Cosmetic composition for preventing vaginitis and improving the vaginal environment containing a mixture of vaginal-derived lactic acid bacteria

Abstract

The present invention relates to a cosmetic composition for preventing vaginitis and improving the vaginal environment, comprising as an active ingredient a mixed strain fermentation product consisting of a culture solution of Lactobacillus crispatus SY-J504 strain, a culture solution of Lactobacillus jensenii strain, and a culture solution of Lactobacillus paraplantarum strain. According to the present invention, a strong antimicrobial synergistic effect against vaginitis-causing pathogens is exhibited by including Lactobacillus strains, including a newly identified strain, in an optimal weight ratio, or by including the fermented product and a cell-crushed fermented lysate in a specific ratio. In addition, it inhibits the production of inflammation-inducing factors, significantly enhances the expression of skin barrier and moisturizing factors, and is non-cytotoxic, so it can be usefully utilized as a safe and effective feminine cleanser and vulvar moisturizer.

Description

Technology Field

[0001] The present invention relates to a cosmetic composition for preventing vaginitis and improving the vaginal environment comprising a mixture of vaginal-derived lactic acid bacteria, specifically a cosmetic composition for preventing vaginitis and improving the vaginal environment comprising, as active ingredients, a fermented product of a strain mixture consisting of a culture solution of Lactobacillus crispatus strain, a culture solution of Lactobacillus gensenii strain, and a culture solution of Lactobacillus paraplantarum strain, and a fermented lysate thereof. Background Technology

[0003] The vaginal microbiome of a healthy woman is mainly Lactobacillus ( Lactobacillus Lactobacilli of the genus ) are dominant, and they play a key role in inhibiting the invasion and proliferation of external pathogens by producing lactic acid and maintaining the vaginal pH at an acidic level of 4.5 or lower. However, if the balance of the normal vaginal flora is disrupted due to various causes such as stress, weakened immunity, excessive cleansing, or antibiotic use, Gardnerella vaginalis ( Gardnerella vaginalis Anaerobic bacteria such as ) or Candida albicans ( Candida albicans Fungi such as ) abnormally overgrow and cause bacterial vaginosis and candidal vaginosis.

[0004] Conventionally, antibiotics and antifungal agents such as metronidazole and clindamycin have been prescribed primarily to treat such vaginitis. However, chemical therapy using antibiotics has fatal limitations, as it kills not only the causative bacteria but also beneficial bacteria in the vagina, such as Lactobacillus, thereby further worsening the vaginal environment, and frequent use leads to the emergence of resistant bacteria and the recurrence of chronic vaginitis.

[0005] To overcome this, recent attempts have been made to improve the vaginal environment using probiotics; however, simply adding known probiotic culture solutions to cosmetics makes it difficult to expect excellent antibacterial efficacy. Furthermore, there are clear technical limitations to relying solely on existing simple fermented products when it comes to rapidly restoring the moisture barrier of the vaginal mucosa damaged by inflammation and effectively suppressing inflammatory mediators. Prior art literature

[0007] Republic of Korea Registered Patent KR 10-2825583 B1 The problem to be solved

[0008] Against this backdrop, the inventors, after making research efforts to overcome the limitations of antibiotic resistance caused by the overuse of conventional antibiotics and the death of beneficial bacteria in the vagina, confirmed that a cosmetic composition containing a mixed fermentation product consisting of Lactobacillus crispatus culture solution, Lactobacillus gensenii culture solution, and Lactobacillus paraplantarum culture solution as an active ingredient can not only effectively suppress the causative bacteria of vaginitis but also have anti-inflammatory and moisturizing improvement effects.

[0009] Accordingly, the present invention aims to discover an optimal combination of lactic acid bacteria including a novel Lactobacillus crispatus SY-J504 strain newly isolated and identified from the female vagina, and to provide an optimal mixing weight ratio in which these mixed strains can exert a synergistic antibacterial effect against the causative bacteria of vaginitis.

[0010] Furthermore, the present invention aims to provide a cosmetic composition for preventing vaginitis and improving the vaginal environment that can drastically alleviate vaginal inflammation and moisturize damaged vaginal mucosa and strengthen the skin barrier by including not only a simple fermented product of the strain mixture but also a fermented lysate from which an active ingredient (PDRN, etc.) is eluted through cell wall disruption in a specific ratio.

[0011] However, the problems that this invention seeks to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by a person skilled in the art from the description below. means of solving the problem

[0013] Accordingly, one example of the present invention is Lactobacillus crispatus ( Lactobacillus crispatus ) strain culture solution, Lactobacillus genseni( Lactobacillus jensenii ) strain culture solution and Lactobacillus paraplantarum( Lactobacillus paraplantarum A cosmetic composition for preventing or improving vaginitis, comprising as an active ingredient a strain mixture fermented product consisting of a strain culture solution,

[0014] The above Lactobacillus crispatus strain is the Lactobacillus crispatus SY-J504 strain deposited under accession number KCTC16719BP, and

[0015] The above strain mixture fermented product is a cosmetic composition for preventing or improving vaginitis, characterized by comprising 55 to 85 parts by weight of Lactobacillus genseni strain culture and 15 to 45 parts by weight of Lactobacillus paraplantarum strain culture per 100 parts by weight of Lactobacillus crispatus strain culture.

[0016] Another example of the present invention is a feminine hygiene product for preventing or improving vaginitis, comprising the above composition.

