JP7521945B2 - Skin preparations - Google Patents

Description

The present invention relates to a skin flora balance improving agent that is a liquid containing molasses or an extract thereof and brewer's lees or an extract thereof, and a liquid skin topical preparation that contains the same.

Various bacteria exist on human skin, and it has been suggested that this resident skin flora is related to skin homeostasis and diseases. For example, in about 90% of patients with atopic dermatitis, Staphylococcus aureus is said to be colonized in the rash area, and a relationship between this bacterium and the worsening of the rash has also been pointed out. On the other hand, Staphylococcus epidermidis is known as a normal epidermal flora, and is said to increase resistance to pathogenic bacteria by promoting the expression of antimicrobial peptides, etc. Therefore, it is important to maintain the number of beneficial skin bacteria on human skin and at the same time suppress the number of harmful bacteria.

For example, Patent Document 1 discloses an agent for improving normal skin flora, which contains 0.01 to 20% by mass of cellooligosaccharides having a cellobiose content of 60% by mass or more and 0.1 to 40% by mass of one or more selected from cellotriose, cellotetraose, cellopentaose, and cellohexaose, and does not bacteriostatically inhibit Staphylococcus epidermidis but bacteriostatically inhibits Staphylococcus aureus and Pseudomonas aeruginosa.

On the other hand, the present inventors have discovered that a liquid derived from natural products, which combines sugars or sugar extracts with brewer's lees or brewer's lees extracts, can provide various favorable effects on the skin and hair when added to topical agents or oral compositions (see Patent Document 2).

Patent No. 5121187 Patent Publication 2019-194175

The problem to be solved by the present invention is to provide a naturally derived ingredient or composition that has the effect of improving the balance of resident human skin bacteria in order to improve skin conditions.

In order to solve the above problems, the present inventors searched for a substance that promotes the growth of Staphylococcus epidermidis and inhibits the growth of Staphylococcus aureus in vitro, and confirmed the relationship with skin parameters. As a result, they discovered that a liquid containing molasses or an extract thereof and brewer's lees or an extract thereof significantly improves the balance of resident skin bacteria when added to an external preparation and applied to the skin.

That is, the present invention includes the following embodiments.
(1) An agent for improving the balance of resident skin bacteria, which is a liquid containing molasses or an extract thereof and liquor brewer's lees or an extract thereof and has a sugar content of 60 to 85.
(2) The agent according to (1), which contains 1% by mass or less of a preservative based on the total weight.
(3) The agent according to (1) or (2), having a viscosity of 2000 mPa·s or less as measured at 20°C using a B-type viscometer.
(4) The agent according to any one of (1) to (3), which has the effect of increasing the proportion of Staphylococcus epidermidis and decreasing the proportion of Staphylococcus aureus among normal skin flora.
(5) A liquid skin topical preparation containing the agent according to any one of (1) to (4).
(6) An external skin preparation according to (5), which, when applied to the skin, has the effect of reducing the proportion of Staphylococcus aureus among normal skin flora.
(7) An external skin preparation according to (5) or (6) for imparting smoothness to the skin of a subject prone to skin inflammation.

The agent of the present invention can improve the balance of normal bacteria on human skin, thereby improving skin conditions.

Fig. 1 shows the results of investigating the effect of the agent of the present invention on the growth of Staphylococcus epidermidis and Staphylococcus aureus in a co-culture system of these bacteria. In Fig. (A), "S. epi" indicates the colony count of Staphylococcus epidermidis, and "S. Aur" indicates the colony count of Staphylococcus aureus. Figure 2 shows the results of comparing skin parameter measurements 0 and 4 weeks after application of the test lotion between the Waminobonbon group and the placebo group in a human clinical trial. Figure 2(A) shows the percentage of Staphylococcus aureus bacteria, Figure 2(B) shows the cell detachment rate, and Figure 2(C) shows the stratum corneum thickness. FIG. 3 shows the results of a questionnaire on subjective effect conducted on subjects four weeks after application of the test lotion containing Waminobonbon. Figure 4 shows micrographs of keratinocytes from skin taken in a tape stripping test, with (A) and (C) showing the results for a subject at week 0, and (B) and (D) showing the results for the same subject at week 4 after application of the test lotion. FIG. 5 shows the results of comparing the percentage of Staphylococcus epidermidis (A) and Staphylococcus aureus (B) bacteria counts at weeks 0 and 4 between the smooth sensation group and the non-smooth sensation group for subjects who applied a test lotion containing Waminobonbon.

