KR102679284B1 - A probiotic composition(InoRexyneTM) containing Lactobacillus plantarum YC-225, which can help with women's vaginal infections or improve vaginal health, is referred to as NU Probiotics - Google Patents

Abstract

The present invention relates to a composition containing a mixed prebiotic strain (InoRexyne ^TM ) for women's vaginal health, which promotes vaginal health, has significantly significant preventive, improving and therapeutic effects on vaginitis, and has cytotoxic effects. Since it does not represent, it can be usefully used in food compositions, pharmaceutical compositions, and cosmetic compositions.

Description

A probiotic composition (InoRexyneTM) containing Lactobacillus plantarum YC-225, which can help with women's vaginal infections or improve vaginal health health, is referred to as NU Probiotics}

The present invention relates to a prebiotic complex composition (InoRexyne ^TM ) for women's vaginal health, and more specifically, to a probiotic (mixed strain) consisting of Lactobacillus crispatus, Lactobacillus fermentum, and Lactobacillus palantharum. It relates to a composition containing.

A woman's vaginal environment can be influenced by many factors, which can affect her quality of life and overall health. The key to a woman's vagina is maintaining the balance of various microorganisms.

A woman's healthy vaginal mucosa is formed by a stratified squamous non-keratinized epithelium of approximately 28 cell layers. It is covered by a mucosal layer that is constantly lubricated by cervical fluid. Additionally, the outermost layer of the vaginal epithelium is made up of dead, noninfectious keratinized cells that act as a protective barrier against pathogens.

If this protective layer is continuously exposed to environmental factors such as antibiotics, stress, and hormonal changes, the balance in the vagina is broken, causing the invasion of pathogens and causing infectious inflammatory diseases. These infectious inflammatory diseases of the female genital tract can be caused by various pathogens such as bacteria, viruses, and molds. Among them, representative ones include bacterial vaginosis and candidal vaginitis. Bacterial vaginosis is caused by the proliferation of bacteria such as Gardnerella vaginalis, and candidal vaginitis (Yeast infection) is a disease caused by the dominant proliferation of yeast bacteria such as Candida albicans. Vaginitis is a disease that is common enough to be called the female cold, but if treatment is missed, it can develop into other diseases such as cystitis and pelvic inflammatory disease, or cause problems in one's life as it is accompanied by itching and pain during defecation, so early treatment and prevention are very important. do.

Republic of Korea Patent Publication No. 10-2020-0001214

Under the above background, the present inventors have made diligent efforts to develop a composition that helps vaginal health, and as a result, it has excellent antibacterial activity against vaginitis-causing bacteria and can prevent, improve, or treat vaginitis by inhibiting the growth of vaginitis-causing bacteria. The present invention was completed by developing mixed strains and compositions containing them.

The purpose of the present invention is to provide a food composition, pharmaceutical composition, and cosmetic composition for preventing, improving, or treating vaginitis containing mixed strains.

The object of the present invention is not limited to the objects mentioned above. The object of the present invention will become clearer from the following description and may be realized by means and combinations thereof as set forth in the claims.

To achieve the above objective, the following solution is provided.

One aspect of the present invention is Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus fermentum LF05 (DSM 32277) and Lactobacillus plantarum YC-225 (KCTC19068P) ) Provides a food composition for preventing or improving vaginitis containing a mixed strain consisting of.

The Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus fermentum LF05 (DSM 32277), and Lactobacillus plantarum YC-225 (KCTC19068P) are 1:0.5. -5: It may be mixed at a cell number ratio of 0.5-5.

The composition may have antibacterial activity against one or more vaginitis-causing bacteria selected from the group consisting of Gardnerella vaginalis and Candida albicans.

Another aspect of the present invention is Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus fermentum LF05 (DSM 32277) and Lactobacillus plantarum YC-225 KCTC19068P. Provided is a pharmaceutical composition for preventing or treating vaginitis comprising a mixed strain.

The vaginitis may be any one or more selected from the group consisting of bacterial vaginitis and candidal vaginitis.

Another aspect of the present invention is Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus fermentum LF05 (DSM 32277) and Lactobacillus illus plantarum YC-225 KCTC19068P Provides a cosmetic composition for preventing or improving vaginitis comprising NU probiotics (mixed strains).

Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus fermentum LF05 (DSM 32277) and Lactobacillus plantarum YC-225 (KCTC19068P) according to the present invention. The mixed strain (NU probiotics) composition promotes vaginal health, has significantly significant preventive, improving and therapeutic effects on vaginitis, and does not show cytotoxicity, so it can be usefully used as food compositions, pharmaceutical compositions and cosmetic compositions. there is.

The effects of the present invention are not limited to the effects mentioned above. The effects of the present invention should be understood to include all effects that can be inferred from the following description.

Figure 1 is a graph confirming the cytotoxicity of RAW 264.7 cells treated with the mixed strain composition prepared from Example 1 and Comparative Examples 1 to 11 diluted to a concentration of 0.005X. In the drawing, 'a-f' indicates significance analyzed through Duncan's multiple range test.
Figure 2 is a graph confirming cytotoxicity by treating RAW 264.7 cells with the mixed strain composition prepared from Example 1 and Comparative Examples 1 to 11 diluted to a concentration of 0.01X. In the drawing, 'a-f' indicates significance analyzed through Duncan's multiple range test.
Figure 3 is a diagram showing the results of confirming the effect of inhibiting nitric oxide (NO) production of the mixed strain composition prepared from Example 1 and Comparative Examples 1 to 11 diluted to a concentration of 0.005X. In the figure, 'a-f' indicates significance analyzed through Duncan's multiple range test.
Figure 4 is a diagram showing the results of confirming the effect of inhibiting nitric oxide (NO) production of the mixed strain composition prepared from Example 1 and Comparative Examples 1 to 11 diluted to a concentration of 0.01X. In the drawing, 'a-f' indicates significance analyzed through Duncan's multiple range test.
Figure 5 is a graph showing the body weight measured from the normal control group (Nor), negative control group (CA), low experimental group, mid experimental group, high experimental group, and positive control group (PC).
Figure 6(a) is a graph showing the NO concentration measured in vaginal tissue separated from the normal control group (Nor), negative control group (CA), low experimental group, mid experimental group, high experimental group, and positive control group (PC). (b) is a graph showing the concentration of IL-6 measured in vaginal tissue isolated from the normal control group (Nor), negative control group (CA), low experimental group, mid experimental group, high experimental group, and positive control group (PC). * P < 0.05, ** P < 0.01, *** P < 0.001 Student t-test.
Figure 7(a) shows the results of Gram staining for diagnosing vaginitis on vaginal tissue isolated from the normal control group (Nor), negative control group (CA), low experimental group, mid experimental group, high experimental group, and positive control group (PC). 7(b) shows the vaginitis-causing bacteria ( Gardnerella vaginalis ) present in vaginal tissues isolated from the normal control group (Nor), negative control group (CA), low experimental group, mid experimental group, high experimental group, and positive control group (PC). ) This is a graph showing the number of viable bacteria (CFU/ml) measured by KCTC5097). **** P < 0.05 Student t-test.
Figure 8(a) shows the results of staining with H&E to confirm the thickness of vaginal tissue separated from the normal control group (Nor), negative control group (CA), low experimental group, mid experimental group, high experimental group, and positive control group (PC), Figure 8(b) is a graph showing the exfoliation scores of vaginal tissues separated from the normal control group (Nor), negative control group (CA), low experimental group, mid experimental group, high experimental group, and positive control group (PC). am. * P < 0.05, ** P < 0.01, *** P < 0.001 Student t-test.
Figure 9(a) is a graph measuring the expression level of ZO-1 protein in vaginal tissues isolated from the normal control group (Nor), negative control group (CA), low experimental group, mid experimental group, high experimental group, and positive control group (PC). , Figure 9(b) is a graph measuring the FOXA1 protein expression level in vaginal tissues isolated from the normal control group (Nor), negative control group (CA), low experimental group, mid experimental group, high experimental group, and positive control group (PC). * P < 0.05, ** P < 0.01, *** P < 0.001 Student t-test.