[0017] Another example of the present invention is Lactobacillus crispatus ( Lactobacillus crispatus ) strain culture solution, Lactobacillus genseni( Lactobacillus jensenii ) strain culture solution and Lactobacillus paraplantarum( Lactobacillus paraplantarum A food composition for preventing or improving vaginitis, comprising as an active ingredient a strain mixture fermented product consisting of a strain culture solution,

[0018] The above Lactobacillus crispatus strain is the Lactobacillus crispatus SY-J504 strain deposited under accession number KCTC16719BP, and

[0019] The above strain mixture fermented product is a food composition for preventing or improving vaginitis, characterized by comprising 55 to 85 parts by weight of Lactobacillus genseni strain culture and 15 to 45 parts by weight of Lactobacillus paraplantarum strain culture per 100 parts by weight of Lactobacillus crispatus strain culture.

[0020] Another example of the present invention is Lactobacillus crispatus ( Lactobacillus crispatus ) strain culture solution, Lactobacillus genseni( Lactobacillus jensenii ) strain culture solution and Lactobacillus paraplantarum( Lactobacillus paraplantarum It is intended for the prevention, improvement, or treatment of vaginitis using a strain mixture fermented product composed of a strain culture solution. Effects of the invention

[0022] The composition according to the present invention exhibits the following excellent effects.

[0023] First, by mixing the newly identified Lactobacillus crispatus SY-J504 strain (KCTC16719BP) in the present invention with the Lactobacillus genseni strain and the Lactobacillus paraplantarum strain in a specific weight ratio, a synergistic antibacterial effect is exerted that strongly inhibits the growth of Gardnerella vaginalis and Candida albicans, which are major causative agents of vaginitis.

[0024] Second, the composition of the present invention has no toxicity to human vaginal epithelial cells, so it can be safely used without side effects even when applied to sensitive vulvar and vaginal mucosa.

[0025] Third, by including a fermented product containing metabolic products of the cell and a fermented lysate containing intracellular active ingredients (PDRN, etc.) through cell wall disruption in an optimal weight ratio of 1:2 to 2:1, it provides an excellent anti-inflammatory effect that significantly reduces the production of inflammatory factors such as nitric oxide (NO) and TNF-α compared to a simple mixture.

[0026] Fourth, it significantly increases the expression of filaggrin, a key moisturizing factor, and aquaporin-3 (AQP3), a water channel protein, thereby supplying moisture to the dry vaginal environment and effectively repairing the damaged mucosal barrier. Specific details for implementing the invention

[0028] Specific structural or functional descriptions of the embodiments are disclosed for illustrative purposes only and may be modified and implemented in various forms. Accordingly, the embodiments are not limited to the specific disclosed forms, and the scope of this specification includes modifications, equivalents, or substitutions that fall within the technical concept.

[0029] Terms such as "first" or "second" may be used to describe various components, but these terms should be interpreted solely for the purpose of distinguishing one component from another. For example, the first component may be named the second component, and similarly, the second component may be named the first component.

[0030] When it is stated that a component is "connected" to another component, it should be understood that it may be directly connected to or joined to that other component, or that there may be other components in between.

[0031] The terms used in the embodiments are for illustrative purposes only and should not be interpreted as intended to be limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as "comprising" or "having" are intended to indicate the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0032] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the embodiments pertain. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application.

[0033] The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0034] In the embodiments of the present invention, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in the embodiments of the present invention.

[0035] Furthermore, in describing the present invention, if it is determined that a detailed description of related prior art could unnecessarily obscure the essence of the present invention, such detailed description is omitted. Where terms such as "includes," "has," or "is made up" are used in this specification, other parts may be added unless "only" is used. Where a component is expressed in the singular, it includes cases where it is included in the plural unless specifically stated otherwise.

[0036] In interpreting the components, they are interpreted to include a margin of error even in the absence of a separate explicit statement.

[0037] The features of each of the various embodiments of the present invention may be combined or combined with one another, either partially or wholly, and as will be fully understood by those skilled in the art, various technical interlocking and operation are possible, and each embodiment may be implemented independently of one another or together in an interlocking relationship.

[0039] Embodiments of the present invention have been described in detail below, but the present invention is not limited thereto.

[0041] One aspect of the present invention is Lactobacillus crispatus ( Lactobacillus crispatus ) strain culture solution, Lactobacillus genseni( Lactobacillus jensenii ) strain culture solution and Lactobacillus paraplantarum( Lactobacillus paraplantarum A cosmetic composition for preventing or improving vaginitis, comprising as an active ingredient a strain mixture fermented product consisting of a strain culture solution,

[0042] The above Lactobacillus crispatus strain is the Lactobacillus crispatus SY-J504 strain deposited under accession number KCTC16719BP, and

[0043] The above strain mixture fermented product is a cosmetic composition for preventing or improving vaginitis, characterized by comprising 55 to 85 parts by weight of Lactobacillus genseni strain culture and 15 to 45 parts by weight of Lactobacillus paraplantarum strain culture per 100 parts by weight of Lactobacillus crispatus strain culture.

[0044] The term "culture solution" in this specification may be used interchangeably with terms such as "culture supernatant," "conditional culture solution," "culture medium," or "adjusted medium," and may refer to the entire medium containing said strain, its metabolites, excess nutrients, etc., obtained by culturing said strain for a certain period in a medium capable of supplying nutrients so that said strain can grow and survive in a test tube. Additionally, said culture solution may refer to a culture solution from which the cells have been removed from a culture solution obtained by culturing the strain. Meanwhile, the liquid from which the cells have been removed from said culture solution is also referred to as "supernatant," and it may be obtained by leaving the culture solution undisturbed for a certain period of time to take only the liquid from the upper layer excluding the part that has settled at the bottom, by removing the cells through filtration, or by centrifuging the culture solution to remove the lower sediment and taking only the upper liquid. The above "bacterial body" refers to the strain of the present invention itself, and includes the strain itself isolated and selected from vaginal secretions or traditional fermented food samples, etc., or the strain isolated from the culture medium by culturing the said strain. The said bacterial body can be obtained by centrifuging the culture medium and taking the portion that settles to the bottom layer, or by letting it sit for a certain period of time and removing the liquid from the top as it sinks to the bottom layer of the culture medium due to gravity.