The following describes the agent and other aspects of the present invention.

(Skin flora balance improver)
In this embodiment, the term "skin resident microbiota balance improver" refers to a preparation or composition that maintains the skin resident microbiota present on healthy skin and provides a barrier function that prevents the invasion of pathogenic bacteria from the outside. When the balance of the skin resident microbiota is disrupted, excessive proliferation of the resident microbiota and invasion and proliferation of other harmful bacteria occur, inducing various skin symptoms. Since Staphylococcus epidermidis, which is one of the skin resident microbiota, has an antagonistic effect against pathogenic bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa and can inhibit the proliferation of such harmful bacteria, the action of promoting the growth of Staphylococcus epidermidis is considered to be one of the functions of the skin resident microbiota balance adjuster. In addition, the preparation or composition may contain a substance that has an antibacterial effect that does not affect the growth of Staphylococcus epidermidis and the like, but suppresses the growth of harmful Staphylococcus aureus and the like. In this embodiment, the balance improvement of the skin resident microbiota can be, for example, increasing the ratio of Staphylococcus epidermidis and decreasing the ratio of Staphylococcus aureus in the skin resident microbiota.

In one embodiment, an agent for improving the balance of resident skin bacteria (hereinafter, sometimes simply referred to as "agent") is provided, which is composed of a liquid containing sugars or an extract thereof and alcohol brewing lees or an extract thereof. The agent in this embodiment preferably has the effect of increasing the proportion of Staphylococcus epidermidis and decreasing the proportion of Staphylococcus aureus in the resident skin bacteria. Here, examples of sugars include glucose, fructose, galactose, sucrose, sugar, brown sugar, molasses, blackstrap molasses, raw sugar, lactose, maltose, etc. Note that brown sugar is a type of molasses, also known as brown sugar, which is a crude black lump sugar that is rich in iron, calcium, and other inorganic substances.

(About molasses)
Molasses is a by-product that is produced when sugar is refined from raw materials (sugar cane, sugar beet), and is a dark brown, viscous liquid that contains sugar and minerals, and is also called blackstrap molasses. Raw sugar is a type of sugar that is in the processing stage. Molasses can be produced, for example, using a process similar to that of brown sugar up to a certain point, but in this method, the juice of sugar cane or sugar beet is pretreated and then crystallized, and the molasses (blackstrap molasses) is separated to a certain extent using a centrifuge or the like, and the remaining crystals become raw sugar. This separated molasses can be used in the present invention. Molasses can be derived from, for example, shiroshitato, wasanbon, etc.

Among the above sugars, it is preferable to use molasses or an extract thereof. In this embodiment, "molasses" is a broad concept meaning a liquid containing the above sugars, and includes, but is not limited to, molasses, blackstrap molasses, and molasses-containing sugar, which are by-products that appear when sugars are refined from raw materials, and an example of this is wasanbon molasses. Molasses extract refers to an extract obtained after alcoholic fermentation of molasses, or an extract obtained by cold or warm infusion of molasses in a solvent such as water, methanol, or ethanol, followed by filtration and concentration.