The objects, other objects, features and advantages of the present invention will be readily understood through the following preferred embodiments in conjunction with the accompanying drawings. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided so that the disclosed content will be thorough and complete and so that the spirit of the present invention can be sufficiently conveyed to those skilled in the art.

In this specification, terms such as “comprise” or “have” are intended to designate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, but are not intended to indicate the presence of one or more other features. It should be understood that this does not exclude in advance the possibility of the existence or addition of elements, numbers, steps, operations, components, parts, or combinations thereof.

In this specification, when a range is written for a variable, the variable will be understood to include all values within the stated range, including the stated end points of the range. For example, the range "5 to 10" includes the values 5, 6, 7, 8, 9, and 10, as well as any subranges such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, etc. It will be understood that it also includes any values between integers that fall within the scope of the stated range, such as 5.5, 6.5, 7.5, 5.5 to 8.5, and 6.5 to 9, etc. Also, for example, the range "10% to 30%" includes values such as 10%, 11%, 12%, 13%, etc. and all integers up to and including 30%, as well as 10% to 15%, 12% to 12%, etc. It will be understood that it includes any subranges, such as 18%, 20% to 30%, etc., and any value between reasonable integers within the range of the stated range, such as 10.5%, 15.5%, 25.5%, etc.

Hereinafter, the present invention will be described in detail.

The present invention consists of Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus fermentum LF05 (DSM 32277), and Lactobacillus plantarum YC-225 (KCTC19068P) It relates to mixed strain compositions and uses thereof. The mixed strain composition according to the present invention is also called “InoRexyne ^TM ”.

More specifically, Lactobacillus plantarum YC-225 strain isolated from kimchi and Lactobacillus crispatus, which have acid resistance, bile resistance, or intestinal adhesion ability, are not hemolytic, and have an anti-inflammatory effect. It relates to a food composition for preventing or improving vaginitis containing a mixed strain composition containing (Lactobacillus crispatus) LCR01 (DSM 24619) and Lactobacillus fermentum LF05 (DSM 32277) as an active ingredient.

In addition, another aspect of the present invention relates to a pharmaceutical composition for preventing or treating vaginitis containing the above mixed strains as an active ingredient.

In addition, another aspect of the present invention relates to a cosmetic composition for preventing or improving vaginitis containing the above mixed strains as an active ingredient.

The mixed strains are Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus fermentum LF05 (DSM 32277), and Lactobacillus plantarum YC-225 (KCTC19068P). If any one of the above strains is changed to another strain or omitted, antibacterial activity against vaginitis-causing bacteria cannot be achieved, so it is preferable that all three strains are included.

Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus fermentum LF05 (DSM 32277) and Lactobacillus plantarum YC-225 (KCTC19068P) of the present invention are 1 : 0.5-5 : 0.5-5 cell number ratio, preferably Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus fermentum LF05 (DSM 32277) and Lactobacillus It may contain Lactobac illus plantarum YC-225 (KCTC19068P) at a cell number ratio of 1:0.5-2:0.5-2.

The composition consists of Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus fermentum LF05 (DSM 32277), and Lactobacillus plantarum YC-225 (KCTC19068P). The mixed strain may be included at a concentration of 1 × 10 ² to 1 × 10 ¹⁰ CFU/ml, preferably 1 × 10 ² to 1 × 10 ⁸ CFU/ml, 1 × 10 ² to 1 × 10 ⁷ CFU. /ml, and may include a concentration of 1 × 10 ⁴ to 1 × 10 ⁷ CFU/ml, but is not limited thereto.

As a result of analyzing the antibacterial activity against Gardnerella vaginalis and Candida albicans through the experiment described later, it was confirmed that the mixed strain according to the present invention had significantly excellent antibacterial activity against various vaginitis strains, as well as vaginitis It was confirmed that it is highly effective in strengthening the physical barrier of vaginal tissue by repairing damage to vaginal tissue and restoring tight junctions between cells in the vaginal mucosa layer.

In addition, the mixed strain can regulate immune function by suppressing the production of NO and IL-6 in the vagina induced by pathogenic microorganisms, thereby maintaining immune homeostasis.

Therefore, the mixed strain can protect the vaginal environment from infection by bacteria or Candida, and even if infected, it can inhibit the growth of vaginitis-causing bacteria and show excellent vaginitis improvement and treatment efficacy. The mixed strain according to the present invention can be used as a probiotic. When developed and used as probiotics, they have excellent acid resistance, bile resistance, and intestinal adhesion ability, and are effectively delivered to the vagina. They can exert the effect of improving vaginal health by protecting, preventing, improving, and treating the vaginal environment and vaginal barrier. .

The " Lactobacillus plantarum YC-225 strain" is a strain isolated from kimchi, a traditional fermented food, deposited at the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center on February 27, 2023 with accession number KCTC19068P, It has acid resistance, bile resistance, and intestinal adhesion characteristics. The Lactobacillus plantarum YC-225 (KCTC19068P) strain is 16S rRNA and includes the base sequence shown in SEQ ID NO: 1. The Lactobacillus plantarum YC-225 strain of the present invention is a new, undisclosed strain, and is comprised of the chromosomal sequence and plasmids 1 and 2 of the Lactobacillus plantarum YC-225 strain of the present invention. The sequences are listed in sequence in SEQ ID NOs: 4 to 6.

In the present invention, the active ingredients of the mixed strain , Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus fermentum LF05 (DSM 32277), and Lactobacillus plantarum YC- 225 (KCTC19068P) may be any one selected from the group consisting of the strain itself, a culture of the strain, or a lysate of the strain.

The culture of the strain is a culture medium and/or a material containing the cultured strain, but is not limited thereto, and may be a culture medium cultured including the strain or the strain itself. The formulation of the culture is not limited, and may be liquid or solid, for example.

In the present invention, the 'culture medium' refers to the cell culture solution or culture medium remaining after culturing the strain, excluding the strain.

In the present invention, the term lysate refers to a cell lysate obtained by lysing the strain by a chemical method or a physical method using a surfactant (detergent) or the like.

The composition of the present invention may be prepared according to conventional probiotic composition preparation methods, and may generally be provided in lyophilized, encapsulated form, culture suspension, or dry powder form.

