Novel lacticaseibacillus rhamnosus strain exhibiting antibacterial, skin moisturizing, skin barrier strengthening, Anti-atopic, Anti-inflammatory and antioxidant activities, and use thereof

Abstract

The present invention relates to a novel Lacticaseibacillus rhamnosus strain exhibiting antibacterial, skin moisturizing, skin barrier strengthening, anti-atopic, anti-inflammatory and antioxidant activities, and use thereof. More specifically, the present invention relates to: a novel Lactisbacillus rhamnosus strain that is isolated from female vaginal fluid and exhibits antibacterial, skin moisturizing, skin barrier strengthening, anti-atopic, anti-inflammatory and antioxidant activities; and use thereof in a skin external preparation, a cosmetic composition, a pharmaceutical composition or a food composition.

Description

Novel Lacticazeibacillus rhamnosus strains having antibacterial, skin moisturizing, skin barrier strengthening, anti-atopic, anti-inflammatory and antioxidant activities and their uses

[0001] The present invention relates to a novel Lacticaseibacillus rhamnosus strain having antibacterial, skin moisturizing, skin barrier strengthening, anti-atopic, anti-inflammatory, and antioxidant activities, and to uses thereof. More specifically, the present invention relates to a novel Lacticaseibacillus rhamnosus strain isolated from a woman's vaginal fluid having antibacterial, skin moisturizing, skin barrier strengthening, anti-atopic, anti-inflammatory, and antioxidant activities, and to uses thereof in external skin preparations, cosmetic compositions, pharmaceutical compositions, or food compositions.

[0002]

[0003] Reactive oxygen species, whether introduced from outside the body or generated within it, can accelerate aging and cause numerous problems, including cancer. Therefore, extensive research and development is being conducted on antioxidants that suppress oxidation caused by reactive oxygen species. Antioxidants are widely distributed throughout the animal and plant kingdoms, and fruits and vegetables are known to contain phenolic compounds, flavonoids, tocopherols, vitamin C, and selenium. However, naturally occurring antioxidants are not effectively effective when applied to the skin. Therefore, synthetic antioxidants, which boast excellent antioxidant properties and are inexpensive, are widely used. However, their use is limited due to safety concerns, including side effects.

[0004]

[0005] Furthermore, the epidermis, the outermost layer of the skin, performs a protective function, defending against various external physical, chemical, and mechanical stimuli and preventing excessive loss of body moisture through the skin. This protective function is possible through the normal formation and maintenance of the stratum corneum, which is composed of keratinocytes. Keratinocytes are cells that are formed through gradual changes in shape and function as basal cells that continuously proliferate in the lowest layer of the epidermis (stratum basale) migrate to the stratum corneum. After a certain period of time, old keratinocytes fall off from the skin, and new keratinocytes that rise from the lowest layer of the epidermis take over their function, repeating the process of epidermal differentiation or keratinization. During this keratinization process, keratinocytes produce natural moisturizing factors (NMF) and intercellular lipids such as ceramides, cholesterol, and fatty acids, so that the stratum corneum acts as a barrier to the outside world, thereby maintaining its function as a skin barrier.

[0006]

[0007] In addition, dry skin, which is considered one of the major diseases in modern society, is a symptom caused by abnormal skin barrier function, and has been increasing recently due to various factors such as environmental pollution, increase in dry environments such as apartments and high-rise buildings, increase in social stress, excessive bathing culture unique to our country, and skin aging, and cases where symptoms are severe and require treatment are also continuously increasing.

[0008]

[0009] Atopic dermatitis, which affects 10% of children, is also known to be primarily caused by dry skin or, more fundamentally, a dysfunction in the skin barrier function. To treat atopic dermatitis, much research has focused on maintaining adequate moisture within the skin, supplying moisture from outside sources or minimizing moisture loss from the body. Indeed, moisturizers with moisture-retaining properties, such as ceramide or its derivatives, have been developed and widely used in pharmaceutical and cosmetic applications. However, most of these moisturizers only provide temporary symptom relief rather than fundamental treatment, failing to demonstrate sufficient efficacy in treating dry skin and skin barrier dysfunction, including atopic dermatitis. Therefore, there is an urgent need to develop substances that fundamentally regenerate damaged skin barriers.

[0010]

[0011] Meanwhile, Lactobacillus rhamnosus was originally considered a subspecies of L. casei, but genetic studies have revealed that it belongs to the L. casei clade, which also includes L. paracasei and L. zeae. Lactobacillus rhamnosus is known to be isolated from various environments, such as the vagina and gastrointestinal tract of animals, including humans, and plants such as blueberries, Korean buckwheat, and ginseng sprouts.

[0012]

[0013] Prior literature

[0014] 1. Republic of Korea Publication Patent No. 10-2018-0124424

[0015]

[0016] The present inventors have made great efforts to develop a component that is safe for the body and exhibits excellent moisturizing effects and skin barrier strengthening activity. As a result, they have confirmed that a novel Lacticaseibacillus rhamnosus strain isolated from a woman's vaginal fluid has antibacterial activity against Staphylococcus aureus or Cutibacterium acnes, and can promote skin moisturizing and barrier strengthening by increasing the expression of HAS3 (Hyaluronan Synthase 3), FLG (Filaggrin), IVL (Involucrin), and LOR (Loricrin) genes in a concentration-dependent manner, and can exhibit anti-atopic and anti-inflammatory effects by inhibiting TARC production and reducing NO production, as well as scavenging DPPH free radicals and inhibiting cell damage caused by UVB. In addition, the inventors of the present invention completed the present invention after confirming that the novel Lacticazeibacillus rhamnosus can improve skin condition by regulating the Firmicutes / Actinobacteria balance and strengthen the skin microbiota balance through beneficial metabolites such as SCFA (short-chain fatty acids).

[0017]

[0018] Accordingly, the present invention aims to provide a novel Lacticaseibacillus rhamnosus strain and a culture medium thereof.

[0019] The present invention also aims to provide an antibacterial, skin moisturizing, skin barrier strengthening, anti-atopic, anti-inflammatory and antioxidant composition comprising the novel Lacticaseibacillus rhamnosus strain as an active ingredient.

[0020]

[0021] However, the problems that the present invention seeks to solve are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0022]

[0023] The present invention provides a novel Lacticaseibacillus rhamnosus strain, and a composition for improving skin for antibacterial, skin moisturizing, skin barrier strengthening, anti-atopic, anti-inflammation and anti-oxidation, including the novel Lacticaseibacillus rhamnosus strain as an active ingredient, for example, a skin external preparation, a cosmetic composition, a pharmaceutical composition or a food composition.

[0024]

[0025] According to the first implementation example,

[0026] A novel Lacticaseibacillus rhamnosus strain or a culture solution thereof is disclosed.

[0027] In the present invention, the novel Lacticazei Bacillus rhamnosus strain may have the deposit number KCTC 15925BP extracted from a woman's vaginal fluid.

[0028] In the present invention, the novel Lacticazeibacillus rhamnosus strain or its culture solution may have antibacterial activity against Staphylococcus aureus or Cutibacterium acnes, skin moisturizing and skin barrier strengthening by increasing the expression of HAS3 (Hyaluronan Synthase 3), FLG (Filaggrin), IVL (Involucrin) and LOR (Loricrin), anti-atopic and anti-inflammation by inhibiting TARC production and reducing NO production, and DPPH free radical scavenging and UVB-induced cell damage suppression activities.

[0029]

[0030] According to the second implementation example,

[0031] A skin external preparation for improving the skin for antibacterial, skin moisturizing, skin barrier strengthening, anti-atopic, anti-inflammation and antioxidant effects is disclosed, comprising the novel Lacticazei Bacillus rhamnosus strain or a culture solution thereof as an active ingredient.

[0032] In the present invention, the external skin preparation may include a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, a drug-containing bandage, a lotion, or a combination thereof.

[0033]

[0034] According to the third implementation example,

[0035] A cosmetic composition for improving skin for antibacterial, skin moisturizing, skin barrier strengthening, anti-atopic, anti-inflammation and anti-oxidation effects is disclosed, comprising the novel Lacticazei Bacillus rhamnosus strain or a culture thereof as an active ingredient.

[0036] In the present invention, the cosmetic composition may be formulated to include a toner (skin lotion), skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, foundation, essence, nourishing essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, suspension, gel, powder, paste, mask pack, or sheet or aerosol composition.

[0037]

[0038] According to the fourth implementation example,

[0039] A pharmaceutical composition for preventing or treating skin diseases is disclosed, comprising the novel Lacticazei Bacillus rhamnosus strain or a culture thereof as an active ingredient.

[0040] In the present invention, the skin disease may include a disease caused by damage to the skin barrier function, skin aging, skin wound, skin scar, or skin inflammation or atopic dermatitis.

[0041] In the present invention,

[0042] The above skin inflammation may include atopic dermatitis, eczema, seborrheic dermatitis, psoriasis or acne.

[0043]

[0044] According to the fifth implementation example,

[0045] A health functional food for improving skin for antibacterial, skin moisturizing, skin barrier strengthening, anti-atopic, anti-inflammation and anti-oxidation is disclosed, comprising the novel Lacticazei Bacillus rhamnosus strain or a culture solution thereof as an active ingredient.

