WO2023080764A1 - Lactic acid bacteria having antibacterial activity against microorganisms causing vaginosis or urinary tract infection and use thereof - Google Patents

Abstract

The present invention provides the lactic acid bacterium strain L. salivarius CLS0420 (KCTC 13815BP) or L. paracasei CLPC0629 (KCTC 14726BP). In addition, the present invention provides a use of the strain for the prevention or treatment of vaginosis or urinary tract infection or a composition comprising the strain for the prevention or treatment of vaginosis or urinary tract infection.

Description

Lactic acid bacteria having antibacterial activity against microorganisms causing vaginitis or urinary tract infections and their uses

This application claims priority based on Korean Patent Application No. 10-2021-0152643 filed on November 8, 2021, and all contents disclosed in the specification and drawings of the application are incorporated into this application. The present invention relates to a lactic acid bacterium having antibacterial activity against vaginitis or urinary tract infection-causing microorganisms, a composition for preventing or treating vaginitis containing the lactic acid bacteria, or a use thereof, and more specifically, antibacterial activity against various bacteria known as vaginitis or urinary tract infection-causing microorganisms. It relates to active lactic acid bacteria, a composition for preventing or treating vaginitis containing the same, or a use thereof.

The female vagina is naturally colonized by a variety of bacteria, yeasts and microorganisms. Under normal conditions, the vaginal flora provides a slightly acidic environment that helps protect against invasion by pathogenic microorganisms. However, under normal conditions as described above, vaginal flora, that is, vaginal equilibrium, can be easily upset by various external factors that ultimately lead to vaginal infections. Vaginal infection is a clinical syndrome, and bacterial vaginosis, Candida vaginitis, and Trichomonas vaginitis have been reported.

The most common disease is bacterial vaginosis, caused by Gardnerella vaginalis. Candida vaginitis, a common disease along with bacterial vaginosis, is known as a disease in which Candida albicans, a fungus, inhabits the moist genital area and causes inflammation.

Treatment is different depending on the causative organism of vaginitis. For bacterial vaginosis, drugs such as metronidazole and clindamycin are used, and for candida vaginitis, azole antifungal agents are used. Such antibiotics may cause resistance problems due to antibiotics, and difficulties in treatment due to resistant bacteria may occur due to long-term treatment.

On the other hand, along with vaginitis, urinary tract infection is one of the most common diseases in women. It is a collective term for infectious diseases that occur in the urinary system. This includes the kidney, bladder, ureter, urethra, etc., and also includes the prostate and epididymis due to their anatomical and physiological structure. Urinary tract infections are caused by urinary tract obstruction due to pregnancy, absenteeism, reflux of the bladder, renal disease, high blood pressure, diabetes, etc., and are easily infected when the immune system is weakened. The most common causative organism is Escherichia coli, but Staphylococcus aureus, streptococcus, fungi, tuberculosis bacillus, and viruses may also cause it. Urinary tract infections are more common in women and children with a relatively short urethra.

The present inventors studied probiotics as an alternative to antibiotics to prevent the occurrence and recurrence of vaginitis or urinary tract infection and maintain women's health, and as a result, new microbial strains having antibacterial activity against one or more various vaginitis or urinary tract infection causing bacteria, Or by confirming its use, the present invention was completed.

Therefore, the problem to be solved by the present invention is to provide a novel lactic acid bacteria that has high antibacterial activity against pathogenic microorganisms and exhibits antibacterial activity against microorganisms that cause vaginitis and/or urinary tract infection in particular.

In order to solve the above problems, the present invention provides a novel lactic acid bacteria that has high antibacterial activity against pathogenic microorganisms and exhibits antibacterial activity against microorganisms that cause vaginitis in particular.

One embodiment of the present invention provides Lactobacillus salivarius strain CLS0420 isolated from the female vagina. The CLS0420 was deposited with the Biological Resources Center of the Korea Research Institute of Bioscience and Biotechnology on February 15, 2019 under the accession number KCTC 13815BP. One embodiment of the present invention provides Lactobacillus paracasei strain CLPC0629 isolated from fruit. The CLPC0629 was deposited with the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center on October 6, 2021 under the accession number KCTC14726BP.

The inventors of the present invention have completed the present invention after confirming that the strains of the present invention have an excellent preventive or therapeutic effect on vaginitis or urinary tract infection. The strains mentioned in this specification have antibacterial activity against all four types of microorganisms known to cause vaginitis or urinary tract infections. Preferably, a composition for preventing or treating vaginitis or urinary tract infection comprising the strain, or a use of the strain for preventing or treating vaginitis or urinary tract infection is provided.

One embodiment of the present invention provides a composition for preventing or treating vaginitis or urinary tract infection comprising one or more strains, or a use of the strain for preventing or treating vaginitis or urinary tract infection.

The Lactobacillus salivarius strain CLS0420 and / or Lactobacillus paracasei strain CLPC0629 is Candida albicans albicans ), Proteus mirabilis ( Proteus mirabilis ), Klebsiella pneumoniae ( Klebsiella pneumoniae ), and Garderella vaginalis , and has antibacterial activity against one or more microorganisms selected from the group consisting of, more preferably, the strain may have antibacterial activity against all of the above microorganisms.