The composition of the present invention can be provided as a food composition for preventing or improving vaginitis for vaginal health.

The 'food composition' contains Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus fermentum LF05 (DSM 32277), and Lactobacillus plantarum YC- as active ingredients. In addition to the mixed strain (InoRexyne ^TM ) consisting of 225 (KCTC19068P), it includes food raw materials that can be used as foods and food additives listed in the Food Additive Code as specified in the Food Standards and Specifications ('Food Code of Commerce') commonly used in food manufacturing. do.

In the present invention, the term "vaginal health" refers to a woman's healthy vaginal mucosa composed of stratified squamous non-keratinized epithelium of about 28 cell layers and covered with a mucosal layer that is continuously lubricated by cervical fluid. The outermost layer of the vaginal epithelium is composed of dead, uninfected keratinized cells and serves as a protective barrier against pathogens. If this protective layer continues to be stimulated by environmental factors such as antibiotics, stress, and hormonal changes, the microbial community that maintains the balance in the vagina is destroyed, the number of lactic acid bacteria decreases, and pH increases, causing infectious inflammation that causes the invasion of pathogens. Disease is caused. Vaginal infections usually appear as bacterial vaginitis and candidal vaginitis, and the causative bacteria of each vaginitis are known to be Gardnerella vaginalis and Candida albicans. The above-mentioned vaginitis occurs in many women and has a high risk of recurrence, so efforts to prevent, improve, and treat it are ongoing.

Consisting of Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus fermentum LF05 (DSM 32277) and Lactobacillus plantarum YC-225 (KCTC19068P) of the present invention The mixed strain composition (InoRexyne ^TM ) effectively settles in the vagina and produces useful components, inhibiting the growth of vaginitis-causing bacteria, alleviating inflammatory reactions in the vagina, and protecting the vaginal barrier (tight junctions between cells of the vaginal mucosa layer). junction)) to maintain the health of the vagina.

Therefore, a mixed strain consisting of Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus fermentum LF05 (DSM 32277), and Lactobacillus plantarum YC-225 (KCTC19068P) (InoRexyne ^TM ) can be used as a composition that can prevent or improve vaginal health.

In particular, the composition of the present invention is useful for vaginitis and can prevent or improve vaginitis. Specifically, the present invention has an excellent growth inhibitory effect on Candida albicans and Gardnerella vaginalis, which are the main causative bacteria of vaginitis that frequently occurs in women, and has excellent antibacterial activity against the vaginitis-causing bacteria. have More specifically, it has antibacterial activity against various vaginitis-causing bacteria, such as Gardnerella vaginalis KCTC5097, Gadnerella vaginalis KCTC5096, and Candida albicans KCTC7122.

There is no need to specifically limit it, but examples include proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. The carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sugar, lactose, etc.; Oligosaccharides or polysaccharides, such as dextrin, starch syrup, cyclodextrin, etc.; Sugar alcohols such as xylitol, sorbitol, erythritol, etc. can be used. The flavoring agent may be a natural flavoring agent (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) or a synthetic flavoring agent (saccharin, aspartame, etc.).

A mixed strain consisting of Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus fermentum LF05 (DSM 32277), and Lactobacillus plantarum YC-225 (KCTC19068P) When manufacturing a food composition with (InoRexyne ^TM ) as an active ingredient, the mixed strain does not need to be specifically limited as long as it has the effect of preventing or improving vaginal health, but for example, 0.1 to 99% by weight, 0.5 to 95% by weight. It may be included in weight%, 1 to 90% by weight, 2 to 80% by weight, 3 to 70% by weight, 4 to 60% by weight, and 5 to 50% by weight.

In the food composition, the active ingredients Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus fermentum LF05 (DSM 32277), and Lactobacillus plantarum YC-225 ( The mixed strain (InoRexyne ^TM ) consisting of KCTC19068P) varies depending on the condition, body weight, and presence, degree, and duration of the disease, but can be appropriately selected by a person skilled in the art. For example, based on the daily dosage, it may be 1 to 5,000 mg, preferably 5 to 2,000 mg, more preferably 10 to 1,000 mg, even more preferably 20 to 800 mg, and most preferably 50 to 500 mg. There is no need to specifically limit the number of administrations, but a skilled technician can adjust it within the range of three times a day to once a week. In the case of long-term intake for health and hygiene purposes or health control, it may be below the above range.

The food composition does not need to be particularly limited, but may be, for example, powder, granule, tablet, capsule, pill, extract, jelly formulation, tea bag formulation, or beverage formulation.

In addition, in order to prevent or improve vaginal health in general foods , Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus fermentum LF05 (DSM 32277), and Lactobacillus plantarum (Lactobacillus illus ) plantarum) YC-225 (KCTC19068P) mixed strain (InoRexyne ^TM ) can be added, and there is no need to specifically limit the foods that can be added, but for example, the food standards and specifications pursuant to Article 7 of the Food Sanitation Act (' Confectionery, bread or rice cake, processed cocoa products or chocolate, processed meat or egg products, processed fish products, tofu or jelly, noodles, tea, coffee, beverages, special-purpose foods, sauces, seasonings, dressings as exemplified in the ‘Food Code of Conduct’) It can be added to seafood, kimchi, salted seafood, pickled foods, stewed foods, alcoholic beverages, dried raisins, and other foods. In addition, it can be added to dairy products, processed meat products, packaged meat, and egg products as exemplified in the livestock product processing standards and ingredient specifications ('Livestock Product Code') pursuant to Article 4 of the Livestock Products Sanitation Management Act.

Meanwhile, the food composition containing the mixed strain (InoRexyne ^TM ) as an active ingredient can be used alone as a “health functional food that helps prevent or improve vaginal health” and a “health functional food that helps prevent vaginitis.” , It can be used as a “health functional food that helps improve vaginitis” or a “health functional food that helps relieve vaginitis.”

The above ‘health functional food’ refers to food manufactured (including processing) in accordance with legal standards using raw materials or ingredients with functional properties useful to the human body (Article 3, Paragraph 1 of the Health Functional Food Act). The terminology or scope of the above ‘health functional food’ may vary depending on the country, but it is also called ‘Dietary Supplement’ in the United States, ‘Food Supplement’ in Europe, ‘Health Functional Food’ in Japan, or ‘Food Supplement’ in Japan. It may be classified as ‘Food for Special Health Use (FoSHU)’ or ‘Health Food’ in China.

The above food composition or health functional food may additionally contain food additives, and its suitability as a food additive is determined by the specifications and standards for the relevant item in accordance with the general provisions of the ‘Food Additive Code’ and general test methods, etc., unless otherwise specified. Follow.

In addition, the health functional food includes Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus fermentum LF05 (DSM 32277), and Lactobacillus illus plantarum YC-225 ( It can be used in combination with a mixed strain (InoRexyne ^TM ) consisting of KCTC19068P) and health functional food materials related to the prevention or improvement of vaginal health, or related to the prevention or improvement of vaginitis.

Additionally, the composition of the present invention can be prepared as a pharmaceutical composition for preventing or treating vaginitis.