[0046]

[0047] The novel Lacticaseibacillus rhamnosus or its culture solution isolated from a woman's vaginal fluid according to the present invention has the following effects:

[0048] 1. Skin improvement effect

[0049] The novel Lacticazei Bacillus rhamnosus according to the present invention can promote skin moisturization and barrier strengthening by concentration-dependently increasing the expression of HAS3, FLG, IVL and LOR genes, and can exhibit anti-atopic and anti-inflammatory effects by inhibiting TARC production and reducing NO production, and can also suppress DPPH free radical scavenging and UVB-induced cell damage.

[0050] 2. Clinical efficacy verification

[0051] The formulation comprising the novel Lacticazeibacillus rhamnosus according to the present invention not only significantly increases skin moisture content and reduces TEWL (teratogenic water loss), but is also effective in improving dry skin, and can improve XMI (Xerosis-Microbiome Index) to regulate skin microbiome balance and contribute to promoting skin health.

[0052] 3. Regulating skin microbiome balance

[0053] The novel Lacticazeibacillus rhamnosus according to the present invention can improve skin condition by regulating the Firmicutes / Actinobacteria balance and strengthen the skin microbiota balance through beneficial metabolites such as SCFA (short-chain fatty acids).

[0054] 4. Safety Assessment

[0055] The novel Lacticazeibacillus rhamnosus according to the present invention has been proven to be a safe strain as it is non-cytotoxic and exhibits γ-hemolysis.

[0056]

[0057] Figure 1 shows the results of whole genome sequencing analysis of candidate strain LP51.

[0058] (A) The LP51 strain genome is shown as a single circular chromosome with a length of 3,003,557 bp. The antisense and sense strands are color-coded according to their respective COG (Orthologous Groups) categories, with the outer edge representing the central branch. The circular map also shows the positions of tRNA and rRNA, colored red and blue, respectively. The inner circle of the map represents GC skew, with yellow and blue representing positive and negative values, respectively, and the GC content highlighted in red and green.

[0059] (B) Functional classification of genes revealed that 2,441 proteins were assigned to the Clusters of Orthologous Groups (COG) family. Of these, 1,761 proteins had known biological functions, but the roles of 680 CDSs remain unknown.

[0060] (C) The genome similarity of LP51 was analyzed using the OrthoANI (Orthologous Average Nucleotide Identity) method and compared with Lacticaseibacillus and non-Lacticaseibacillus strains. The data showed that LP51 had a high similarity of 99.74% to Lacticaseibacillus rhamnosus CP-1, and 79.58%, 77.21%, and 66.68% to other Lacticaseibacillus strains, such as LC130, HL182, and YH-lac23, respectively.

[0061] (D) As a result of comparing the chromosomal characteristics of various L. rhamnosus strains, the results strongly suggest that the newly discovered strain LP51 is a new strain of L. rhamnosus. In this study, the NCBI accession number of the candidate strain Lacticaseibacillus rhamnosus LABIO-PMC-51 (LP51) is PRJNA1123863, and the strains and their NCBI RefSeq assemblies used for genome comparison were Hsryfm 1301 (GCF_008727835.1), TK-F8B (GCF_015377485.1), LR-B1 (GCF_004010975.1), LDTM7511 (GCF_017795605.1), SN21-1 (GCF_033802705.1), and VSI43 (GCF_029011275.1).

[0062] Figure 2 shows the results of in vitro testing of LP51 culture filtrate on various skin-related indicators. The effects of LP51 on skin hydration, skin barrier strengthening, anti-atopic effects, anti-inflammatory effects, antioxidant effects, and cytotoxicity were confirmed through various in vitro tests.

[0063] (A) The effect of LP51 culture filtrate on skin barrier strengthening and moisturizing ability was evaluated by measuring the transcription level of HAS3 (hyaluronan synthase 3) in HaCaT (human keratinocyte) cells, and the results showed a significant increase at a concentration of 0.75% or higher, and similar results to hEGF (human epidermal growth factor, 1 μg / mL) were shown at a concentration of 3%.

[0064] The skin barrier strengthening effect was evaluated by measuring the transcript levels of (B) FLG (filaggrin), (C) IVL (involucrin), and (D) LOR (loricrin) in the same cell line, and showed a significant increase in a concentration-dependent manner at 1.5% and 3% concentrations of LP51.

[0065] (E) The anti-atopic effect was evaluated by analyzing the change in TARC (thymus and activation-regulated chemokine) production, and LP51 1% significantly reduced TARC levels in cells treated with TNF-α (10 ng / mL) and IFN-γ (20 ng / mL), and showed better results than dexamethasone (10 μg / mL), an immunosuppressive corticosteroid.

[0066] (F) The anti-inflammatory effect was investigated by measuring NO (carbon monoxide) levels induced by LPS (lipopolysaccharide, 1 μg / mL) using RAW 264.7 macrophages, and it was found that NO levels were significantly reduced in a concentration-dependent manner at concentrations of LP51 0.25% or higher.

[0067] (G) The antioxidant effect was confirmed through DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity analysis, and showed an effect similar to that of L-ascorbic acid (50 μg / mL) at a concentration of LP51 15% or higher.

[0068] (H) Additionally, LP51 was found to play a role in restoring cell viability damaged by UVB (ultraviolet B) exposure in HaCaT cells.

[0069] Results are presented as mean and standard deviation. Statistical significance between groups was analyzed using Student's t-test and is indicated as (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

[0070] Figure 3 presents the results of a double-blind clinical trial evaluating the efficacy of an LP51 formulation for dry skin. To evaluate efficacy, a 2.9% LP51 formulation was applied to the flexor forearm area twice daily (morning and evening) for 4 weeks. Evaluations were conducted at baseline (week 0), weeks 2, and 4, and compared with a placebo group that applied a formulation containing no LP51.

[0071] (A, B) Skin hydration levels significantly increased at weeks 2 and 4 compared to baseline, and showed a statistically significant increase compared to the placebo group.

[0072] (C, D) Transepidermal water loss (TEWL) was significantly reduced at week 4 compared to baseline, and showed a significant reduction compared to the placebo group.

[0073] (E, F) The severity of pruritus measured by visual analogue scale (VAS) and the visual appearance evaluated by ESIF scale (erythema, keratin, induration, cracking) showed a tendency to decrease, but no statistically significant difference was observed compared to the placebo group.

[0074] Results are expressed as mean and standard deviation, and statistical significance between groups was analyzed using repeated measures ANOVA for VAS scores, and Mann-Whitney U test using delta values ​​was applied for other comparisons (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

[0075] (G, H) Photographs of the treatment area were taken at baseline (week 0), weeks 2, and weeks 4 using a Folliscope equipped with a digital camera and a 40x magnification lens.

[0076] (I, J) The stability of the formulation was tested, and “N” indicates Normal, and the corresponding images showing the results were included.

[0077] Figure 4 shows the results of skin metagenome analysis based on the dryness-microbiome index (XMI) of LP51.

[0078] (A) Skin metagenome analysis was performed during a 4-week clinical trial to investigate changes in microbial composition before and after application. LP51 significantly reduced Actinobacteria and increased Firmicutes. Significant differences between microbial communities were confirmed using the Wilcoxon signed-rank test (**, p < 0.01; ***, p < 0.001).

[0079] (B) As a result, LP51 significantly increased the gut microbiome index (XMI), a newly proposed index based on the ratio of Firmicutes / Actinobacteria, compared to the placebo group. LP51 for 4 weeks showed a significant effect of increasing the fold change of XMI compared to the placebo.

[0080] (C) In linear regression analysis, XMI showed a positive correlation with skin hydration level, and LP51 increased the skin hydration slope compared to XMI.

[0081] (D) Transepidermal water loss (TEWL) showed a negative correlation with XMI, and LP51 was found to effectively reduce water loss by lowering the y-intercept of TEWL.

[0082] (E) In vitro antibacterial test results showed that LP51 exhibited a superior inhibitory effect against Propionibacterium acnes (Actinobacteria) than against Staphylococcus aureus (Firmicutes), further supporting the observed XMI results.

[0083] (F) These XMI regulatory effects are thought to be due to short-chain fatty acids (SCFAs) produced by the LP51 strain.

[0084] (G) In addition, alpha and beta diversity analyses showed that LP51 did not cause dysbiosis (disruption of balance) in the skin microbiome, thereby proving the safety of the microbiome.

[0085] Figure 5 shows the safety profile of LP51.

[0086] (AC) LP51 did not exhibit cytotoxicity under any conditions, similar to Lactobacillus rhamnosus (KCTC 5033), in the WST-1 assay using mouse macrophage RAW 264.7 cells and immortalized human keratinocyte HaCaT cells, and in the LDH assay using human colorectal adenocarcinoma HT-29 cells. This is in contrast to the significant cytotoxicity observed with the positive control (lysis buffer) and a pathogenic E. coli strain (NCCP 14780).

[0087] (D) The safety of LP51 was confirmed as compared to the reference strain L. rhamnosus, as D-lactate production was not significantly increased in LP51-treated cells.

[0088] (E) In addition, LP51 showed γ-hemolysis (no hemolysis) on blood agar medium, similar to L. rhamnosus, in contrast to the β-hemolysis shown by S. aureus (ATCC 6538).