(About brewery lees)
In this embodiment, the brewery lees of alcoholic beverages refers to the brewery lees of sake, synthetic sake, powdered sake, miscellaneous alcoholic beverages, shochu, cooking sake, mirin, fruit alcoholic beverages, whiskey, spirits, liqueurs, beer, and low-malt alcoholic beverages. The brewery lees of alcoholic beverages refers to the brewery lees of alcoholic beverages specified in the Liquor Tax Act, i.e., sake, synthetic sake, shochu, mitsuo, beer, fruit alcoholic beverages, whiskey, spirits, liqueurs, and miscellaneous alcoholic beverages, and refers to the filtration residue of various edible raw materials containing sugars and starches that are fermented into alcohol by microorganisms. In addition, any residue after saccharification of various raw materials (rice, wheat, sweet potato, potato, corn, kouriyan, potato, malt, hops, grapes, various fruits, various grains, etc.) by microorganisms and then alcoholic fermentation may be used. The brewer's lees extract refers to an extract obtained by cold or digesting the brewer's lees in a solvent such as water, methanol, or ethanol, followed by filtration and concentration.

In a preferred embodiment of the present invention, the agent is a liquid containing molasses or an extract thereof and brewer's lees or an extract thereof, and having a sugar content of 60 to 85. This liquid is rich in nutrients such as sugars, amino acids, and vitamins, and therefore, from the viewpoint of preventing contamination by microorganisms, the sugar content is preferably 60 or more, more preferably 65 or more, and even more preferably 70 or more. On the other hand, if the sugar content is too high, for example, the viscosity of the liquid increases, making it difficult to handle when blending it into an external agent or various compositions, so a sugar content of 85 or less is preferred. More preferably, the sugar content is 80 or less, and even more preferably, the sugar content is 75 or less. One specific liquid included in this embodiment is sold by Ichimaru Pharcos Co., Ltd. under the product name "Waminobonbon (registered trademark)".

(About sugar content)
In this embodiment, sugar content means Brix value. Here, Brix value is a unit for measuring the amount of soluble solids (such as sugars) in grams contained in 100 g of solution. Brix value can be measured using a commercially available refractometer or saccharometer. In detail, when 1 gram of sucrose is dissolved in 100 grams of aqueous solution at 20°C, the Brix value of the solution is said to be 1 degree, and the Brix value of a solution that shows the same sugar content refractometer value as this sucrose solution is defined as 1 degree. According to this definition, Brix value does not necessarily mean the mass percentage of sucrose in the sample solution.

(About preservatives)
Furthermore, the agent in this embodiment preferably contains a preservative in an amount of 1% by mass or less relative to the total mass. Here, the preservative is not particularly limited as long as it is a preservative that is added to a skin topical agent described later, but there are problems such as skin irritation and stickiness when added to a skin topical agent, and it is desired to reduce the amount used as much as possible. The agent in this embodiment has a high sugar content, so the growth of microorganisms is suppressed, and a preservative may not be added when stored at low temperature, but the use of a low concentration of a preservative ensures the desired preservative properties of the liquid even when stored at room temperature. Therefore, it is possible to produce an external agent composition that can be used without feeling irritation even by people with sensitive skin who feel skin irritation with the conventional amount of preservative. Therefore, there is no limit to the lower limit concentration when a preservative is added, and it is sufficient if it exceeds 0% by mass. The upper limit concentration is 1% by mass, preferably 0.5% by mass, and more preferably 0.1% by mass. Preservatives that have an antiseptic effect at these low concentrations are, for example, parabens, benzoic acid and its salts, and more preferably sodium benzoate.

(Regarding the form of the skin flora balance improver)
The skin resident bacteria balance improving agent in this embodiment is a substance that is fluid at room temperature, and includes paste-like or slurry-like substances. The agent in this embodiment is preferably adjusted to have a viscosity at 20°C of 10,000 mPa·s or less, preferably 7,000 mPa·s or less, preferably 5,000 mPa·s or less, preferably 2,000 mPa·s or less, and more preferably 1,500 mPa·s or less. This viscosity is determined, for example, by measuring the liquid in this embodiment at 20°C using a B-type viscometer (e.g., B8M, manufactured by Tokyo Keiki Co., Ltd.) with rotor No. 2 and a rotation speed of 12 rpm for 5 minutes. The lower limit of the viscosity of this liquid is not particularly limited, but since it contains a large amount of sugar, it is usually 500 mPa·s or more. As the viscosity of a liquid increases, it becomes more difficult to handle when added to a cosmetic composition or a food or beverage composition; however, the liquid of this embodiment is preferably adjusted to have a viscosity of 2000 mPa·s or less, making it easier to handle and more likely to be mixed uniformly with various materials.