In the present invention, the term "vaginitis" is not particularly limited as long as it is an infectious inflammatory disease induced by abnormal growth in the vagina. For example, it may be any one or more selected from bacterial vaginitis and candida vaginitis, and specifically Candida albicans ( It may be induced by one or more selected from the group consisting of Candida albicans ) and Gardnerella vaginalis .

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable” in the present invention means that the composition exhibits non-toxic properties to cells or humans exposed to the composition.

The carrier may be used without limitation as long as it is known in the art, such as a buffer, preservative, analgesic agent, solubilizer, isotonic agent, stabilizer, base, excipient, lubricant, etc.

In addition, the pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods. there is. Furthermore, it can be used in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel, or external skin preparation in the form of a gel.

Meanwhile, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "administration" of the present invention means introducing a predetermined substance into an individual by an appropriate method, and the composition may be administered through any general route as long as it can reach the target tissue. Specifically, it may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, locally, intranasally, intrapulmonaryly, or rectally, but is not limited thereto.

The term “individual” refers to all animals, including rats, mice, and livestock, including humans. Preferably, it may be a mammal, including humans.

The term "pharmaceutically effective amount" means an amount that is sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is determined by the patient's gender, age, and weight. , factors including health status, type and severity of the disease, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration, and excretion rate, duration of treatment, drugs combined or used simultaneously, and other well-known fields of medicine. It can be readily determined by a person skilled in the art based on known factors.

In addition, according to the present invention, the composition may be manufactured as a pharmaceutical composition or quasi-drug composition for preventing, improving, or treating vaginitis.

The term "quasi-drug" used in the present invention refers to products with a milder effect than pharmaceuticals among products used for the purpose of diagnosing, treating, improving, alleviating, treating or preventing diseases in humans or animals. For example, according to the Pharmaceutical Affairs Act. According to it, quasi-drugs exclude products used for medicinal purposes and include products used to treat or prevent diseases in humans and animals, and products that have a mild or no direct effect on the human body.

The quasi-drug composition is from the group consisting of body cleanser, disinfectant cleaner, detergent, kitchen cleaner, cleaning agent, toothpaste, mouthwash, wet tissue, detergent, soap, hand wash, hair cleaner, hair softener, humidifier filler, mask, ointment and filter filler. It can be prepared in a selected formulation, but is not limited thereto.

In addition, according to the present invention, the composition can be prepared as a cosmetic composition for preventing or improving vaginitis.

The cosmetic composition may have, for example, an softening lotion, a nourishing lotion, a massage cream, a nourishing cream, an essence, a pack, a gel, an ampoule, or a skin-adhesive cosmetic formulation.

Ingredients included in the cosmetic composition may include ingredients commonly used in cosmetic compositions in addition to the composition as an active ingredient, for example, conventional auxiliaries and carriers such as stabilizers, solubilizers, vitamins, pigments, and fragrances. may include.

Additionally, the composition may be a composition for external skin application.

The external skin agent may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal delivery patch, drug-containing bandage, lotion, or a combination thereof. The above skin external preparations include ingredients commonly used in external skin preparations such as cosmetics and medicines, such as water-based ingredients, oil-based ingredients, powder ingredients, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, and fragrances. , colorants, various skin nutrients, or a combination thereof may be appropriately mixed according to need. The skin external preparations include metal sequestrants such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belafamil, licorice extract, glablidin, and calin. Hot water extract of fruit, various herbal medicines, drugs such as tocopherol acetate, glytylitinic acid, tranexamic acid and its derivatives or salts, vitamin C, magnesium ascorbate phosphate, ascorbate glucoside, arbutin, kojic acid, glucose, fructose, Sugars such as trehalose can also be appropriately mixed.

Another aspect of the invention provides a method of preventing, ameliorating, or treating a condition in a subject comprising treating or administering an effective amount of the composition described above to the subject in need thereof.

The condition of the subject may be a skin-related condition.

As used herein, the terms “administering,” “introducing,” and “implanting” are used interchangeably and are used interchangeably to introduce a composition into an individual by a method or route that results in at least partial localization to the desired site according to one embodiment. It may refer to the arrangement of the composition according to one embodiment of.

Administration can be done by methods known in the art. Administration may be administered directly to the subject by any means, for example, intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. You can. The administration may be administered systemically or locally.

The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be an individual in need of improving vaginal health, for example, strengthening the vaginal barrier, relieving vaginal inflammation, treating vaginitis, or improving vaginitis.

The administration of the composition according to one embodiment is 0.1 mg to 1,000 mg, 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 mg per day. 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. However, the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, gender, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity, and those skilled in the art will Taking these factors into consideration, the dosage can be adjusted appropriately. The frequency of administration can be once a day or more than twice within the range of clinically acceptable side effects, and can be administered at one or two or more locations, daily or at intervals of 2 to 5 days. The number of days of administration can be from 1 to 30 days per treatment. If necessary, the same treatment can be repeated after an appropriate period. For animals other than humans, the dosage per kg is the same as that for humans, or the above dosage is converted into, for example, the volume ratio (e.g., average value) of the organs (heart, etc.) between the target animal and the human. One dose can be administered.

Hereinafter, the present invention will be described in more detail through the examples below. However, the following examples are only examples to easily explain the content and scope of the technical idea of the present invention, and the technical scope of the present invention is not limited or changed thereby. Additionally, based on these examples, it can be easily determined by those skilled in the art that various modifications and changes are possible within the scope of the technical idea of the present invention.

Preparation Example 1. Strain isolation and identification

Lactobacillus plantarum YC-225 (KCTC19068P) strain, which has excellent acid resistance, bile resistance, and intestinal adhesion, was isolated from traditional fermented foods and deposited after performing 16S rRNA analysis.

Among strains isolated from traditional fermented foods, the Lactobacillus illus plantarum YC-178 strain, which has lower acid resistance, bile resistance, and intestinal adhesion than the YC-225 strain, was selected and used as a comparative strain.

Hemolysis analysis was performed on the Lactobacillus illus plantarum YC-225 (KCTC19068P) strain, and the results confirmed that it was γ-hemolytic (i.e., no hemolytic activity was observed).

Hemolysis analysis was performed using tryptic agar (BD Bioscience, NJ, USA) plates (MBCell, Seoul, Korea) containing 5% (w/v) sheep blood. After smearing a line on the medium, the cells were cultured at 37°C for 24 and 48 hours, and then the area around the colonies was observed to evaluate hemolysis.

[SEQ ID NO: 1] 16S rRNA of YC-225

GACGACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGGCGAACTGGTGAGTAACACGTGGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGCTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGG GTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATG GAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAG CCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGG TAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTG AAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGA GGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAA ACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAG TTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTTAGGAACCAGCCGCCTAAGGTGGGACAGATGATTAGGGTG

Example 1. Mixed strain composition (InoRexyne ^TM )

Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus fermentum LF05 (DSM 32277), and Lactobacillus plantarum YC-225 (KCTC19068P) were each used in MRS liquid medium. After shaking culture at 37°C for 48 hours, prepare a ^strain culture dilution diluted to a concentration of 1 A mixed strain composition was prepared by mixing.