[0045] The above culture medium may include, but is not limited to, the culture medium itself, its concentrate, or freeze-dried product obtained by culturing the strain, or the culture supernatant, its concentrate, or freeze-dried product obtained by removing the strain from the culture medium.

[0046] The above culture medium may be obtained by culturing a strain of the genus Lactobacillus in a medium (e.g., liquid Lactobacilli MRS broth medium, etc.) at a temperature of 10 to 40°C for a certain period of time, e.g., 4 to 72 hours, but is not limited thereto.

[0047] The culture medium and culture conditions for culturing the above-mentioned Lactobacillus strains may be appropriately selected or modified and used by a person with ordinary knowledge in the art.

[0048] In the present invention, the Lactobacillus crispatus strain may be the Lactobacillus crispatus SY-J504 strain deposited under accession number KCTC16719BP.

[0049] In the present invention, the Lactobacillus genseni strain may be the Lactobacillus genseni strain deposited under deposit number KCTC3599 and the Lactobacillus genseni FO-C822 strain, but is not limited thereto. In one embodiment, the Lactobacillus genseni strain may be the Lactobacillus genseni strain deposited under deposit number KCTC3599 or the Lactobacillus genseni FO-C822 strain, but it is obvious that known Lactobacillus genseni strains may also be included.

[0050] In the present invention, the Lactobacillus paraplantarum strain may be the Lactobacillus paraplantarum strain deposited under accession number KCCM11826P and the Lactobacillus paraplantarum IN-J111 strain, but is not limited thereto. In one embodiment, the Lactobacillus paraplantarum strain may be the Lactobacillus paraplantarum strain deposited under accession number KCCM11826P or the Lactobacillus paraplantarum IN-J111 strain, but it is obvious that known Lactobacillus paraplantarum strains may also be included.

[0051] In the present invention, the strain mixture fermented product may comprise 55 to 85 parts by weight, 55 to 80 parts by weight, 55 to 75 parts by weight, 55 to 70 parts by weight, 60 to 85 parts by weight, 60 to 80 parts by weight, 60 to 75 parts by weight, 60 to 70 parts by weight, 65 to 85 parts by weight, 65 to 80 parts by weight, 65 to 75 parts by weight, 65 to 70 parts by weight, 70 to 85 parts by weight, and 70 to 80 parts by weight, based on 100 parts by weight of the Lactobacillus crispatus strain culture. Specifically, it may comprise 70 to 75 parts by weight, and more specifically, it may comprise 70 parts by weight.

[0052] In the present invention, the strain mixture fermented product may comprise 15 to 45 parts by weight, 15 to 40 parts by weight, 15 to 35 parts by weight, 15 to 30 parts by weight, 20 to 45 parts by weight, 20 to 40 parts by weight, 20 to 35 parts by weight, 20 to 30 parts by weight, 25 to 45 parts by weight, 25 to 40 parts by weight, 25 to 35 parts by weight, 25 to 30 parts by weight, 30 to 45 parts by weight, and 30 to 40 parts by weight, based on 100 parts by weight of the Lactobacillus crispatus strain culture. Specifically, it may comprise 30 to 35 parts by weight, and more specifically, it may comprise 30 parts by weight.

[0053] In the present invention, the vaginitis may be bacterial vaginitis, candidal vaginitis, atrophic vaginitis, or trichomonas vaginitis, and specifically may be bacterial vaginitis or candidal vaginitis, but is not limited thereto.

[0054] The term "included as an active ingredient" as used in this specification means including a sufficient amount of the strain mixture fermented product in the composition to achieve the desired effect. In this case, the "desired effect" in the present invention may be the prevention, improvement, or treatment of vaginitis, and the sufficient amount to achieve such desired effect may be appropriately adjusted by the target, form, route of application, etc.

[0055] In the present invention, the composition may further comprise a lysate obtained by dissolving the strain mixture fermented product, and may comprise the strain mixture fermented product and the lysate in a weight ratio of 1:2 to 2:1. For example, the composition may comprise the strain mixture fermented product and the lysate thereof in a weight ratio of 1:2 to 2:1, and specifically may comprise them in a weight ratio of 1:1.

[0056] In the present invention, the composition is the Lactobacillus crispatus ( Lactobacillus crispatus ) strain, Lactobacillus genseni( Lactobacillus jensenii ) strain or Lactobacillus paraplantarum( Lactobacillus paraplantarum It may further contain polydeoxyribonucleotide (PDRN) derived from the strain.

[0057] In the present invention, the composition may be one or more formulations selected from the group consisting of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, powder foundation, emulsion foundation, and spray, but is not limited thereto.

[0058] For example, the cosmetic composition of the present invention may be prepared in any one formulation selected from 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, soap, shampoo, rinse, cleansing foam, cleansing lotion, cleansing cream, body lotion, and body cleanser, but is not limited thereto.

[0059] The cosmetic composition of the present invention may additionally include one or more cosmetically acceptable carriers that are incorporated into general skin cosmetics, and may appropriately incorporate, for example, oil, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, pigments, preservatives, fragrances, etc. as conventional ingredients, but is not limited thereto.

[0060] Cosmetically acceptable carriers included in the cosmetic composition of the present invention may vary depending on the formulation of the cosmetic composition.

[0061] For example, when the formulation of the present invention is an ointment, paste, cream, or gel, animal oil components, vegetable oil components, wax, paraffin, starch, tracanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. may be used as carrier components, but are not limited thereto, and these may be used alone or mixed in two or more types.

