JP2023046282A - Candida-suppressing composition - Google Patents

Abstract

To provide a Candida-suppressing composition that can alleviate smell, itching and discharge from inside the woman's vagina.SOLUTION: The present composition is a Candida-suppressing composition that is characterized by containing, as an active ingredient, a lactic acid bacterium belonging to the genus Enterococcus. Preferably, the lactic acid bacterium is Enterococcus faecalis bacterium of nanoECF-9 (accession number NITE BP-02477). Further, preferably, the Enterococcus faecalis bacterium is contained by 100 billion to 10 trillion per gram. According to this, when the present composition is orally ingested by a woman as a food or the like, the growth of Candida, which causes vaginitis in women, is suppressed, and the smell, itching and discharge from inside the woman's vagina can be alleviated.SELECTED DRAWING: Figure 3

Description

TECHNICAL FIELD The present invention relates to a composition for inhibiting Candida that inhibits the growth of Candida, which occurs mainly in the vagina of women and causes vaginitis.

Conventionally, lactic acid bacteria have been widely used in cosmetics and foods (see Patent Documents 1 to 7).

For example, Patent Literature 1 discloses cosmetics and foods containing compositions containing dead lactic acid bacteria or processed products thereof.

Further, Patent Document 2 discloses a composition for promoting hyaluronic acid production that restores the amount of hyaluronic acid in the skin by containing lactic acid bacteria belonging to the genus Enterococcus faecalis.

Further, Patent Document 3 discloses an aqueous composition in which the dispersion stability of the dead lactic acid bacteria is improved by the combined use of dead lactic acid bacteria, xanthan gum and bittern.

In addition, Patent Document 4 discloses a lactic acid bacteria formulation for immunostimulation, in which a fermented wheat extract and Enterococcus faecalis bactericidal cells are mixed and which has anti-inflammatory, analgesic, and allergy-improving effects.

In addition, Patent Document 5 discloses a glycerol production promoter derived from S. epidermidis that contains dead cells of Enterococcus faecalis as an active ingredient, prevents skin dryness, and protects the skin from harmful bacteria on the skin. there is

In addition, Patent Document 6 discloses a moisturizing agent that enhances the moisturizing effect and eliminates skin irritation by using, as an active ingredient, a fermentation product obtained by allowing one or more types of lactic acid bacteria to act on aloe vera.

Further, in Patent Document 7, a culture containing β-glucan obtained by culturing a bacterium belonging to the genus Aureobasidium and heat-treated cells of lactic acid bacterium Enterococcus faecalis are contained as active ingredients, and have a moisturizing effect. BACKGROUND ART Skin moisturizing agents are known which are excellent in persistence and feeling of use.

Republished 2018-155660 Japanese Unexamined Patent Application Publication No. 2020-105100 Japanese Patent Application Laid-Open No. 2020-050598 Japanese Patent No. 5207313 Japanese Patent No. 6185041 Japanese Patent No. 3795011 Japanese Patent No. 4000078

"Functional food expected to improve oral candidiasis" Teikyo University Medical Mycology Research Vol.2 No.1:15-20,2011Issn1883-3195

However, all of these conventional compositions focus only on the effects of lactic acid bacteria on the outside of the skin, such as skin moisturizing effects, anti-inflammatory effects, and analgesic effects. No attention has been paid to alleviating the resulting odor, itching and discharge.

The present invention has been made in view of the technical background, and aims to provide a Candida-inhibiting composition capable of alleviating vaginal odor, itching, and vaginal discharge of women caused by vaginal candidiasis. and

The present applicant has conducted extensive research to reduce the odor, itching, and vaginal discharge caused by vaginal candidiasis in women, and discovered that lactic acid bacteria are effective against oral candidiasis. bottom. Oral candidiasis is caused by overgrowth of the Candida bacteria that live in the human oral cavity, and if the symptoms worsen, there is a risk of causing pneumonia and oral cancer. It has been studied that a functional food containing it improves this disease (for example, see Non-Patent Document 1 above).

The applicant conjectures that among the lactic acid bacteria that are effective against oral candidiasis, there may be those that are equally effective against vaginal Candida bacteria that cause vaginitis in women. As a result of verifying whether it can reduce the odor, itching, and vaginal discharge of women, the present invention was achieved.