When the agent of this embodiment is incorporated into a skin topical preparation, the amount of incorporation is not particularly limited as long as it is within a range in which the effect can be confirmed, but may be, for example, in the range of 0.01 mg/g to 400 mg/g (the denominator indicates the weight of the preparation), preferably 0.1 mg/g to 100 mg/g, and more preferably 1 mg/g to 50 mg/g.

(External skin preparations)
When the skin flora balance improving agent of the present invention is used as a cosmetic, topical medicine, quasi-drug, or other topical agent composition, it is prepared in the form of a general skin topical agent using a liquid containing sugars or an extract thereof and alcohol brewing lees or an extract thereof, together with a suitable pharmaceutical carrier that is pharmaceutical acceptable, and used in practice. Examples of such pharmaceutical carriers include moisturizing agents such as glycerin, petrolatum, urea, hyaluronic acid, and heparin; PABA derivatives (para-aminobenzoic acid, Escarol 507, etc.), cinnamic acid derivatives (Neo Heliopan, Parsol MCX, Sungard B, etc.), salicylic acid derivatives (octyl salicylate, etc.), benzophenone derivatives (ASL-24, ASL-24S, etc.), dibenzoylmethane derivatives (Parsol A, Parsol DAM, etc.), heterocyclic derivatives (Tinuvin series, etc.), and ultraviolet absorbing/scattering agents such as titanium oxide; disodium edetate, trisodium edetate, citric acid, and the like. Sequestering agents such as acid, sodium citrate, tartaric acid, sodium tartrate, lactic acid, malic acid, sodium polyphosphate, sodium metaphosphate, gluconic acid, etc.; sebum suppressants such as salicylic acid, sulfur, caffeine, tannin, etc.; bactericides and disinfectants such as benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, etc.; anti-inflammatory agents such as diphenhydramine hydrochloride, tranexamic acid, guaiazulene, azulene, allantoin, hinokitiol, glycyrrhizic acid and its salts, glycyrrhizic acid derivatives, glycyrrhetinic acid, etc.; vitamin A, vitamin B group (B1, B2, B6, Vitamins such as vitamin B12, B15), folic acid, nicotinic acid, pantothenic acid, biotin, vitamin C, vitamin D group (D2, D3), vitamin E, ubiquinones, and vitamin K (K1, K2, K3, K4); amino acids and derivatives thereof such as aspartic acid, glutamic acid, alanine, lysine, glycine, glutamine, serine, cysteine, cystine, tyrosine, proline, arginine, and pyrrolidone carboxylic acid; retinol, tocopherol acetate, magnesium ascorbyl phosphate, ascorbyl glucoside, arbutin, kojic acid, ellagic acid, and placenta extract. Examples of suitable extracts include whitening agents; antioxidants such as butylhydroxytoluene, butylhydroxyanisole, and propyl gallate; astringents such as zinc chloride, zinc sulfate, zinc phenocarbonate, zinc oxide, and potassium aluminum sulfate; sugars such as glucose, fructose, maltose, sucrose, trehalose, erythritol, mannitol, xylitol, and lactitol; various plant extracts such as licorice, chamomile, horse chestnut, saxifrage, peony, quince, Scutellaria Root, Phellodendron Bark, Coptis Rhizome, Jew's Root, and ginkgo leaf, as well as oily components, surfactants, thickeners, alcohols, powdery components, and pigments. These may be added by carrying out processing that is conventionally carried out according to the type and form of the product to which they are to be added (for example, a combination of any of the following processing steps: pulverization, milling, washing, hydrolysis, fermentation, purification, squeezing, extraction, fractionation, filtration, drying, powderization, granulation, dissolution, sterilization, pH adjustment, deodorization, decolorization, etc.), and then arbitrarily selecting from various materials.