[0062] As another example, when the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. may be used as carrier components, and in particular, in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether may be additionally included, but is not limited thereto, and these may be used alone or in a mixture of two or more types.

[0063] As another example, when the formulation of the present invention is a solution or an emulsion, a solvent, a solubilizing agent, or an emulsifying agent may be used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, etc. may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan may be used, but are not limited thereto, and these may be used alone or mixed in two or more types.

[0064] As another example, when the formulation of the present invention is a suspension, liquid diluents such as water, ethanol, or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used as carrier components, but are not limited thereto, and these may be used alone or in combination of two or more.

[0065] As another example, when the formulation of the present invention is soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolyzates, isethionates, lanolin derivatives, aliphatic alcohols, vegetable oil components, glycerol, sugars, etc. may be used as carrier components, but are not limited thereto, and these may be used alone or mixed in two or more types.

[0066] The composition of the present invention is harmless to the human body as demonstrated by cytotoxicity tests, effectively prevents cosmetics from spoiling due to harmful microorganisms, and does not impair the quality of the cosmetic material upon addition, making it suitable for use in cosmetic compositions. Furthermore, since it has the function of improving the vaginal environment or alleviating vaginitis in women, it is desirable to use it in functional cosmetic compositions.

[0068] Another aspect of the present invention is a feminine hygiene product for preventing or improving vaginitis, comprising the above composition.

[0069] The terms composition, vaginitis, etc., are as described in the aforementioned embodiments.

[0071] Another aspect of the present invention is Lactobacillus crispatus ( Lactobacillus crispatus ) strain culture solution, Lactobacillus genseni( Lactobacillus jensenii ) strain culture solution and Lactobacillus paraplantarum( Lactobacillus paraplantarum A food composition for preventing or improving vaginitis, comprising as an active ingredient a strain mixture fermented product consisting of a strain culture solution,

[0072] The above Lactobacillus crispatus strain is the Lactobacillus crispatus SY-J504 strain deposited under accession number KCTC16719BP, and

[0073] The above strain mixture fermented product is a food composition for preventing or improving vaginitis, characterized by comprising 55 to 85 parts by weight of Lactobacillus genseni strain culture and 15 to 45 parts by weight of Lactobacillus paraplantarum strain culture per 100 parts by weight of Lactobacillus crispatus strain culture.

[0074] The food composition of the present invention may include a health functional food.

[0075] The term "health functional food" as used in this specification refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., using raw materials or ingredients that have functional properties useful to the human body. Here, "functionality" means obtaining effects useful for health purposes, such as regulating nutrients or physiological actions regarding the structure and function of the human body, and in the present invention, refers to the functions of improving the vaginal environment, anti-inflammatory effects, and skin moisturizing. The health functional food of the present invention can be manufactured using methods commonly used in the art, and can be manufactured by adding raw materials and ingredients commonly added in the art.

[0077] Another aspect of the present invention is Lactobacillus crispatus ( Lactobacillus crispatus ) strain culture solution, Lactobacillus genseni( Lactobacillus jensenii ) strain culture solution and Lactobacillus paraplantarum( Lactobacillus paraplantarum A pharmaceutical composition for the prevention or treatment of vaginitis comprising, as an active ingredient, a strain mixture fermented product consisting of a strain culture solution,

[0078] The above Lactobacillus crispatus strain is the Lactobacillus crispatus SY-J504 strain deposited under accession number KCTC16719BP, and

[0079] The above strain mixture fermented product is a pharmaceutical composition for preventing or treating vaginitis, characterized by comprising 55 to 85 parts by weight of Lactobacillus genseni strain culture and 15 to 45 parts by weight of Lactobacillus paraplantarum strain culture per 100 parts by weight of Lactobacillus crispatus strain culture.

[0081] Another aspect of the present invention is Lactobacillus crispatus ( Lactobacillus crispatus ) strain culture solution, Lactobacillus genseni( Lactobacillus jensenii ) strain culture solution and Lactobacillus paraplantarum( Lactobacillus paraplantarum It is intended for the prevention, improvement, or treatment of vaginitis using a strain mixture fermented product composed of a strain culture solution.

[0083] Hereinafter, the structure of the present invention and the resulting effects are to be explained in more detail through specific embodiments and comparative examples. However, these embodiments are intended to explain the present invention more specifically, and the scope of the present invention is not limited to these embodiments.

[0085] [Preparation Example]

[0087] Isolation and Identification of Lactobacillus crispatus SY-J504 Strain

[0088] Vaginal secretion samples from healthy Korean women without vaginal disease were suspended in 0.1% peptone water and plated on MRS agar medium (De Man, Rogosa and Sharpe agar) supplemented with 0.05% L-cysteine. These samples were placed in an anaerobic jar and incubated at 37°C for 48 hours. Among the colonies formed, only Gram-positive and Catalase-negative bacilli-shaped colonies were initially selected. Subsequently, the formed colonies were isolated, and PCR amplification was performed using known primers 27F and 1492R, followed by 16S rRNA sequence identification. The sequences of the determined 16S rRNA regions among the isolated cultured colonies were compared and analyzed with other strains registered using the BLAST program provided on the website of the National Center for Biotechnology Information (NCBI). Only novel species with 97% or less homology were selected and used, and among them, a novel microorganism with 94% or less homology was named Lactobacillus crispatus SY-J504. The above SY-J504 strain was deposited at the National Institute of Biological Resources of the Korea Research Institute of Biotechnology and Bioengineering on March 23, 2026, and was assigned deposit number KCTC16719BP. Additionally, a Lactobacillus genseni strain isolated and identified was named Lactobacillus genseni FO-C822.

[0090] [Preparation Example]

[0092] 1. Preparation of a mixed fermented product of Lactobacillus strains

[0093] Lactobacillus crispatus ( Lactobacillus crispatus ) strain, Lactobacillus genseni( Lactobacillus jensenii ) strains and Lactobacillus paraplantarum( Lactobacillus paraplantarum ) strains were prepared separately.