[0089] (F) LP51 did not grow on bile acid agar medium, similar to the reference strain L. rhamnosus (KCTC 5033), and, unlike the positive control L. plantarum (KCTC 3105), did not show taurodeoxycholic acid (TDCA) hydrolysis activity.

[0090] (G) In addition, LP51 was proven safe in an antibiotic resistance evaluation according to the European Food Safety Authority (EFSA) standards.

[0091] Statistical significance was analyzed using Student's t-test compared to the control group and was expressed as **p < 0.01; ***p < 0.001.

[0092]

[0093] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0094] The term "antibacterial" as used herein means killing microorganisms or inhibiting the growth of microorganisms. In one embodiment, it may be used in the present specification to mean killing a specific bacteria or inhibiting the growth of a specific bacteria. In a specific embodiment, it may be used in the present specification to mean killing a bacteria belonging to Staphylococcus aureus or Cutibacterium acnes or inhibiting the growth thereof. It may be used in the meaning of killing one or more bacteria selected from the group consisting of or inhibiting the growth thereof.

[0095] The term “skin barrier” used herein refers to the function of the stratum corneum, formed through repeated epidermal differentiation or keratinization in the epidermis, which performs a defensive function against various external physical, chemical, and mechanical stimuli, as a barrier to the outside world. Therefore, by treating the effective ingredient of the present invention, the differentiation of keratinocytes in the epidermal layer is increased through increased expression of HAS3 (Hyaluronan Synthase 3), FLG (Filaggrin), IVL (Involucrin), and LOR (Loricrin), thereby making the skin surface healthy, thereby generating a “skin barrier strengthening effect.”

[0096] The term "skin moisturizing" as used herein refers to inhibiting or suppressing the decrease in skin moisture or increasing the skin's moisture content to smooth the skin surface and impart a glow. Preferably, the moisturizing effect is related to filaggrin, a protein that plays a key role in the formation of the stratum corneum, and a decrease in the expression of filaggrin causes changes in the skin barrier function and skin moisturizing. Therefore, by treating the effective ingredient of the present invention, the expression of filaggrin, which is related to skin moisturizing, can be promoted, thereby significantly enhancing the skin moisturizing effect.

[0097] The term "anti-inflammatory" as used herein may be used interchangeably with "inhibiting or improving inflammation" and may refer to any action that alleviates the immune response and inhibits NO production.

[0098] The term “anti-atopic” used herein refers to an effect of preventing and treating skin diseases such as atopy and dry skin, and refers to suppressing, inhibiting, or alleviating atopic symptoms. Preferably, the atopic improvement effect is related to PPAR, which plays an important role in maintaining and restoring homeostasis of skin barrier function, moisturizing ability, and expression of the inflammatory healing process. Therefore, by treating the effective ingredient of the present invention, the activity of PPAR alpha, a key regulator of atopic dermatitis, is enhanced, thereby regulating skin conditions such as regulating skin barrier permeability, inhibiting epidermal proliferation, and inducing differentiation, thereby exhibiting a remarkable effect in improving atopy.

[0099] The term “antioxidant” as used herein refers to the inhibition of oxidation of cells by highly reactive free radicals or reactive oxygen species (ROS) due to oxidative stress caused by intracellular metabolism or ultraviolet rays, and includes the reduction of damage to cells caused by removing free radicals or reactive oxygen species.

[0100] The term "improvement" as used herein may mean any action that at least reduces the severity of a symptom, for example, a parameter associated with alleviating or treating a condition.

[0101]

[0102] I Novel strain of Lacticazei Bacillus rhamnosus

[0103] The present invention aims to provide a novel Lacticaseibacillus rhamnosus strain.

[0104] The novel Lacticaseibacillus rhamnosus strain according to the present invention may have the deposit number KCTC 15925BP isolated from a woman's vaginal fluid.

[0105]

[0106] II. Composition for skin improvement

[0107] The present invention provides a composition for improving skin for antibacterial, skin moisturizing, skin barrier strengthening, anti-atopic, anti-inflammation and anti-oxidation, comprising a novel Lacticaseibacillus rhamnosus strain or a culture thereof as an active ingredient.

[0108] In the composition for improving skin according to the present invention, the culture solution may be a culture solution obtained by culturing a strain and removing the strain.

[0109] In the composition for improving skin according to the present invention, the culture medium is present in an amount of 0.001 wt% to 80 wt%, for example, 0.01 wt% to 60 wt%, 0.01 wt% to 40 wt%, 0.01 wt% to 30 wt%, 0.01 wt% to 20 wt%, 0.01 wt% to 10 wt%, 0.01 wt% to 5 wt%, 0.05 wt% to 60 wt%, 0.05 wt% to 40 wt%, 0.05 wt% to 30 wt%, 0.05 wt% to 20 wt%, 0.05 wt% to 10 wt%, 0.05 wt% to 5 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 40 wt%, 0.1 wt% to 30 wt%, It may be included in an amount of 0.1 wt% to 20 wt%, 0.1 wt% to 10 wt%, 0.1 wt% to 5 wt%, 0.1 wt% to 3 wt%, or 0.1 wt% to 2 wt%.

[0110] In one exemplary embodiment, the skin improvement composition may exhibit a skin barrier strengthening effect by increasing the expression of FLG (filagrin), IVL (involucrin), and LOR (loricrin).

[0111] In one exemplary embodiment, the skin improvement composition may increase the expression of hyaluronan synthases, ceramide synthase 3, b-glucocerebrosidase, or AQP3 (Aquaporin 3), and specifically may exhibit a skin moisturizing effect by increasing the expression of hyaluronan synthase.

[0112] In one exemplary embodiment, the skin improvement composition may inhibit the expression of IL-1a, IL-6, TARC (thymus and activation-regulated chemokine) or TSLP (thymic stromal lymphopoietin), and specifically may exhibit an anti-inflammatory or anti-atopic effect by inhibiting the expression of TARC.

[0113] In one exemplary embodiment, the skin improvement composition may exhibit an anti-inflammatory effect by inhibiting NO production.

[0114] In one exemplary embodiment, the skin improvement composition may exhibit an antioxidant effect by inhibiting oxidation of cells by free radicals or reactive oxygen species (ROS).

[0115] In the composition for improving skin according to the present invention, the composition may contain the culture medium as an effective amount or as an effective ingredient. The effective amount may be appropriately selected depending on the individual. It may be determined based on factors including the severity of the disease or condition, the individual's age, weight, health, and sex, the individual's sensitivity to the extract, the time of administration, the route of administration and excretion rate, the duration of administration, other compositions combined with or used concurrently with the composition, and other factors well known in the fields of physiology and medicine.

[0116] In the composition for improving skin according to the present invention, the composition may further include a cosmetically, food-wise, or pharmaceutically acceptable excipient or carrier. The carrier may be an excipient, a disintegrant, a binder, a glidant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be sodium starch glycolate, calcium hydrogen phosphate anhydrous, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose, or a combination thereof. The glidant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

[0117] In the composition for improving skin according to the present invention, the composition may be formulated as a parenteral administration dosage form. The parenteral administration dosage form may be an injection or a skin external preparation. The skin external preparation may be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, a drug-containing bandage, a lotion, or a combination thereof. The skin external preparation may appropriately incorporate ingredients commonly used in skin external preparations such as cosmetics or pharmaceuticals, such as aqueous ingredients, oily ingredients, powder ingredients, alcohols, moisturizers, thickeners, UV absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, and various skin nutrients, as needed. The above skin external preparation may also appropriately contain metal sequestrants such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; caffeine, tannin, bellapamil, licorice extract, glabridin, hot water extract of the fruit of Calin; various herbal medicines; tocopheryl acetate, glycyrrhizic acid, tranexamic acid and derivatives or salts thereof; and sugars such as vitamin C, magnesium ascorbic acid phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, and trehalose.

[0118]

[0119] 1. Cosmetic composition

[0120] According to one exemplary embodiment, the skin improvement composition according to the present invention may be a cosmetic composition.

[0121] In a cosmetic composition according to an exemplary embodiment, the culture medium may be prepared in a formulation including a toner (skin lotion), a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisture lotion, a nourishing lotion, a massage cream, a nourishing cream, a moisture cream, a hand cream, a foundation, an essence, a nourishing essence, a pack, a soap, a cleansing foam, a cleansing lotion, a cleansing cream, a body lotion, a body cleanser, a suspension, a gel, a powder, a paste, a mask pack, or a sheet or aerosol composition. A composition in such a formulation may be prepared according to a method conventional in the art. The cosmetic composition may further include a preservative, a stabilizer, a surfactant, a solubilizer, a moisturizer, an emollient, an ultraviolet absorber, an antiseptic, a bactericide, an antioxidant, a pH adjuster, an organic and inorganic pigment, a fragrance, a cooling agent or an antiperspirant, etc. The mixing amount of the additional ingredients such as the above moisturizer can be easily selected by a person skilled in the art within a range that does not impair the purpose and effect of the present invention, and the mixing amount may be 0.001 to 5 wt%, specifically 0.01 to 3 wt%, based on the total weight of the composition.

