KR20240081161A - Novel strains having antibacterial activity against candida albicans and their uses - Google Patents

Abstract

The present invention relates to strains that inhibit the growth and activity of Candida albicans, which causes vaginitis, specifically Clostridium , Enterococcus , Streptococcus , It relates to a strain of the genus Bacteroides , and includes at least one selected from the group consisting of the strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, an extract of the culture, and dead cells of the strain. It relates to a pharmaceutical composition for preventing or treating vaginitis containing it as an active ingredient, and a health functional food composition for preventing or improving vaginitis.

Description

Novel strain with antibacterial activity against Candida albicans and its use {NOVEL STRAINS HAVING ANTIBACTERIAL ACTIVITY AGAINST CANDIDA ALBICANS AND THEIR USES}

The present invention relates to strains that inhibit the growth and activity of Candida albicans, which causes vaginitis, specifically Clostridium , Enterococcus , Streptococcus , It relates to a strain of the genus Bacteroides , and includes at least one selected from the group consisting of the strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, an extract of the culture, and dead cells of the strain. It relates to a pharmaceutical composition for preventing or treating vaginitis containing it as an active ingredient, and a health functional food composition for preventing or improving vaginitis.

The vaginal acidity of healthy women is pH 3.8 to 4.5. The useful lactic acid bacteria in the vagina maintain the pH acidic and protect the inside of the vagina from external infectious agents. Lactobacillus crispatus , Lactobacillus jensenii , Lactobacillus gasseri , and Lactobacillus coleohominis are known as useful vaginal lactic acid bacteria.

When changes occur in the vaginal flora, the pH increases and the probability of vaginitis infection increases. When vaginitis is infected, antibiotics are mainly used. Treatments for bacterial vaginosis include metronidazole, tinidazole, and clindamycin, and miconazole and terconazole are used for candidal vaginitis. Although they treat vaginitis, they cause antibiotic-resistant bacteria and at the same time affect useful lactic acid bacteria in the vagina. It's crazy.

Accordingly, interest in plant-based natural products with fewer side effects has increased, and many studies on antibacterial activity have been reported. Natural plant extracts prevent and treat diseases by activating phagocytosis within the human body and increasing immunity by promoting the production of antibodies.

Vaginitis is a disease that women get as often as a cold, and is one of the diseases that occurs when the immune system is weakened. Additionally, because it is a disease with a high recurrence rate, women are always exposed to the risk of vaginitis. Vaginitis is largely divided into bacterial vaginosis, candida vaginitis, and trichomonas vaginitis (Van 2010). Bacteria that cause bacterial vaginosis attach to vaginal epithelial cells and form a biofilm, and Candida albicans, which causes candida vaginitis, form biofilms in vaginal epithelial cells and infect the host.

Republic of Korea Patent No. 10-2053744

The present invention relates to Clostridium butyricum BM-02 strain (KCTC 14663BP), Enterococcus faecium LCM001 strain (KCTC 14669BP), and Enterococcus lati ( Enterococcus ratti ) BM-05 strain (KCTC 14665BP), Streptococcus hyointestinalis BM-08 strain (KCTC 14668BP), or Bacteroides suis BM-01 strain (KCTC 14662BP). We would like to provide

In another embodiment of the present invention, the active ingredient includes at least one selected from the group consisting of the strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, an extract of the culture, and dead cells of the strain. The aim is to provide pharmaceutical compositions, food compositions, and health functional food compositions for preventing, improving, or treating vaginitis.

However, the problem to be solved by the present application is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

In order to achieve the above object, the present invention provides Clostridium butyricum BM-02 strain, which is deposited under the accession number KCTC 14663BP and has antibacterial activity against vaginitis-causing bacteria.

In another embodiment, the present invention provides the Enterococcus faecium LCM001 strain, which is deposited under the accession number KCTC 14669BP and has antibacterial activity against vaginitis-causing bacteria.

In another embodiment, the present invention provides Enterococcus ratti BM-05 strain, which is deposited under the accession number KCTC 14665BP and has antibacterial activity against vaginitis-causing bacteria.

In another embodiment, the present invention provides a strain of Streptococcus hyointestinalis BM-08, which is deposited under the accession number KCTC 14668BP and has antibacterial activity against bacteria that cause vaginitis.