Comparative Examples 1 to 11. Mixed strain composition

Prepare lactic acid bacteria known to have an effect on vaginal health, culture them in MRS liquid medium at 37°C with shaking for 48 hours, and then prepare a strain culture dilution diluted to a concentration of 1 × 10 ⁶ cfu/ml, which is incubated at 13,000 rpm. After preparing the supernatant by centrifuging for 10 minutes, a mixed strain composition was prepared by mixing at the mixing weight ratio shown in Table 1 below. LCR01 and LF05 are the same strains as Example 1, and the specific names will be omitted.

'LA02' is Lactobacillus acidophilus LA02 (DSM 21717), 'LP01' is Lactobacillus plantarum LP01 (LMG P-21021), and 'LP YC-178' is Lactobacillus plantarum (Lactobacillus plantarum) YC-225 (KCTC19068P) is a candidate strain with antibacterial activity against vaginitis-causing bacteria recovered during the selection process.

In addition, Lactobacillus reuteri RC-14® (LRT RC-), a lactic acid bacteria strain related to vaginal health, manufactured by Christian Hansen (Chr.Hansen), a global lactic acid bacteria company, was selected as an individually recognized functional raw material for improving vaginal health. 14) and Lactobacillus rhamnosus GR-1® (LR GR-1) were also used.

division Mixed weight ratio of mixed strains LCR01 LF05 LP YC-225 LP01 LP YC-178 LA02 LRT RC-14 LR GR-1 Example 1 One One One - - - - - Comparative Example 1 One One - One - - - - Comparative Example 2 One One - - One - - - Comparative Example 3 One One - - - One - - Comparative Example 4 - One One One - - - - Comparative Example 5 - - One One One - - - Comparative Example 6 - - - One One One - - Comparative Example 7 1.5 1.5 - - - - - - Comparative Example 8 - 1.5 1.5 - - - - - Comparative Example 9 - - 1.5 1.5 - - - - Comparative Example 10 - - - 1.5 1.5 - - - Comparative Example 11
(Urex) - - - - - - 1.5 1.5

Experimental Example 1. Mixed strain (InoRexyne ^TM ) Confirmed antibacterial activity of vaginitis-causing bacteria

Pathogenic microorganisms that cause vaginitis, such as Gardnerella vaginalis KCTC5097 or KCTC5096 or Candida albicans KCTC7122, were grown anaerobically in 5 ml of Reinforced Clostridium Medium (Difco) or 5 ml of YPD medium. The cells were pre-cultured in an incubator at 37°C. The culture was terminated when the absorbance of the culture medium was measured at 0.8 at 660 nm. 1% of the pathogenic microorganism pre-culture was inoculated into the MRS liquid medium, and the main culture was prepared by shaking and culturing at 37°C for 3 hours.

2 ml of the mixed strain composition prepared in Example 1 and Comparative Examples 1 to 11 was added to each of the above pathogenic microorganism main cultures and cultured for 48 hours under anaerobic conditions, and then the number of viable pathogenic microorganisms was measured to determine the degree of antibacterial activity. was confirmed. At this time, the antibacterial activity was analyzed using Equation 1 below, and the results are shown in Table 2. The control group is the number of viable pathogenic microorganisms in a culture medium cultured in the same manner by adding 2 ml of MRS liquid medium instead of the sample. Antibacterial activity in the experiment refers to the amount (%) of pathogenic microorganisms killed compared to the control group.

[Equation 1]

Antibacterial activity (%) = (1-(A/B))×100

In Equation 1, A is the number of viable pathogenic microorganisms in the experimental group or comparison group, and B is the number of viable pathogenic microorganisms in the control group.

division G. Vaginalis
KCTC5096 G. Vaginalis
KCTC5097 C. albicans KCTC7122 Antibacterial activity (%) Antibacterial activity (%) Antibacterial activity (%) experimental group 87.84 ± 24.30* 50.46 ± 7.79* 99.05 ± 0.21* Comparison group 1 23.91 ± 2.79 11.50 ± 3.07 83.22 ± 4.92 Comparison group 2 23.16 ± 1.60 12.99 ± 3.03 81.60 ± 2.95 Comparison group 3 23.97 ± 5.81 14.96 ± 1.91 79.27 ± 4.48 Comparison group 4 27.84 ± 4.30 10.46 ± 7.79 77.14 ± 4.71 Comparison group 5 16.72 ± 3.49 10.56 ± 1.47 79.39 ± 1.28 Comparison group 6 19.49 ± 5.01 9.33 ± 0.55 82.60 ± 3.51 Comparison group 7 18.12 ± 2.11 18.96 ± 1.51 83.68 ± 6.29 Comparison group 8 20.30 ± 1.83 18.02 ± 2.47 81.48 ± 4.64 Comparison group 9 17.71 ± 2.52 15.01 ± 2.90 77.82 ± 1.71 Comparison group 10 21.50 ± 2.25 18.30 ± 2.93 81.73 ± 1.75 Comparison group 11
(Urex) 42.99 ± 9.38 33.97 ± 5.85 86.03 ± 1.82

* Student T-test P < 0.05 compared to Urex

As shown in Table 2, comparative analysis of antibacterial activity against vaginitis-causing strains ( Gadnerella vaginalis KCTC5097, Gadnerella vaginalis KCTC5096, and Candida albicans KCTC7122) As a result, among the mixed strains , Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus fermentum LF05 (DSM 32277), and Lactobacillus illus plantarum YC-225 ( It was confirmed that the mixed strain composition (InoRexyne ^TM ) consisting of KCTC19068P) had the highest antibacterial activity. In particular, a mixed strain of Lactobacillus rhamnosus GR-1® and Lactobacillus reuteri RC-14®, which are UREX probiotics (No. 2014-27), which are individually approved functional ingredients related to improving vaginal health. It was confirmed that it exhibited significantly higher antibacterial activity (P < 0.05).

Experimental Example 2. Confirmation of cytotoxicity

Using RAW 264.7 cells, which are mouse macrophages, it was attempted to determine whether the mixed strain composition (experimental group) according to the present invention exhibits toxicity to cells. First, RAW 264.7 cells are seeded in a 96 well plate at a concentration of 2×10 ⁴ cells/well and cultured at 5% CO ₂ and 37°C for 24 hours.

The mixed strain compositions prepared from Example 1 and Comparative Examples 1 to 11 were diluted to concentrations of 0.005X and 0.01X, and then 20 μL of them were treated in each well.

After 24 hours, 10 μL of WST-1 reagent (Daeil Lab Service, Seoul, South Korea) was added to each well, and 2 hours later, 450 nm was measured using a VERSAmax microplate spectrometer (VERSAmax, Molecular Devices, Sunnyvale, CA). The absorbance was measured. Cell viability was confirmed using the following equation.

[Equation 2]

Cell viability (%) = (OD ₄₅₀ (experimental group) - OD ₄₅₀ (blank))/(OD ₄₅₀ (control group) - OD ₄₅₀ (blank)) × 100

At this time, the blank is only medium used, and the control is RAW 264.7 cells treated with μL of liquid medium instead of the sample.