Specifically, Candida is a resident fungus that inhabits the human skin and vagina, but it is a type of fungus, and in a harsh environment, it exhibits a round trait called yeast type and becomes dormant. Since it takes However, administration of anticancer drugs, antibacterial drugs, steroids, etc. disturbs the homeostasis of the human immune system and bacteria, and when placed in a moist environment at a temperature of 37 degrees, Candida becomes a highly pathogenic mycelium. It develops into a morphology and proliferates actively. Here, the inventors discovered that administration of lactic acid bacteria has the effect of suppressing the growth of highly pathogenic mycelium Candida, although the effect of suppressing the number of Candida bacteria is hardly observed.

That is, in order to achieve the above object, the composition for inhibiting Candida according to the present invention is a lactic acid bacterium belonging to the genus Enterococcus, preferably a lactic acid bacterium of Enterococcus faecalis, more preferably nanoECF-9 (accession number NITE BP-02477) ) as an active ingredient, and inhibits the growth of mycelial Candida to prevent or protect against vaginal candidiasis.

According to this, when a woman orally ingests the present composition, for example, as a food, it prevents or protects against vaginal candidiasis by suppressing the growth of Candida bacteria that cause vaginitis in women, and as a result, It can reduce odor, itching and discharge from the vagina of women.

Moreover, the Enterococcus faecalis bacterium may be heat-killed cells. This is due to the discovery that even non-living heat-killed cells of Enterococcus faecalis can suppress the growth of highly pathogenic mycelium Candida. (100 billion to 10 trillion) of the Enterococcus faecalis bacteria can be stably contained in the bath agent.

In addition, when 100 billion to 10 trillion Enterococcus faecalis bacteria are contained per 1 gram, it is possible to further reduce odor, itching and vaginal discharge in women.

According to the present invention, when a woman orally ingests the present composition, for example, as a food, it prevents or protects against vaginal candidiasis by suppressing the growth of Candida bacteria in the woman's vagina. It can reduce odor, itching, and discharge from the vagina.

In addition, since the present composition contains Enterococcus faecalis bacteria as an ingredient, in addition to the effect of reducing the odor, itching, and vaginal discharge of women described above, it also improves body odor and skin condition. , Intestinal regulation effect and premenstrual mental stability (relief of irritation), Allergy (hay fever, asthma, etc.) and / or atopy reduction by immunostimulatory effect, Reduction of viral infections through biological defense function, Metabolic The effect of suppressing the risk of syndrome can be obtained.

1 is a table showing the composition ratio of nanoECF-9, which is a material of the present composition. 1 is a table showing test results of nutritional analysis of nanoECF-9. 2 is a table showing the test results of nanoECF-9 product standards. FIG. 2 shows a method for producing nanoECF-9. FIG. 2 shows the experimental schedule for vaginal candidiasis mouse model. It is a figure which shows the C.albicans viable count in each sample administration mouse vaginal striatum. FIG. 1 shows C. albicans and leukocyte micrographs during mouse vaginal lavage. Fig. 10 shows the viable C.albicans count (retry) during vaginal lavage of each sample-administered mouse.

(First embodiment)
Next, a first embodiment of a composition for inhibiting Candida (hereinafter referred to as the present composition) according to the present invention will be described with reference to FIGS. 1 to 4. FIG.

The composition is an orally ingested granular health supplement containing lactic acid bacteria belonging to the genus Enterococcus as an active ingredient. In this embodiment, in addition to lactic acid bacteria belonging to the genus Enterococcus, collagen/tripeptides, indigestible dextrin, fermented milk powder, ceramide, vitamin C, citric acid, fragrance, and stevia are blended. In addition, other components may be included.

This lactic acid bacterium belonging to the genus Enterococcus is generally called enterococcus, and can be ordered from domestic and international distribution organizations such as ATCC, IFO, and JCM. Since this lactic acid bacterium is lactic acid bacterium commonly present in food, lactic acid bacterium used in food production, or lactic acid bacterium isolated from the feces of healthy individuals, there is no risk of side effects. In addition, since it can be easily obtained in large amounts by culturing, the use of microbial cells obtained by culturing is economical with low production costs.

Lactic acid bacteria belonging to the genus Enterococcus include Enterococcus faecalis and Enterococcus faecium. Among them, Enterococcus faecalis is preferably used. 02477) is used.

This nanoECF-9 (acceptance number NITE BP-02477) is mainly composed of lactococcal powder and dextrin, and has the composition ratio shown in FIG.