In another embodiment, the present invention provides the Bacteroides suis BM-01 strain, which is deposited under the accession number KCTC 14662BP and has antibacterial activity against vaginitis-causing bacteria.

The bacteria causing vaginitis is Candida albicans.

In another embodiment of the present invention, the active ingredient includes at least one selected from the group consisting of the strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, an extract of the culture, and dead cells of the strain. Provides pharmaceutical compositions, food compositions, and health functional food compositions for alleviating or treating vaginitis.

Therefore, the strain of the present invention having the above effects can be usefully used as a composition for preventing, improving, or treating vaginitis by inhibiting the proliferation and activity of Candida albicans, which causes vaginitis.

The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

Figure 1 is a graph showing the inhibition rate of Candida albicans proliferation of Clostridium butyricum BM-02 strain (KCTC 14663BP).
Figure 2 is a graph showing the inhibition rate of Candida albicans proliferation of Enterococcus faecium LCM001 strain (KCTC 14669BP).
Figure 3 is a graph showing the inhibition rate of Candida albicans proliferation of Enterococcus ratti BM-05 strain (KCTC 14665BP).
Figure 4 is a graph showing the inhibition rate of Candida albicans proliferation of Streptococcus hyointestinalis BM-08 strain (KCTC 14668BP).
Figure 5 is a graph showing the inhibition rate of Candida albicans proliferation of Bacteroides suis BM-01 strain (KCTC 14662BP).

Specific structural and functional descriptions of the embodiments of the present invention disclosed in the present specification or application are merely illustrative for the purpose of explaining the embodiments according to the present invention, and the embodiments according to the present invention may be implemented in various forms. and should not be construed as limited to the embodiments described in this specification or application.

Since the embodiments according to the present invention can make various changes and have various forms, specific embodiments will be illustrated in the drawings and described in detail in the specification or application. However, this is not intended to limit the embodiments according to the concept of the present invention to a specific disclosed form, and should be understood to include all changes, equivalents, and substitutes included in the spirit and technical scope of the present invention.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the technical field to which the present invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and unless clearly defined in this specification, should not be interpreted as having an ideal or excessively formal meaning. .

The present invention relates to Clostridium butyricum BM-02 strain (KCTC 14663BP), Enterococcus faecium LCM001 strain (KCTC 14669BP), and Enterococcus lati ( Enterococcus ratti ) BM-05 strain (KCTC 14665BP), Streptococcus hyointestinalis BM-08 strain (KCTC 14668BP), or Bacteroides suis BM-01 strain (KCTC 14662BP). to provide.

The present inventors identified and identified microorganisms that have a relieving effect on vaginitis, including Clostridium butyricum BM-02 strain (KCTC 14663BP) and Enterococcus faecium LCM001 strain (KCTC 14669BP). , Enterococcus ratti BM-05 strain (KCTC 14665BP), Streptococcus hyointestinalis BM-08 strain (KCTC 14668BP), or Bacteroides suis BM-01 strain. (KCTC 14662BP), it was discovered that the culture of the strain, the concentrate of the culture, the dried product of the culture, the extract of the culture, and the dead cells of the strain were effective in preventing, improving, or treating vaginitis.

The bacterium that causes vaginitis is Candida albicans, and the strains exhibit an effect of inhibiting the growth of Candida albicans, that is, an antibacterial effect.

Accordingly, the present inventors analyzed the 16s rRNA base sequence of the novel strain, analyzed the genetic phylogenetic tree, and sent the strains to the Biological Resource Center (KCTC).

Clostridium butyricum BM-02 strain was deposited with the deposit number KCTC 14663BP on August 23, 2021.

Enterococcus faecium LCM001 strain was deposited with deposit number KCTC 14669BP on August 23, 2021.

Enterococcus ratti BM-05 strain was deposited on August 23, 2021 with the deposit number KCTC 14665BP.

Streptococcus hyointestinalis BM-08 strain was deposited on August 23, 2021 with deposit number KCTC 14668BP.

Bacteroides suis BM-01 strain was deposited on August 23, 2021 with deposit number KCTC 14662BP.

