KR20190102498A - Lactobacillus sp. strain having inhibitory effect against microorganisms causing vaginosis uses thereof - Google Patents

Abstract

The present invention provides Lactobacillus sp. strains having a proliferation inhibitory effect against microorganisms causing vaginosis, wherein the strains can be Lactobacillus sakei ATG-K3, Lactobacillus sakei ATG-K14, Lactobacillus plantarum HAC01, and Lactobacillus rhamnosus BFE5264.

Description

Combination of Lactobacillus strains having proliferation inhibitory activity against the causative agent of vaginitis and products containing the same as active ingredients {Lactobacillus sp. strain having inhibitory effect against microorganisms causing vaginosis uses according to

The present invention relates to a combination of Lactobacillus strains having a proliferation inhibitory activity against the causative agent of vaginitis and a product containing the same as an active ingredient.

Three-quarters of the world's female population suffers from infections of inflammatory female diseases, including bacterial vaginosis, of which 50% are reported to relapse. In addition, more than 60% of women's yeast infections are asymptomatic infections, and antibiotics, stress, hormonal changes after menopause, and sensitivity to vaginitis increases significantly.

Vaginitis caused by infection is divided into three main causes: bacterial vaginosis (BV), candidiasis (Candida vaginitis (CV)) and trichomonas vaginalis (Trichomonas vaginalis). Among these, bacterial vaginosis has recently been recognized as the most common vaginitis, and vaginal secretions isolated from patients with bacterial vaginosis have been found to include Atopobium vaginae, Megasphaera, Gardnerella vaginalis, Eggerthella, Clostridium-like, Prevotella bivia, and Peptostreptococcus micros. In particular, Gardnerella vaginalis is known as a major causative agent of bacterial vaginosis.

Women's vaginal microbial community is a major factor in maintaining vaginal health, where a variety of bacteria, yeasts and other microorganisms coexist in balance. The dominant species of healthy vaginal flora is Lactobacillus lactic acid bacteria, which are known to inhibit various pathogens causing vaginitis and female diseases by performing functions such as maintaining vaginal acidity, generating hydrogen peroxide, and activating mucosal immune system.

Existing treatment for vaginitis with antibiotics involves various side effects such as increased recurrence rate, decreased accompanied by Lactobacillus flora, and inflammation caused by other antibiotic resistant strains besides Gardnerella and Candida. The importance of suppressing proliferation of vaginitis pathogens and restoring vaginal flora has been highlighted.

The present invention screens the inhibitory activity of vaginal pathogenic microorganisms against Lactobacillus strains, and discovers probiotic Lactobacillus strains with novel activity through the search for Lactobacillus strains having proliferation inhibitory activity against vaginitis causative bacteria and vaginitis. A comprehensive inhibitory effect on development was identified.

Accordingly, it is an object of the present invention to provide a strain of the genus Lactobacillus having an activity of inhibiting the growth of pathogenic microorganisms in the vagina.

Still another object of the present invention is to provide a pharmaceutical composition for treating and / or preventing diseases related to the proliferation of pathogenic microorganisms in the vagina containing the strain of the genus Lactobacillus as an active ingredient.

Still another object of the present invention is to provide a disease related to the proliferation of pathogenic microorganisms in the vagina containing the Lactobacillus strain as an active ingredient, such as nutraceuticals for improving, treating and / or preventing health functional foods or cleaning products.

The present invention provides a strain of Lactobacillus genus having a proliferation inhibitory activity against a causative agent of vaginitis. At this time, the strain may be Lactobacillus Sakei ATG-K3 (Accession No. KCTC 18667P). The strain may be Lactobacillus sakei ATG-K14 (Accession No. KCTC 18668P). The strain may be Lactobacillus plantarum HAC01. The strain may be Lactobacillus rhamnosus BFE5264.

The present invention provides a composition combining two or more selected from the group comprising Lactobacillus sakei ATG-K3, Lactobacillus sakei ATG-K14, Lactobacillus plantarum HAC01 and Lactobacillus rhamnosus BFE5264.

The present invention provides a probiotic formulation comprising the strain or composition as an active ingredient.

The present invention provides a nutraceutical composition comprising the strain or the composition as an active ingredient.

The present invention provides a pharmaceutical composition comprising the strain or composition as an active ingredient.

The present invention provides a dietary supplement comprising the strain or composition according to claims 1 to 6 as an active ingredient.