In addition, when this nanoECF-9 (acceptance number NITE BP-02477) was tested for nutritional analysis, as shown in Fig. 2, the water content per 100 g product was 4.0 g (atmospheric pressure drying method), and the protein content was 56.4 g (combustion method), 3.3 g of fat (acid decomposition method), 8.1 g of ash (direct incineration method), 28.2 g of carbohydrate (calculation based on food labeling standards), 7.5 g of sugar 7g (calculation formula according to food labeling standards), dietary fiber 20.5g (enzyme-gravimetric method), energy 327kcal (conversion factor according to food labeling standards), sodium 1.75g (atomic absorption spectrophotometry), salt equivalent has 4.45 g (calculated formula) of nutrients.

In addition, when nanoECF-9 (acceptance number NITE BP-02477) was tested for product specifications, as shown in FIG. (5 trillion) cells/g or more (DAPI staining method), general viable count of 3000 cells/g or less (standard agar medium culture method), less than 100 cells/g of fungi (potato dextrose agar medium culture method), E. coli Group negative (BGLB method), moisture content less than 7.0% (loss on drying method), arsenic (as As2O3) less than 2 ppm (ICP-OES), lead (as Pb) less than 20 ppm (colorimetric method) , the IL-12 induction activity was a measured value with a specific activity of 1.0 or more (in-house measurement method).

In addition, although the method for producing nanoECF-9 (accession number NITE BP-02477) is not particularly limited, for example, as shown in FIG. conduct an inspection. Next, after adjusting the medium of the original lactic acid bacterium, it is ingested and cultured under aerobic conditions (for example, by shaking culture or aeration stirring culture), and after the medium is sterilized, ingestion and culture are performed. Next, the cultured lactic acid bacteria are extracted by centrifugation, and an excipient is added to disperse them in the diluent. Next, after sterilizing and cooling the diluted liquid, it is pulverized by spray drying, and the resulting powder is sieved. Finally, the sieved powder is filled into containers, subjected to product testing, and then packaged and stored.

In addition, collagen tripeptide promotes the synthesis of calcium, which is the main component of bone, and thus has the effect of early healing of cartilage damage. In addition, since it has a smaller molecular structure than general collagen, it is quickly absorbed into the body and has a very high effect of repairing the above-mentioned cartilage damage.

Also, indigestible dextrin is a degradation product of indigestible starch that is not digested by human digestive enzymes. However, it can be broken down by the human intestinal flora and partly converted into energy. It is abundant in roasted dextrin, and the reducing terminal group of glucose is intramolecularly dehydrated during the thermal decomposition process of dextrin, and the dissociated glucose residue is randomly transferred to other hydroxyl groups. Generated as a result of a join. It is a type of water-soluble dietary fiber that is naturally contained in ripe fruits and the like, and when taken orally, it suppresses the absorption of sugar and fat, and suppresses the rapid rise in blood sugar level after meals. In the food industry, indigestible dextrin produced from the hydrolyzate of corn starch is distributed.

Fermented milk powder is obtained by freeze-drying fermented milk fermented with lactic acid bacteria and powdering it.

In addition, since ceramide is a major component of intercellular lipids and has a barrier function, it has the effect of protecting the skin from stimulation and the effect of moisturizing the skin. In addition, in this embodiment, rice-derived glucosylceramide is blended as ceramide.

In addition, since vitamin C is an essential nutrient for the synthesis of collagen, it has the effect of keeping the skin beautiful. In addition, since it is a nutrient that helps absorb iron, it also has the effect of preventing anemia.

Citric acid is an organic compound contained in citrus fruits and the like, and is one of hydroxy acids. Due to its refreshing acidity, it is often used as a food additive. The sourness of citrus fruits is mostly due to the taste of citric acid. It is also contained in large amounts in pickled plums. The aqueous solution exhibits weak acidity. It is a colorless or white solid at room temperature, and contains anhydride and monohydrate crystals. Both are non-volatile and odorless. The monohydrate melts at 100°C when heated and becomes the anhydrate with a melting point of 153°C when kept at 130°C. Above 175°C, it becomes aconitic acid through intramolecular dehydration. When citric acid is ingested, anaerobic respiration does not occur during exercise, so lactic acid is not produced and fatigue is reduced. It is also believed to aid in the decomposition of ethanol. It is generally known that an increase in anionic electrolytes in body fluid components such as citric acid and lactic acid causes a diuretic effect.