Other embodiments of the present invention include Lactobacillus paracasei strain CLPC0629 and Lactobacillus salivarius strain CLS0420, Candida albicans, Proteus mirabilis ( Proteus mirabilis ), Klebsiella pneumoniae ( Klebsiella pneumoniae ), and Gardnerella vaginalis ( Garderella vaginalis ) to provide a composition for preventing or treating vaginitis or urinary tract infection, having antibacterial activity against one or more microorganisms selected from the group consisting of, more preferably, all four microorganisms.

The Proteus Mirabilis ( Proteus mirabilis ), and Klebsiella pneumoniae are also known to cause urinary tract infections. Another embodiment of the present invention provides a composition for treating or preventing vaginitis and/or urinary tract infection comprising CLPC0629 and/or CLS0420.

The composition may include the CLS0420 strain and the CLPC0629 strain in a weight ratio of 1: 0.6 to 2.5 (CLS0420: CLPC0629), more preferably in a weight ratio of 1: 0.7 to 2.3, and even more preferably in a weight ratio of 1: 0.8 to 2.2. can include When included in the above content ratio, antibacterial activity against all of the above-mentioned microorganisms causing vaginitis or urinary tract infection may be excellent.

In one embodiment, each of the strains or the total sum of the two strains in the composition is 10 ² to 10 ²⁰ CFU / ml, 10 ³ to 10 ¹⁵ CFU / ml, 10 ⁴ to 10 ¹⁴ CFU / ml, 10 ⁵ to 10 ¹³ CFU/ml, 10 ⁶ to 10 ¹² CFU/ml, 10 ⁷ to 10 ¹¹ CFU/ml, or 10 ⁸ to 10 ¹⁰ CFU/ml.

Vaginosis (diseases of vagina, vaginosis) as defined herein is a concept encompassing all types of vaginitis called bacterial vaginosis, fungal vaginitis and tricomonas vaginitis, which are caused by infection with microorganisms. Vaginitis can be all included.

Urinary tract infection, as defined herein, refers to a situation in which bacteria enter the bladder or kidney and multiply. The cause of urinary tract infection may be the same as or different from the cause of vaginitis.

The present invention is a causative agent of vaginitis or urinary tract infection Candida albicans ( Candida albicans ), Proteus mirabilis ( Proteus mirabilis ), Klebsiella pneumoniae ), and/or Gardnerella vaginalis ( Garderella vaginalis ) provides a composition for preventing or treating infection. The composition may be provided as a pharmaceutical composition, a health functional food composition, a quasi-drug composition, and the like.

As used herein, the term 'prevention' may refer to any action that suppresses or delays the onset of infection or vaginitis caused by vaginitis-causing bacteria by administering the composition according to the present invention to a subject.

As used herein, the term 'treatment' may refer to all activities that can improve symptoms of infection or vaginitis caused by vaginitis-causing bacteria by administering the composition according to the present invention to a subject.

The terms of the present application, 'for antibacterial use' or 'for antiviral use' refer to an activity that inhibits or inhibits the growth of harmful bacteria or harmful viruses. Preferably, the harmful microorganism may be a microorganism causing various diseases related to women's health, and in particular, the disease may be vaginitis or urinary tract infection.

The CLS0420 strain and the CLPC0629 strain of the present invention have excellent inhibitory effects on vaginitis-inducing harmful bacteria, and thus can be provided as probiotics for preventing or treating vaginitis.

The 'probiotic' is a living microorganism, that is, a microorganism capable of remaining in the gastrointestinal tract and surviving when ingested by a human or animal, and refers to a microbial agent effective in preventing or treating a specific pathological condition. In general, probiotics have the effect of treating and improving various symptoms caused by abnormal fermentation of intestinal flora. It lowers the intestinal pH and inhibits the growth of harmful microorganisms. In addition, the administered probiotic plays a role of helping the activity of the intestinal villi responsible for the absorption of nutrients.

The above-described compositions may contain the disrupted cell wall fraction of the lactic acid bacteria according to the present invention, live bacteria, dead bacteria, dried bacteria, or a culture thereof, or a cell-free supernatant thereof as an active ingredient, and may further include a suitable excipient or carrier. can The culture is a culture solution obtained by culturing the lactic acid bacteria according to the present invention in a suitable liquid medium, a filtrate obtained by filtering or centrifuging the culture solution to remove the strain (filtrate or centrifuged supernatant), sonicating the culture solution or in the culture solution. It includes, but is not limited to, cell disruption solution obtained by treatment with lysozyme.

In addition, the composition may be prepared and administered in various formulations and methods according to methods known in the art. For example, the lactic acid bacteria or culture thereof of the present invention is mixed with carriers commonly used in the pharmaceutical field to form tablets, troches, capsules, elixirs, syrups , It may be prepared and administered in the form of a powder, suspension, or granule. Examples of the carrier include, but are not limited to, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, and flavoring agents.

For example, a powder may be prepared by simply mixing the lactic acid bacteria of the present invention with suitable pharmaceutically acceptable excipients such as lactose, starch, and microcrystalline cellulose. The granule is the lactic acid bacteria of the present invention; suitable pharmaceutically acceptable excipients; and a suitable pharmaceutically acceptable binder such as polyvinylpyrrolidone and hydroxypropyl cellulose, followed by a wet granulation method using a solvent such as water, ethanol, isopropanol, or the like, or a dry granulation method using compression force. can In addition, tablets may be prepared by mixing the granules with an appropriate pharmaceutically acceptable lubricant such as magnesium stearate and then tableting them using a tableting machine.

