WO2023075357A1

WO2023075357A1 - Lactobacillus crispatus strain and composition for prevention or treatment of vaginitis comprising same

Info

Publication number: WO2023075357A1
Application number: PCT/KR2022/016340
Authority: WO
Inventors: 이성희; 김영후; 박재완; 은수현; 이동훈; 전우진; 한치영
Original assignee: 일동제약(주)
Priority date: 2021-10-26
Filing date: 2022-10-25
Publication date: 2023-05-04
Also published as: TW202334391A; KR20230059753A

Abstract

The present invention relates to a novel Lactobacillus sp. strain, a culture thereof, and a composition for treating vaginitis, comprising same and, in particular, to a novel Lactobacillus crispatus strain isolated from the vagina of a healthy woman, a culture thereof, and a pharmaceutical composition and a health functional food composition for the prevention or treatment of vaginitis, comprising same. According to the present invention, three types of novel Lactobacillus crispatus strains were isolated on the basis of the ability to use glycogen, which is a major bacterial energy source in the vagina, antibacterial activity against harmful microorganisms, and whether to inhibit harmful bacteria in the vaginal environment, and the isolated strains suppress vaginitis-causing bacteria, thereby being able to exhibit a desired antibacterial effect required for the prevention or treatment of vaginitis.

Description

Lactobacillus crispatus strain and composition for preventing or treating vaginitis containing the same

The present invention relates to a novel Lactobacillus genus strain, a culture thereof, and a composition for treating vaginitis containing the same, and specifically, a novel Lactobacillus crispatus strain isolated from the vagina of a healthy woman, a culture thereof, It relates to a pharmaceutical composition and a health functional food composition for the prevention or treatment of vaginitis comprising.

Under normal conditions, a woman's vagina is a slightly acidic environment due to many lactic acid bacteria inside the vagina, which plays a role in inhibiting the proliferation of pathogenic and harmful microorganisms. The slightly acidic environment in the vagina is caused by external factors such as excessive vaginal washing, long-term administration of antibiotics or contraceptives, disruption of hormone balance due to pregnancy, childbirth, menopause, etc. If changes occur, it can be broken, which is known to cause vaginitis that causes discharge, itching, and pain. Vaginitis is one of the common female gynecological diseases and is often accompanied by symptoms such as itching, burning, unpleasant odor and abnormal vaginal discharge.

Among vaginitis occurring in children of childbearing age, bacterial vaginosis (BV), the most common, accounts for 40-50%, followed by vulvovaginal candidiasis (VVC) (20-25%) and Trichomoniasis (15 -20%), and cases of non-infectious vaginitis may also exist. Chlamydia or gonorrhea mainly causes cervicitis and is a major causative agent of pelvic inflammatory disease, and vaginal secretion may increase due to an increase in purulent secretion. Bacterial vaginosis or Trichomonas vaginitis increase the risk of premature amniotic membrane rupture and preterm labor obstetrically, and are associated with an increase in postoperative complications such as infections. appropriate treatment is essential.

Drugs mainly used in the treatment of vaginitis include sulfonamides, antibiotics, disinfectants by chemical synthesis, etc., but the use of these drugs is fatal not only to harmful bacteria but also to beneficial lactic acid bacteria. In particular, the use of antibiotics is an inevitable cause of antibiotic resistance. In addition, there is a problem in that long-term use may cause systemic toxicity due to absorption of antibiotics through the vagina. In addition, chemically synthesized disinfectants, such as povidone-iodine, may cause hypersensitivity symptoms such as iodine or itching, burning sensation, and dermatitis due to mucosal damage, and hypothyroidism due to increased iodine levels (Int J Environ Res Public Health. 2019 Oct; 16(20): 3859.).

As a supplement to these problems, studies are being conducted on ways to prevent recurrence of vaginitis by improving the vaginal environment as well as removing harmful bacteria, and in particular, a method using probiotics is receiving attention.

The normal vaginal flora is mostly aerobic and consists of an average of six different bacteria, the most common of which is Lactobacillus, which secretes lactic acid and hydrogen peroxide. The vaginal microflora is determined by various factors that affect the survival of bacteria, including the acidity of the vagina and nutrients necessary for the metabolism of bacteria.

Normal vaginal acidity is less than 4.5 and is maintained mainly by lactic acid produced by lactic acid bacteria. Vaginal epithelium is induced by estrogen and is rich in glycogen, which is decomposed into monosaccharides or lactic acid by lactic acid bacteria.