Flavoring agents are also food additives that impart some of the aroma and taste to foods. It is made by blending a large number of natural fragrances extracted from various plants and some animals, or chemically synthesized synthetic fragrances. Most natural fragrances are essential oils and resins extracted from plants. Steam distillation is the most commonly used method for extracting essential oils. However, some essential oils that are unstable against heat are obtained by squeezing the skins of citrus fruits or by extracting them with an organic solvent, such as essential oils of flowers. Synthetic fragrances are components of natural fragrances or chemically synthesized compounds that do not exist in nature but are effective as fragrances. Since it consists of a single compound, it is sometimes called a single fragrance as opposed to a compounded fragrance, or an aroma chemical because it is chemically synthesized. Raw materials for synthetic fragrances include ethylene and acetylene obtained from petroleum, as well as terpene compounds separated from essential oils and fatty acids obtained from fats and oils. Synthetic fragrances are obtained by chemically reacting these compounds.

Also, stevia is a perennial plant belonging to the Asteraceae family and genus Stevia, which is native to South America including Paraguay. The plant is 50 cm to 1 m tall, and the stem is covered with fine white hairs. Small white flowers bloom at the tips of branches from summer to autumn. Also known as Amahastevia. Since it contains terpenoid glycosides such as stevioside and rebaudioside A as sweetening components, it is used as a sweetener. Stevioside is also called "Stevioside". Stevioside is 300 times sweeter than sucrose, and has long been used in Paraguay, one of the countries of origin, to add sweetness to mate tea and as a medicinal herb. Even today, it is sometimes used as a substitute for sugar in diet foods and menus for diabetics. Orally ingested stevioside is not absorbed in the small intestine and is degraded to steviol by intestinal bacteria. This steviol is the final product and is excreted unchanged.

The composition is packaged in single-use type stick-shaped packaging bags, and each packaging bag is opened and used.

Thus, when the composition is orally ingested by a woman as food or the like, it suppresses the growth of Candida, which causes vaginitis in women, and reduces the odor, itching, and vaginal discharge produced in the woman's vagina. can do.

In addition, since the present composition contains Enterococcus faecalis bacteria as an ingredient, in addition to reducing the above-described odor, itching, and vaginal discharge, body odor, skin condition, and intestinal regulation can be improved. Action and its premenstrual mental stability (relief of irritation), allergy (hay fever, asthma, etc.) and / or atopy reduction by immunostimulatory action, reduction of viral infections through biological defense function, risk of metabolic syndrome A suppression effect is obtained.

In the above embodiment, the composition contains 5.0×10 (5 trillion) nanoECF-9 (accession number NITE BP-02477) per gram. It is sufficient if it contains 1 to 10 trillion.

(Second embodiment)
Next, a second embodiment of the composition for suppressing Candida according to the present invention will be described. In the following, only configurations different from the above embodiment will be described, and descriptions of the same configurations will be omitted.

The composition is a syrup that is orally ingested by being sprinkled over vegetables and fruits, and contains lactic acid bacteria belonging to the genus Enterococcus as an active ingredient. In addition to lactic acid bacteria, galacto-oligosaccharides, water-soluble dietary fiber, acidulant, grapefruit seed extract, and fragrance are added.

Among these, lactic acid bacteria, in addition to the above effects, increase IgA (immune globulin), which neutralizes bacteria and viruses, by ingesting vegetables and fruits covered with syrup, further improving immune function. Let

In addition, since galacto-oligosaccharides are difficult to be degraded by gastric acid and digestive enzymes, they can easily reach the intestines, increase bifidobacteria in the intestines, create an environment where lactic acid bacteria can work easily, relieve constipation, and have a diet effect and beautiful skin effect. Let

In addition, water-soluble dietary fiber serves as nutrients for lactic acid bacteria in the intestine, and along with galacto-oligosaccharides, creates an environment where lactic acid bacteria can easily work. In addition, it suppresses the rapid increase in blood sugar level after meals.

An acidulant is a food additive that imparts a sour taste to food. As of June 2020 in the Consumer Affairs Agency's "Food Labeling Standards", there are 25 types of food additives called "acidulants", such as acetic acid, sodium acetate, citric acid, succinic acid, gluconic acid, gluconic acid, Nodeltalactone, tartaric acid, lactic acid, malic acid, phosphoric acid and the like are included.

Grapefruit seed extract is also a liquid extract of grapefruit seeds, pulp and tunica. Grapefruit seeds, juice-free pulp are ground and then mixed with glycerin. The commercial version is made by grinding together seeds, pulp, and glycerin.

Flavoring agents, as mentioned above, are food additives that impart some of the aroma and taste to foods.