[0094] Specifically, the Lactobacillus crispatus strain was prepared as the known strain Lactobacillus crispatus KCTC0860BP and the Lactobacillus crispatus SY-J504 strain isolated in the above preparation example, the Lactobacillus genseni strain was prepared as the known strain Lactobacillus genseni KCTC3599 and the Lactobacillus genseni FO-C822 strain isolated in the above preparation example, and the Lactobacillus paraplantarum strain was prepared as the known strain Lactobacillus paraplantarum KCCM11826P and the Lactobacillus paraplantarum IN-J111 strain isolated from kimchi, a traditional fermented food, respectively.

[0095] After shaking-cultured the above-prepared Lactobacillus crispatus, Lactobacillus genseni, and Lactobacillus paraplantarum in MRS liquid medium at 37°C for 48 hours, 1 x 10⁶ 8 A strain culture solution diluted to a concentration of cfu / mL was prepared, and the supernatant was collected by centrifuging it at 13,000 rpm for 10 minutes. The strain culture solutions were then mixed according to the weight ratios in Table 1 below to prepare a mixed strain fermentation product.

[0096] division Lactobacillus crispatus culture (g) Lactobacillus genseni culture (g) Lactobacillus paraplantarum culture (g) Weight ratio of each strain culture solution KCTC0860BP SY-J504 KCTC3599 FO-C822 KCCM11826P IN-J111 Example 1 - 100 60 - 20 - 10:6:2 Example 2 - 100 - 80 20 - 10:8:2 Example 3 - 100 80 - - 40 10:8:4 Example 4 - 100 - 60 - 40 10:6:4 Example 5 - 100 - 70 - 30 10:7:3 Comparative Example 1 - 100 50 - 20 - 10:5:2 Comparative Example 2 - 100 - 90 20 - 10:9:2 Comparative Example 3 - 100 50 - - 50 10:5:5 Comparative Example 4 - 100 - 90 - 50 10:9:5 Comparative Example 5 - 100 60 - 10 - 10:6:1 Comparative Example 6 - 100 80 - - 10 10:8:1 Comparative Example 7 100 - - 80 20 - 10:8:2 Comparative Example 8 100 - - 60 - 40 10:6:4

[0098] 2. Preparation of a lysate from a mixed fermentation of Lactobacillus strains

[0099] The Lactobacillus strain mixed fermentation product of Example 5 (see Table 1) was centrifuged at 4°C and 8,000 rpm for 15 minutes to remove the supernatant and recover only the cell pellets. To completely remove the remaining culture medium components and impurities from the obtained cell mixture, sterile phosphate-buffered physiological saline (PBS, pH 7.2) was added and washed three times.

[0100] The washed cell mixture was uniformly suspended in sterile distilled water or 0.9% physiological saline at a concentration of 10 to 30% (w / v). Subsequently, physical disruption using a sonicator was performed to effectively break down the cell walls and extract intracellular active ingredients (PDRN, enzymes, peptides, etc.). At this time, to prevent denaturation and destruction of active ingredients caused by frictional heat generated during the sonication process, the sample was kept immersed in an ice bath. The cells were completely disrupted by repeating a cycle of 3 minutes of disruption followed by 1 minute of rest (pulse) a total of 10 to 15 times under conditions of a frequency of 20 kHz and an output of 300 W.

[0101] The ultrasonically treated lysis solution was heat-treated in an 80°C water bath for 30 minutes to inactivate intracellular autolysin and ensure safety when applied to the skin.

[0102] Subsequently, to remove uncrushed residual cells and cell debris, the lysate was centrifuged at 4°C and 10,000 rpm for 20 minutes to recover only the supernatant. The recovered supernatant was passed through a 0.22 µm diameter membrane filter to remove particulates and perform sterilization filtration, thereby finally obtaining a clear liquid fermentation lysate of the strain mixture. (If necessary, the obtained fermentation lysate filtrate was frozen at -80°C and then dehydrated using a freeze dryer to produce a powder for use.)

[0104] 3. Preparation of composition

[0105] A composition was prepared by mixing the Lactobacillus strain mixed fermentation product of Example 5 (see Table 1) and the lysate obtained by dissolving the same in the weight ratio according to Table 2 below, and adding an appropriate amount of 1,2-hexanediol and other additives.

[0106] division Strain mixed fermentation product (g) Soluble product of fermented product (g) 1,2-Hexanediol (g) Weight ratio of fermented product and dissolved product Preparation Example 1 40 80 3 1:2 Preparation Example 2 60 60 3 1:1 Preparation Example 3 80 40 3 2:1 Comparative Manufacturing Example 1 30 90 3 1:3 Comparative Manufacturing Example 2 90 30 3 3:1 Comparative Manufacturing Example 3 24 96 3 1:4 Comparative Manufacturing Example 4 96 24 3 4:1 Comparative Manufacturing Example 5 120 - 3 -

[0108] [Experimental Example]

[0110] 1. Confirmation of antibacterial activity against causative bacteria of vaginitis

[0111] Gardnerella vaginalis, known as a major causative agent of vaginitis ( Gardnerella vaginalis , KCTC5096 or KCTC5097) and Candida albicans ( Candida albicans The inhibitory effect on , KCTC7122 or KCTC7965) was confirmed as follows.

[0112] (1) Confirmation of antibacterial activity against causative agents of vaginitis according to strain combination

[0113] Each pathogen was pre-cultured in 5 ml of Reinforced Clostridium Medium (Difco) or 5 ml of YPD medium under anaerobic conditions in an incubator at 37°C. Culture was terminated when the absorbance of the culture solution at 660 nm reached 0.8. 1% of the pathogenic microbial pre-culture solution was inoculated into MRS liquid medium, and the main culture solution was prepared by shaking incubation at 37°C for 3 hours.