The term "culture" of the present invention includes the strain, fungal extract, its metabolites, extra nutrients, etc. obtained by culturing the strain for a certain period of time in a medium that can supply nutrients for the strain to grow and survive. It refers to the entire medium, but also includes the culture medium from which the strain is removed after culturing the strain.

The culture medium may be left alone for a certain period of time to take only the liquid in the upper layer excluding the part that has settled in the lower layer, the bacteria may be removed through filtration, or the culture medium may be centrifuged to remove sediment in the lower layer and only the liquid in the upper layer. This culture medium can also be concentrated by a conventional method and used as a concentrate.

The culture medium or concentrate of the culture medium can be dried in a conventional manner and provided as a dried product.

Unless otherwise specified, the term “culture” referred to herein includes the culture medium of the above-mentioned bacterial cells, the concentrate of the culture medium, and the dried product of the culture medium or concentrate. The culture may or may not contain bacteria depending on the case, and it does not matter whether bacteria are included or not.

The culture is preferably obtained by inoculating the strain in sterilized MRS broth medium and culturing for more than 12 hours.

The dead cells are prepared by 1) cultivating the strain; 2) autoclaving the culture medium; 3) It is characterized in that it is manufactured by a method comprising the steps of concentrating, drying, and powdering using a membrane filtration concentrator.

In the culturing step, it is preferable to inoculate the strain into sterilized MRS broth medium and culture for at least 12 hours.

The culture medium is preferably autoclaved at 100 to 121°C for 30 to 60 minutes.

The membrane filtration concentrator may have a filtration membrane size of 0.1 to 1㎛, but is not limited thereto.

The ^dead cell powder may have ^a concentration of 4.0

In another embodiment of the present invention, the active ingredient includes at least one selected from the group consisting of the strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, an extract of the culture, and dead cells of the strain. Provided is a pharmaceutical composition for preventing or treating vaginitis.

In another embodiment of the present invention, the active ingredient includes at least one selected from the group consisting of the strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, an extract of the culture, and dead cells of the strain. Provided is a food composition for preventing or improving vaginitis.

In another embodiment of the present invention, the active ingredient includes at least one selected from the group consisting of the strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, an extract of the culture, and dead cells of the strain. Provides a health functional food composition for preventing or improving vaginitis.

The vaginitis is characterized as being caused by Candida albicans.

The pharmaceutical composition for preventing or treating vaginitis is the main ingredient of the strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, an extract of the culture, and at least one effective agent selected from the group consisting of dead cells of the strain. The composition can be prepared in a conventional dosage form by selecting an effective amount of ingredients and one or two or more conventional pharmaceutically acceptable carriers or one or two or more additives.

The carrier can be one or two or more selected from diluents, lubricants, binders, disintegrants, sweeteners, stabilizers and preservatives, and the additives can be one or two or more selected from flavorings, vitamins and antioxidants. You can use it.

In the present invention, any pharmaceutically acceptable carrier and additive can be used. Specifically, diluents include lactose monohydrate, trehalose, cornstarch, and soybean oil. , microcrystalline cellulose or mannitol (D-mannitol) is preferred, magnesium stearate or talc is preferred as a lubricant, and polyvinyl pyrolidone (PVP) or talc is preferred as a binder. It is preferable to choose among hydroxypropylcellulose (HPC). In addition, the disintegrant is preferably selected from carboxymethylcellulose calcium (Ca-CMC), sodium starchglycolate, polacrylin potassium, or cross-linked polyvinylpyrrolidone. , the sweetener is selected from white sugar, fructose, sorbitol, or aspartame, and the stabilizer is carboxymethylcellulosesodium (Na-CMC), beta-cyclodextrin, and white lead. Select from bee's wax or xanthan gum, and preservatives include methyl p-hydroxy benzoate (methylparaben), propylp-hydroxybenzoate (propylparaben), or potassium sorbate. It is preferable to select from among, but is not limited to.

The pharmaceutical composition of the present invention can be administered to a patient in a single dose, or can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. In the above, 'pharmacologically effective amount' refers to an amount that shows a greater response than the negative control, and preferably refers to an amount sufficient to prevent or treat inflammatory diseases. In addition, the pharmaceutically effective amount may vary appropriately depending on various factors such as the disease and its severity, the patient's age, weight, health condition, gender, administration route, and treatment period.