In the above embodiment, the present composition was explained as a syrup, but it is not limited to this, and is intended for all foods that are orally ingested. For example, beverages such as soft drinks (e.g. juice, smoothies, coffee, tea), fermented foods such as natto, yogurt, cheese, semi-solid foods such as dietary supplements, confectionery (e.g. candy, snacks, gum, baked goods) , fresh confectionery, etc.), powdered foods such as dried vegetable powder, dried grain powder, dried seaweed powder, and the like.

(Third embodiment)
Next, a third embodiment of the composition for suppressing Candida according to the present invention will be described.

The present composition is a granular health supplement that is orally ingested by being sprinkled or mixed with food, and contains lactic acid bacteria belonging to the genus Enterococcus as an active ingredient. In addition to lactic acid bacteria, it contains lactose, dextrin, fructo-oligosaccharide, tricalcium phosphate, and guar gum.

Among them, lactose is a disaccharide low-intensity sweetener and has a sweetness 0.4 times that of sucrose. It is hydrolyzed to galactose and glucose by β-galactosidase. Some of the lactose contained in breast milk reaches the large intestine without being degraded, and acts to increase bifidobacteria in the infant's intestines.

Dextrins are also low molecular weight carbohydrates obtained from the hydrolysis of starch or glycogen. It has a molecular structure in which α-glucose is polymerized through α-glycosidic bonds. It is classified as a polysaccharide, intermediate between starch and maltose, and has many hydroxy groups in its structure, making it water-soluble. However, the solubility in water decreases as the molecular weight increases. In vivo, it is degraded to maltose by amylase and finally to glucose, but there are some components that are difficult to decompose by amylase, and purification of these components yields indigestible dextrin.

Fructo-oligosaccharides are indigestible oligosaccharides in which 2 to 4 molecules of fructose are bound to glucose. When taken orally, it is not degraded by human digestive enzymes, so it reaches the large intestine as it is without being decomposed in the stomach or small intestine. It has about half the calories of sugar and is found in vegetables such as fresh onions and burdock.

Also, tricalcium phosphate is a salt of phosphoric acid and calcium. It occurs naturally as apatite, is widely distributed in soil, and is an essential component for plant growth. It is also the substance obtained when burning bones.

Guar gum is a water-soluble natural polysaccharide obtained from the so-called endosperm (more precisely, cotyledons) of guar beans. It is a polysaccharide having two molecules of mannose bound in a straight chain and one molecule of galactose as a side chain, and has a molecular weight of 200,000 to 300,000. It is recognized as a food additive and is widely used as a thickener, stabilizer, and gelling agent. It is known to have physiological effects such as an action to suppress blood sugar level elevation, a cholesterol-lowering action, and an improvement in bowel movements.

In the above embodiments, the present composition was described as a granular health supplement, but the present composition is not limited to this, and is a powdered health supplement, a solid block-shaped health supplement, a liquid health supplement, and a gel. It may be in the form of health supplements or the like, or may be orally ingestible other than health supplements. The present composition may also be used as a skin-contacting agent such as a coating agent for the skin (excluding bathing agents).

Although the embodiments of the present invention have been described above with reference to the drawings, the present invention is not limited to the illustrated embodiments. Various modifications and variations can be added to the illustrated embodiment within the same scope as the present invention or within an equivalent scope.

<Checking the operation of the experimental system>
When 6.0×10 ⁶ cfu of Candida albicans was inoculated into the vagina of mice, an average of about 2.36×10 ⁶ cfu/mouse of C. It was confirmed that C. albicans was growing. Clotrimazole 1.0mg/mouse, an antifungal agent, was administered intravaginally three times for three days from the day after intravaginal inoculation of C. albicans. The results were performed as expected and the experimental system was found to be reliable.

<Action of nanoECF>
Under the above conditions, even if 20 microliters of each concentration of nanoECF was administered four times for 4 days from 3 days before intravaginal inoculation of C. albicans to about 2 hours before inoculation, the number of C. albicans in the vaginal washings was significantly increased. It was found that no significant decrease was observed. In the high-concentration nanoECF (300mg/ml) intravaginal administration group, the C. albicans count was shown to be slightly lower in this study, so a retest was performed.
No significant reduction in albicans count was observed. The lactic acid bacterium used in this experiment was nanoECF-9.

