1 was set to 100, and in Table 9, the value (average) for group 8 was set to 100, showing the values ​​(tensile strength) in Tables 8 and 9. Generally, the tensile strength was higher in the group treated with the Example 1 aqueous solution compared to the placebo water treatment group.

[0090] [Table 8]

[0091] [Table 9] [Industrial applicability]

[0092] This invention can be used for topical skin preparations and methods for producing them.

Claims

1. A topical skin preparation containing a lactic acid fermented product produced from aloe vera leaves, The lactic acid bacterium is Lactiplantibacillus plantarum FL-781 strain (accession number: NITE ABP-04146), and the preparation is for topical application to the skin.

2. A method for producing a topical skin preparation containing a lactic acid fermented product produced from the juice of aloe vera leaves, The process for producing the aforementioned lactic acid fermented product is as follows: A method for producing a topical skin preparation, comprising a fermentation step of fermenting the aforementioned liquid with Lactiplantibacillus plantarum FL-781 strain (accession number: NITE ABP-04146).

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