Figure 1 is a graph confirming cytotoxicity by treating RAW 264.7 cells with mixed strain compositions prepared from Example 1 and Comparative Examples 1 to 11 diluted to 0.005X concentration, and Figure 2 is Example 1 diluted to 0.01X concentration. , This is a graph confirming cytotoxicity by treating RAW 264.7 cells with the mixed strain composition prepared from Comparative Examples 1 to 11. In the drawing, 'a-f' indicates significance analyzed through Duncan's multiple range test.

As shown in Figures 1 and 2, the mixed strain composition prepared in Example 1 was confirmed to be a safe composition that is not toxic to RAW 264.7 cells. On the other hand, it was confirmed that the mixed strain composition prepared in Comparative Example 6 and the mixed strain composition prepared in Comparative Example 11 (UREX) exhibited cell toxicity at the same concentration.

Experimental Example 3. Confirmation of anti-inflammatory effect

To analyze the anti-inflammatory effect, NO production ability was analyzed. RAW 264.7 cells, which are mouse macrophages, were distributed in a 24-well plate at 5×10 ⁵ cells/well and then cultured at 5% CO ₂ and 37°C for 24 hours. The mixed strain compositions prepared from Example 1 and Comparative Examples 1 to 11, in which 24-hour culture was completed, were diluted to a concentration of 0.005X or 0.01X, and then 20 μL was applied to each well. After culturing for 24 hours, the cells were treated with 1 μg/mL of LPS (Lipopolysaccharide) and cultured for an additional 24 hours. The nitric oxide (NO) production activity of the culture medium obtained after cultivation was measured by absorbance at 540 nm using a Nitric Oxide (NO) detection kit (iNtRON).

A control group with no treatment (No treatment) and a negative control group with only LPS treatment (LPS) were prepared and prepared in the same manner as above except for the samples. MRS is obtained by adding only strain-free MRS culture medium to the negative control group (LPS).

Figure 3 is a diagram showing the results of confirming the effect of inhibiting nitric oxide (NO) production of the mixed strain composition prepared from Example 1 and Comparative Examples 1 to 11 diluted to a concentration of 0.005 This figure shows the results of confirming the effect of inhibiting nitric oxide (NO) production of the mixed strain composition prepared from Example 1 and Comparative Examples 1 to 11. In the figure, 'a-f' indicates significance analyzed through Duncan's multiple range test.

As shown in Figures 3 and 4, it was confirmed that the effect of inhibiting nitric oxide production in the group treated with the mixed strain composition prepared in Example 1 was significantly superior. In addition, the mixed strain composition composed of other lactic acid bacteria (Comparative Examples 1-11, No. 11 is Urex) has little effect in suppressing NO production, whereas the mixed strain composition of the present invention (Example 1) inhibits NO production and has excellent anti-inflammatory properties. It was confirmed to be active (NO production inhibition efficacy of more than 50%). In other words, it can be seen that the mixed strain according to the present invention has an anti-inflammatory prevention and treatment effect against inflammatory substances (LPS) secreted from pathogenic microorganisms. Unlike other mixed strains, the NU probiotic mixed strain according to the present invention has vaginitis This means that it can be usefully used for the prevention or treatment of.

Experimental Example 4. Vaginitis treatment effect of mixed strain composition (InoRexyne ^TM ) in animal model

Preparation of vaginitis animal model

C57BL/6 female mice (5 weeks old, weighing 19-22g) were purchased from Orient Bio (Seongnam, Korea), acclimatized for a week in a breeding farm, and then used in experiments. Animal rearing was conducted under conditions of temperature 21±1℃, humidity 50±10%, light/dark cycle (12 hours day/12 hours night), and illumination intensity of 300 Lux, and feed and water were provided ad libitum (No. EEGJ36060, Purina, Seongnam, Korea).

The experiment was conducted by randomly distributing adaptively bred C57BL/6 female mice into 5 groups (n=6/group). 0.5 mg of β-estradiol-3-benoate (Sigma) dissolved in 100 ml of olive oil was subcutaneously injected into the mouse. Three days later, Candida albicans ( Candida albicans ) KCTC7122) (2 × 10 ⁷ CFU/mouse) was implanted into the vagina of mice to prepare a vaginitis animal model.

Experimental group preparation

^NU probiotic mixed strain composition of Example 1 (LOW: total ¹ /mouse) ( ^High : total ⁵ The experiment was performed by sacrificing the animal model 24 hours after the final administration of lactic acid bacteria on the 14th day (16th day after transplantation).

Each experimental group was given the following name.

Normal control group (Nor): Normal animal model without any treatment

Negative control group (CA): Animal model in which vaginitis was induced by ^vaginal administration of Candida albicans (CA) KCTC7122 2

Low experimental group: CA-induced vaginitis animal model in which NU probiotic mixed strain composition (Example 1) was orally administered once daily for 2 weeks at a total concentration of 1 × 10 ⁹ CFU/mouse.

Mid experimental group: CA-induced vaginitis animal model administered orally once daily for 2 weeks with a probiotic mixed strain composition (InoRexyne ^TM ) (Example 1) at a total concentration of 2 × 10 ⁹ CFU/mouse.

High experimental group: CA-induced vaginitis animal model administered orally once daily for 2 weeks with a probiotic mixed strain composition (InoRexyne ^TM ) (Example 1) at a total concentration of 5 × 10 ⁹ CFU/mouse.

Positive control (PC): CA-induced vaginitis animal model in which the Urex composition (Example 1) was orally administered once daily for 2 weeks at a total concentration of 1 × 10 ⁹ CFU/mouse instead of the mixed strain composition.

Check your weight change

Before sacrifice, the body weight of each animal model was measured and compared.

Figure 5 is a graph showing the body weight measured from the normal control group (Nor), negative control group (CA), low experimental group, mid experimental group, high experimental group, and positive control group (PC). According to this, all animal models showed significant differences in body weight. Not observed.

Check whether inflammatory factors occur in the vagina

Concentrations of inflammatory factors (NO, IL-6) were analyzed from the vaginal tissues of the sacrificed animal models (normal control group (Nor), negative control group (CA), low experimental group, mid experimental group, high experimental group, positive control group (PC)). . The content of inflammatory cytokines (IL-6, NO) in the solvent of vaginal tissue from each group was measured using an ELISA kit.

Figure 6(a) is a graph showing the NO concentration measured in vaginal tissue separated from the normal control group (Nor), negative control group (CA), low experimental group, mid experimental group, high experimental group, and positive control group (PC). (b) is a graph showing the concentration of IL-6 measured in vaginal tissue isolated from the normal control group (Nor), negative control group (CA), low experimental group, mid experimental group, high experimental group, and positive control group (PC). * P < 0.05, ** P < 0.01, *** P < 0.001 Student t-test.

As shown in Figure 6, the negative control group (CA) injected with the vaginitis-causing bacteria was confirmed to have a significant increase in the concentration of inflammatory factors (NO, IL-6) compared to the normal control group (Nor). In other words, it can be seen that inflammation within vaginal tissue increases due to the bacteria that cause vaginitis.

In the Low experimental group, Mid experimental group, and High experimental group, the concentration of inflammatory factors (NO, IL-6) was significantly decreased compared to the negative control group (CA) (P < 0.05). Through this, it was confirmed that the mixed strain composition of Example 1 reduced the level of inflammation increased by vaginitis-causing bacteria to a normal level regardless of concentration.