The composition may be administered by various parenteral methods including oral administration or intravenous, intramuscular, subcutaneous, topical, sublingual, intranasal, intraperitoneal, intrarectal, and the like.

In addition, the administration dose can be appropriately selected according to the degree of absorption of the active ingredient in the body, the rate of inactivity, the rate of excretion, the age, sex, condition of the recipient, the severity of the disease, and the like. In the case of a composition for probiotics, the number of viable cells in the composition may be administered to 1Х10 ⁵ to 1Х10 ¹¹ cells/g, preferably 1Х10 ⁶ cells/g or more.

In addition, the strain or a composition containing the same may be prepared by including it in addition to all commercially available feminine hygiene products known in the art to which the present invention pertains, as long as the effects provided by the present invention are not impaired. The sanitary products may include, for example, vaginal washes and vaginal tablets.

The composition containing the strain may contain, if necessary, natural carbohydrates and the like as additional components within a range that does not impair the object of the present invention. At this time, examples of natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides, for example, conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol, fructooligosaccharide, isomaltooligosaccharide, galactooligosaccharide, maltooligosaccharide, xyl and oligosaccharides such as rooligosaccharides. In addition, natural flavors (thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) may be used as additional components. In addition, the strain Compositions containing various nutrients, vitamins, minerals (electrolytes), colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonated beverages It can be produced as a health functional food containing a carbonation agent, etc. The strains of the present application are excellent in acid resistance and bile resistance, so they can be provided as probiotics.

The strain or culture thereof of the present application; Alternatively, a composition containing the same may reduce the production of inflammatory cytokines, tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interferon-gamma (IFN-γ). said strain or culture thereof; Alternatively, a composition containing the same may act to reduce the production of inflammatory cytokines, tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interferon-gamma (IFN-γ). The strain or culture thereof of the present application; Alternatively, a composition containing the same may reduce the production of inflammatory cytokines, tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interferon-gamma (IFN-γ), thereby improving vaginitis or urinary tract infection. It can represent, and use for improving vaginitis or urinary tract infection, and a method for improving vaginitis or urinary tract infection can be provided.

In another embodiment of the present invention, the composition may further include auxiliary additives within a range that does not impair the object of the present invention. A person skilled in the art can select and adjust auxiliary additives as needed. In certain embodiments, the additive may be selected from the group consisting of other nutrients, minerals, vitamins, or any combination thereof. In certain embodiments, the other nutrients are dietary fiber, prebiotics, proteins (eg enzymes), carbohydrates, lipids (eg fats), minerals, vitamins, plant components (eg plant extracts) , amino acids, immunomodulatory agents, milk substitutes, or any combination thereof. The mineral may be selected from the group consisting of iron, zinc, potassium, sodium, calcium, magnesium, and any combination thereof, but is not limited thereto. In certain embodiments, the vitamin may be selected from the group consisting of vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, and any combination thereof; Not limited to this.

Another embodiment of the present invention is a therapeutically effective amount of Lactobacillus salivarius CLS0420 ( Lactobacillus salivarius CLS0420) KCTC 13815BP or a culture thereof; or a therapeutically effective amount of Lactobacillus paracasei CLPC0629 CLPC0629) Provides a method for improving, preventing or treating vaginitis or urinary tract infection comprising administering KCTC 14726BP or a culture thereof to a subject in need thereof. The composition comprises at least one microorganism selected from the group consisting of Candida albicans, Proteus mirabilis, Klebsiella pneumoniae, and Garderella vaginalis. may have growth inhibitory activity against

Another embodiment is a therapeutically effective amount of Lactobacillus salivarius CLS0420 ( Lactobacillus salivarius CLS0420) KCTC 13815BP or a culture thereof; Or a therapeutically effective amount of Lactobacillus paracasei CLPC0629 ( Lactobacillus paracasei CLPC0629) KCTC 14726BP or a culture thereof, comprising administering to a subject in need thereof Candida albicans, Proteus mirabilis ( Proteus mirabilis), Klebsiella pneumoniae, and Garderella vaginalis. Vaginitis or urinary tract infection, characterized in that the method reduces the production of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and/or interferon-gamma (IFN-γ) in the body. Improvement, prevention or treatment methods are provided. All contents described in the above composition can be equally applied to the improvement, prevention or treatment method.

An embodiment of the present invention is Lactobacillus salivarius CLS0420 ( Lactobacillus salivarius CLS0420) KCTC 13815BP or its culture; or Lactobacillus paracasei CLPC0629 CLPC0629) KCTC 14726BP or a culture thereof for the improvement, prevention or treatment of vaginitis or urinary tract infection in women. Another embodiment is Lactobacillus salivarius CLS0420 ( Lactobacillus salivarius CLS0420) KCTC 13815BP or a culture thereof; or Lactobacillus paracasei CLPC0629 CLPC0629) KCTC 14726BP or its culture in the group consisting of Candida albicans, Proteus mirabilis, Klebsiella pneumoniae, and Garderella vaginalis It provides a use for killing or inhibiting the growth of one or more selected microorganisms.