The composition of the present invention can be formulated in various ways depending on the route of administration by methods known in the art along with the pharmaceutically acceptable carrier. As used above, “pharmaceutically acceptable” means a non-toxic composition that is physiologically acceptable and does not inhibit the action of the active ingredient when administered to humans and does not usually cause gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions. says The composition of the present invention can be formulated in various ways depending on the route of administration by methods known in the art along with the pharmaceutically acceptable carrier. The route of administration is not limited to this, but may be administered orally or parenterally.

According to another embodiment of the present invention, at least one selected from the group consisting of the strain, the culture of the strain, the concentrate of the culture, the dried product of the culture, the extract of the culture, and the dead cell body of the strain as an active ingredient. Provided is a food composition for preventing or improving vaginitis, comprising:

According to another embodiment of the present invention, at least one selected from the group consisting of the strain, the culture of the strain, the concentrate of the culture, the dried product of the culture, the extract of the culture, and the dead cell body of the strain as an active ingredient Provides a health functional food composition for preventing or improving vaginitis, comprising:

The food composition or health functional food of the present invention includes beverages containing the food composition (including tea or alcoholic beverages), fruits and processed foods thereof (e.g. canned fruit, bottled food, jam, mamalade, etc.), fish, meat and Processed foods (e.g. ham, sausage, corned beef, etc.), breads and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese) etc.), edible vegetable oil, margarine, vegetable protein, retort food, frozen food, various seasonings (e.g. soybean paste, soy sauce, sauce, etc.).

Additionally, the food composition or health functional food of the present invention may be formulated into tablets, pills, powders, granules, powders, capsules, liquid formulations, etc. They may be formulated further including one or more of carriers, diluents, excipients, and additives.

Specific examples of the carriers, excipients, diluents and additives include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, erythritol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium phosphate, calcium. Silicates, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water, sugar syrup, methylcellulose, methyl hydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate. It is preferred that at least one selected from the group consisting of mineral oil is used.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example. Antibacterial activity test against Candida albicans

1. Reagents and Materials

Candida albicans (ATCC ^® 11006 ^TM ) was purchased from the Korea Center for Microbial Conservation (KCCM, Korea) and cultured using LB Broth Low Salt (MB cell, Korea), Agar, bacteriological (DEAJUNG, Korea). .

2. Candida albicans ( Candida albicans ) Screening for strains with antibacterial activity against

The antibacterial activity test for Candida albicans was measured using the disk diffusion method.

(1) Microbial culture

10ml of LB Broth Low Salt media was used by sterilizing with high-pressure steam at 121℃ for 15min, and a single colony of microorganisms cultured on an LB agar plate was inoculated and cultured in an incubator at 37℃ for 48 hours at 150rpm.

(2) Sample and manufacturing

The selected microorganisms were used by sterilizing 10 ml of LB Broth Low Salt media at 121°C for 15 minutes with high-pressure steam, and a single colony of cultured microorganisms was inoculated on an LB Agar plate and cultured at 150 rpm for 48 hours in a 37°C incubator. The cultured microorganisms and culture medium were centrifuged at 3000 rpm to separate them into microorganisms and cell-free supernatant, and the cell-free supernatant was filtered using a PVDF filter with a pore size of 0.22um.

(3) Disk diffusion test

After preparing YM agar (1.5%) and sterilizing it with high-pressure steam at 121°C for 15 minutes, dispense the sterilized YM agar media into a 100 petri dish and place it on a flat surface to wait until it hardens. Afterwards, YM broth is sterilized by high-pressure steam at 121℃ for 15 minutes to inoculate Candida albicans single colony, and then cultured in a 25℃ incubator at 150rpm for 24 hours.

When the culture is complete, 0.7% agar excluding the media is sterilized by high-pressure steam at 121°C for 15 minutes. Then, 1% of the cultured Candida albicans is inoculated and mixed well with the 0.7% agar. Then, the solidified YM agar (1.5% ) Dispense 3ml on top and spread thinly. When the 0.7% agar containing Candida albicans hardened to a certain extent, 30 ul of the filtered culture solution was dispensed onto a sterilized pater dish and cultured for 72 hours in an incubator at 25°C.