When extracts from various plants (herbal medicines) or additives derived from animal raw materials are added to the topical preparation of this embodiment, in addition to protecting the skin and hair, moisturizing, improving feel and texture, imparting softness, easing irritation, relieving stress through fragrance, activating cells (preventing cell aging), suppressing inflammation, improving skin and hair quality, preventing and improving rough skin, promoting hair growth, preventing hair loss, imparting shine, cleansing effects, relieving fatigue, promoting blood flow, and providing a hot bath effect, effects such as fragrance, deodorization, thickening, preservation, and buffering can also be expected.

Furthermore, by combining the various cosmetic and medicinal effects of each of the raw materials known to date, it is possible to create a product that is expected to have multifunctional effects in addition to the desired effect of improving the balance of resident skin bacteria.

Specific examples of topical skin preparations include cosmetic creams, milky lotions, lotions, packs, skin milk (emulsions), gels, powders, lip balms, lipsticks, under-makeup, foundations, sun care, bath products, body shampoos, body rinses, soaps, cleansing foams, ointments, patches, jellies, aerosols, etc.

(Beauty methods)
Another embodiment of the present invention relates to a cosmetic method using the above-mentioned skin topical preparation. The cosmetic method of this embodiment is characterized in that the above-mentioned skin topical preparation is applied to a subject to improve the balance of skin resident bacteria and improve the skin condition. The application method is, for example, application to the skin of a desired part of the subject. Application to the skin may be performed, for example, once or multiple times a day. The method can be appropriately selected depending on the type of topical preparation. In general, when applied as a topical preparation prepared as a lotion, emulsion, gel, cream, or ointment, it is preferable to apply it to the skin of the face or the like about once or twice a day. The skin topical preparation of the present invention can also be used as a cosmetic, and can be applied to lotions, emulsions, beauty essences, creams, liquid foundations, powder foundations, lipsticks, and the like. In this embodiment, the cosmetic method is not only a method that is simply performed personally, but also includes a method that is provided as a cosmetic prescription tailored to the customer when a beauty-related product is provided, and is provided by a cosmetics salesperson or esthetician other than a doctor. It also includes a method provided as a method of using the product in the instructions (package insert, etc.) of the beauty-related product.

The cosmetic method of this embodiment can be used, for example, to improve skin conditions where an improvement in the balance of resident skin bacteria is desired. For example, it can be used to impart smoothness to the skin of subjects who are prone to skin inflammation. The term "smoothness" refers to a smooth, moist, and fresh skin condition. It is believed that improving the balance of resident skin bacteria improves rough skin and other disorders caused by mild inflammation of the skin and disturbance of the stratum corneum, and imparts smoothness.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. In the following examples, the unit % of the numerical values showing the amount of each component added means mass %.

(Production Example 1)
100 g of molasses (Baikodo Co., Ltd.) was mixed, stirred and dispersed in 250 g of purified water (about 30° C.), and then filtered to obtain about 300 g of molasses extract.

(Production Example 2)
100 g of sake lees (Nakano BC Co., Ltd.) were mixed and dispersed in 150 g of purified water (about 30° C.) with stirring, and then filtered to obtain about 200 g of sake lees extract.

(Production Example 3)
100 g of the molasses extract of Production Example 1 and 100 g of the sake lees extract of Production Example 2 were mixed, stirred and dispersed, and then filtered to obtain about 150 g of a molasses and sake lees mixed extract.

(Production Example 4)
100 g of molasses (Baikodo Co., Ltd.) and 25 g of sake lees (Nakano BC Co., Ltd.) were mixed, stirred and dispersed, and then filtered to obtain approximately 100 g of a molasses and sake lees mixed extract.