Check the number of vaginitis-causing bacteria in vaginal tissue

The presence and number of viable bacteria in the vaginal tissues of the sacrificed animal models (normal control group (Nor), negative control group (CA), low experimental group, mid experimental group, high experimental group, positive control group (PC)) were analyzed. . The degree of attachment of Candida albicans (CA) in the vaginal tissue isolated from each group was analyzed by PAS (Periodic Acid-Schiff) staining, and the presence of Candida albicans (CA) in the vaginal wash obtained from each group was analyzed. ), the number of viable bacteria of Candida albicans (CA) was quantified by qPCR analysis using primers for a specific sequence.

16s rRNA

Forward: ACTCCTACGGGAGGCAG (SEQ ID 2)

Reverse: ATTACCGCGGCTGCTGG (SEQ ID NO: 3)

Figure 7(a) shows the results of Gram staining for diagnosing vaginitis on vaginal tissue isolated from the normal control group (Nor), negative control group (CA), low experimental group, mid experimental group, high experimental group, and positive control group (PC). 7(b) shows the vaginitis-causing bacteria ( Gardnerella vaginalis ) present in vaginal tissues isolated from the normal control group (Nor), negative control group (CA), low experimental group, mid experimental group, high experimental group, and positive control group (PC). ) This is a graph showing the number of viable bacteria (CFU/ml) measured by KCTC5097). **** P < 0.05 Student t-test

As shown in Figure 7, the negative control group (CA) injected with the vaginitis-causing bacteria was confirmed to have a significant increase in the live bacterial count of the vaginitis-causing bacteria ( Gardnerella vaginalis KCTC5097) compared to the normal control group (Nor). In other words, it can be seen that the vaginitis-causing bacteria ( Gardnerella vaginalis KCTC5097) significantly proliferate within the vaginal tissue due to the injection of the vaginitis-causing bacteria.

The number of vaginitis-causing bacteria in the Low experimental group, Mid experimental group, and High experimental group was significantly reduced compared to the negative control group (CA) (P < 0.05). Through this, it was confirmed that the mixed strain composition of Example 1 can not only prevent vaginitis by inhibiting the growth of vaginitis-causing bacteria, but also improve and treat vaginitis by removing proliferated vaginitis-causing bacteria.

Check vaginal tissue condition

Characteristic changes in lesions such as vaginal tissue thickness and vaginal detachment were confirmed in the sacrificed animal models (normal control group (Nor), negative control group (CA), low experimental group, mid experimental group, high experimental group, positive control group (PC)). I wanted to do it. Generally, superficial symptoms of vaginitis include vaginal erythema, congestion, and epidermal detachment, and the progress of the disease can be confirmed through the thickness and degree of detachment of vaginal tissue.

First, the vaginal tissue isolated from each group was washed with PBS and fixed in 10% neutral buffered formalin for 24 hours at room temperature. The tissue was dehydrated in alcohol and xylene and then placed in paraffin. Paraffin-embedded specimens were cut, stained with hematoxylin & eosin (H&E), and observed under a digital light microscope.

Based on optical photographs stained with H&E, the degree of keratinization of vaginal tissue was calculated according to the severity of keratin described in PASI (Psoriasis Area and Severity Index) (0 to 4).

Figure 8(a) shows the results of staining with H&E to confirm the thickness of vaginal tissue separated from the normal control group (Nor), negative control group (CA), low experimental group, mid experimental group, high experimental group, and positive control group (PC), Figure 8(b) is a graph showing the exfoliation scores of vaginal tissues separated from the normal control group (Nor), negative control group (CA), low experimental group, mid experimental group, high experimental group, and positive control group (PC). am. * P < 0.05, ** P < 0.01, *** P < 0.001 Student t-test.

As shown in Figure 8, the negative control group (CA) injected with the vaginitis-causing bacteria was confirmed to have a significant increase in the thickness of vaginal tissue compared to the normal control group (Nor). In other words, it can be seen that infection with the causative bacteria of vaginitis ( Gardnerella vaginalis KCTC5097) causes inflammation in the vagina, and the thickness of the subepithelial tissue thickens due to tissue edema and infiltration of inflammatory cells.

In the Low experimental group, Mid experimental group, and High experimental group, the thickness and peeling of vaginal tissue were significantly reduced compared to the negative control group (CA) (P < 0.05). Through this, it can be confirmed that the mixed strain composition of Example 1 has antibacterial activity against vaginitis-causing bacteria and inhibits their growth and proliferation, so that the symptoms of increased thickness and peeling of vaginal tissue, which are pathological findings, are restored to normal levels. . That is, it can be seen that the mixed strain composition according to the present invention (Example 1) has excellent preventive, improvement, or treatment efficacy against the etiology of vaginitis.

Confirmation of tight junction-related gene expression in vaginal tissue

ZO-1, a tight junction protein, in vaginal tissue of the sacrificed animal models (normal control group (Nor), negative control group (CA), low experimental group, mid experimental group, high experimental group, positive control group (PC)) and FOXA1 expression levels were measured by immunoblotting. The outermost epidermal layer of vaginal tissue forms a physical barrier through tight junctions, which prevents the invasion of foreign substances. In particular, it performs the function of preventing liquid from moving through the space between cells. When the barrier is weakened, it cannot protect against external substances or stimuli, causing inflammatory or immune reactions.

Vaginal tissue was isolated from each group. Total RNA extraction was performed from isolated vaginal tissue. Specifically, the RNeasy mini kit (Qiagen, USA) was used and performed according to the manufacturer's manual. The absorbance of extracted total RNA was measured at 260 nm and 280 nm using NanoQuant PlateTM. The quantified RNA was used for quantitative real-time PCR. Quantitative real-time PCR was performed using an ABI prism 7500 cycler (Applied Biosystem USA). The final volume of the PCR mixture was 15 ㎕ by adding 10 ㎕ of Power SYBRⓡ Green PCR Master Mix (Applied Biosystem, USA), 1 ㎕ of forward primer, 1 ㎕ of reverse primer, and 1 ㎕ of distilled water. 15 ㎕ of PCR mixture and 5 cDNA. ㎕ was added to a 96 well optical reaction plate, making the final volume of the PCR reaction 20 ㎕. The plate was covered with optical adhesive covers (Applied Biosystem, USA) and PCR reaction was performed using a real-time PCR machine (Applied Biosystem, USA). The reaction conditions were the first step at 50°C for 2 minutes and 95°C for 10 minutes, and the second step at 95°C for 15 seconds, 58°C for 30 seconds, and 72°C for 33 seconds, repeated 40 times. . The results were analyzed and quantified using Microsoft Excel, and the comparative Ct (threshold cycle) method was used as the analysis method.

Figure 9 is a graph measuring the expression level of ZO-1 protein in vaginal tissues isolated from the normal control group (Nor), negative control group (CA), low experimental group, mid experimental group, high experimental group, and positive control group (PC). * P < 0.05, ** P < 0.01, *** P < 0.001 Student t-test.