[0122]

[0123] 2. Food composition

[0124] According to one exemplary embodiment, the skin improvement composition according to the present invention may be a food composition.

[0125] In a food composition according to an exemplary embodiment, the food composition may be formulated into a formulation of a conventional health functional food known in the art. The food composition may be manufactured into a general formulation such as a powder, granule, tablet, pill, capsule, suspension, emulsion, syrup, infusion, liquid, or extract, and may be manufactured into any health food form such as dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, jelly, ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes. A food-wise acceptable carrier or additive may be used to formulate the health food, and any carrier or additive known to be usable in the art may be used to manufacture the formulation to be manufactured. The above additives may include various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition may contain fruit pulp for producing natural fruit juice, fruit juice drinks, and vegetable drinks. These additive components may be used independently or in combination, and the proportion of the additives may be 0.001 to 5 wt%, specifically 0.01 to 3 wt%, based on the total weight of the composition.

[0126] In a food composition according to an exemplary embodiment, the content of the culture medium in the food composition may be suitably determined depending on the intended use (prevention or improvement). Generally, it may be included in an amount of 0.01 to 15 wt% of the total food weight, and when manufactured as a beverage, it may be included in an amount of 0.02 to 10 g, specifically 0.3 to 1 g, based on 100 mL. The beverage may further include other ingredients in addition to the extract, and may further contain various flavorings or natural carbohydrates typically used in beverages. The natural carbohydrates may include conventional sugars such as monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), polysaccharides (e.g., dextrin, cyclodextrin, etc.), and sugar alcohols such as xylitol, sorbitol, and erythritol. Additionally, the beverage may contain natural flavoring agents (e.g., thaumatin, stevia extract, etc.) and synthetic flavoring agents (e.g., saccharin, aspartame, etc.). The amount of the natural carbohydrates may be generally about 1 to 20 g, specifically about 5 to 12 g, per 100 mL of the beverage.

[0127]

[0128] 3. Pharmaceutical composition

[0129] According to one exemplary embodiment, the skin improvement composition according to the present invention may be a pharmaceutical composition.

[0130] According to an exemplary embodiment, the pharmaceutical composition may be a pharmaceutical composition for preventing or treating a skin disease. The skin disease may be a disease caused by impaired skin barrier function, skin aging, skin wounds, skin scars, skin inflammation, or atopic dermatitis. The term "prevention" includes suppressing the occurrence of a disease. The term "treatment" includes suppressing, alleviating, or eliminating the development of a disease. The impaired skin barrier function may refer to any change that appears on the skin due to a decrease or damage to the skin barrier function.

[0131] In a pharmaceutical composition according to an exemplary embodiment, the skin inflammation may refer to any disease caused by immune stimulation. Examples include, but are not limited to, atopic dermatitis, eczema, seborrheic dermatitis, psoriasis, and acne.

[0132]

[0133] III. How to improve skin

[0134] The present invention

[0135] A step of administering to a subject a skin improvement composition for antibacterial, skin moisturizing, skin barrier strengthening, anti-atopic, anti-inflammatory and anti-oxidation, comprising a novel Lactobacillus strain as an active ingredient;

[0136] The present invention aims to provide a method for improving the skin of an object including the composition. The composition is the same as that described above in II. Composition for improving skin.

[0137] In the skin improvement method according to the present invention, the method may be a method for strengthening the skin barrier function of an individual, a method for maintaining skin moisture or effectively preventing moisture loss of an individual, a method for enhancing antioxidant activity in an individual, a method for improving or treating inflammation of an individual, or a method for improving, treating, or preventing atopic dermatitis of an individual.

[0138] In the skin improvement method according to the present invention, the administration may be directly administered to the subject by any means, such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal, or subcutaneous administration. The administration may be systemic or local. The terms "administering," "introducing," and "implanting" are used interchangeably and may refer to the placement of the composition according to one embodiment into the subject by a method or route that results in at least partial localization of the composition according to one embodiment to the desired site. The administration may be by any suitable route that delivers the culture medium or at least a portion of the culture medium component of the composition according to one embodiment to the desired site in the viable subject.

[0139] In the skin improvement method according to the present invention, the subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be an object in need of skin beauty improvement, such as skin barrier strengthening, skin moisturizing, antioxidant, anti-inflammatory, or anti-atopic effects.

[0140] In the skin improvement method according to the present invention, the administration is 0.1 mg to 1,000 mg of the novel Lactobacillus strain culture solution per day per individual, for example, 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg. It may be administered in mg. However, the dosage may vary depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and those skilled in the art can appropriately adjust the dosage by considering these factors. The frequency of administration may be once a day or twice or more within the range of clinically acceptable side effects, and the administration site may be administered in one or more sites, and the total number of administration days may be from 1 to 30 days per treatment, daily or at intervals of 2 to 5 days. If necessary, the same treatment may be repeated after an appropriate period. For animals other than humans, the same dosage per kg as for humans may be administered, or the above dosage may be converted into an amount based on the volume ratio (e.g., average value) of the organs (heart, etc.) of the target animal and humans.

[0141]

[0142] Hereinafter, the present invention will be described in detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0143]

[0144] Materials and Methods

[0145] 1. Culturomics-based study to isolate candidate strains from healthy vaginal fluid.

[0146] Lactobacillus strains were isolated from healthy vaginal microbiota using a culturomics approach combined with 16S rRNA sequencing technology. Specimens were collected from healthy Korean women according to the method reported in [Kim, S.; Seo, H.; Rahim, MA; Tajdozian, H.; Kim, YS; Song, HY. Characteristics of Vaginal Microbiome in Women with Pelvic Inflammatory Disease in Korea. Pol. J. Microbiol. 2021, 70, 345-357.]. The collected vaginal swab samples were preserved, diluted through a freeze-thaw process, and inoculated onto modified MRS agar and Rogosa SL agar containing bromocresol purple (0.12 g / L, Sigma Aldrich, Steinheim, Germany) and L-cystine HCl (0.1%, Sigma Aldrich, Germany). Cultivation was performed at 37°C in aerobic, anaerobic, and microaerophilic chambers (Baker Ruskin, Rotherham, UK) for 0–48 h, respectively. After incubation, the plates were inspected, and colonies were selected based on size, shape, color, and morphology. The selected colonies were purified through subculture to obtain pure strains. After securing pure strains of interest, they were cultured on appropriate media and submitted to a company performing base sequence analysis to confirm the strain identity. Simultaneously, the isolated strains were suspended in a 30% glycerol solution and stored at 80°C for preservation for future experiments.

[0147]

[0148] 2. Identification of isolated probiotic strains through 16S rRNA gene sequencing

[0149] The isolated probiotic strains were identified using 16S rRNA gene-sequencing technology. After DNA extraction, the 16S rRNA gene region was amplified by PCR using the primer pair 27F (5'-AGA GTT TGA TCC TGG CTC AG-3') and 1492R (5'-GGT TAC CTT GTT ACG ACT T-3'). The PCR-amplified products were purified using an ABI PRISM 3730XL DNA Analyzer (Applied Biosystems, Waltham, MA, USA) and then sequenced. The generated sequence data were compared with the NCBI (National Center for Biotechnology Information) GenBank database using the basic local alignment search tool (BLAST) to identify the strains.

[0150]

[0151] 3. Whole genome sequencing of probiotic strains

[0152] Whole-genome sequencing (WGS) was performed to accurately identify the candidate probiotic strains at the strain level. Genomic DNA was extracted using the QIAamp DNA Extraction Kit (Qiagen, Germany) according to the manufacturer's instructions, and the extracted DNA was submitted to a WGS service provider (Chunlab Inc., Republic of Korea). PacBio sequencing data were compiled using the HGAP2 protocol in SMRTLink (13.1.0), and the generated contigs were circularized using Circlator 1.4.0 (Sanger Institute, Hinxton, UK). Protein coding sequences (CDSs) were predicted using Prodigal 2.6.2, and the predicted CDSs were classified into direct homologous groups based on EggNOG 4.5 (http: / / eggnogdb.embl.de, as of November 22, 2024). Additionally, tRNAs were identified using tRNAscan-SE 1.3.1, and rRNAs and other non-coding RNAs were identified using covariance model searches against the Rfam 12.0 database. Inter-genome comparisons were performed using the OrthoANIu algorithm-based Average Nucleotide Identity (ANI) calculator.