The present invention provides a novel lactic acid bacteria strain having an antibacterial effect against microorganisms known to cause vaginitis or urinary tract infections. The strain of the present invention, or a culture thereof, or a composition containing the same has an effect of preventing or treating vaginitis or urinary tract infection. The strain of the present invention, or a culture thereof, or a composition comprising the same has an effect of reducing inflammatory cytokines TNF-α, IL-1β, and/or IFN-γ.

The following drawings attached to this specification illustrate preferred embodiments of the present invention, and serve to further understand the technical idea of the present invention together with the contents of the above-described invention, so the present invention is limited to those described in the drawings. It should not be construed as limiting.

Figure 1 shows the results of confirming the antibacterial activity of L. paracasei CLPC0629.

Figure 2 shows the results of confirming the antibacterial activity of L. salivarius CLS0420.

Figure 3 shows the results of confirming the antibacterial activity of the mixture containing CLPC0629 and CLS0420.

4 is a result of confirming the expression level of tumor necrosis factor-alpha in Raw264.7 (Tumor necrosis factor-alpha in macrophage (Raw264.7)).

5 is a result of confirming the expression level of Interleukin-1 beta in Raw264.7 (Interleukin-1 beta in macrophage (Raw264.7))

6 is a result of confirming the expression level of tumor necrosis factor-alpha in PBMC (Tumor necrosis factor-alpha in PBMC).

7 is a result of confirming the expression level of Interleukin-1 beta in PBMC (Interleukin-1 beta in PBMC).

8 is a result of confirming the expression level of interferon-gamma in PBMC (Interferon-gamma in PBMC).

Hereinafter, examples and the like will be described in detail to aid understanding of the present invention. However, the embodiments according to the present invention can be modified in many different forms, and the scope of the present invention should not be construed as being limited to the following examples. Embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

1. Primary selection of lactic acid bacteria with high antibacterial activity

In order to select lactic acid bacteria with high antibacterial activity against pathogenic microorganisms and vaginitis-inducing microorganisms, the antibacterial activity against pathogenic microorganisms was confirmed for bacteria whose productivity was confirmed among the lactic acid bacteria possessed. The test method is as follows.

Each lactic acid bacteria is incubated overnight at 37 ℃ after inoculation in MRS broth. The cultured lactic acid bacteria are centrifuged, washed twice with PBS (pH 7.0), and then prepared by resuspension in 10ml PBS.

Each pathogenic microorganism is prepared by culturing overnight after inoculation in a suitable medium.

The antibacterial activity test was conducted in the following manner.

After mixing the lactic acid bacteria prepared above with MRS agar (agar 0.8%) at a ratio of 1: 1, drop 20ul on the MRS agar plate, leave it to stand at room temperature until it hardens, and incubate overnight at 37 ° C anaerobic conditions. After preparing Top agar media (agar concentration: 0.8%) by selecting an appropriate medium for each pathogenic microorganism, culturing overnight in 4ml of Top-agar media, adding 100 μl of the prepared pathogenic microorganism culture medium, and then dispensing on the MRA agar plate in which lactic acid bacteria were cultured. Shake to spread well. After leaving it at room temperature until the top-agar hardens, it is incubated overnight at the growth temperature of the pathogen.

The antibacterial activity was confirmed by checking the clearing zone on the cultured plate. Depending on the size of the clear zone, it was marked with +, ++, +++, and when antibacterial activity was not observed, it was marked with -. Most of the lactic acid bacteria tested showed antibacterial activity against S.aureus, E.cloacea, and E.coli, but there were differences in the degree, and lactic acid bacteria that did not show antibacterial activity against C.albicans, a fungus known to cause vaginitis. there was also Accordingly, strains exhibiting antibacterial activity against Candida albicans among the tested lactic acid bacteria were first selected.

Confirmation of antibacterial activity against pathogenic microorganisms

Strain ID	Species	antibacterial activity
		S. aureus	C. albicans	E. cloacea	E. coli
LA-PBF	Lactobacillus acidophilus	++	-	++	+++
CLPC0629	Lactobacillus paracasei	+++	++	+++	+++
LPC-PBF	Lactobacillus paracasei	+++	+	+++	+++
LL-PBF	Lactococcus lactis	++	-	+++	+++
SNZ1969	Bacillus coagulans	++	-	++	+++
LS3	Lactobacillus salivarius	++	-	++	+++
KE99	Lactobacillus paracasei	+++	-	++	+++
G101	Lactobacillus brevis	++	+	+++	+
CLC0629	Lactobacillus casei	+	-	+	++
CLS0420	Lactobacillus salivarius	+++	++	+++	+++
CBC0843	Bacillus coagulans	++	+	++	+++

2. Candida albicans Selection of strains exhibiting antibacterial activity against

The antibacterial activity against Candida albicans was reconfirmed for the strains selected for the first time in the same way as above, and Lactobacillus paracasei CLPC0629 and Lactobacillus salivarius CLS0420 (accession number: KCTC13815) showed excellent antibacterial activity against C.albicans in two repeated experiments. was finally selected.