Under such a technical background, the inventors of the present application completed the present invention by isolating a novel strain of the genus Lactobacillus for the prevention or treatment of vaginitis, and confirming the effect of preventing or treating vaginitis thereby.

발명의 요약Summary of Invention

An object of the present invention is to recognize the problems of the prior art mentioned above, and to provide a novel strain of the genus Lactobacillus that can be used for the treatment of vaginitis.

An object of the present invention is to provide a culture of a strain of the genus Lactobacillus.

An object of the present invention is to provide a composition for preventing or treating vaginitis comprising a Lactobacillus genus strain or a culture thereof.

In order to achieve the above object, the present invention provides a Lactobacillus crispatus ID-B02 strain of Accession No. KCTC 14466BP.

The present invention also provides a Lactobacillus crispatus ID-B05 strain of accession number KCTC 14469BP.

The present invention also provides a Lactobacillus crispatus ID-A07 strain of Accession No. KCTC 14470BP.

The present invention also provides two or more Lactobacillus crispatus strains selected from the group consisting of:

Accession No. KCTC 14466BP Lactobacillus crispatus ( Lactobacillus crispatus ) ID-B02 strain;

Accession No. KCTC 14469BP Lactobacillus crispatus ( Lactobacillus crispatus ) ID-B05 strain; and

Accession No. KCTC 14470BP Lactobacillus crispatus ( Lactobacillus crispatus ) ID-A07 strain.

The present invention also provides a culture of two or more Lactobacillus crispatus strains selected from the group consisting of:

The present invention also provides a pharmaceutical composition for preventing or treating vaginitis comprising the Lactobacillus crispatus strain or culture thereof.

The present invention further provides a health functional food composition for preventing or treating vaginitis comprising the Lactobacillus crispatus strain or culture thereof.

Figure 1 shows the results of culturing strains using glucose and glycogen as carbon sources.

발명의 상세한 설명 및 구체적인 구현예DETAILED DESCRIPTION OF THE INVENTION AND SPECIFIC EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.

Vaginitis includes vaginitis and atrophic vaginitis caused by infection with harmful microorganisms, and bacterial, fungal and protozoal vaginitis caused by infection with harmful microorganisms account for most of them. Therefore, it is important to identify and suppress pathogenic microorganisms that cause bacterial, fungal and protozoal vaginitis. Most vaginitis treatments currently on the market are composed of chemicals such as antibiotics, and have excellent short-term effects, but have a high possibility of recurrence.

Accordingly, the inventors of the present application isolated beneficial bacteria from vaginal samples of healthy women, and then selected bacteria that can show high viability in the vaginal environment and inhibit vaginitis-causing bacteria, and then selected three novel Lactobacillus crispatus strains. was derived.

Evaluation of glycogen availability related to the viability of the strain to reveal the mechanism of inhibition of vaginitis-causing bacteria, and evaluation of hydrogen peroxide generation ability, growth inhibition of harmful microorganisms related to vaginitis, and biofilm removal ability to determine whether there is competition/inhibition against harmful bacteria. Through this, the characteristics of the strain were studied.

Specifically, the main bacterial energy source in the female vagina is glycogen present in the vaginal mucosa, and accordingly, glycogen utilization was confirmed. In addition, the ability to generate hydrogen peroxide was confirmed to reveal the antibacterial mechanism related to the ability to compete/inhibit harmful bacteria. Furthermore, it was confirmed whether the desired antibacterial/antibiotic ability against aerobic and/or anaerobic bacteria, fungi, and protozoa that cause infectious vaginitis appears.

Based on this, the present invention provides the following novel Lactobacillus crispatus strain in one aspect:

Accession No. KCTC 14469BP Lactobacillus crispatus ( Lactobacillus crispatus ) ID-B05 strain; or

In another aspect, the present invention provides a culture of the following novel Lactobacillus crispatus strain.

In another aspect, the present invention provides two or more Lactobacillus crispatus strains selected from the group consisting of:

In another aspect, the present invention provides a culture of two or more Lactobacillus crispatus strains selected from the group consisting of the following.