[0153]

[0154] 4. Confirmation of expression of HAS3 (Hyaluronan Synthase 3), FLG (Filaggrin), IVL (Involucrin), and LOR (Loricrin)

[0155] The effects of LP51 culture filtrate on skin barrier function and moisturizing properties were evaluated using human keratinocytes (HaCaT). HaCaT cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium, Welgene, Gyeongsan, Republic of Korea) supplemented with 10% FBS (fetal bovine serum, Welgene, Republic of Korea) and 1% penicillin / streptomycin (Gibco, Waltham, MA, USA) and maintained in a humidified atmosphere at 37°C and 5% CO₂. Before the experiment, cells were seeded in 6-well plates at a concentration of 5 × 10 cells / mL and cultured overnight. Afterwards, the medium was replaced with serum-free medium and cultured for an additional 24 h. After culture, cells were treated with LP51 culture filtrate at concentrations of 0.75%, 1.5%, and 3% for 6 h. After treatment, cells were washed with DPBS (Dulbecco's Phosphate-Buffered Saline, Welgene, Republic of Korea), and RNA was extracted using the TaKaRa Mini BEST Universal RNA Extraction Kit (Takara, Kusatsu, Japan). In this experiment, 1 μg / mL of human epidermal growth factor (hEGF, JForCell, JFC-GF008, Daejeon, Republic of Korea) was used as a positive control. After quantifying the concentration of the extracted RNA, cDNA was synthesized, and real-time PCR was performed using TaqMan master mix and SYBR Green reagent (BioRad, Hercules, CA, USA) using the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).HAS3 and FLG expression were analyzed using TaqMan primers (HAS3: Hs00193436_m1, FLG: Hs01566408_m1, GAPDH: Hs02758991_g1; Life Technologies, Waltham, MA, USA). IVL and LOR expression were analyzed by PCR using SYBR Green reagent, and all results were evaluated for differences in each gene expression.

[0156]

[0157] 5. Measurement of TARC (Thymus and Activation-Regulated Chemokine) production

[0158] The anti-atopic effect of LP51 culture filtrate was evaluated using human keratinocytes (HaCaT). HaCaT cells were seeded in 24-well plates and cultured until a confluent monolayer was formed. The cells were then treated with 10 ng / mL tumor necrosis factor-α (TNF-α, R&D Systems, 210-TA-005 / CF, Minneapolis, MN, USA), 20 ng / mL interferon-γ (IFN-γ, 285-IF-100 / CF, R&D Systems, USA), 10 μg / mL dexamethasone (D4902, Sigma-Aldrich, St. Louis, MO, USA), and a mixture of LP51 culture filtrate at concentrations of 0.25%, 0.5%, and 1%. The treated cells were then cultured for an additional 24 hours. After the culture was completed, the culture medium was collected and centrifuged at 4°C to recover the supernatant. The recovered supernatant was quantitatively measured for TARC production using an ELISA kit provided by R&D Systems (USA).

[0159]

[0160] 6. Evaluation of nitric oxide (NO) production

[0161] The anti-inflammatory effect of LP51 culture filtrate was evaluated using RAW 264.7 macrophages. RAW 264.7 cells were seeded at a density of 2.5 × 10 cells / well in 96-well plates and cultured overnight in a humidified atmosphere containing 5% CO₂ at 37°C. After overnight culture, the medium was replaced with serum-free medium and cultured for an additional 24 h. The cells were then treated with LP51 culture filtrate at concentrations of 0.25%, 0.5%, or 1%, 20 μM quercetin (Q4951, Sigma, USA), and / or 1 μg / mL lipopolysaccharide (LPS, L4391, Sigma, USA) for 24 h. After treatment, 100 μL of the cell culture medium was transferred to a new 96-well plate and treated with Griess reagent (Sigma-Aldrich, St. Louis, MO, USA). After incubation at room temperature for 20 minutes, the absorbance was measured at 540 nm using a multiplate reader (Perkin Elmer, Waltham, MA, USA). This quantitatively assessed the amount of NO synthesized in the cells.

[0162]

[0163] 7. Evaluation of DPPH scavenging ability

[0164] The antioxidant activity of the LP51 culture filtrate was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay (Sigma-Aldrich, USA). Briefly, 100 μL of a 0.2 mM DPPH solution was mixed with 100 μL of diluted LP51 culture filtrate (concentrations of 1.875%, 3.75%, 7.5%, 15%, and 30%) or 50 μg / mL L-ascorbic acid (vitamin C) and incubated for 30 min in the dark at room temperature. The control group was a DPPH solution mixed with an equal volume of ethanol without the test substance. After incubation, the absorbance was measured at 520 nm using an ELISA reader (Thermo Labsystems, Basingstoke, UK), and the DPPH scavenging activity was calculated based on the absorbance.

[0165]

[0166] 8. In vitro UV blocking ability evaluation

[0167] The UV blocking ability of LP51 culture filtrate was evaluated using human keratinocyte HaCaT cells. Cells were seeded at 1 × 10 in 96-well black plates. 4 Cells were seeded at a concentration of 1 cell / well and cultured overnight. After culture, the cells were washed with Hank's Balanced Salt Solution (HBSS) and treated with various concentrations of LP51 culture filtrate for 24 h. The cells were then exposed to UVB irradiation at 20 mJ / cm² and cultured for an additional 24 h. Cell viability was measured using the EZ-Cytox Cell Viability Assay Kit (DoGenBio, Seoul, Republic of Korea).

[0168]

[0169] 9. Preparation of test formulation using LP51 culture filtrate for clinical trials

[0170] An LP51 formulation was prepared to investigate the efficacy on dry skin using LP51 culture filtrate as the main ingredient. The test formulation consisted of the following ingredients: purified water (86.55%), glycerin (3%), carbomer (0.4%), xanthan gum (0.05%), caprylic / capric triglyceride (1%), cetyl alcohol (0.7%), glyceryl stearate (0.7%), PEG-75 stearate (0.3%), ceteth-20 (0.15%), steareth-20 (0.15%), 1,2-hexanediol (2.1%), propanediol (2%), and LP51 culture filtrate (2.9%). A placebo cream was prepared as a control group for the clinical trial using the same ingredients except for the active ingredient, LP51 culture filtrate. The concentration of LP51 was selected to effectively demonstrate efficacy while maintaining the viscosity and consistency of the formulation, and optimal performance was confirmed through observation at various concentrations.

[0171]

[0172] 10. Stability test of manufactured formulations

[0173] Thermal stability tests were conducted on the formulated product at various temperatures. The product was stored at refrigerated temperature (4°C), room temperature (23 ± 2°C), elevated temperature (45°C), and cyclic temperature (4°C→°C→°C). In the cyclic temperature test, the product was stored at each temperature for 8 hours. During the storage period, phase separation, odor, and color were observed for 0, 1, 2, 4, 9, and 11 weeks.

[0174]

[0175] 11. LP51 Clinical Trial Subject Selection Criteria for Dry Skin

[0176] A four-week clinical trial was conducted to evaluate the effects of LP51 culture filtrate on dry skin. A total of 46 Korean adults (male and female) who met the inclusion and exclusion criteria were included in the trial. The selection criteria were as follows:

[0177] Inclusion criteria: Healthy men and women aged 19 to 70 years, without acute or chronic diseases (except atopic dermatitis), experiencing dry skin and itching at the test site (inner elbow), objectively proven skin barrier damage with a transepidermal water loss (TEWL) value ≥12 g / h / m² and skin water content <35 (as measured by corneometry), voluntarily giving informed consent, and willing to faithfully participate in the study visits and observation schedule.

[0178] Exclusion criteria: Those with active skin disease requiring treatment or an ESIF (erythema, scaling, induration, fissure) score of more than 6; those who used antibiotics, steroids, immunosuppressants, antihistamines, retinoids, or phototherapy within the past 4 weeks; those who used skin disease-related supplements or moisturizers within the past 2 weeks; those who participated in another clinical trial within the past 4 weeks; those with frequent ultraviolet (UV) exposure; women who were pregnant, breastfeeding, or planning to become pregnant during the study; and those with other factors deemed unsuitable by the investigator.

[0179] This study was conducted in compliance with the ethical principles set forth in the Declaration of Helsinki (1964).

[0180]

[0181] 12. Clinical Trial Study Design

[0182] A 4-week, double-blind clinical trial was conducted to evaluate the effects of Lactobarriome 5% cream containing LP51 culture filtrate on dry skin. Participants were screened based on inclusion and exclusion criteria and randomly assigned to the test group (test formulation) or control group (placebo) using a simple randomization method.

[0183] Product Use: Participants applied the product to the proximal and distal 3 cm areas of the inner elbow twice daily, in the morning and evening.

[0184] Evaluation schedule: Clinical indicators were evaluated over a total of three visits: baseline, 2 weeks after use, and 4 weeks after use.

[0185] Evaluation items and measurement methods: Visual assessment, subjective itching assessment, skin moisture measurement, and TEWL measurement were performed by a dermatologist at all visits. Additionally, magnified photographs were taken using a Folliscope, and skin samples were collected to assess microbiological changes after treatment. Before testing, the target areas were washed with water and allowed to rest for 30 minutes under constant conditions (22 ± 2°C, 50 ± 10% RH).

[0186]

[0187] 13. Assessment of skin hydration, transepidermal water loss (TEWL), subjective itching severity, and visual appearance

[0188] Skin hydration was measured at baseline, 2 weeks, and 4 weeks using a Corneometer (Courage and Khazaka, Köln, Germany). The device was gently pressed onto the test area, and measurements were taken three times, with a 5-second interval between measurements. Each measurement assessed a depth of 10–20 μm below the stratum corneum.

[0189] Transepidermal water loss (TEWL) measurement: To evaluate the effect of LP51 on the stratum corneum permeability, a Tewameter (Courage + Khazaka electronic GmbH, Koln, Germany) was used. The probe was pressed against the test site with constant pressure for 30 seconds. The measurement was repeated three times and the average of the last three values ​​was recorded.