The present invention relates to a novel Lactobacillus sp. Strain having intravaginal pathogenic microbial growth inhibitory activity, and a pharmaceutical composition, a health functional food, and a cleaning product containing the same as an active ingredient. Therefore, the present invention exhibits a proliferation inhibitory effect on pathogens associated with the development of infection and bacterial vaginitis, and thus can be used for the prevention and treatment of female vaginitis.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the present invention is not limited or limited by the embodiments, and those skilled in the art can easily change these embodiments. Also, like reference numerals in the drawings denote like elements.

Also, the terminology used herein is a term used to properly express a preferred embodiment of the present invention, which may vary depending on a viewer, an operator's intention, or customs in the field to which the present invention belongs. Therefore, the definitions of the terms should be made based on the contents throughout the specification.

Overview of strain

BFE5264 strain is a strain of the genus Lactobacillus isolated from Maasai milk (Kule naoto) (Mathara et al, Int. J. Food Microbiol. 126: 57-64, 2008).

 HAC01 strain is a strain deposited with the accession number KCTC 12647BP (deposited August 6, 2014 to the Korea Research Institute of Bioscience and Biotechnology), it was described by Korea Patent Registration No. 10-1500974.

The Lactobacillus sakei ATG-K3 strain is a strain deposited as accession number KCTC 18667P.

The Lactobacillus sakei ATG-K1 strain is a strain deposited as accession number KCTC 18668P.

Isolation and sequencing of the BFE5264 strain and the HAC01 strain are described in the paper (Mathara et al, Int. J. Food Microbiol. 126: 57-64, 2008) and Korean Patent No. 10-1500974, which are omitted here. Shall be.

1. Isolation and Identification of Strains

Lactobacillus sakei ATG-K3 and Lactobacillus sakei ATG-K14 can be separated from kimchi.

Applicants put 90 mL of physiological saline (0.85% NaCl / L), put 10 g of kimchi samples in a high-pressure sterilized Erlenmeyer flask, mix them uniformly, and dilute step by 10-fold to smear BCP plate medium. In addition, the Applicant first isolates the colonies of the bacillus shape by gram staining by separating the yellow colonies grown by growing at 37 ℃ for 24 hours. At this time, Applicants have identified taxonomic species by analyzing 16S rDNA gene sequences to identify selected strains. As a result, both strains were identified as Lactobacillus sakei (see FIGS. 1A and 1B), and these were named as ATG-K3 and ATG-K14, respectively, and were deposited with the Korea Institute of Bioscience and Biotechnology, and deposited with the patent numbers KCTC 18667P and KCTC. 18668P.

2. Acid and bile resistance test

Acid and bile resistance tests were performed to determine whether the isolates were capable of surviving the stomach and duodenum, which secrete strong gastric acid and bile. The isolated strains were incubated in MRS liquid medium at 37 ° C. for 18 hours and then diluted to determine the number of viable cells. Thereafter, 1 mL of the culture solution was centrifuged at 12,000 xg for 10 minutes, and the cells were washed twice with physiological saline and then physiological saline was removed. Precipitated cells were thoroughly mixed with 10 mL of MRS liquid medium adjusted to pH 3.0, and then incubated at 37 ° C for 1 hour, 1 mL of the suspension was diluted to 10 ml, and the number of viable cells was measured by plating on MRS plate medium. The remaining 9 mL was again centrifuged at 12,000 xg for 10 minutes, and then the cell pellet was mixed with 4 mL of bile acid solution (10 g oxgall in 100 mL of distilled water and autoclaved) and 17 mL of synthetic duodenum juice (6.4 g / L NaHCO _3). , 0.239 g / L KCl and 1.28 g / L NaCl were mixed well in distilled water and then used by autoclaving to adjust the pH to 7.4). After 2 hours of incubation at 37 ° C. incubator, each step was diluted 10 times and plated on MRS solid medium to count the number of viable bacteria. In addition, the number of viable cells counted and the number of viable cells after 1 hour and 3 hours were expressed as survival rates. Total survival is shown in Table 1 below.