As shown in Figure 9, the negative control group (CA) injected with the vaginitis-causing bacteria was confirmed to have a significant decrease in the expression level of ZO-1, a tight junction protein, compared to the normal control group (Nor). In other words, it can be confirmed that tight junctions between cells in the vaginal mucosa layer are weakened by infection with the vaginitis-causing bacteria ( Gardnerella vaginalis KCTC5097).

On the other hand, the expression level of tight junction-related proteins between cells in the vaginal mucosa layer in the Low, Mid, and High experimental groups significantly increased compared to the negative control group (CA) (P < 0.05). Through this, it can be confirmed that the mixed strain composition of Example 1 not only has antibacterial activity against bacteria causing vaginitis, but also restores the tight junction structure between cells in the vaginal mucosa layer to the normal level.

In particular, the mixed strain composition of Example 1 is most effective when administered at 2 × 10 ⁹ CFU/mouse, and the mixed strain composition according to the present invention (Example 1) can prevent, improve, or treat infection by pathogenic microorganisms. In addition, it can be seen that it has the effect of strengthening the tight junction structure between vaginal tissue cells.

Through the above experiments, Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus fermentum LF05 (DSM 32277) and Lactobacillus plantarum YC- according to the present invention. InoRexyne ^TM , composed of 225 (KCTC19068P), can be used as a freeze-dried powder for culture media excluding strains as well as live bacteria. It is economical due to its excellent productivity and has a remarkable anti-inflammatory effect. It is used as a probiotic for intestinal health and vaginal health. It was confirmed that it can be used in a variety of anti-inflammatory compositions as a probiotic.

Formulation Example 1: Preparation of pharmaceutical composition

In the manufacture of the pharmaceutical composition below, the mixed strain composition includes Lactobacillus crispatus LCR01 (DSM 24619) strain culture, Lactobacillus fermentum LF05 (DSM 32277) strain culture, and Lactobacillus plantarum. (Lactobac illus plantarum) YC-225 (KCTC19068P) strain cultures were mixed at a cell number ratio of 1:0.5-5:0.5-5.

Formulation Example 1-1: Preparation of powder

Mixed strain composition 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients were mixed and filled into an airtight bubble to prepare a powder.

Formulation Example 1-2: Preparation of tablets

Mixed strain composition 10 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above ingredients, tablets were manufactured by tableting according to a conventional tablet manufacturing method.

Formulation Example 1-3: Preparation of capsules

Mixed strain composition 10 mg

Crystalline cellulose 3 mg

Lactose 15 mg

Magnesium stearate 0.2 mg

After mixing the above ingredients, a capsule was prepared by filling a gelatin capsule according to a typical capsule manufacturing method.

Formulation Example 1-4: Preparation of pills

Mixed strain composition 10 mg

Lactose 150 mg

Glycerin 100 mg

Xylitol 50 mg

After mixing the above ingredients, it was prepared in an amount of 4 g per ring according to a conventional method.

Formulation Example 1-5: Preparation of granules

Mixed strain composition 15 mg

Soybean extract 50 mg

Dextrose 200 mg

Starch 600 mg

After mixing the above ingredients, 100 mg of 30% ethanol was added and dried at 60°C to form granules, which were then filled into bags.

Formulation Example 1-6: Preparation of injection

Mixed strain composition 10 mg

Sodium metabisulfite 3.0 mg

Methylparaben 0.8 mg

Propylparaben 0.1 mg

Proper amount of sterilized distilled water for injection

After mixing the above ingredients, 2 ml of them was filled into an ampoule and sterilized to prepare an injection.

Formulation Example 2: Preparation of food composition

In the production of the following food composition, the mixed strain composition includes Lactobacillus crispatus LCR01 (DSM 24619) strain culture, Lactobacillus fermentum LF05 (DSM 32277) strain culture, and Lactobacillus plantarum. (Lactobac illus plantarum) YC-225 (KCTC19068P) strain cultures were mixed at a cell number ratio of 1:0.5-5:0.5-5.

Formulation Example 2-1: Production of flour food

0.5 parts by weight of the mixed strain composition was added to 100 parts by weight of flour, and bread, cakes, cookies, crackers, and noodles were manufactured using this mixture.

Formulation Example 2-2: Manufacturing of dairy products

0.5 parts by weight of the mixed strain composition was added to 100 parts by weight of milk, and various dairy products such as butter and ice cream were manufactured using the milk.

Formulation Example 2-3: Preparation of Sunsik

Brown rice, barley, glutinous rice, and coix seed were gelatinized and dried using a known method, roasted, and then made into powder with a particle size of 60 mesh using a grinder.

Black beans, black sesame seeds, and perilla seeds were steamed and dried using a known method, roasted, and then made into powder with a particle size of 60 mesh using a grinder.

The grains, seeds, and mixed strain composition prepared above were mixed in the following ratio.

Grains (30 parts by weight of brown rice, 17 parts by weight of coix radish, 20 parts by weight of barley),

Seeds (7 parts by weight perilla seeds, 8 parts by weight black beans, 7 parts by weight black sesame seeds),

Mixed strain composition (1 part by weight),

Reishi (0.5 parts by weight),

Rehmannia glutinosa (0.5 parts by weight).

Formulation Example 2-4: Preparation of health drink

Mix 1 g of the mixed strain composition with auxiliary ingredients such as high fructose corn syrup (0.5 g), oligosaccharide (4 g), sugar (2 g), table salt (0.5 g), and water (77 g) homogeneously, flash sterilize it, and then sterilize it. It was manufactured by packaging it in small containers such as glass bottles and PET bottles.

Formulation Example 2-5: Preparation of vegetable juice

Vegetable juice was prepared by adding 2 g of the mixed strain composition to 1,000 ml of tomato or carrot juice.

Formulation Example 2-6: Preparation of fruit juice

Fruit juice was prepared by adding 1 g of the mixed strain composition to 1,000 ml of apple or grape juice.

Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC19068P 20230102

Sequence list electronic file attached

Claims (6)

A mixed strain consisting of Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus fermentum LF05 (DSM 32277), and Lactobacillus plantarum YC-225 (KCTC19068P) A food composition for preventing or improving vaginitis containing the food composition. According to paragraph 1,
The Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus fermentum LF05 (DSM 32277), and Lactobacillus plantarum YC-225 (KCTC19068P) are 1:0.5. A food composition for preventing or improving vaginitis, characterized in that it is mixed at a cell number ratio of -5:0.5-5. According to paragraph 1,
The composition is a food composition for preventing or improving vaginitis, characterized in that it has antibacterial activity against one or more vaginitis-causing bacteria selected from the group consisting of Gardnerella vaginalis and Candida albicans. Vaginitis prevention or treatment comprising a mixed strain consisting of Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus plantarum LF05, and Lactobacillus illus plantarum YC-225 KCTC19068P For pharmaceutical compositions. According to clause 4,
A pharmaceutical composition for preventing or treating vaginitis, wherein the vaginitis is at least one selected from the group consisting of bacterial vaginitis and candida vaginitis. Prevention of vaginitis comprising a mixed strain consisting of Lactobacillus crispatus LCR01 (DSM 24619), Lactobacillus plantarum LF05 and Lactobacillus illus plantarum YC-225 KCTC19068P or Cosmetic composition for improvement.