[0190] Subjective itching assessment: The subjective itching severity was assessed using a 10 cm visual analogue scale (VAS), and participants self-reported their itching severity on a scale from 0 to 10 (0 points: no itching, 10 points: very severe itching).

[0191] Visual Assessment: At each visit, a dermatologist visually assessed the skin for erythema, scaling, induration, and fissuring, with a total score ranging from 0 to 12. Additionally, the condition of the test areas was photographed using a digital camera (Canon, Tokyo, Japan) and a Folliscope (LeadM, Seoul, Republic of Korea).

[0192]

[0193] 14. Skin Metagenomics Analysis

[0194] To evaluate the effect of the test formulation on changes in the microbial composition of treated skin, 16S-rRNA-based amplicon sequencing was performed. For this purpose, skin samples were collected by gently rubbing a 3 cm area (3 × 3 cm² test site) near the inner elbow using the NBgene-SKIN kit (Noble Bio, Hwaseong, Republic of Korea). The collected samples were transported to the Human Microbiome Medical Research Center (HM-MRC) and stored at -80°C until further analysis. To extract skin DNA from the collected samples, beads were beating, centrifuged sufficiently, and the supernatant free of visible particles was collected. The quality of the extracted DNA was assessed by agarose gel electrophoresis. Additionally, DNA concentration was quantified using the Qubit dsDNA HS Assay Kit (Invitrogen, Waltham, MA, USA).

[0195] The V4 region of the 16S rRNA gene was amplified using the Illumina 16S bacterial rRNA amplicon primer set (16S-F: TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG, 16S-R: GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC), 10 ng of template DNA, and KAPA HiFi HotStart ReadyMix (Kapa Biosystems, Wilmington, MA, USA). PCR was performed according to a previously described protocol. PCR was performed on all samples, including negative controls (no template DNA) and positive controls (5 ng of skin DNA). PCR was performed in a Veriti 96-well Thermal Cycler (Applied Biosystems, USA). PCR conditions included an initial denaturation at 95 °C for 3 min, followed by 25 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s. The final extension was performed at 72 °C for 5 min. PCR products were purified using AMPure beads (Beckman Coulter, South Kraemer Boulevard Brea, CA, USA) according to the manufacturer's instructions. 5 μL of the purified amplicon PCR product was used for indexing, and indexing PCR was performed using the Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA, USA). AMPure beads were then used for further purification. The final DNA concentration of each sample was adjusted to 1 nM with H₂O. 5 μL of each sample was pooled, and the pooled library (50 pMol) was spiked with 30% PhiX and sequenced on the iSeq100 platform (Illumina, USA).

[0196] Sequence analysis was performed using Quantitative Insights Into Microbial Ecology (QIIME) software (version 1.9.1), and sequences were assigned to operational taxonomic units (OTUs) using a 97% similarity threshold. Representative sequences from each OUT were selected, and taxonomic data were assigned using the RDP classifier. These representative sequences were mapped to a taxonomic hierarchy from phylum to family based on the Human Microbiome Database. Taxonomic assignments were performed using a Bayesian approach with a 97% confidence threshold. To assess the richness and diversity of bacterial populations within samples, α-diversity indices, including observed OTUs, Chao1, ACE, Simpson, Shannon, and Fisher indices, were calculated. β-diversity indices (Bray-Curtis, Jansen-Shannon, and Jaccard) were also used to analyze changes in bacterial communities between samples. Permutational multivariate analysis of variance (PERMANOVA) was applied to evaluate the significance of β-diversity between clusters.

[0197]

[0198] 15. Determination of MIC and MBC of LP51 against skin pathogens

[0199] To determine the MIC and MBC of LP51 against skin pathogens, the antibacterial efficacy of LP51 culture filtrate was evaluated. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays were performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines for skin pathogens, including Staphylococcus aureus KCTC 3881 and Propionibacterium acnes KCTC 3314. First, S. aureus and P. acnes were cultured in tryptic soy broth and brain heart infusion broth, respectively, at 37°C for 24 h. Then, 100 μL of the inoculum suspension containing the test substance was added to each well of a 96-well plate and incubated for 24 h. The MIC value was determined as the lowest filtrate concentration that completely inhibited visible bacterial growth. After the MIC was determined, 20 μL was aliquoted from wells without visible bacterial growth, plated on the corresponding agar medium, and incubated at 37°C for 24 h. The MBC was defined as the lowest filtrate concentration at which no bacterial growth was observed after incubation.

[0200]

[0201] 16. Cytotoxicity test of LP51

[0202] The cytotoxicity of LP51 was evaluated in RAW 264.7 and HaCaT cells using the EZCytox cell viability assay kit (DoGenBio, Republic of Korea). Cells were cultured in DMEM containing 10% FBS and 1% penicillin / streptomycin at 37°C in a 5% CO₂ incubator. RAW 264.7 and HaCaT cells (2.5 × 10 cells / well) were seeded in 96-well plates (SPL Life Sciences, Pocheon, Republic of Korea) and cultured overnight. After the cells reached approximately 75–85% confluency, RAW 264.7 cells were treated with 0.05%, 0.5%, and 1% LP51 culture filtrate / medium, and HaCaT cells were treated with 1.5% and 3% LP51 culture filtrate / medium, and cultured for 24 h. After incubation, 20 μL of WST solution was added to each well and incubated for an additional 2 hours. Cytotoxicity was evaluated by measuring the absorbance at 570 nm using a multiplate reader (Perkin Elmer, USA). The cytotoxicity of LP51 was also evaluated using a lactate dehydrogenase (LDH) release assay in HT29 cells. HT29 cells were cultured under the same conditions and seeded in 96-well plates at 1 × 10⁴ cells / well. After overnight incubation, the cells were washed and treated with LP51 at 1 × 10, 1 × 10, and 1 × 10 CFU / well for 24 hours. LDH released into the medium was measured using a cytotoxicity detection kit (Roche Diagnostics, Milan, Italy), and the absorbance was measured at 490 nm using a multiplate reader.In this assay, Lacticaseibacillus rhamnosus KCTC5033 (Korean Collection for Type Cultures, KCTC, Jeongeup, Republic of Korea) and Escherichia coli NCCP 14538 (National Culture Collection for Pathogens, NCCP, Cheongju, Republic of Korea) were used as reference strains, and lysis buffer was used as a positive control.

[0203]

[0204] 17. D-lactate production test

[0205] D-lactate production by LP51 was evaluated using a D-lactate colorimetric assay kit (Abcam, Waltham, MA, USA). Probiotic strains were cultured in an anaerobic incubator for 24 h, and reaction samples, including a standard curve, were prepared according to the manufacturer's instructions. LP51 culture filtrates were treated to contain 1 × 10 , 1 × 10 , and 1 × 10 CFU, respectively. The reaction mixtures were then added to the test samples and incubated for 30 min. D-lactate production was evaluated by measuring the absorbance at 450 nm using a multiplate reader. L. rhamnosus KCTC5033 (Korean Collection for Type Cultures, KCTC, Jeongeup, Republic of Korea) was used as a negative control in this assay.

[0206]

[0207] 18. Hemolytic activity and bile salt debinding test

[0208] The hemolytic activity of LP51 was evaluated according to the protocol of the American Society for Microbiology (ASM). The strain was plated on blood agar (MB Cells, Seoul, Republic of Korea) and incubated under anaerobic conditions at 37°C for 48 h. Hemolysis was assessed by observing the area around the bacterial cultures: complete hemolysis (β), partial hemolysis (α), or no hemolysis (γ). L. rhamnosus KCTC5033 and S. aureus NCCP 14780 were used as reference strains in this assay. The bile salt debinding ability of LP51 was tested according to a previously established protocol. The probiotic strains were plated on agar plates containing taurodeoxycholic acid (TDCA) sodium salt (Sigma-Aldrich, St. Louis, MO, USA) and incubated under anaerobic conditions at 37°C for 5 days. The plates were then examined to determine the bile salt debinding ability of the strains. L. rhamnosus KCTC5033 was used as a control strain.

[0209]

[0210] 19. Antibiotic susceptibility testing (E-test)

[0211] The E-test was performed to assess the antibiotic susceptibility of candidate probiotic strains, as recommended by the European Food Safety Authority (EFSA) for safety assessment. Bacterial suspensions were inoculated onto Mueller-Hinton agar plates. E-test strips (Liofilchem, Roseto degli Abruzzi, Teramo, Italy) were impregnated with a gradient of various antibiotics (ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol), placed on the agar, and incubated at 37°C under anaerobic conditions for 48 h. The minimum inhibitory concentration (MIC) that completely inhibited bacterial growth was determined and compared with the EFSA MIC cutoff criteria.

[0212]

[0213] 20. SCFA Analysis Using GC-MS: Reagents and Sample Handling

[0214] Acetic acid (CAS no. 64-19-7, 99.9%), propionic acid (CAS no. 1979-09-4, 99.9%), and butyric acid (CAS no. 107-92-6, 99.7%) were purchased from TCI, and valeric acid (CAS no. 109-52-4, 99.4%) was purchased from Sigma-Aldrich. Sodium chloride (CAS no. 7647-14-5) and sulfuric acid (CAS no. 7664-93-9) were supplied from Daejeon (Seoul, Republic of Korea). For analysis, 2.5 g of NaCl, 5 mL of sample, and 1 mL of 2% sulfuric acid were prepared in vials and analyzed using a gas chromatography-mass spectrometry system (Perkin Elmer Clarus 690 GC, Clarus SQ8-GC-MS, TurboMatrix Headspace). Standard solutions were processed in a similar manner, and acetic acid was diluted to concentrations of 1, 2, 5, 10, 20, 50, 100, 200, 500, and 1000 mg / L, and propionic acid, butyric acid, and valeric acid were diluted to concentrations of 1, 2, 5, 10, 20, 50, and 100 mg / L and analyzed under the same conditions.