Confirmation of antibacterial activity against C.albicans

Strain ID	Species	Isolation source	1st experiment		2nd experiment
			clearing zone(mm)	Antibacterial activity evaluation	clearing zone(mm)	Antibacterial activity evaluation
CLPC0629	Lactobacillus paracasei	fruit	8 a /12 b	++	7/16	+++
LPC-PBF	Lactobacillus paracasei	fermented milk	10/15	++	7/11	+
G101	Lactobacillus brevis	kimchi	9/10	+	7/9	+
CLS0420	Lactobacillus salivarius	human body (vagina)	8/13	++	7/18	+++
CBC0843	Bacillus coagulans	fruit	7/9	+	7/10	+

a: Growth ring diameter of inoculated bacteria (mm); b: Diameter of clear zone (mm) Evaluation of antibacterial activity: The higher the b-a value, the better the antibacterial activity

+ : b - a < 5 ; ++: 5≤b-a<9; +++ : 9≤b-a

3. Selected 2 species Lactobacillus Confirmation of antibacterial activity against vaginitis microorganisms

The antibacterial activity of the finally selected L. paracasei CLPC0629 and L. salivarius CLS0420 against vaginitis-related microorganisms was confirmed. In addition to S.aureus, E.cloacae, E.coli, and Candida albicans used in the strain selection process, antibacterial activity against Proteus mirabilis, Klebsiella pneumoniae, and Garderella vaginalis known to be related to vaginitis was confirmed by the same test method as before. It was confirmed that there is antibacterial activity against all pathogenic microorganisms. The results are shown in Table 3 and Figs. 1-2.

Confirmation of antibacterial activity against strains of Proteus mirabilis, Klebsiella pneumoniae, and Gardnerella vaginalis

Strain ID	Species	Proteus mirabilis		Klebsiella pneumoniae		Gardnerella vaginalis
		Clearing zone(mm)	Antibacterial activity evaluation	Clearing zone(mm)	Antibacterial activity evaluation	Clearing zone(mm)	Antibacterial activity evaluation
CLPC0629	Lactobacillus paracasei	9 a /20 b	+++	10/25	+++	10/22	+++
CLS0420	Lactobacillus salivarius	10/24	+++	9/27	+++	10/25	+++

a: Diameter of the growth ring of the inoculated bacteria (mm); b: Diameter of clear zone (mm)

+ : b-a<5 ; ++ : 5≤b-a<9 ; +++ : 9≤b-a

As can be confirmed from the above results, it was confirmed that the CLPC0629 and CLS0420 strains of the present invention have antibacterial activity against all microorganisms known to cause vaginitis or urinary tract infections. Therefore, the new strain of the present invention can show excellent treatment effects of vaginitis and urinary tract infections.

4. Comparative evaluation of antibacterial activity with commercially available lactic acid bacteria products

The antibacterial activity of the two finally selected strains was compared with those of lactobacillus strains known to have an inhibitory effect on female vaginitis. Lactobacillus strains were isolated and identified from commercially available products, and after confirming whether the indicated strains were correct, they were cultured and used in experiments.

The antibacterial activity against Candida albicans and Gardnerella vaginalis as microorganisms related to vaginitis was compared as follows. As a result of the comparison, CLPC0629 and CLS0420 showed better antibacterial activity than previously known L.reuteri and L.rhamnosus.

Comparative evaluation of antibacterial activity against Candida albicans

Strain ID	Species	1st experiment			2nd experiment
		clearing zone(mm)	Antibacterial activity evaluation		clearing zone(mm)	Antibacterial activity evaluation
			b-a (mm)	evaluation		b-a (mm)	evaluation
CLPC0629	Lactobacillus paracasei	12 a /20 b	8	++	13/21	8	++
CLS0420	Lactobacillus salivarius	12/21	9	+++	12/22	10	+++
RC-14	Lactobacillus reuteri	13/20	7	++	13/21	8	++
GR-1	Lactobacillus rhamnosus	13/20	7	++	13/20	7	++

a: Growth ring diameter of inoculated bacteria (mm); b: diameter of clear zone (mm) Evaluation of antibacterial activity: the higher the b-a value, the better the antibacterial activity

+ : b - a < 5 ; ++: 5≤b-a<9; +++ : 9≤b-a

Comparative evaluation of antibacterial activity against Gardnerella vaginalis

Strain ID	Species	1st experiment			2nd experiment
		clearing zone(mm)	Antibacterial activity evaluation		clearing zone(mm)	Antibacterial activity evaluation
			b-a (mm)	evaluation		b-a (mm)	evaluation
CLPC0629	Lactobacillus paracasei	12 a /39 b	27	+++	13/41	28	+++
CLS0420	Lactobacillus salivarius	12/42	30	+++	14/41	27	+++
RC-14	Lactobacillus reuteri	14/40	26	+++	13/38	25	+++
GR-1	Lactobacillus rhamnosus	13/39	26	+++	12/39	27	+++

a: Growth ring diameter of inoculated bacteria (mm); b: Diameter of clear zone (mm) Evaluation of antibacterial activity: The higher the b-a value, the better the antibacterial activity

+ : b - a < 5 ; ++: 5≤b-a<9; +++ : 9≤b-a

5. Evaluation of antibacterial activity when mixed

Antibacterial activity against vaginitis-related microorganisms was confirmed when the two selected strains, CLPC0629 and CLS0420, were used alone or mixed. The test method is as follows.