[Example 1] Construction of a co-culture system of Staphylococcus epidermidis and Staphylococcus aureus and sample evaluation In order to screen for a substance that promotes the growth of Staphylococcus epidermidis and inhibits the growth of Staphylococcus aureus in vitro, a co-culture system of Staphylococcus aureus and Staphylococcus epidermidis was constructed. The media and strains used in this co-culture are as follows:
- NB liquid medium (Nutrient Broth No. 2 + 0.1% NaCl (pH 6.0)) is prepared by dissolving NB medium (Kanto Chemical, 711067-5 Nutrient Broth No. 2) and NaCl in water, adjusting the pH to 5.5 to 6.5 with HCl, and sterilizing in an autoclave. The dissolution was performed at a ratio of 25 g of NB medium to 1000 ml of water.
- NB agar medium (Nutrient Broth No. 2) is NB medium to which agar has been added so that the concentration becomes 1.5% and then solidified.
・Staphylococcus epidermidis (S. epidermidis): ATCC12228
Staphylococcus aureus (S. aureus): ATCC 6538

(Culture preparation)
A frozen stock of the bacteria to be used was prepared in advance. This frozen stock was appropriately diluted with NB liquid medium and spread on an agar medium, and the viable bacterial concentration contained in the original stock was calculated from the number of colonies. A frozen stock of approximately 10 ⁹ CFU/mL was obtained. Next, NB liquid medium was added to a 24-well plate, followed by an appropriate amount of the sample to be used in the test. In this example, the molasses sake lees mixed extract (Waminobonbon, Ichimaru Pharcos Co., Ltd.) prepared in Production Example 4 was added to a final concentration of 0.01% or 1.00%, respectively, and the final liquid volume was adjusted to 500 μL. As a control, only NB liquid medium was used.

(Preparation, addition, and cultivation of a mixture of Staphylococcus epidermidis and Staphylococcus aureus)
The frozen stocks of bacteria were thawed and cooled on ice. Staphylococcus epidermidis and Staphylococcus aureus were appropriately diluted and mixed, and then an appropriate amount of the bacterial mixture was added to the prepared 24-well plate. In this example, the final concentration of Staphylococcus epidermidis was adjusted to 4.5 x 10 ⁵ CFU/mL, and the final concentration of Staphylococcus aureus was adjusted to 0.5 x 10 ⁵ CFU/mL. The 24-well plate containing the bacterial mixture was gently shaken to mix, and then cultured at 37°C for 16 hours.

(Plating of culture medium)
After 16 hours of culture, the 24-well plate was removed from 37°C and cooled on ice. The culture fluid in each well was transferred to a new 1.5 mL tube and cooled on ice. The bacterial culture fluid was diluted with new NB liquid medium. An appropriate amount of the diluted culture fluid was dropped onto the NB agar medium, spread with a conlarger, and cultured overnight at 37°C.

(Tallying up the results)
After overnight incubation, the number of colonies grown on the NB agar medium was counted. Since the color of the colonies can be used to distinguish between Staphylococcus epidermidis (white colonies) and Staphylococcus aureus (yellow colonies), the number of bacteria of each species was counted, and the proportion of Staphylococcus epidermidis in the total number of bacteria was calculated.

The results are shown in Table 1 and Figure 1. The counting was performed three times for each group (each addition group). The values shown in Table 1 and Figure 1 are the average values of the results of the three countings.

As shown in Table 1 and Figure 1 (A), it was found that the number of Staphylococcus aureus was significantly reduced when at least 1.00% Waminobonbon was added compared to when Waminobonbon was not added. When 1.00% Waminobonbon was added, the total number of bacteria was also reduced compared to when no Waminobonbon was added, so the proportion (%) of Staphylococcus epidermidis in the total number of bacteria was calculated, and the results are shown in Figure 1 (B). As shown in Figure 1 (B), the group without Waminobonbon added (labeled 0% in Figure 1 (B)) was 59.9%, the group with 0.01% Waminobonbon added (labeled 0.01% in Figure 1 (B)) was 52.9%, and the group with 1.00% Waminobonbon added (labeled 1.00% in Figure 1 (B)) was 90.7%. The results shown in Figure 1 (B) indicate that at least in the group where 1.00% Waminobonbon was added, the number of Staphylococcus epidermidis out of the total number of bacteria was significantly increased compared to the group where Waminobonbon was not added, suggesting that Staphylococcus aureus was selectively reduced.