[0114] 2 ml of the strain mixture fermented product of Examples 1, 2, 3, 4, Comparative Example 7, or Comparative Example 8 prepared above was added to each of the above-mentioned main cultures of pathogenic microorganisms and incubated under anaerobic conditions for 48 hours. Subsequently, the number of viable pathogenic microorganisms was measured to confirm the degree of antimicrobial activity. The antimicrobial activity was analyzed using Formula 1 below, and the results are shown in Table 3 below. The control group is the number of viable pathogenic microorganisms in a culture medium in which 2 ml of MRS liquid medium was added instead of the strain mixture fermented product and incubated in the same manner. In this experiment, antimicrobial activity refers to the percentage of pathogenic microorganisms killed relative to the control group.

[0115] [Formula 1]

[0116] Antimicrobial activity (%) = (1-(A / B))X100

[0117] A: Number of viable pathogenic microorganisms in the example or comparative example

[0118] B: Number of viable pathogenic microorganisms in the control group

[0119] division Gardnerella vaginalis G. vaginalis Candida albicans C. albicans KCTC5096 KCTC5097 KCTC7122 KCTC7965 Antimicrobial activity (%) Example 1 87.9±0.5 86.1±2.5 85.8±4.3 85.3±5.8 Example 2 88.2±0.8 87.5±2.7 89.2±0.4 86.2±7.2 Example 3 90.1±4.9 88.4±4.6 87.5±5.1 88.6±1.8 Example 4 91.7±5.5 90.7±3.5 93.6±1.7 89.4±9.1 Comparative Example 7 68.1±3.1 62.9±6.3 46.9±2.9 52.6±4.7 Comparative Example 8 65.4±4.6 62.5±1.4 48.3±3.4 56.7±1.8

[0120] As can be seen in Table 3 above, strains that cause vaginitis ( G. vaginalis KCTC5096, KCTC5097, C. albicansAs a result of comparative analysis of the antibacterial activity against KCTC7122 and KCTC7965, it was confirmed that among the mixed strain fermentation products, Lactobacillus gensenii strain or Lactobacillus paraplantarum strain both had high antibacterial activity of 86% or higher without any difference in antibacterial activity depending on the type (Examples 1 to 4).

[0121] On the other hand, among the mixed strain fermentation products, there were significant differences in antibacterial activity depending on the type of Lactobacillus crispatus strain, and in particular, even when mixed in the same weight ratio, it was confirmed that Examples 2 and 4 containing Lactobacillus crispatus SY-J504 strain showed significantly increased antibacterial activity against causative agents of vaginitis compared to Comparative Examples 7 and 8 containing Lactobacillus crispatus KCTC0860BP strain.

[0122] Through the above results, it was experimentally confirmed that even among Lactobacillus crispatus strains of the same genus and species, a strain mixture fermented product containing the Lactobacillus crispatus SY-J504 strain identified and isolated in the present invention has a more effective effect in preventing or improving vaginitis.

[0124] (2) Confirmation of antibacterial activity according to strain mixing ratio

[0125] Using the same experimental method as in (1) above, the antibacterial activity against causative agents of vaginitis was further analyzed using the mixed fermented product of strains of Example 5 and Comparative Examples 1 to 6, and the results are shown in Table 4 below.

[0126] division Gardnerella vaginalis G. vaginalis Candida albicans C. albicans KCTC5096 KCTC5097 KCTC7122 KCTC7965 Antimicrobial activity (%) Example 1 87.9±0.5 86.1±2.5 85.8±4.3 85.3±5.8 Example 2 88.2±0.8 87.5±2.7 89.2±0.4 86.2±7.2 Example 3 90.1±4.9 88.4±4.6 87.5±5.1 88.6±1.8 Example 4 91.7±5.5 90.7±3.5 93.6±1.7 89.4±9.1 Example 5 92.1±7.2 91.7±6.8 95.9±7.1 91.8±1.7 Comparative Example 1 72.8±3.4 70.7±1.0 73.9±8.3 70.3±1.7 Comparative Example 2 71.5±0.5 70.4±2.9 67.9±1.6 67.5±2.1 Comparative Example 3 73.7±4.3 74.1±1.7 68.9±5.8 67.8±3.0 Comparative Example 4 72.2±5.0 69.0±7.7 65.8±1.4 66.2±2.1 Comparative Example 5 72.4±4.7 71.9±6.3 68.9±1.5 69.4±5.5 Comparative Example 6 73.4±6.6 72.8±3.1 69.4±1.8 68.2±2.1

[0127] As can be seen in Table 4 above, strains that cause vaginitis ( G. vaginalis KCTC5096, KCTC5097, C. albicansAs a result of comparative analysis of the antimicrobial activity of KCTC7122 and KCTC7965, it was confirmed that among the mixed strain fermented products, the mixed strain fermented product containing Lactobacillus crispatus culture, Lactobacillus genseni culture, and Lactobacillus paraplantarum culture in a weight ratio of 10:6 to 8:2 to 4 exhibited a high antimicrobial activity of at least 85%. In particular, it was confirmed that the mixed strain fermented product containing Lactobacillus crispatus culture, Lactobacillus genseni culture, and Lactobacillus paraplantarum culture in a weight ratio of 10:7:4 exhibited the highest antimicrobial activity (at least 91%), thereby confirming that the above weight ratio is the optimal weight ratio for the mixed strain fermented product (Examples 1 to 5).