Each of the two strains was inoculated into MRS broth and incubated overnight at 37°C. After culturing, samples were prepared by mixing the two strains, CLS0420 and CLPC0629, at ratios of 1:1, 1:2, and 2:1.

E.coli, E.cloacae, S.aureus, K.penumoniae, and C.albicans among the pathogenic microorganisms previously used in the antibacterial activity confirmation experiment were inoculated into suitable media and then cultured overnight.

After adjusting the OD values of lactic acid bacteria and pathogenic microorganisms to be 0.2 to 0.3, each was inoculated with 3ml (1:1), and after culturing overnight, the number of viable pathogenic microorganisms was determined immediately.

All experimental groups showed more than 90% antibacterial activity, and all of the experimental groups in which CLS0420 and CLPC0629 were mixed 1:1 and 1:2 showed more than 99% antibacterial activity.

experimental group	Antibacterial activity (%)
	E. coli	E. cloacae	K. pneumoniae	S. aureus	C. albicans
Control	0%	0%	0%	0%	0%
CLS0420	99.95%	99.99%	99.89%	99.99%	99.89%
CLPC0629	99.74%	99.94%	99.86%	99.99%	99.30%
CLS:CLPC=1:1	99.99%	99.99%	99.97%	99.99%	99.89%
CLS:CLPC=1:2	99.99%	99.97%	99.99%	99.99%	99.86%
CLS:CLPC=2:1	99.99%	99.99%	99.97%	99.97%	93.83%

The case with the lowest antibacterial activity was also 93% or more, confirming the excellent antibacterial effect of the mixed microorganisms.

6. acid resistance and Bile acid resistance evaluation

Acid resistance and bile acid resistance are important characteristics of probiotics because probiotics are exposed to an acidic environment by gastric acid in the stomach and bile salts in the small intestine after ingestion. To be used as probiotics, resistance to gastric juice to survive low pH and enzymes, and tolerance to bile fluid to survive in the extreme environment of the intestine are essential. Accordingly, the acid resistance and bile acid resistance of the selected strains were evaluated.

For acid resistance evaluation, the selected lactic acid strain was inoculated into MRS broth, cultured for 24 hours, and then used as a test solution. Inoculate 100ul of test solution into 10ml of artificial gastric fluid (pH 2) and incubate at 37℃. The number of bacteria was measured by sampling at 0 hour, 30 minutes, 1 hour, and 2 hours while stirring using a rotator. It was confirmed that the strain had excellent acid resistance by maintaining a viable cell count of 10 ⁹ CFU/mL or more after 2 hours at pH 2.

Acid resistance evaluation

	L. paracasei CLPC0629	L. salivarius CLS0420
	viable count (CFU/ml)	viable count (CFU/ml)
0 hours	3.85Х10 9	3.45Х10 9
30 minutes	2.73Х10 9	3.60Х10 9
1 hours	2.72Х10 9	2.67Х10 9
2 hours	1.61Х10 9	2.47Х10 9

To evaluate bile acid resistance, prepare MRS agar and MRS agar with 0.3% oxgall added. The overnight cultured lactic acid bacteria culture medium was diluted by an appropriate factor, spread on the two previously prepared media, cultured at 37°C, and then the colony formation in both media was compared to evaluate bile acid resistance. Even in the artificial bile solution resistance test, it was confirmed that the viable cell count was maintained at 10 ⁸ CFU/mL or more, resulting in very good resistance.

Bile acid resistance evaluation

	L. paracasei CLPC0629	L. salivarius CLS0420
	CFU/ml	CFU/ml
MRS agar	3.80Х10 9	3.90Х10 9
MRS agar with 0.3% oxgall	1.84Х10 9	5.10Х10 8

7. Assessment of antibiotic resistance

Due to the overuse of antibiotics, the social problem of antibiotic-resistant bacteria is becoming serious. Accordingly, antibiotic resistance to probiotic bacteria has become an important issue for the use and safety of bacteria. Antibiotic resistance was evaluated for the selected CLPC0629 and CLS0420 according to the EFSA guideline (2012) as follows. As a result, both strains were evaluated as safe because they did not have antibiotic resistance.

MIC evaluation result of L.paracasei CLPC0629

Antibiotics	MIC Cut-off value (mg/L) by EFSA guide	MIC Result(mg/L)	Susceptible /Resistant
Ampicillin	4	One	S
Vancomycin	not required
Gentamycin	32	0.5	S
Kanamycin	64	8	S
Streptomycin	64	2	S
Erythromycin	One	0.5	S
Clindamycin	One	0.5	S
Tetracycline	4	0.5	S
Chlormaphenicol	4	4	S

* Criterion MIC>cut-off value: evaluated as Resistant(R);

MIC≤cut-off value: Evaluated as Susceptible (S)

MIC evaluation results of L.salivarius CLS0420

Antibiotics	MIC Cut-off value (mg/L) by EFSA guide	MIC Result(mg/L)	Susceptible /Resistant
Ampicillin	4	0.5	S
Vancomycin	not required
Gentamycin	16	0.5	S
Kanamycin	64	0.5	S
Streptomycin	64	0.5	S
Erythromycin	One	0.5	S
Clindamycin	One	0.5	S
Tetracycline	8	One	S
Chlormaphenicol	4	4	S

* Criterion MIC>cut-off value: evaluated as Resistant(R);

MIC≤cut-off value: Evaluated as Susceptible (S)

8. Characteristics of strains

(1) 16S rDNA analyze

16S rDNA sequencing was performed on the two lactic acid bacteria used in the experiment, and the results are shown in SEQ ID NOs: 1 and 2, respectively. As a result of 16S rDNA homology analysis, it was confirmed that the lactic acid bacteria CLS0420 of the present invention was Lactobacillus salivarius, and CLPC0629 was confirmed again to be Lactobacillus paracasei.