Three strains of Lactobacillus crispatus according to the present invention were deposited with the Korea Research Institute of Bioscience and Biotechnology as of February 04, 2021. The Lactobacillus crispatus strain of accession number KCTC 14466BP is described as Lactobacillus crispatus ID-B02, and the Lactobacillus crispatus strain of accession number KCTC 14469BP is described as Lactobacillus crispatus ID-B05 And, the Lactobacillus crispatus strain of accession number KCTC 14470BP is described as Lactobacillus crispatus ID-A07.

Regarding the evaluation of glycogen availability related to the viability of the isolated strains, it was confirmed that the three finally selected strains had excellent glycogen utilization ability, which is a major bacterial energy source in the female vagina, and could proliferate in the vaginal environment.

In addition, with respect to the ability to compete/inhibit harmful bacteria, it was confirmed that the three finally selected strains exhibited excellent hydrogen peroxide generating ability and thus could act advantageously in the removal of harmful bacteria in the vaginal environment. Moreover, it was confirmed that antibacterial ability was exhibited against 10 types of vaginitis-causing bacteria, and excellent antibiotic ability could be exhibited against fungal vaginitis-causing bacteria.

In another aspect, the present invention provides a culture of two or more Lactobacillus crispatus strains selected from the group consisting of:

In the present specification, "culture" may mean that each of the three strains or a strain containing one or more of the three strains is cultured in a culture medium or culture medium. The culture may or may not contain each of the three strains or one or more of the three strains. The culture may be a liquid or solid formulation, but is not limited thereto.

Various forms of the culture may be included, for example, a concentrate, dried product or extract of the culture.

The extract may be extracted using water, an organic solvent, and the like, for example, water, lower alcohol having 1 to 4 carbon atoms, hexane, chloroform, ethyl acetate, or a mixed solvent thereof.

In another aspect, the present invention provides a composition for preventing or treating vaginitis comprising the Lactobacillus crispatus strain or culture thereof. Various forms of the culture may be included, for example, a concentrate, dried product or extract of the culture.

The strains may be transported in pharmaceutically acceptable carriers such as colloidal suspensions, powders, saline solutions, lipids, liposomes, microspheres, or nanospheres. They may be complexed with or associated with the delivery vehicle and are known in the art such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation reagents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances or fatty acids. It can be delivered in vivo using known delivery systems.

The composition for preventing or treating vaginitis may be a pharmaceutical composition. Based on this, the present invention provides a pharmaceutical composition for preventing or treating vaginitis comprising the Lactobacillus crispatus strain or culture thereof. In this case, the vaginitis may be induced by at least one microorganism selected from the group consisting of bacterial, fungal and protozoal microorganisms.

The pharmaceutical composition may be prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art, or It can be prepared by incorporating into a dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet, capsule, or gel (eg, hydrogel), and may additionally contain a dispersing agent or a stabilizer. there is.

Pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrol, commonly used in formulations. lidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. In addition to the above components, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like may be further included.

The pharmaceutical composition can be administered orally or parenterally and can be used in the form of a general pharmaceutical preparation. That is, the pharmaceutical composition of the present invention can be administered in various oral and parenteral formulations at the time of actual clinical administration. When formulated, commonly used fillers, extenders, binders, wetting agents, disintegrants, It is prepared using excipients. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc. do. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, Witepsol, Macrogol, Tween 61, cacao butter, laurin paper, glycerol, gelatin, and the like may be used.

The concentration of the active ingredient included in the composition may be determined in consideration of the purpose of treatment, the condition of the patient, the required period, and the like, and is not limited to a specific range of concentration. The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, 'pharmaceutically effective amount' means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is based on the type, severity, activity of the drug, drug It may be determined according to factors including sensitivity to , time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent, or in combination with other therapeutic agents, and may be administered simultaneously, separately, or sequentially with conventional therapeutic agents, and may be administered single or multiple times. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors, which can be easily determined by those skilled in the art. The effective amount may vary depending on the patient's age, sex, condition, weight, absorption rate, inactivation rate, excretion rate, disease type, concomitant drug, administration route, severity of vaginitis, gender, weight, age, etc. may increase or decrease accordingly.

The composition for preventing or treating vaginitis may be a quasi-drug composition.

"Quasi-drug" refers to items that are less active than pharmaceuticals among items used for the purpose of diagnosing, treating, improving, mitigating, treating or preventing human or animal diseases. Products used for the treatment or prevention of diseases in humans and animals, products with minor or no direct action on the human body, etc. may be included.