Table 1 Results of Acid and Bile Resistance Analysis of Lactobacillus Sakei ATG-K3 and Lactobacillus Sakei ATG-K14

3. Antibiotic Resistance Test

Minimal inhibitory concentrations (MICs) were measured for antibiotics E-test (tetracycline, ampicillin, chloramphenicol, erythromycin) to determine antibiotic resistance. Minimum inhibitory concentrations were measured by the E-test (bioMeFrance) method and evaluated for susceptibility to antibiotics compared to the breakpoint suggested by Danielsen & Wind. In the experimental method, colonies incubated in MRS solid medium for 18 hours were diluted with 1X PBS to McFarland No. 0.5, and then plated onto MRS plate medium using a sterile swab. The strip kit containing each antibiotic on it was measured by incubating at 37 ℃ for 18 to 24 hours. If the measured minimum inhibitory concentration was lower than the suggested breakpoint, it was considered not to be resistant to the antibiotic. Both Lactobacillus SAKAY ATG-K3 and Lactobacillus SAKAY ATG-K14 showed no resistance to the tested antibiotics. 2).

TABLE 2 Antibiotic Resistance Survey Results of Lactobacillus Sakei ATG-K3 and Lactobacillus Sakei ATG-K14

4. Hemolytic Activity Assessment

Since bacteria may exhibit hemolysis that destroys red blood cells in the blood, in this example, evaluation of hemolytic activity was performed to confirm pathogenicity of lactic acid bacteria isolated from kimchi. MRS medium was plated with 5% sheep blood and two strains were plated and incubated at 37 ° C. for 24 hours to check for hemolysis. As a result, Lactobacillus Sakei ATG-K3 did not show a hemolytic action, Lactobacillus Sakei ATG-K14 was confirmed as a safe lactic acid bacteria because hemolysis did not appear (Fig. 2).

Lactobacillus Sakei ATG-K3 and Lactobacillus Sakei ATG-K14 strains with probiotic activity were isolated from kimchi, and the existing probiotic strains, Lactobacillus plantarum HAC01 and Lactobacillus rhamnosus BFE5264, were isolated. Together, we examined the inhibitory effect on the vaginal infection bacteria Candida albicans.

5. Inhibition test

Lactobacillus sakei ATG-K3, Lactobacillus sakei ATG-K14, Lactobacillus plantarum HAC01, Lactobacillus rhamnosus BFE5264 four strains were determined whether the antimicrobial activity against vaginitis bacteria. First, four lactobacillus strains of Lactobacillus sakei ATG-K3, Lactobacillus sakei ATG-K14, Lactobacillus rhamnosus BFE5264, and Lactobacillus plantarum HAC01 were incubated in 20 mL MRS broth for 18 hours, and then 3,000 rpm, 10 The supernatant obtained by centrifugation for a minute was lyophilized. The lyophilized culture was dissolved in PBS and filtered and sterilized with a 0.2 μm filter to be used for antibacterial activity. Candida albicans colonies incubated in YM medium for 24 hours were diluted with 1X PBS to Mcfarland No. 0.5, and then plated on YM plate medium. The lyophilized solution prepared above was put on each of the disks, and then cultured for 18 to 24 hours to determine whether the suppressed ring was formed. As shown in Figure 3, all four strains produced a suppression ring, indicating that it inhibits the proliferation of Candida albicans.

6. Antibacterial Activity of Lactic Acid Bacteria on Vaginitis Bacteria

Candida albicans incubated in YM broth was incubated for 18 hours and diluted using the same medium to 1 × cfu / mL. Four lactobacillus strains of Lactobacillus sakei ATG-K3, Lactobacillus sakei ATG-K14, Lactobacillus rhamnosus BFE5264, and Lactobacillus plantarum HAC01 were incubated in 20 mL MRS broth for 18 hours, followed by 3,000 rpm and 10 minutes. The supernatant obtained by centrifugation was lyophilized. The lyophilized culture solution was dissolved in PBS and filtered and sterilized with a 0.2 μm filter. 100 μL of 1 × 10 ⁶ cfu / mL Candida albicans culture solution and each lactic acid strain lyophilized solution was added to a 96 well plate and cultured at 37 ° C. for 24 hours. As a result, all four strains showed antimicrobial activity against Candida albicans, except that Lactobacillus sakei ATG-K3 showed lower antimicrobial activity than the other three strains (Table 3).

Table 3 Antimicrobial Activity against Candida Albicans of Four Strains

7. Co-aggregation Activity of Four Lactic Acid Bacteria Against Vaginitis Bacteria

Using McDarland No. 1 colony of Candida albicans incubated on YM agar plate. A suspension of 4 was made. For the four lactic acid strains to be tested, suspensions of the same concentration were prepared using colony cultured on an MRS agar plate. A suspension of vaginitis bacteria and a suspension of each lactic acid strain were added to a well of 6 mL in 6 mL plate and incubated at 37 ° C. for 4 hours. On the other hand, a portion of the culture solution was smeared on a slide glass and stained with crystal violet and observed under a microscope.