[0215]

[0216] <Result>

[0217] 1. Culturomics results for isolating strains from healthy female vaginal fluid.

[0218] In this study, we used culturomics-based techniques to isolate Lactobacillus strains from healthy vaginal fluid using various agar media. Bacterial growth was observed in 9 of 30 vaginal samples, yielding 72 bacterial colonies exhibiting characteristics similar to those of Lactobacillus. The isolated strains were identified by 16S rRNA gene sequencing. Thirty-four of the selected colonies were identified as belonging to the genus Lactobacillus, while the remaining strains were classified as non-Lactobacillus. The candidate strain LP51 in this study was found to have 99% similarity to L. rhamnosus strain JCM 1136 based on 16S rRNA gene sequencing (Table 1). Furthermore, the sequence showed 97–99% similarity to several species of Lacticaseibacillus. Based on these results, the candidate strain was confirmed to belong to the genus Lactobacillus. Further details were confirmed through whole-genome sequencing.

[0219] NCBI RefOrganismLength (bp)ScoreIdentityGapNR_043408.1Lacticaseibacillus rhamnosus strain JCM 113615212641 bits (1430)1435 / 1438 (99%)1 / 1438 (0%)NR_037122.1Lacticaseibacillus zeae strain RIA 48215222580 bits (1397)1424 / 1438 (99%)0 / 1438 (0%)NR_179712.1Lacticaseibacillus chiayiensis strain BCRC 8106215362575 bits (1394)1424 / 1439 (99%)2 / 1439 (0%)NR_113337.1Lacticaseibacillus paracasei strain NBRC 1588914952564 bits (1388)1421 / 1438 (99%)0 / 1438 (0%)NR_025880.1Lacticaseibacillus paracasei strain R09415222562 bits (1387)1421 / 1438 (99%)0 / 1438 (0%)NR_113333.1Lacticaseibacillus casei strain NBRC 1588314952560 bits (1386)1421 / 1439 (99%)2 / 1439 (0%)NR_041893.1Lacticaseibacillus casei DSM 2001115172556 bits (1384)1422 / 1440 (99%)3 / 1440 (0%)NR_113823.1Lacticaseibacillus paracasei strain NBRC 1590614972553 bits (1382)1421 / 1440 (99%)2 / 1440 (0%)NR_117987.1Lacticaseibacillus paracasei strain ATCC 2530214412551 bits (1381)1418 / 1436 (99%)2 / 1436 (0%)NR_115322.1Lacticaseibacillus casei strain BCRC1069715282527 bits (1368)1419 / 1443 (98%)6 / 1443 (0%)NR_179363.1Lacticaseibacillus mingshuiensis strain 117-114482350 bits (1272)1366 / 1411 (97%)7 / 1411 (0%)NR_180281.1Lacticaseibacillus yichunensis strain 33-114422344 bits (1269)1365 / 1411 (97%)7 / 1411 (0%).

[0220] 2. Analysis of strain whole genome sequencing results

[0221] The major genomic characteristics of the LP51 strain are shown in Figure 1. LP51 contained a single circular chromosome with an average GC content of 46.7% and a genome length of 3,003,557 bp (Figure 1A). 2,794 coding sequences (CDSs) were identified in the genome and classified using the Clusters of Orthologous Groups (COG) system (Figure 1B). Of these CDSs, 2,441 proteins were assigned to COG families, 1,761 proteins had known biological functions, and 680 CDSs corresponded to conserved proteins with unknown functions in other organisms. Additionally, 2,855 hypothetical proteins did not match known proteins in the database. Additionally, 62 tRNA and 19 rRNA genes were predicted. The NCBI accession number for LP51 is PRJNA1123863.

[0222] Similarity analysis of strains with similar 16S rRNA sequences was performed using the OrthoANI method utilizing the whole genome sequencing data of LP51 (Fig. 1C). Comparison with all published L. rhamnosus genomes revealed that LP51 shared 99.74% similarity with L. rhamnosus CP-1, significantly exceeding the 95% threshold for species differentiation. In addition, LP51 was also compared with other strains belonging to the same genus but different species, including LC130, HL182, YH-lac23, SRCM217410, LG542, and MFPC41A2801. The similarities among these strains were 79.5%, 77.2%, 66.6%, 67.4%, 67.2%, and 66.9%, respectively, all of which were less than 80%. This confirmed that LP51 is a strain belonging to the L. rhamnosus species (KCTC 15925BP).

[0223] Furthermore, to confirm whether the isolated strain was a newly discovered strain, the genome information of LP51 was compared with that of other L. rhamnosus strains, such as Hsryfm 1301, TKF8B, LR-B1, LDTM7511, SN21-1, and VSI43 (Fig. 1D). Although these strains all belong to the same species, they differ in origin, genome size, G + C content, CDS, rRNA, and tRNA numbers. This confirmed that strain LP51 is a new strain of L. rhamnosus.

[0224]

[0225] 3. In vitro test results for improving skin moisture, strengthening the skin barrier, anti-atopic, anti-inflammatory, antioxidant, and cytotoxic effects.

[0226] Various in vitro experiments were performed to evaluate the effects of candidate strain LP51 on improving skin moisture, strengthening the skin barrier, anti-atopic, anti-inflammatory, antioxidant, and cytotoxic effects (Fig. 2).

[0227] First, the effect of LP51 culture filtrate on skin barrier strength and moisturizing capacity was evaluated by measuring the transcription level of HAS3 in HaCaT cells (Fig. 2A). As a result, HAS3 expression significantly increased in 0.75% LP51 treatment compared to untreated cells. At higher concentrations, the expression level further increased, reaching a level similar to that of the positive control, human epidermal growth factor (hEGF), particularly at a 3% concentration.

[0228] Simultaneously, the effect of the candidate strains on skin barrier enhancement was assessed by measuring the transcript level of FLG in the same cell line (Fig. 2B). The results showed that FLG expression significantly increased in a concentration-dependent manner when treated with 1.5% and 3% LP51 culture filtrate. IVL expression levels also significantly increased at 1.5% and 3% LP51 culture filtrate concentrations (Fig. 2C). Furthermore, LOR transcript levels were significantly increased when treated with 3% LP51 culture filtrate (Fig. 2D).

[0229] The anti-atopic effects of the candidate strains were assessed by analyzing changes in TARC production in the same cell lines (Fig. 3E). In cells treated with TNF-α + IFN-γ (positive control), TARC levels were significantly reduced by 1% LP51 treatment compared to untreated cells, demonstrating superior results compared to the immunosuppressant dexamethasone.

[0230] The anti-inflammatory effect of LP51 culture filtrate was evaluated using RAW 264.7 macrophages (Fig. 2F). LPS-treated cells exhibited significant NO production, and treatment with LP51 culture filtrate at concentrations greater than 0.25% significantly decreased NO production in a dose-dependent manner.

[0231] The free radical-scavenging effect of LP51 was evaluated using the DPPH reagent (Fig. 2G). As a result, it exhibited excellent antioxidant effects in a concentration-dependent manner, with an effect similar to that of L-ascorbic acid at a concentration of 30%.

[0232] The effect of LP51 on UVB protection was evaluated using HaCaT cells (Fig. 2H). Results showed that compared to the control group, the viability of cells exposed to UVB was significantly reduced, but cell viability was restored at concentrations of 0.003% or higher when treated with LP51.

[0233]

[0234] 4. Clinical evaluation of LP51 formulation in dry skin

[0235] The efficacy of the LP51 formulation for dry skin was evaluated in a double-blind clinical trial and compared to a placebo group receiving an LP51-free formulation (Figure 3).

[0236] In participants treated with the LP51 formulation, skin moisture levels increased from 25.0 before application to 30.6 after 2 weeks and 35.0 after 4 weeks (Fig. 3A). This represented a significant increase of 22.6% after 2 weeks (p < 0.001) and 40.1% after 4 weeks (p < 0.001). In the placebo group, skin moisture levels also significantly increased from 26.8 before application to 29.6 after 2 weeks and 31.7 after 4 weeks, but the increases in the LP51 group were significantly greater than those in the placebo group at 2 weeks (p < 0.05) and 4 weeks (p < 0.01) (Fig. 3B).

[0237] The effect on TEWL was also evaluated (Fig. 3C). In the LP51 group, TEWL decreased from 20.4 before application to 18.5 after 2 weeks and 16.0 after 4 weeks, a significant decrease of 21.5% (p < 0.05) was observed after 4 weeks. In contrast, the placebo group showed no significant change in TEWL values, with values ​​of 17.7 before application, 18.3 after 2 weeks, and 18.6 after 4 weeks. The decrease in the LP51 group was statistically significant compared to the placebo group (p < 0.05) (Fig. 3D).