The composition for preventing or treating vaginitis may be a health functional food composition. Based on this, the present invention provides a health functional food composition for preventing or treating vaginitis comprising the Lactobacillus crispatus strain or culture thereof. In this case, the vaginitis may be induced by at least one microorganism selected from the group consisting of bacterial, fungal and protozoal microorganisms.

When the composition is used as a food additive, the health functional food composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods. The amount of the active ingredient can be appropriately used depending on the purpose of its use (prevention or improvement).

There is no particular limitation on the type of health functional food. Examples of foods to which the health functional food composition can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea There are drinks, alcoholic beverages, vitamin complexes, and the like, and may include all health foods in a conventional sense.

The health functional food composition may be prepared as a food, particularly a functional food. The functional food of the present invention includes components commonly added during food preparation, and may include, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when prepared as a drink, natural carbohydrates or flavoring agents may be included as additional ingredients in addition to active ingredients. The natural carbohydrates are monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), oligosaccharides, polysaccharides (eg, dextrins, cyclodextrins, etc.) or sugar alcohols (eg, maltose, sucrose, etc.) , xylitol, sorbitol, erythritol, etc.) are preferred. As the flavoring agent, natural flavoring agents (eg, thaumatin, stevia extract, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.) may be used.

In addition to the health functional food composition, various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonic acid A carbonation agent used in beverages may be further included.

The composition for preventing or treating vaginitis may be a food composition.

The composition for preventing or treating vaginitis may be a parenteral composition.

The composition for preventing or treating vaginitis may be a detergent composition.

The composition for preventing or treating vaginitis may be a cosmetic composition. The cosmetic composition may be prepared in general emulsified and solubilized formulations. For example, cosmetic lotion, such as softening lotion or nourishing lotion; emulsions such as facial lotion and body lotion; creams such as nourishing creams, moisture creams, and eye creams; essence; cosmetic ointment; spray; gel; pack; sunscreen; makeup base; foundations such as liquid type, solid type or spray type; powder; makeup removers such as cleansing creams, cleansing lotions, and cleansing oils; Alternatively, it may be formulated as a cleanser such as a cleansing foam, soap, body wash, etc., but is not limited thereto.

The composition for preventing or treating vaginitis may be a composition for external application. It can be used in appropriate combination with common ingredients formulated in the composition for external application for skin, for example, antibiotics, binders, disintegrants, diluents, lubricants, stabilizers, preservatives, or flavoring agents. The skin external preparation may be formulated as an ointment, patch, gel, cream or spray, but is not limited thereto.

The composition for preventing or treating vaginitis may be a composition for inhibiting the growth of at least one microorganism selected from the group consisting of bacterial, fungal and protozoal microorganisms.

In another aspect, the present invention provides a medical device for preventing or treating vaginitis comprising the Lactobacillus crispatus strain or culture thereof.

The medical devices are gynecological medical devices for vaginitis, such as colposcopes, gynecological selfchecking colposcopes, vaginal dilators, vaginal speculums, and pelvic inflammatory diseases. It may include a treatment device, a gynecological irrigator, or an antibacterial device for external gynecological use, but is not limited thereto.

In addition, the present invention provides a disinfectant product for preventing or treating vaginitis containing the Lactobacillus crispatus strain or culture thereof. The above disinfection products are gynecological disinfection products for vaginitis, such as mucosa disinfectant, mucosal disinfection ointment, gynecological disinfection protection pad, gynecological disinfection tissue, and antibacterial gel for gynecological external use. , antibacterial ointment for gynecological external use, or gynecological antiseptic, but is not limited thereto.

In addition, the present invention provides a feminine cleanser for preventing or treating vaginitis comprising the Lactobacillus crispatus strain or culture thereof.

Example

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Isolation and Screening of Vaginal Microorganisms

A total of 285 strains were isolated from vaginal fluid samples taken from healthy subjects at Eulji University Hospital. Then, from the vaginal-derived microbial library, bacteria expected to be related to vaginitis prevention and treatment were selected through microbiome analysis. The selection criterion was to ensure novelty and originality as much as possible by considering it as the same strain if it was derived from the same sample regardless of the separation conditions (media, culture method, etc.). The isolated strain was prepared as a glycerol stock and stored at -80 ℃.