[Example 2] Evaluation by human clinical trial A one-month continuous use test was conducted using a lotion (shown in Table 2 below) containing the molasses lees mixed extract (Waminobonbon, Ichimaru Pharcos Co., Ltd.) prepared in Production Example 4, and a placebo lotion in which the molasses lees mixed extract was replaced with water. The subjects (Waminobonbon group: 11 subjects (1 dropout), placebo group: 11 subjects) were healthy Japanese women in their 20s to 50s who were fully informed of the purpose and contents of the experiment. Approximately six months before this test, the skin bacterial flora of 82 subjects was collected and analyzed, and 22 subjects in whom Staphylococcus aureus was detected were selected and assigned to two groups. Four of them had a history of atopic dermatitis.

At the start of the study, and after two and four weeks, stratum corneum moisture content, transepidermal water loss (TEWL), sebum content, pH, melanin index, hemoglobin index, and image analysis using VISIA (registered trademark) were performed. At the start of the study and after four weeks, in addition to measuring skin parameters, skin bacterial flora samples were also taken and stratum corneum cells were sampled by tape stripping. After measurements two and four weeks after the start of the study, participants were asked to complete a questionnaire on their perception of effectiveness. For statistical analysis, the variance of the data was examined, and a Student's t-test was used if the variances were equal, and a Welch's t-test was used if the variances were unequal. A paired t-test was used to compare weeks 0 and 4 of the same group.

Details of the main test methods are as follows:
(Collection and analysis of subjects' skin microbiota)
The skin surface of the subject's cheek was rubbed with a wet swab (a cotton swab soaked in phosphate-buffered saline) to obtain a sample containing the bacterial flora. The changes in the bacterial flora were analyzed by the Next Generation Sequencing (NGS) method. DNA samples for NGS analysis were prepared from each swab with bacterial flora attached using a UCP Pathogen Mini Kit (QIAGEN) with a mechanical prelysis protocol and a spin protocol, and eluted in a volume of 30 μL. 1st PCR was performed on this DNA sample using Taq HS Perfect Mix (Takara) and a primer for the bacterial 16S rRNA v3 region.

Fw primer: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGAGGCAGCAG-3' (SEQ ID NO: 1)
Rv primer: 5'-GTCTCGTGGGGCTCGGAGATGTGTATAAGAGACAGATTACCGCGGCTGCTGG-3' (SEQ ID NO: 2)

The resulting DNA fragments were purified using AMPure XP (BECKMAN COULTER). The purified DNA fragments were subjected to 2nd PCR using Taq HS Perfect Mix (Takara) and Nextera XT Index Kit v2 (Illumina). Both 1st PCR and 2nd PCR were performed using MiniAmp (Thermo Fisher) as the PCR device. The resulting DNA fragments were purified using AMPure XP (BECKMAN COULTER) and subjected to NGS analysis using Miniseq (Illumina).

BLAST Analysis The FASTq files obtained from Miniseq were subjected to BLAST processing using homology analysis tool v3 (World Fusion), and then bacterial species were determined using Metagenome@Kin (World Fusion).

(Analysis by tape stripping method)
Using commercially available cellophane tape, keratinocytes were stripped from the subject's face and cheeks. The strips were then attached to a polyvinyl chloride plate. The plate was then immersed in hexane, and the tape was removed to transfer the cells onto a slide. The slide was stained with gentian violet and brilliant green. The specimens were observed under a microscope, and the area and shape of the cells were measured using a combination of a drawing device and a digitizer.

The cell detachment rate was calculated as the percentage of the number of pixels excluding white areas (areas without stratum corneum) to the total number of pixels using image analysis software (Adobe Photoshop) for images of the stratum corneum taken from the above specimens. The degree of stratification was determined by checking the brightness of the stratified areas in several places in six images for each person using the same image analysis software (Adobe Photoshop), determining the standard brightness for determining that the area is stratified, extracting only the stratum corneum from these images, and the ratio of the extracted stratum corneum that was below the standard brightness was regarded as the degree of stratification.