(2) sugar availability check

The following sugar availability confirmation results are shown below.

One) CLS0420 sugar availability check

sugars	usability	sugars	usability
Control (Negative)	-	Esculin	+
Glycerol	-	Salicin	-
Erythritol	-	Cellobiose	+
D-arabinose	-	Maltose	+
L-arabinose	-	Lactose	+
D-ribose	-	Melibiose	+
D-xylose	-	Sucrose	+
L-xylose	-	Trehalose	+
D-adonitol	-	Inulin	-
Methyl-βD-xylopyranoside	-	Melezitose	+
D-galactose	+	Raffinose	+
D-glucose	+	Starch	-
D-fructose	+	Glycogen	-
D-Mannose	+	Xylitol	-
L-sorbose	-	Gentiobiose	-
Rhamnose	+	D-turanose	-
Dulcitol	-	D-lyxose	-
Inositol	-	D-tagatose	-
Mannitol	+	D-fructose	-
Sorbitol	+	L-fructose	-
α-methyl-D-mannoside	-	D-arabitol	-
α-methyl-D-glucoside	-	L-arabitol	-
N-acethyl-glucosamine	+	Gluconate	-
Amygdalin	-	2-keto-gluconate	-
Arbutin	-	5-keto-gluconate	-

-: no reaction, +: reaction

2) CLPC0629 sugar availability check

sugars	usability	sugars	usability
Control (Negative)	-	Esculin	+
Glycerol	-	Salicin	+
Erythritol	-	Cellobiose	+
D-arabinose	-	Maltose	+
L-arabinose	-	Lactose	+
D-ribose	+	Melibiose	-
D-xylose	-	Sucrose	v
L-xylose	-	Trehalose	+
D-adonitol	-	Inulin	-
Methyl-βD-xylopyranoside	-	Melezitose	+
D-galactose	+	Raffinose	-
D-glucose	+	Starch	-
D-fructose	+	Glycogen	-
D-Mannose	+	Xylitol	-
L-sorbose	+	Gentiobiose	-
Rhamnose	+	D-turanose	+
Dulcitol	v	D-lyxose	v
Inositol	-	D-tagatose	+
Mannitol	+	D-fructose	-
Sorbitol	+	L-fructose	-
α-methyl-D-mannoside	-	D-arabitol	-
α-methyl-D-glucoside	-	L-arabitol	-
N-acethyl-glucosamine	+	Gluconate	v
Amygdalin	-	2-keto-gluconate	-
Arbutin	-	5-keto-gluconate	-

-: no reaction (negative), +: reaction (positive), v: weak positive

9. Hypersensitivity immune reaction mitigation test of lactic acid bacteria

1) Using PBMC (peripheral blood mononuclear cell), an immune cell isolated from mice, and Raw264.7, a macrophage cell line, the hyperimmune response of lactic acid bacteria was evaluated.

2) The test group consisted of a normal control group not treated with stimulant, a negative control group treated with stimulant, a positive control group (Lactobacillus rhamnosus GG) treatment group, and a positive control group (Dexamethasone). ) treatment group, test substances (Lactobacillus salivarius CLS0420, Lactobacillus paracasei CLPC0629).

All test groups except for the normal control group were treated with 5x10 ⁷ CFU or 10x10 ⁷ CFU of stimulant and test substance, and cultured for 24 to 96 hours.

<Test result>

1) In the case of Raw264.7, TNF-α and IL-1β were significantly increased in the negative control group treated with LPS, a stimulant, compared to the normal control group, confirming that hyperimmune response was induced. As a result of treatment with the test substance under this condition, TNF-α and IL-1β showed a significant decrease in all test groups, showing an effect equal to or better than that of the positive control substance.

2) In the case of PBMC, compared to the normal control group, the negative control group treated with LPS, a stimulant, showed a significant increase in TNF-α, IL-1β, or IFN-γ, confirming that hyperimmune response was induced. As a result of measuring the change of each cytokine by treating the test substance under this condition, TNF-α, IL-1β, and IFN-γ were significantly decreased in all test substance treatment groups compared to the negative control group.

[Entrusted institution]

Name of Depositary Institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC13815BP

Entrusted date: 20190215

Name of Depositary Institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC14726BP

Entrusted date: 20211006

The present invention can provide health functional food, medicine, etc. for preventing or treating vaginitis or urinary tract infection. The present invention may be provided for use in inhibiting the growth or proliferation of the causative bacteria of vaginitis or urinary tract infections, or killing them. The present invention can be used for improving, preventing, or treating vaginitis or urinary tract infection, and can provide a method for improving, preventing, or treating vaginitis or urinary tract infection.