In order to select strains with vaginitis-inducing bacteria suppression function, glycogen utilization related to viability was evaluated, and hydrogen peroxide generation ability, antibacterial ability, biofilm removal ability, and in vivo vaginal infectious bacteria were evaluated to determine whether there was competition/inhibition against harmful bacteria. A removal capacity evaluation was performed. As a result, a total of three strains were derived and named as ID-A07, ID-B02 and ID-B05, respectively.

Example 2: Identification of 16S rDNA of the three strains selected in the present invention

Molecular biological identification of the three strains selected in the present invention was performed using 27F universal primer (5'-AGA GTT TGA TCM TGG CTC AG-3') and 1492R universal primer (5'-TAC GGY TAC CTT GTT ACG ACT T-3') After amplifying the 16S rRNA gene by PCR (polymerase chain reaction) method, 785F universal primer (5'-GGA TTA GAT ACC CTG GTA-3') and 907R universal primer (5'-CCG TCA ATT CMT TTR AGT TT-3') was used to analyze the nucleotide sequence of the PCR amplification product previously obtained. After that, the analyzed base sequence is identified using the Nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) database of NCBI (National Center for Biotechnology Information) using the homology of the base sequence. got As a result, ID-B02, ID-B05, and ID-A07 strains showed 99%, 100%, and 99% homology with the Lactobacillus crispatus ATCC 33820 (Accession No. NR_041800) strain, respectively, and all three strains were Lactobacillus crispatus. (Lactobacillus crispatus). Novel Lactobacillus crispatus ID-A07, ID-B02 and ID-B05 strains according to the present invention were deposited with the Korea Center for Biological Resources (Table 1).

Example 3: Hydrogen peroxide generating ability evaluation

To evaluate the hydrogen peroxide generating ability, the test strains were inoculated into MRS (de Man, Rogosa and Sharpe) medium, incubated at 37°C for 24 hours, and then plated on Brucella agar medium containing TMB (tetramethyl-benzidine) and horseradish peroxidase at 37°C. were cultured anaerobically for 2 days. Table 2 shows the results of visually observing the degree of blue exposure after exposure to the air for 30 minutes or more after the end of the culture.

As shown in Table 2, as a result of confirming the amount of hydrogen peroxide production for a total of 13 strains, excellent hydrogen peroxide activity was shown in a total of 8 strains of Lactobacillus crispatus, including 3 strains finally selected.

Example 4: Evaluation of glycogen utilization

The test strains were inoculated into NYC III (New York City III) medium, incubated at 37 ° C for 24 hours, washed twice with PBS (Phosphate-buffered saline), suspended in NYC III medium, and 0.5% glycogen was used as a carbon source. Inoculated in the included NYC III medium and cultured for 48 hours, absorbance was measured at 600 nm and shown in Table 3.

As a result of culturing using glycogen as a carbon source, the three final selected strains among the tested strains showed the highest level of glycogen utilization.

Example 5: Inhibiting the growth of vaginitis-related harmful microorganisms

The ability to inhibit the growth of anaerobic bacteria, aerobic bacteria, and fungi related to the induction of vaginitis was evaluated. Each test strain was inoculated into MRS medium, cultured at 37° C. for 24 hours, and the supernatant from which bacteria were removed was collected by filtering. GAM (Gifu Anaerobic Medium) with 5% horse serum added (bacterial culture) and YM (Yeast Malt) medium (fungal culture) were prepared at twice the concentration, respectively, and mixed 1:1 with the supernatant of each test strain. Harmful bacteria were inoculated and then incubated at 37° C. for 24 hours, and the degree of proliferation of each microorganism was confirmed by measuring absorbance at 600 nm. Table 4 shows the degree of inhibition of the growth of each harmful bacteria by the supernatant of each test strain in percentage (%).

As a result of experiments on vaginitis-related bacteria and fungi, it was confirmed that the final selected strains showed high growth inhibitory activity on average, and in particular, not only bacteria but also fungi showed higher inhibitory activity than other strains tested.