[0128] On the other hand, when the weight ratio of the Lactobacillus gensenii strain culture solution included in the mixed strain fermentation product was low or excessively high, or when the weight ratio of the Lactobacillus paraplantarum strain culture solution was low or excessively high, that is, when deviating from the optimal weight ratio, the antibacterial activity was measured to be a maximum of 74%, confirming that it could not effectively inhibit the causative bacteria of vaginitis compared to the examples (Comparative Examples 1 to 4).

[0129] Through the above results, it was confirmed that in order to suppress the causative bacteria of vaginitis, including the Lactobacillus crispatus SY-J504 strain isolated and identified in the present invention in the strain mixture fermented product is an important factor, but the weight ratio in which the culture solutions of Lactobacillus crispatus, Lactobacillus genseni, and Lactobacillus paraplantarum are mixed is also a very important factor. Specifically, it was experimentally confirmed that the causative bacteria of vaginitis can be most effectively suppressed when the strain mixture fermented product contains the culture solutions of Lactobacillus crispatus, Lactobacillus genseni, and Lactobacillus paraplantarum in a weight ratio of 10:6 to 8:2 to 4.

[0131] 2. Confirmation of cytotoxicity

[0132] The cytotoxicity of the Lactobacillus strain mixed fermentation product was confirmed as follows.

[0133] Specifically, human vaginal epithelial cell line VK2 / E6E7 cells were seeded into a 96-well plate at a concentration of 1 x 10⁴ cells / well and cultured for 24 hours. Subsequently, the medium was changed to a 2% FBS-containing medium, and the mixed culture of the strains from Examples 1 to 3 was diluted with serum-free medium to achieve final concentrations of 1%, 5%, and 10% (v / v), followed by an additional 24 hours of culture. After the culture was complete, MTT solution (5 mg / mL) was added to each well and reacted for 4 hours; the supernatant was then removed, and 100 µL of DMSO was added to dissolve the formazan crystals. The absorbance was then measured at 570 nm using a microplate reader. The control group consisted of VK2 / E6E7 cells treated only with MTT solution without any sample addition, and the results are shown in Table 5 below.

[0134] division Cell viability (%) according to sample concentration 1%(v / v) 5%(v / v) 10%(v / v) Unprocessed 100 Example 1 101.4 99.9 87.5 Example 2 100.9 98.8 88.6 Example 3 102.8 99.4 88.7

[0135] As can be seen in Table 5 above, the mixed fermented strains of Examples 1 to 3 showed a cell viability of over 100% at 1% (v / v) and over 87% at 10% (v / v), experimentally confirming that they are safe and free from cytotoxicity even when applied to sensitive areas such as the vulva and vaginal mucosa.

[0137] 3. Confirmation of anti-inflammatory effects according to the mixing ratio of fermented and lysed products

[0138] In order to confirm the effect of the compositions of Preparation Examples 1 to 3 and Comparative Preparation Examples 1 to 4, prepared by varying the mixing ratio of the fermented product and the lysate in the above preparation examples, on inflammation relief, the inhibition rate of the production of inflammatory mediators nitric oxide (NO) and pro-inflammatory cytokine (TNF-α) was evaluated using mouse-derived macrophage cell line RAW 264.7 cells.

[0139] Specifically, RAW 264.7 cells were plated in 96-well plates at a rate of 5 x 10⁶ using DMEM medium containing 10% FBS. 4 Cells were dispensed at a concentration of cells / well and cultured for 24 hours in a 37°C, 5% CO₂ incubator. Subsequently, the inflammatory agent LPS (Lipopolysaccharide) was treated at a concentration of 1 μg / ml, and at the same time, the compositions of Preparation Examples 1 to 3 and Comparative Preparation Examples 1 to 4 were each treated at a concentration of 5% (v / v) and cultured for an additional 24 hours.

[0140] After the completion of culture, the supernatant was collected and the amount of NO produced was measured through the Griess reagent reaction, and the protein expression level of TNF-α was quantified using an ELISA kit (R&D Systems). The amount produced by the LPS-alone treatment group (control group) was converted to 100%, and the inhibitory activity of each composition is shown in Table 6 below.

[0141] division NO production amount (%) TNF-α production (%) Normal group (LPS untreated) 5.2 8.1 Control group (LPS alone treatment) 100.0 100.0 Preparation Example 1 28.4 31.5 Preparation Example 2 32.1 35.2 Preparation Example 3 30.8 33.9 Comparative Manufacturing Example 1 48.5 52.3 Comparative Manufacturing Example 2 51.2 45.6 Comparative Manufacturing Example 3 45.4 48.1 Comparative Manufacturing Example 4 52.4 50.1 Comparative Manufacturing Example 5 61.9 59.5

[0142] As can be seen in Table 6 above, in the case of Preparation Examples 1 to 3, which contain a strain mixture fermented product and a lysate thereof in a weight ratio of 1:2 to 2:1, it was confirmed that there is an excellent anti-inflammatory effect by rapidly reducing the amount of NO and TNF-α produced to about 30% compared to the control group, thus confirming that the above weight ratio is the optimal mixing ratio of the fermented product and the lysate to exert an anti-inflammatory effect.

[0143] On the other hand, it was confirmed that in the case of Comparative Preparation Example 5, which contains only the strain mixed fermentation product, or Comparative Preparation Examples 1 to 4, which contain an excessive amount of the strain mixed fermentation product or its lysate outside the above mixing ratio, the inhibitory effect on NO and TNF-α production was not significantly higher compared to the preparation examples.

[0144] Through the above results, it is suggested that the lysate obtained by dissolving the Lactobacillus strain mixed fermentation product according to the present invention can exhibit an excellent anti-inflammatory effect when mixed with the strain mixed fermentation product, including not only extracellular metabolites present in the fermentation product but also intracellular active ingredients (cytoplasm, nucleic acids (PDRN, etc.), intracellular enzymes) released as the cell membrane is destroyed and degraded cell wall / cell membrane components. In particular, it was experimentally confirmed that a powerful synergistic effect in inhibiting inflammatory factors can be expressed when the strain mixed fermentation product and its lysate are mixed in a specific weight ratio.