In the Diffiquick-stained photographs of the vaginal lavage fluid, in the Saline-administered control group, hypha-like masses and yeast-like C. albicans were observed around the hyphae in the vaginal mucosa. On the other hand, in some mice intravaginally administered with high concentration nanoECF (300mg/ml), short mycelium C. albicans and many monocytes and neutrophil-like leukocytes were observed in the lavage fluid. Regarding the presence or absence of Candida hyphae observed in the stained specimens of both groups, there was a tendency that there were more individuals in the nanoECF 300mg/ml intravaginal administration group in which mycelial growth was not observed compared to the control group. Although no significant difference was observed between the two in the retest, statistical processing using stained specimens, including the main test and the retest, showed a significant difference between the two (chi-square test, p=0.021).

These results suggest that nanoECF may inhibit Candida hyphal development and exhibit preventive and protective effects against vaginal candidiasis mouse models.

In this study, intravaginal administration of nanoECF did not reduce viable Candida counts in lavage fluids of a mouse model of vaginal candidiasis, and it was not possible to conclude that there was a clear protective effect. However, monocytes and neutrophil-like leukocytes were observed in the vaginal washings of mice administered 300 mg/ml nanoECF, suggesting biological reactions. In the Saline-administered control group, such a reaction was poor, and mycelium-like masses of C. albicans were observed in some specimens. C. albicans is known to be highly tissue invasive and highly pathogenic when it exhibits hyphal growth, suggesting that nanoECF may have protective effects against vaginal candidiasis. In the future, it will be necessary to examine in detail the images of C. albicans in vaginal tissue sections and washings. A discussion of additional experiments to obtain clearer conclusions is also included.

<Background>
We have previously reported that heat-killed Enterococcus faecalis cells protect against systemic Candida infection and also show protective effects against oral candidiasis in a mouse model. Since animal models and human intervention studies have already reported the protective effect of vaginal administration and oral intake of lactic acid bacteria against vaginal candidiasis, it is possible that Enterococcus nanoECF can also protect against vaginal candidiasis. I want to clarify.

<Purpose>
To investigate whether intravaginal pre-administration of nanoECF inhibits colonization and growth of C. albicans in the vagina.

<Materials and methods>
1. Materials
For each test, 20 mg of clotrimazole reagent (Fuji Film Wako Pure Chemical Industries, Ltd.), which is used as an azole antifungal drug, was dissolved in 20 µl of 100% ethanol in advance, and then dissolved in 180 µl of Physiological saline was added to adjust the concentration to 50 mg/ml. As enterococci powder, nanoECF manufactured by RAPHAS JAPAN Co., Ltd. was diluted with physiological saline to each concentration and used.

2. Bacterial strains used
For Candida albicans, the TIMM2640 clinical isolate (-80°C cryopreservation) stored at Teikyo University Medical Mycology Research Center was thawed according to the prescribed method and used for each test.

3. Animals
BALB/c mice (Oriental Yeast), 6 weeks old, female, 5-10 per group were used for infection experiments. After moving into the shed, the mice were allowed to freely ingest solid diet for mice (MF diet, oriental yeast), and after preliminarily feeding them for one week, they were subjected to experiments. This animal experiment has been approved by the Ethics Committee on Animal Experiments.

4. Measuring the effect of nanoECF using a mouse model of vaginal candidiasis
4-1. Main Test Each concentration of nanoECF was administered intravaginally to mice according to a protocol for a predetermined vaginal candidiasis model. The effect of nanoECF was evaluated by the number of viable C. albicans cells in the vaginal washings and microscopic cytological images. The actual time schedule is shown in Figure 5 (Fig.1). From one week before infection with C. albicans, tap water containing 4.13 mg/ml of chlortetracycline hydrochloride (CTC soluble powder “SP”, Sumika Enviroscience Co., Ltd.) was given ad libitum during the experiment. 0.125 mg of β-estradiol-17-valerate (Tokyo Kasei Kogyo Co., Ltd.) was subcutaneously injected twice, 3 days before and 1 day after infection, to bring the mice into estrus. From 3 days before C. albicans infection to 4 times on the day of infection, nanoECF 20 µl diluted to each concentration was intravaginally administered to each mouse. In the control group, the same amount of physiological saline was intravaginally administered. C. albicans was suspended in RPMI1640 medium containing 2.5% fetal calf serum (FCS) at 3×10 ⁸ cells/ml and inoculated intravaginally at 20 μl (6×10 ⁶ cells/mouse). A group of positive control mice received intravaginally 20 µl of clotrimazole (1.0 mg/mouse) at a concentration of 50 mg/ml three times, 1 to 3 days after infection with C. albicans. Four days after infection, the vagina was washed with physiological saline. The vaginal wash was diluted, applied to Candida GS medium, and cultured at 37°C for about 18 hours. On the next day, the number of colonies was measured, and the viable Candida count in the vaginal wash was calculated.