Example 6: Removal of vaginitis-inducing harmful bacteria biofilm

The biofilm produced by Gardnerella vaginalis (GV) and Candida albicans (CA), which are representative harmful bacteria that cause vaginitis, was treated with the test strain to confirm the ability to remove them. In a 96 well plate, 200 μL of each solution inoculated into each strain medium (GV: BHI (Brain heart infusion), CA: YM) was dispensed and cultured by standing at 37 ° C. for 24 hours under anaerobic conditions. Thereafter, the CA was washed twice with PBS (phosphate-buffered saline) after removing the culture medium, and then 200 μL of the supernatant of each test strain was dispensed and treated at 37° C. for 24 hours. After removing the culture supernatant of GV, fresh medium was added and cultured for an additional 24 hours, and then the supernatant of the finally selected strain was treated in the same manner. After treatment with the supernatant, the cells were washed three times with PBS, and then 100 μL of 0.1% crystal violet solution was added and treated at room temperature for 20 minutes to stain the remaining biofilm. The stained biofilm was washed three times with clean water, treated with 200 μL of 20% acetic acid solution, left at room temperature for 20 minutes to decolorize, and absorbance was measured at 570 nm. As a control, PBS was treated instead of the test material, and the biofilm removal ability for each test strain for the control group is shown in Table 5.

For each biofilm produced by the two vaginitis-inducing strains, GV and CA, it was confirmed that the supernatant of the final selected strain could reduce the already produced biofilm as shown in Table 5 above. Considering the above results, it was confirmed that the three selected strains derived from the present invention can exhibit preventive and therapeutic effects on vaginitis.

Example 7: Confirmation of Novelty through Whole Genome Analysis of Three Selected Strains

Full-length genome sequencing was commissioned by Macrogen, and genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Germany), and then using PacBio RS II (Pacific Biosciences, USA) and Illumina MiSeq (Illumina, USA) and proceeded with a hybrid sequencing method. Confirmation of novelty using whole genome information is based on the whole genome sequences of 18 species of Lactobacillus crispatus, ID-A07, ID-B02 and ID-A07, ID-B02 and ID- ANI (Average nucleotide identity) analysis was performed using the full-length genome sequence of B05, and ANI analysis was performed using the Blast algorithm (ANIb) in JspeciesWS (http://jspecies.ribohost.com/jspeciesws/). As a result, the ANIb results between ID-A07, ID-B02 and ID-B05 strains and other Lactobacillus crispatus strains are all over 95%, so ID-A07, ID-B02 and ID-B05 strains are all genome-based. Even among those identified as Lactobacillus crispatus at the species level, there were no identical results with ANIb results greater than 99.9%. Among the three selected strains, the ANIb value between ID-A07 and ID-B05 was very high at 99.96%, but the aligned percentage was only 94.93%, so it was confirmed that they were not the same strains. Therefore, as a result of analysis based on the whole genome, the novelty of the ID-A07, ID-B02 and ID-B05 strains as individual strains was confirmed.

Example 8: Patent strain efficacy comparison experiment

Lactobacillus crispatus strains AB70 (accession number: KCTC12976BP) and SNUV220 (accession number: KCTC18374P), selected in Example 2 for glycogen utilization and vaginal harmful microorganisms Inhibitory efficacy was compared. As representative harmful microorganisms related to vaginitis, Gardnerella vaginalis (GV), Candida albicans (CA), and Trichomonas vaginalis (TV) were used. As negative control groups, the test strains showed relatively poor glycogen utilization and ability to inhibit harmful microorganisms in the previous experiment. Lactobacillus crispatus V22 strain, which was not selected by the above method, was used.

Example 8-1: Glycogen availability

In order to confirm the glycogen utilization ability of each strain, a glucose-free MRS medium was prepared, and then each strain was inoculated into a medium supplemented with 0.5% glucose and 0.5% and 1% glycogen as carbon sources, and cultured at 37°C for 18 hours. . The absorbance of each culture end solution was measured, the absorbance of the culture solution without a carbon source was subtracted, and then the proliferation rate by the corresponding carbon source for each strain was compared.

1 shows the results of culturing each strain using glucose and glycogen as carbon sources. It was confirmed that all strains could grow above a certain level in a medium containing glucose. However, in the medium containing glycogen, there was a difference in utilization ability depending on the strain. In the case of the V22 strain, which had the lowest glycogen availability in the screening process of the present invention, it actually showed little growth in the glycogen medium. Among the patented strains, AB70 showed almost no use of glycogen like V22. In the case of another patented strain, SNUV220, and three selected strains of the present invention, they were grown using glycogen, and the proliferation rate of the selected strains was higher than that of SNUV220. In particular, it was found that the selected strains showed a higher growth rate in a medium containing 1% glycogen than in a medium containing 0.5% glucose, whereas SNUV220 did not. Therefore, it can be seen that the strains selected in the present invention are superior to the previously patented strains in terms of their ability to use glycogen as a carbon source. This excellent glycogen availability is advantageous for the formation of colonies by proliferating through the glycogen present in the vagina, so it seems to have a high vaginal survival rate.