(4) Results

Five strains with antibacterial activity against Candida albicans were selected using the disk diffusion test; Clostridium butyricum BM-02 strain (KCTC 14663BP), Enterococcus faecium LCM001 strain (KCTC 14669BP), Enterococcus ratti BM-05 strain (KCTC 14665BP), Streptococcus hyointestinalis BM-08 strain (KCTC 14668BP) or Bacteroides suis BM-01 strain (KCTC 14662BP).

3. Candida albicans proliferation inhibition rate analysis

In order to analyze the inhibition rate of Candida albicans growth of the five selected strains, the inhibition rate of Candida growth was analyzed by measuring the absorbance at 600 nm using a multi-reader (TECAN, Switzerland).

(1) Microbial culture

10ml of LB Broth Low Salt media was used by sterilizing with high pressure steam at 121°C for 15 minutes, and a single colony of cultured microorganisms was inoculated on an LB Agar plate and cultured in an incubator at 37°C for 48 hours.

(2) Sample and manufacturing

The selected microorganisms were used by sterilizing 10 ml of LB Broth Low Salt media at 121°C for 15 minutes with high-pressure steam, and a single colony of cultured microorganisms was inoculated on an LB Agar plate and cultured at 150 rpm for 48 hours in a 37°C incubator. The cultured microorganisms and culture medium were freeze-dried (-80°C, vacuum) and made into powder.

(3) Absorbance measurement

Candida albicans in progress in cultivation was dispensed equally at 100uL each, and the freeze-dried microbial culture solution was treated with each dispensed Candida albicans culture solution at different concentrations (0.1, 0.25. 0.5, 1. 2 mg/ml) for 48 hours. Culturing in a copper incubator confirms the proliferation and inhibition of Candida albicans. When the 48-hour incubation was completed, the absorbance was immediately measured at 600 nm.

(4) Results

Referring to Figures 1 to 5, it was confirmed that all of the five selected strains inhibited the growth of Candida albicans as the concentration increased, and the five strains were Candida albicans, which causes vaginitis. It was confirmed that it can be usefully used as a composition for preventing, improving, or treating vaginitis by inhibiting the proliferation and activity of .

In this specification, the present invention has been described focusing on limited embodiments, but various embodiments are possible within the spirit and scope of the present invention. Additionally, although not described, equivalent means may also be considered to be incorporated into the present invention. Therefore, the true scope of protection of the present invention should be determined by the following claims.

Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC14663BP 20210823 Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC14662BP 20210823 Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC14665BP 20210823 Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC14668BP 20210823 Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC14669BP 20210823

Claims (8)

Streptococcus hyointestinalis BM-08 strain (KCTC 14668BP), which has antibacterial activity against vaginitis-causing bacteria.
According to paragraph 1,
A strain characterized in that the vaginitis-causing bacteria is Candida albicans.
At least one selected from the group consisting of the strain according to any one of claims 1 or 2, a culture of the strain, a concentrate of the culture, a dried product of the culture, an extract of the culture, and dead cells of the strain. A pharmaceutical composition for preventing or treating vaginitis containing an active ingredient.
According to paragraph 3,
A pharmaceutical composition for preventing or treating vaginitis, wherein the vaginitis is caused by Candida albicans.
At least one selected from the group consisting of the strain according to any one of claims 1 or 2, a culture of the strain, a concentrate of the culture, a dried product of the culture, an extract of the culture, and dead cells of the strain. A food composition for preventing or improving vaginitis containing an active ingredient.
According to paragraph 3,
A food composition for preventing or improving vaginitis, wherein the vaginitis is caused by Candida albicans.
At least one selected from the group consisting of the strain according to any one of claims 1 or 2, a culture of the strain, a concentrate of the culture, a dried product of the culture, an extract of the culture, and dead cells of the strain. A health functional food composition for preventing or improving vaginitis containing as an active ingredient.
In clause 7,
A health functional food composition for preventing or improving vaginitis, characterized in that the vaginitis is caused by Candida albicans.