4-2. Retest
In this test described in 4-1., weight loss was observed in some mice, and some mice were excluded from evaluation on the final day of the test. One of the reasons for this was thought to be a decrease in the amount of drinking water, so the same test was conducted with the amount of chlortetracycline hydrochloride added to the drinking water bottle changed to half the concentration of this test.

5. Preparation of vaginal wash smears and examination of mycelial growth
Vaginal lavage fluid collected 4 days after C. albicans infection was centrifuged at 1500 rpm for 5 minutes, the supernatant was removed, fetal calf serum (FCS) was added to the sediment, and a smear was prepared. After fixation with methanol, Diff-Quick staining (Sysmex Corporation), which is a rapid and simple method of May-Giemsa staining, was performed, and histopathological observation was performed. In addition, specimens from each mouse were examined to determine the presence or absence of hyphae. The efficacy of nanoECF was evaluated from the number of viable Candida bacteria in the vagina and the microscopic image clarified in this experiment.

6. Statistical processing
All viable cell count values for each animal group were expressed as mean±standard deviation (mean±SD). Dunnett t-test was used for comparison test. Fisher's exact method and chi-square test were used for comparative tests on the presence or absence of mycelial growth. In both cases, a risk rate of 5% or less (p < 0.05) was considered statistically significant.

<Results>
(Protective effect of nanoECF in mouse vaginal candidiasis model experimental system)
This test was conducted according to a predetermined mouse vaginal candidiasis model, and the infection-preventing action of nanoECF at each concentration was examined. Figure 6 (Fig.2) shows the number of viable C. albicans bacteria in vaginal washes 4 days after C. albicans infection. The average number of viable C. albicans bacteria per mouse in the saline-administered Control group was 2.36 × 10 ⁶ cfu, while the clotrimazole-administered positive Control group averaged 3.21 × 10 ⁵ cfu and vaginal lavage fluid. A significant decrease in viable C. albicans count was observed in the medium (*p<0.05). Since the effect of clotrimazole was clearly seen, it can be said that the reliability of the evaluation system of this test system was recognized.

We investigated the effectiveness of nanoECF in the above test system. Number of viable C. albicans cells per mouse in vaginal washes of groups of mice treated with nanoECF suspension diluted to concentrations of 3 mg/ml, 30 mg/ml and 300 mg/ml prior to infection with C. albicans. averaged 2.78×10 ⁶ cfu, 2.27×10 ⁶ cfu and 1.32×10 ⁶ cfu, respectively. In the 300 mg/ml nanoECF-administered group, the viable C. albicans count in the vaginal lavage fluid of 3 out of 7 mice was 6.00-6.90 × 10 ⁵ cfu, lower than 10 ⁶ cfu. No statistically significant difference was observed.

Fig. 3 shows typical photographic images of vaginal wash smear staining specimens of Saline-administered and nanoECF 300 mg/ml-administered mice. In the vaginal lavage fluid (a) of Saline-treated mice, there were distinct hyphae (black arrows) and yeast-shaped C. albicans images (dotted black arrows) around the hyphae. On the other hand, in the vaginal lavage of mice treated with 300 mg/ml of nanoECF, short hyphae of C. albicans were observed in some cases, but large hyphal clusters such as a) were not observed. In some cases, many images were observed (b). In order to statistically evaluate this observation result, slide specimens were examined for the Saline-administered group and the nanoECF 300 mg/ml-administered group to confirm the presence or absence of mycelial C. albicans images. The results are shown in Table.1. Mycelium was observed in 7 out of 9 specimens of the control group, but only 2 out of 7 specimens of the vaginal washes from the mouse group administered 300 mg/ml of nanoECF. When these significant differences were examined, it was found that there was a tendency that mycelium C. albicans was not observed when nanoECF 300 mg/ml was administered, although no significant difference was observed. (The chi-square test showed a significant difference at p<0.05, but Fisher's exact test did not show a significant difference. P = 0.126),

Based on the above, in the vaginal lavage of mice treated with high-concentration nanoECF (300 mg/ml), clear C. albicans hyphae tended to be absent, and neutrophil-like white blood cells were observed. However, no significant reduction in the number of viable bacteria in the vaginal washes was observed, indicating that a clear protective effect against the mouse model of vaginal candidiasis could not be confirmed.