[0146] 4. Confirmation of skin moisturizing activity according to the mixing ratio of fermented extract and soluble extract

[0147] In order to confirm whether the compositions of Preparation Examples 1 to 3 and Comparative Preparation Examples 1 to 4, prepared by varying the mixing ratio of the fermented product and the lysate in the above preparation examples, have moisturizing activity capable of improving dryness of the vulva and vaginal mucosa, changes in mRNA expression of filaggrin, a key factor in skin barrier and moisturization, and aquaporin-3 (AQP3), a water channel protein, were confirmed using human keratinocytes (HaCaT).

[0148] Specifically, 2 x 10 HaCaT cells in a 6-well plate 5 After dispensing into cells / well and culturing for 24 hours, the compositions of Preparation Examples 1 to 3 and Comparative Preparation Examples 1 to 4 were diluted to a concentration of 5% (v / v) in serum-free medium and treated for 24 hours. Subsequently, cells were collected, total RNA was extracted using TRIzol reagent, cDNA was synthesized using reverse transcriptase, and Real-time PCR was performed using specific primers. The relative expression levels of each gene were normalized relative to the endogenous control (GAPDH), and the expression levels of the untreated group (normal group) were converted to 1.0. The results are shown in Table 7 below.

[0149] division Filaggrin relative expression (%) AQP-3 relative expression level (%) Normal group (untreated) 1.00 1.00 Preparation Example 1 2.85 2.92 Preparation Example 2 3.12 3.05 Preparation Example 3 2.68 2.74 Comparative Manufacturing Example 1 1.25 1.30 Comparative Manufacturing Example 2 1.42 1.55 Comparative Manufacturing Example 3 1.85 1.72 Comparative Manufacturing Example 4 1.15 1.20 Comparative Manufacturing Example 5 1.08 1.11

[0150] As can be seen in Table 7 above, in the case of Preparation Examples 1 to 3 containing a strain mixture fermented product and its lysate in a weight ratio of 1:2 to 2:1, it was confirmed that the gene expression of filaggrin and aquaporin-3, which are key factors determining moisture retention in the skin, increased significantly by about 2.6 to 3.1 times compared to the normal group, and in particular, the moisturizing factor expression ability was best in Preparation Example 2 (weight ratio of 1:1), in which the weight ratio of the lysate to the fermented product was relatively high.

[0151] On the other hand, in the case of Comparative Example 5, which did not contain a lysate of the fermented product from which the intracellular active ingredient (PDRN, etc.) was extracted, the moisturizing effect remained at the level of the normal group, and in the case of Comparative Example 1 and Comparative Example 3, which contained an excessive amount of the fermented lysate, it was confirmed that the moisturizing activity actually decreased.

[0152] Accordingly, it was experimentally confirmed that a composition comprising the strain mixture fermented product and the lysate thereof according to the present invention in a weight ratio of 1:2 to 2:1 can produce a significant moisture barrier strengthening effect greater than the effect of simply mixing the two components.

[0154] The various embodiments described above may be embodied in other specific forms without departing from the technical idea and essential features. Accordingly, the above detailed description should not be interpreted restrictively in all respects but should be considered exemplary. The scope of the various embodiments shall be determined by a reasonable interpretation of the appended claims, and all modifications within the equivalent scope of the various embodiments are included within the scope of the various embodiments. Furthermore, embodiments may be constructed by combining claims that do not have an explicit citation relationship in the patent claims, or they may be included as new claims through amendments made after filing.

[0156] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC) Trustee Number: KCTC16719BP Date of Deposit: 2026-03-23

Claims

Claim 1 Lactobacillus crispatus ( Lactobacillus crispatus ) strain culture solution, Lactobacillus genseni( Lactobacillus jensenii ) strain culture solution and Lactobacillus paraplantarum( Lactobacillus paraplantarum A cosmetic composition for preventing or improving vaginitis, comprising as an active ingredient a strain mixed fermentation product consisting of a strain culture solution, wherein the Lactobacillus crispatus strain is the Lactobacillus crispatus SY-J504 strain deposited under accession number KCTC16719BP, and the strain mixed fermentation product comprises 60 to 80 parts by weight of a Lactobacillus genseni strain culture solution and 20 to 40 parts by weight of a Lactobacillus paraplantarum strain culture solution per 100 parts by weight of the Lactobacillus crispatus strain culture solution, and the composition further comprises a lysate obtained by dissolving the strain mixed fermentation product, and is characterized by comprising the strain mixed fermentation product and the lysate in a weight ratio of 1:2 to 2:

1. Claim 2 A cosmetic composition for preventing or improving vaginitis, characterized in that, in claim 1, the composition comprises the strain mixed fermented product and the lysate in a weight ratio of 1:

1. Claim 3 In claim 1, the composition is the Lactobacillus crispatus ( Lactobacillus crispatus ) strain, Lactobacillus genseni( Lactobacillus jensenii ) strain or Lactobacillus paraplantarum( Lactobacillus paraplantarum A cosmetic composition for preventing or improving vaginitis, characterized by further including polydeoxyribonucleotide (PDRN) derived from a strain. Claim 4 A cosmetic composition for preventing or improving vaginitis, characterized in that, in claim 1, the composition is one or more formulations selected from the group consisting of solution, suspension, emulsion, paste, gel, cream, lotion, powder, powder foundation, emulsion foundation, and spray. Claim 5 A feminine hygiene product for preventing or improving vaginitis, comprising a composition of any one of claims 1 to 4.

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