Example 8-2: Inhibition of vaginitis-related harmful microorganisms

In order to evaluate the inhibitory effect of each strain on vaginitis-related harmful microorganisms in the vaginal environment, each strain was inoculated and cultured in MRS medium containing 1% glycogen as a carbon source, and the supernatant was collected by centrifugation. Harmful microorganisms were cultured in a medium containing 50% of the culture supernatant of the strain to confirm the growth inhibitory ability. The types of harmful microorganisms used in the test, culture conditions, and growth measurement methods are shown in Table 6 below.

It was cultured until the time when each harmful microorganism was sufficiently grown, and the ratio of the growth rate of the supernatant treatment group of each strain to the growth rate of harmful microorganisms of the control group not treated with the supernatant was expressed as a percentage. The results are shown in Table 7 below.

In the case of V22, which is a dropout strain that was not selected in the present invention, it showed the lowest inhibition rate against all harmful microorganisms. Among the patented strains, SNUV220 showed a higher inhibition rate than AB70 in the culture of all harmful microorganisms, and all three selected strains showed higher inhibition rates than these two patented strains. In the case of TV, the growth was completely inhibited when the supernatant of the selected strain was treated, which seems to have a high overall inhibition rate because TV has sensitive culture conditions. However, it seems sufficient to compare the growth inhibition ability between strains.

Therefore, it was confirmed that the selected strains of the present invention have better glycogen utilization ability and vaginal harmful microorganism inhibition ability than the previously invented Lactobacillus crispatus strains under conditions where glycogen can be used as a carbon source, such as in the vagina.

Through the present invention, three novel Lactobacillus crispatus strains are classified based on glycogen availability, which is a major bacterial energy source in the vagina, antibacterial activity against harmful microorganisms, and whether the growth of harmful bacteria can be inhibited in the vaginal environment. was isolated, and the isolated strain can inhibit vaginitis-causing bacteria to exhibit the antibacterial effect required for the prevention or treatment of vaginitis.

As above, specific parts of the content of the present invention have been described in detail, and for those skilled in the art, these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. It will be clear. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims

Accession No. KCTC 14466BP Lactobacillus crispatus ( Lactobacillus crispatus ) ID-B02 strain or its culture.
Accession No. KCTC 14469BP Lactobacillus crispatus ( Lactobacillus crispatus ) ID-B05 strain or its culture.
Accession No. KCTC 14470BP Lactobacillus crispatus ( Lactobacillus crispatus ) ID-A07 strain or its culture.
Claims 1 to 3 according to any one of the Lactobacillus crispatus ( Lactobacillus crispatus ) A pharmaceutical composition for preventing or treating vaginitis comprising a strain or a culture thereof.
The pharmaceutical composition according to claim 4, wherein the vaginitis is induced by at least one microorganism selected from the group consisting of bacterial, fungal and protozoal microorganisms.
A health functional food composition for preventing or treating vaginitis, comprising the Lactobacillus crispatus strain or culture thereof according to any one of claims 1 to 3.
The health functional food composition according to claim 6, wherein the vaginitis is induced by at least one microorganism selected from the group consisting of bacterial, fungal and protozoal microorganisms.
Two or more Lactobacillus crispatus strains or cultures thereof selected from the group consisting of:

Accession No. KCTC 14466BP Lactobacillus crispatus ( Lactobacillus crispatus ) ID-B02 strain;

Accession No. KCTC 14469BP Lactobacillus crispatus ( Lactobacillus crispatus ) ID-B05 strain; and

Accession No. KCTC 14470BP Lactobacillus crispatus ( Lactobacillus crispatus ) ID-A07 strain.
A pharmaceutical composition for preventing or treating vaginitis comprising the strain or culture thereof according to claim 8.
The pharmaceutical composition according to claim 9, wherein the vaginitis is induced by at least one microorganism selected from the group consisting of bacterial, fungal and protozoal microorganisms.
A health functional food composition for preventing or treating vaginitis comprising the strain or culture thereof according to claim 8.
[Claim 11] The functional food composition according to claim 11, wherein the vaginitis is induced by at least one microorganism selected from the group consisting of bacterial, fungal and protozoal microorganisms.