(Re-examination of protective effect of nanoECF in mouse vaginal candidiasis model experimental system)
From the above results, it was found that vaginal lavage specimens from mice treated with nanoECF 300mg/ml tended to lack clear mycelial growth of C. albicans, suggesting that a protective effect against vaginal candidiasis mouse models can be expected. Therefore, I retested. In the above-mentioned test, the mice did not drink tetracycline-added drinking water very much, and some mice showed significant weight loss. Figure 8 (Fig. 4) shows the results of viable counts of C. albicans in mouse vaginal washes.

In this retest, it was found that the number of C. alibcans in the vaginal washings was about 1/10 lower in the control mice that received intravaginal administration of Saline under the conditions of the retest compared to Fig. 6 (Fig.2). In addition, there was also a large variation between groups, and C. albicans was not detected in the vaginal washings of all 5 mice in 2 cages (5 mice per cage) with 30 mg/ml nanoECF. In the clotrimazole-administered group, no bacteria were detected in the vaginal lavage fluid of one of the four mice, but the other three mice had an average of 33 colonies (1.6×10 ⁵ cells/ml) in the 1000-fold dilution. There was no significant difference between the groups in the number of viable Candida bacteria detected.

When the presence or absence of mycelium-shaped C. albicans images in the slide specimens of the Saline-administered group and the nanoECF 300mg/ml-administered group was examined, 7 out of 10 specimens of the control group had mycelium-shaped C. albicans images. No significant difference was observed in 4 out of 10 vaginal lavage specimens from mice treated with 1/ml. Therefore, pretest and retest sample specimens were summarized and evaluated for the presence or absence of the mycelium form of C. albicans. The results are shown in Table.2. In the Control group, 14 out of 19 specimens had hyphal forms. On the other hand, 6 out of 17 vaginal lavage fluid specimens of the mouse group administered nanoECF 300 mg/ml showed a significant difference (p=0.021 chi-square test).

Based on the above, the administration of 300 mg/ml of nanoECF did not reduce the number of viable bacteria in the vaginal washes of the vaginal candidiasis mouse model in the retest. However, in vaginal lavage specimens from a combination of the above-mentioned test and retest, administration of nanoECF 300 mg/ml significantly reduced mycelium-shaped C. albicans images in the vaginal lavage, suggesting that the vaginal candidiasis mouse model was found to have a protective effect against

<Discussion>
In this study, intravaginal administration of nanoECF prior to C. albicans infection did not reduce the number of viable C. albicans bacteria in vaginal washes in a mouse model of vaginal candidiasis, but inhibited the growth of mycelial C. albicans. made it clear that The effective dose of nanoECF for bacterial count suppression was estimated from Fig. 6 (Fig.2) to be about 300 mg/ml, but as shown in Fig. 8 (Fig.4), this trend was not observed in the retest. I didn't. On the other hand, as a result of detailed observation of the pathological specimens of the vaginal washings, as can be seen from Tables 1 and 2, it was found that the 300mg/ml nanoECF-administered group did not show clear hyphae compared to the Saline-administered control group. This is the most important finding obtained in this study.

Mycelial development of C. albicans is considered to be one of the conditions for C. albicans to enter the mucous membrane and to be an indicator of disease aggravation. It has been reported that it has a therapeutic effect on We have already reported that heat-killed cells of E. faecalis ameliorate tongue symptoms and suppress hyphal development against C. albicans infection in a mouse model of oral candidiasis. . In the oral cavity, there is a constant flow of saliva, and yeast-like bacteria are swept away, so the conditions are somewhat different from vaginal infections. .

Claims (5)

A composition for inhibiting Candida, comprising a lactic acid bacterium belonging to the genus Enterococcus as an active ingredient, and preventing or protecting against vaginal candidiasis by inhibiting the growth of mycelial Candida. The composition for inhibiting Candida according to claim 1, wherein the lactic acid bacterium is Enterococcus faecalis. The composition for inhibiting Candida according to claim 2, wherein the Enterococcus faecalis is nanoECF-9 (accession number NITE BP-02477). 4. The composition for suppressing Candida according to claim 2 or 3, wherein the Enterococcus faecalis is heat-killed cells. 5. The composition for inhibiting Candida according to claim 4, wherein said Enterococcus faecalis bacterium contains 100 billion to 10 trillion per gram.

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