(result)
Figure 2 shows the results of comparing skin parameter measurements taken by subjects 0 and 4 weeks after application of the test lotion between the Waminobonbon group and the placebo group. (A) shows the percentage (%) of the number of leads of Staphylococcus aureus when the number of leads of all bacteria is set to 100, (B) shows the cell detachment rate, and (C) shows the degree of stratum corneum thickness. From the results of Figure 2 (A), in the Waminobonbon group, the ratio of Staphylococcus aureus at 4 weeks was 0.74%, a significant decrease (p = 0.01), compared to the ratio of Staphylococcus aureus at 0 weeks, which was 1.36%, but there was no change in the placebo group (p = 0.66).

The cell detachment rate, which is an index of the uniformity of cell detachment as the percentage of stratum corneum cells collected with tape in the entire visual field, significantly increased from 45.9% at week 0 to 50.9% at week 4 in the Waminobonbon group (p=0.034), but did not change in the placebo group (p=0.56). The degree of layering decreased in both groups, which was thought to be due to the effects of other ingredients in the lotion rather than the effects of Waminobonbon.

Figure 3 shows the results of a questionnaire on subjective effects administered to the subjects four weeks after applying the test lotion containing Waminobonbon. Responses were obtained by selecting multiple items that applied to each item listed on the horizontal axis. As shown in Figure 3, in the Waminobonbon group, six out of ten subjects responded that their skin had "become smoother," compared to two out of eleven subjects in the placebo group.

Figure 4 shows micrographs of keratinocytes from skin taken in a tape stripping test. (A) and (C) are photographs of the subject at week 0, and (B) and (D) are photographs of the same subject at week 4 after application of the test lotion. The magnifications of (A) and (B) are 100x, and (C) and (D) are 1000x. (A) and (C) and (B) and (D) are photographs of the same stratum corneum taken at different magnifications. It can be seen that the cell detachment rate is higher and the degree of stratification is lower in (B) and (D) compared to (A) and (C).

Exfoliation of keratinocytes means that the adhesive mechanism between epidermal keratinocytes weakens, causing old keratinocytes to peel off from the skin surface. Exfoliation is promoted by breaking down proteins called desmosomes, which adhere keratinocytes to each other. However, when the activity of this proteolytic enzyme decreases, the breakdown of desmosomes is inhibited, making it difficult for the keratin to peel off smoothly and causing stratification. This is thought to cause skin problems such as dullness, roughness, and uneven texture. Therefore, it is thought that promoting the exfoliation of keratinocytes and inhibiting stratification will improve dullness and make the skin smoother.

Figure 5 shows the results of comparing the bacterial counts of Staphylococcus epidermidis (A) and Staphylococcus aureus (B) at 0 and 4 weeks between the smooth sensation group (6 subjects) and non-sensation group (4 subjects) for subjects who applied the test lotion containing Waminobonbon. In the smooth sensation group, the proportion of Staphylococcus epidermidis increased from 1.66% at week 0 to 3.12% at 4 weeks after application of the test lotion, and the proportion of Staphylococcus aureus decreased from 1.84% at week 0 to 1.02% at 4 weeks, whereas no such tendency was observed in the non-sensation group.

It has been confirmed that application of a lotion containing the agent of the present invention is effective in providing a feeling of smoothness to the skin, improving indicators of keratin, and controlling Staphylococcus aureus, which is related to these. Therefore, the topical skin agent of the present invention may be used as a cosmetic product that improves rough skin and other skin disorders.

Claims (4)

A liquid that contains molasses or its extract and liquor lees or its extract, and has a sugar content of 60 to 85, and is an agent for improving the balance of resident skin bacteria. The agent according to claim 1, which contains 1% by mass or less of a preservative based on the total weight. The agent according to claim 1 or 2, which has a viscosity of 2000 mPa·s or less as measured with a B-type viscometer at 20°C. The agent according to any one of claims 1 to 3, which has the effect of increasing the proportion of Staphylococcus epidermidis and decreasing the proportion of Staphylococcus aureus among normal skin flora.