Co-aggregation (%) =

The ability of lactic acid bacteria to coagulate against vaginal bacterium plays an important role in suppressing the onset of vaginal bacterium because it plays a role in preventing vaginal bacteria from adhering to vaginal cells. Experimental results (Table 4), Lactobacillus sakei against Candida albicans ATG-K14 showed the highest degree of aggregation, and Lactobacillus sakei ATG-K3, Lactobacillus plantarum HAC01, and Lactobacillus rhamnosus BFE5264 also showed an aggregation rate of more than 30%. In addition, in the results of microscopic observation of the aggregation degree between lactic acid bacteria and Candida albicans (FIG. 4), all four lactic acid bacteria used in this experiment were Candida albicans. It was observed to adhere well to bacteria, and in particular to hyphae, suggesting that it would affect the growth of Candida albicans.

TABLE 4 Antibacterial activity against Candida albicans of four strains

Through the above experiments, it was shown that the four strains used in the experiment inhibit the proliferation of Candida albicans or adhere to vaginal cells. Inhibition and coagulation ability of vaginitis bacteria were different according to strains of lactic acid bacteria, in particular, Lactobacillus sakei In the case of ATG-K3, the antibacterial activity against Candida albicans was relatively low, but the aggregation ability against Candida albicans was the highest among the tested strains. Therefore, in consideration of the characteristics of each strain was expected to have a high effect on vaginitis bacteria when used in combination of several strains.

8. Animal test results

The following experiment was conducted to confirm the effects of four lactic acid strains on the proliferation of vaginitis bacteria through animal experiments. Seven week-old female mice (BALB / c mice) were purchased and allowed to acclimate for one week, weighed, negative control group administered only PBS instead of candida albicans and lactic acid culture mixture, administered Candida albicans and PBS instead of lactic acid culture mixture. The positive control group, Candida albicans and lactobacillus culture mixture was divided into experimental groups. In each group, 0.1 mg of beta-esteradiol 3-benzonate (Sigma E8515) dissolved in soybean oil (Sigma E7381) was administered intraperitoneally three days before the start of the experiment and at the end of the experiment every two days. Hormone regulation and estrous cycle. Candida albicans was directly infected into the vagina at the concentration of 1 × 10 ⁵ cfu / 10 uL at the start of the experiment, and then 1 × 10 ⁶ cfu / 10 uL of lactic acid bacteria culture mixture was administered. Lactobacillus culture mixture is Lactobacillus sakei ATG-K3, Lactobacillus sakei Four strain cultures of ATG-K14, Lactobacillus plantarum HAC01 and Lactobacillus rhamnosus BFE5264 were mixed and used for the experiment. Lactobacillus culture mixture was administered intravaginally once a day for 4 days from the beginning of the experiment. On the 4th day, the uterus and vagina were dissected, and then crushed with an ultrasonic crusher, centrifuged at 13,000 rpm for 10 minutes, and only the supernatant was separated. . After that, DNA was extracted from the supernatant and quantitatively analyzed using a real time PCR kit for Candida albicans detection. If the Ct value was 35 or more according to the kit manufacturer's manual, it was negatively determined.

As a result of the real time PCR experiment (FIG. 5), the candida albicans was detected in all the experimental mice in the positive control group which was not administered the lactic acid bacteria culture mixture, while the detection amount of the Candida albicans was decreased in the experimental group administered the culture mixture of the four lactic acid bacteria. It was. As a result of the quantitative analysis, about 2.6x10 ⁵ ± 0.03 cfu of Candida albicans was detected in the positive control group, while the experimental group detected about 2.5x10 ⁴ ± 0.33 cfu of bacteria and showed a significant difference (p = 0.007). It was confirmed in animal experiments that the culture mixture of the four lactic acid strains used in this experiment could inhibit the proliferation of Candida albicans, a vaginitis bacterium. In the negative control, Candida albicans was not detected as the Ct value of all the mice was 35 or more.

In addition, the present invention may provide a probiotic formulation comprising at least one selected from the group consisting of the strain, the combination of the strains, the culture of the strain, the concentrate and dried product of the culture as an active ingredient.

The probiotic formulations can be prepared and administered in a variety of formulations and methods according to methods known in the art. For example, the strains of the present invention, their cultures, their concentrates or dried products thereof can be mixed with carriers commonly used in the pharmaceutical field to provide powders, liquids and solutions, tablets, capsules. It may be prepared and administered in the form of a capsule, syrup, suspension or granule. The carrier may be, for example, but is not limited to, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a dye, a perfume, and the like. In addition, the dosage may be appropriately selected depending on the degree of absorption, inactivation rate, excretion rate, age, sex, breeder, condition, and severity of disease of the active ingredient in the body.