Claims (18)

1. Lactobacillus salivarius CLS0420 ( Lactobacillus salivarius CLS0420) KCTC 13815BP.
2. Lactobacillus salivarius CLS0420 ( Lactobacillus salivarius CLS0420) A composition for preventing or treating vaginitis or urinary tract infection comprising a KCTC 13815BP strain or a culture thereof.
3. The method of claim 2, wherein the strain or its culture is Candida albicans, Proteus mirabilis, Klebsiella pneumoniae, and Garderella vaginalis. ) A composition for preventing or treating vaginitis or urinary tract infection having antibacterial activity against at least one microorganism selected from the group consisting of.
4. The method of claim 2, wherein the strain or its culture is a vaginitis that reduces the production of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), or interferon-gamma (IFN-γ), or A composition for preventing or treating urinary tract infections.
5. Lactobacillus paracasei CLPC0629 ( Lactobacillus paracasei CLPC0629) KCTC 14726BP.
6. Lactobacillus paracasei CLPC0629 ( Lactobacillus paracasei A composition for preventing or treating vaginitis or urinary tract infection comprising a CLPC0629) KCTC 14726BP strain or a culture thereof.
7. The method of claim 6, wherein the strain or its culture is Candida albicans, Proteus mirabilis, Klebsiella pneumoniae, and Garderella vaginalis. ) A composition for preventing or treating vaginitis or urinary tract infection having antibacterial activity against at least one microorganism selected from the group consisting of.
8. The method of claim 6, wherein the strain or its culture is tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), or interferon-gamma (IFN-γ) to reduce the production of vaginitis or A composition for preventing or treating urinary tract infection.
9. Lactobacillus salivarius CLS0420 ( Lactobacillus salivarius CLS0420) KCTC 13815BP strain and Lactobacillus paracasei CLPC0629 ( Lactobacillus paracasei CLPC0629) KCTC 14726BP strain,

   Antibacterial against one or more microorganisms selected from the group consisting of Candida albicans, Proteus mirabilis, Klebsiella pneumoniae, and Garderella vaginalis An active composition for preventing or treating vaginitis or urinary tract infection.
10. The composition for preventing or treating vaginitis or urinary tract infection according to claim 9, wherein the composition comprises the CLS0420 strain and the CLPC0629 strain in a weight ratio of 1: 0.6 to 2.5 (CLS0420: CLPC0629).
11. Lactobacillus salivarius CLS0420 ( Lactobacillus salivarius CLS0420) KCTC 13815BP strain and Lactobacillus paracasei CLPC0629 ( Lactobacillus paracasei CLPC0629) KCTC 14726BP strain,

    Growth against one or more microorganisms selected from the group consisting of Candida albicans, Proteus mirabilis, Klebsiella pneumoniae, and Garderella vaginalis Having an inhibitory activity, antibacterial or antiviral composition.
12. 12. A composition according to any one of claims 1 to 11, further comprising at least one additive.
13. A therapeutically effective amount of Lactobacillus salivarius CLS0420 ( Lactobacillus salivarius CLS0420) KCTC 13815BP or a culture thereof; or

    A method for improving, preventing or treating vaginitis or urinary tract infection comprising the step of administering a therapeutically effective amount of Lactobacillus paracasei CLPC0629 ( Lactobacillus paracasei CLPC0629) KCTC 14726BP or a culture thereof to a subject in need thereof.
14. 14. The method of claim 13, wherein the composition is selected from the group consisting of Candida albicans, Proteus mirabilis, Klebsiella pneumoniae, and Garderella vaginalis. A method for improving, preventing or treating vaginitis or urinary tract infection, having a growth inhibitory activity against one or more microorganisms selected from.
15. The method of claim 13, wherein the method reduces the production of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), or interferon-gamma (IFN-γ) in the body. A method for improving, preventing or treating vaginitis or urinary tract infection.
16. A therapeutically effective amount of Lactobacillus salivarius CLS0420 ( Lactobacillus salivarius CLS0420) KCTC 13815BP or a culture thereof; or

    Candida albicans, Proteus mirabilis, including the step of administering a therapeutically effective amount of Lactobacillus paracasei CLPC0629 ( Lactobacillus paracasei CLPC0629) KCTC 14726BP or a culture thereof to a subject in need thereof mirabilis), Klebsiella pneumoniae, and Garderella vaginalis. A method for killing or inhibiting the growth of at least one microorganism selected from the group consisting of.
17. Lactobacillus salivarius CLS0420 ( Lactobacillus salivarius CLS0420) KCTC 13815BP or a culture thereof; or Lactobacillus paracasei CLPC0629 CLPC0629) Use of KCTC 14726BP or a culture thereof for the improvement, prevention or treatment of vaginitis or urinary tract infection in women.
18. Lactobacillus salivarius CLS0420 ( Lactobacillus salivarius CLS0420) KCTC 13815BP or a culture thereof; or Lactobacillus paracasei CLPC0629 CLPC0629) KCTC 14726BP or its culture

    Killing of one or more microorganisms selected from the group consisting of Candida albicans, Proteus mirabilis, Klebsiella pneumoniae, and Garderella vaginalis; Use for growth inhibition.

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