[0238] The severity of subjective pruritus was assessed using a visual analog scale (VAS) (Fig. 3E). In the LP51 treatment group, the pruritus score decreased from 4.3 before treatment to 3.5 after 2 weeks (19.4% decrease, p < 0.05), and significantly decreased to 1.6 after 4 weeks (62.8% decrease, p < 0.001). The placebo group also showed a significant decrease in the pruritus score from 4.3 before treatment to 2.9 after 2 weeks and 1.4 after 4 weeks, but there was no significant difference between the groups. Skin appearance was assessed using the ESIF scale (erythema, keratinization, induration, fissures), and although visual appearance improved, there was no statistically significant difference compared to the placebo group (Fig. 3F). At each assessment point, photographs were taken with a digital camera (Fig. 3G), and magnified images were captured using a Folliscope (Fig. 3H).

[0239] To verify product stability, the test product was stored for 11 weeks under various conditions: refrigerated (4°C), room temperature (23 ± 2°C), accelerated (45°C), and cyclic conditions of 4°C → 25°C → 45°C (Fig. 3I, J). The formulation maintained uniformity without phase separation. Regarding the overall color change, no noticeable color change was observed during the storage period. No odor was detected during the study period. These results indicate that the manufactured formulation was physically stable during the 11-week storage period.

[0240]

[0241] 5. Results of LP51-based skin metagenome analysis using the Xerosis-Microbiome Index

[0242] Metagenomic analysis of the skin microbiome was performed during a 4-week clinical trial to observe changes in the microbial composition in the target area before and after treatment (Fig. 4). LP51 significantly reduced Actinobacteria and markedly increased Firmicutes (Fig. 4A). This change in microbial balance resulted in a significant increase in the Xerosis-Microbiome Index (XMI), which is the ratio of Firmicutes to Actinobacteria, compared to the placebo group (Fig. 4B). Over the 4-week period, LP51 increased the XMI by two times more than the placebo group, confirming its effectiveness in improving microbial balance and creating a healthier skin condition.

[0243] Linear regression analysis showed that XMI had a direct positive correlation with skin moisture levels (Fig. 4C). LP51 increased the slope of this relationship, indicating that LP51 amplified the effect of skin moisture more than the placebo group as XMI increased. Conversely, TEWL showed an inverse correlation with XMI (Fig. 4D). LP51 not only decreased TEWL as XMI increased, but also lowered the y-intercept of the regression line, indicating an overall decrease in basal water loss through the skin.

[0244] In vitro antibacterial tests showed that LP51 exhibited a stronger inhibitory effect against P. acnes (Actinobacteria) than against S. aureus (Firmicutes) (Fig. 4E). These results support the above findings and highlight the role of LP51 in promoting the Firmicutes / Actinobacteria balance observed in XMI.

[0245] Additionally, the culture filtrate of LP51 was found to contain 328.65 μg / mL of acetic acid, 0.81 μg / mL of butyric acid, 0.73 μg / mL of propionic acid, and 0.08 μg / mL of valeric acid (Fig. 4F). These SCFAs are considered to be key regulators in XMI regulation.

[0246] Finally, both alpha and beta diversity analyses demonstrated that LP51 did not induce microbial imbalance (Fig. 4G). Indices such as the Shannon, Simpson, and Fisher indices for alpha diversity, and the Bray-Curtis, Jaccard, and UniFrac indices for beta diversity, did not show significant changes between the LP51 and placebo groups. This suggests that LP51 maintains a stable and healthy skin microbiome, supporting the classification of a safe microbiome.

[0247] In conclusion, LP51 treatment was demonstrated to significantly modulate the skin microbiome, increasing beneficial bacteria and maintaining overall microbial diversity while improving skin hydration and reducing TEWL.

[0248]

[0249] 6. Safety evaluation: cytotoxicity, D-lactate production, hemolysis, bile salt deconjugation, and antibiotic susceptibility.

[0250] The safety evaluation of the LP51 strain began with cytotoxicity assessment using the water-soluble tetrazolium salt-1 (WST-1) assay and the lactate dehydrogenase (LDH) release test. The WST-1 assay was performed in RAW 264.7 cells (Fig. 5A) and HaCaT cells (Fig. 5B), and the LDH test was performed in HaCaT cells (Fig. 5C). LP51 was not cytotoxic at all tested concentrations, whereas the positive control lysis buffer and pathogenic E. coli strain exhibited significant cytotoxicity.

[0251] Regarding D-lactate production, LP51 did not show a significant increase compared to the control group, and showed a level similar to that of the reference strain L. rhamnosus (Fig. 5D). In addition, LP51 showed γ-hemolysis (no hemolysis) on blood agar medium, confirming the absence of β-hemolysis (complete hemolysis) or α-hemolysis (partial hemolysis), and showed results similar to that of the reference strain L. rhamnosus (Fig. 5E). In contrast, S. aureus was confirmed to exhibit hemolytic activity by forming a clear zone around the colonies. Furthermore, LP51 showed growth on agar medium containing bile salts, and it exhibited bile salt unbinding activity similar to that of the reference strain L. rhamnosus (Fig. 5F).

[0252] In terms of antibiotic susceptibility, the LP51 strain was sensitive to ampicillin (MIC 0.75 μg / mL), erythromycin (MIC 0.12 μg / mL), clindamycin (MIC 0.19 μg / mL), and tetracycline (MIC 0.5 μg / mL), whereas it was resistant to gentamicin (MIC 32 μg / mL), kanamycin (MIC 128 μg / mL), streptomycin (MIC 64 μg / mL), and chloramphenicol (MIC 12 μg / mL) (Fig. 5G).

[0253] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred implementation examples and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0254]

[0255] The novel Lacticazeibacillus rhamnosus according to the present invention or its culture solution has various skin improvement effects including skin moisturizing, barrier strengthening, anti-atopic, anti-inflammatory, antioxidant and UVB protection effects, and as clinical safety and efficacy have been verified, it is expected that it can be used as a new probiotic formulation for skin health.

[0256]

[0257] Accession number

[0258] Name of depositor: Korea Research Institute of Bioscience and Biotechnology

[0259] Accession number: KCTC 15925BP

[0260] Date of acceptance: 20240605

Claims

1. A novel strain of Lacticaseibacillus rhamnosus or a culture solution thereof, A novel Lacticaseibacillus rhamnosus strain or a culture solution thereof, characterized in that the Lacticaseibacillus rhamnosus strain has the deposit number KCTC 15925BP extracted from a woman's vaginal fluid.

2. In paragraph 1, The novel Lacticazei Bacillus rhamnosus strain or its culture solution is characterized by having antibacterial activity against Staphylococcus aureus or Cutibacterium acnes, skin moisturizing and skin barrier strengthening by increasing the expression of HAS3 (Hyaluronan Synthase 3), FLG (Filaggrin), IVL (Involucrin), and LOR (Loricrin), anti-atopic and anti-inflammation by inhibiting TARC production and reducing NO production, and DPPH free radical scavenging and UVB-induced cell damage suppression activity.

3. A skin improvement external preparation for antibacterial, skin moisturizing, skin barrier strengthening, anti-atopic, anti-inflammation and anti-oxidation, containing a novel Lacticazeibacillus rhamnosus strain or a culture solution thereof according to paragraph 1 or 2 as an active ingredient.

4. In paragraph 3, A skin external preparation characterized in that the above skin external preparation comprises a cream, gel, ointment, skin emulsifier, skin suspension, transdermal delivery patch, drug-containing bandage, lotion, or a combination thereof.

5. A cosmetic composition for improving skin for antibacterial, skin moisturizing, skin barrier strengthening, anti-atopic, anti-inflammation and anti-oxidation, comprising a novel Lacticazeibacillus rhamnosus strain or a culture solution thereof according to paragraph 1 or 2 as an active ingredient.

6. In paragraph 5, The cosmetic composition is characterized in that it is formulated to include a toner (skin lotion), a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nourishing lotion, a massage cream, a nourishing cream, a moisturizing cream, a hand cream, a foundation, an essence, a nourishing essence, a pack, a soap, a cleansing foam, a cleansing lotion, a cleansing cream, a body lotion, a body cleanser, a suspension, a gel, a powder, a paste, a mask pack, or a sheet or aerosol composition.

7. A pharmaceutical composition for the prevention or treatment of skin diseases, comprising a novel Lacticazeibacillus rhamnosus strain or a culture solution thereof according to paragraph 1 or 2 as an active ingredient.

8. In paragraph 7, A pharmaceutical composition, wherein the skin disease is characterized by including a disease caused by damage to the skin barrier function, skin aging, skin wound, skin scar, or skin inflammation or atopic dermatitis.

9. In paragraph 7, A pharmaceutical composition characterized in that the above skin inflammation includes atopic dermatitis, eczema, seborrheic dermatitis, psoriasis or acne.

10. A health functional food for improving skin for antibacterial, skin moisturizing, skin barrier strengthening, anti-atopic, anti-inflammation and anti-oxidation, containing a novel Lacticazeibacillus rhamnosus strain or a culture solution thereof according to paragraph 1 or 2 as an active ingredient.

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