In addition, the present invention provides a health functional food composition for preventing or improving obesity comprising at least one selected from the group consisting of the strain, the combination of the strains, the culture of the strain, the concentrate of the culture and dried products as an active ingredient. to provide.

The dietary supplement composition of the present invention may be prepared in any one formulation selected from the group consisting of tablets, granules, powders, capsules, liquid solutions and rings. Health functional food composition according to the present invention may be formulated in the form of powder, liquid, tablets, soft capsules, granules, tea bags, instant tea or drink including the strain as an active ingredient. The content of the strain as an active ingredient can be suitably determined according to the purpose of use (prevention or improvement). In addition, the food composition according to the present invention may contain, in addition to the strain, other components that can give a synergistic effect to the main effect, preferably within the range of not impairing the main effect of the present invention.

The food composition formulated in such a form may be added to the food as it is, or used with other foods or food ingredients, and may be appropriately used according to conventional methods. Examples of foods include drinks, meats, sausages, breads, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, dairy products including other noodles, gums, ice creams, various soups, beverages, alcoholic beverages and vitamin complexes. , Dairy products and dairy products, and all functional foods in the conventional sense.

When the food composition of the present invention is a beverage, it contains the strain as an essential ingredient in the indicated ratio, and other components used for the purpose of preparing other beverages are not particularly limited, and various flavors or natural ingredients such as ordinary beverages are used. Carbohydrates and the like may be included as additional components. Examples of such natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a flavoring agent other than the above-mentioned, a natural flavoring agent, a synthetic flavoring agent, etc. can be used. The proportion of such natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition, the food composition of the present invention is a variety of nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, colorants and enhancers (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and salts thereof , Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the food composition of the present invention may contain a pulp for producing natural fruit juice and fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0.1 to about 20 parts by weight per part by weight of the strain of the invention.

The present invention also provides a pharmaceutical composition for the prevention or treatment of obesity, containing at least one selected from the group consisting of a culture of the strain, a concentrate of the culture and a dried product as an active ingredient.

The throughput of the pharmaceutical composition for preventing or treating obesity used in the present invention should be a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level may refer to an individual type and severity, age, sex, It can be determined according to the type of virus infected, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, factors including the drug used concurrently, and other factors well known in the medical arts. Effective amounts may vary depending on the route of treatment, the use of excipients, and the possibility of use with other agents, as will be appreciated by those skilled in the art.

Pharmaceutical compositions for the prevention or treatment of obesity of the present invention can be prepared in pharmaceutical formulations using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. In the preparation of the formulation, it is preferred that the active ingredient is mixed or diluted with the carrier or enclosed in a carrier in the form of a container.

Accordingly, the pharmaceutical composition for preventing or treating obesity of the present invention may be formulated in the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and the like, external preparations and patches according to a conventional method. It may be used, and may further comprise a suitable carrier, excipient or diluent conventionally used in the preparation of the composition.

For example, carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium Phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used.

In addition, the strains described herein may be contained in various forms of products, such as dietary supplements, beverages, fermented foods including fermented dairy products, and the like.

Claims (10)

Lactobacillus strain having proliferative inhibitory activity against vaginitis causative bacteria. The method of claim 1,
The strain is Lactobacillus strain Lactobacillus Skei ATG-K3 (Accession No. KCTC 18667P). The method of claim 1,
The strain is Lactobacillus strain Lactobacillus Skei ATG-K14 (Accession No. KCTC 18668P). The method of claim 1,
The strain is Lactobacillus genus strain, characterized in that Lactobacillus plantarum HAC01. The method of claim 1,
The strain is Lactobacillus genus strain, characterized in that Lactobacillus rhamnosus BFE5264. A composition combining two or more selected from the group consisting of Lactobacillus sakei ATG-K3, Lactobacillus sakei ATG-K14, Lactobacillus plantarum HAC01 and Lactobacillus rhamnosus BFE5264. A probiotic formulation comprising the strain or composition according to claim 1 as an active ingredient. A health functional food composition comprising the strain or composition according to claim 1 as an active ingredient. A pharmaceutical composition comprising the strain or composition according to claim 1 as an active ingredient. A health supplement comprising the strain or composition according to claim 1 as an active ingredient.

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