WO2022255569A1 - Powdered feminine cleanser containing lactobacillus rhamnosus and lactobacillus acidophilus - Google Patents

Abstract

The present invention relates to a powdered feminine cleanser comprising Lactobacillus rhamnosus and Lactobacillus acidophilus at a predetermined ratio plus rice starch, betulin, alpha-glucan oligosaccharide, and silica. The cleanser removes vulvar odor and relieves itching by inhibiting Candida albicans strain, and improves skin elasticity, moisture, luster, clarity, and density, thereby contributing to an improvement in women's health and quality of life.

Description

Powder type feminine cleanser containing Lactobacillus rhamnosus and Lactobacillus acidophilus

The present invention relates to a powder type feminine cleanser containing Lactobacillus rhamnosus and Lactobacillus acidophilus.

The microbial environment in a woman's vagina is very important to women's health. In particular, because the female vulva is close to the anus and a moist and warm environment is maintained by secretions, it is a very good environment for pathogens to grow. Lactic acid bacteria reside inside the vagina and release lactic acid and other antibacterial substances during the growth process to maintain an optimal vaginal pH of 4.5 or less to suppress the growth of pathogenic strains. However, when these lactic acid bacteria lose their dominance by various bacteria such as anaerobic bacteria or candida, the balance of the microbial environment in the vagina is disturbed, increasing the possibility of getting vaginitis.

Vaginitis causes premature birth, low birth weight, and weakens the immune system. Vaginitis in women is largely classified into three types: Bacterial vaginosis caused by bacterial infection, Vulvovaginal candidiasis caused by Candida fungi, and Trichomonas vaginitis caused by infection with the protozoan Trichomonas. Bacterial vaginosis is a disease in which the acidity of the vagina increases and the microbial environment changes due to the growth of specific bacteria in the female vagina. It is one of the most common inflammations in women of childbearing age, usually with increased white or off-white vaginal discharge and odor.

As antibiotics used to treat bacterial vaginosis, metronidazole and clindamycin, which are susceptible to anaerobic strains, are commonly used. Metronidazole affects the DNA replication mechanism, and clindamycin affects the protein synthesis process, preventing bacteria from multiplying. However, the use of these antibiotics may affect the survival of not only pathogenic bacteria but also beneficial lactic acid bacteria. In particular, care should be taken as it affects the lactobacilli in the vagina.

Candida vaginitis is a common disease that 75% of women experience at least once in their lifetime, and 45% of women experience it at least twice a year. Candida albicans, a fungus, is the causative agent, and when antibiotics are used for a long time, or when there is pregnancy or diabetes, the concentration of lactic acid bacteria and normal vaginal bacteria in the vagina is lowered, resulting in overgrowth of fungi, resulting in this disease. Symptoms of candida vaginitis include itching of the vulva, vaginal discharge with a bad odor, pain during sexual intercourse, pain during urination, etc., and are treated by taking antifungal drugs or inserting vaginal tablets. May cause complications, etc. In addition, Candida vaginitis often recurs, in which case persistent vulvar itching occurs.

Recently, orally administered lactic acid bacteria have been used to prevent vaginitis and improve vaginal health. If you take lactic acid bacteria regularly, the lactic acid bacteria survives through the intestines and into the vagina, and after settling, proliferates as beneficial bacteria necessary for vaginal health. However, since the ingested lactic acid bacteria must pass through the digestive tract, there is a problem in that the number of guaranteed bacteria settled in the vagina is small compared to the number of injected bacteria.

In addition, in order to create a stable vaginal microbial environment and effective sterilization of pathogenic bacteria, above all, the number of lactobacillus species, dominant species, core species and their colony ratio in the vaginal flora, and the total number of lactic acid bacteria that are particularly effective against vaginitis-causing bacteria such as Candida A study on the characteristics is needed.

On the other hand, Lactobacillus rhamnosus is a Gram-positive lactic acid bacterium most commonly found in healthy female genital tract-urinary tract. It is known as a strain that helps to grow, and also contributes to basic colon health. However, Lactobacillus rhamnosus alone has a problem in that its antibacterial activity against vaginitis-causing bacteria such as Candida is limited.

Korean Registered Patent No. 2139687 discloses Lactobacillus rhamnosus MG4288 strain (Accession No. KCTC13823BP) having antibacterial activity against Gardnerella vaginalis and Candida albicans , strain culture medium and A cell-free supernatant of the strain is described. In addition, Korean Patent Registration No. 1098241 discloses Lactobacillus sp. having an activity to inhibit urinary tract infection and candida vaginitis. H. Y 7801 and products containing it as an active ingredient are described, and Korean Patent Registration No. 1784847 relates to a composition containing salt, sugar and lactic acid bacteria, salt selected from molten salt, refined salt or sun salt; (2) sugars such as xylose and arabinose; And (3) Lactobacillus acidophilus (KCTC No. 3164), Bifidobacterium longum subsp. longum (KCTC No. 3128), Streptococcus sp; KCTC No. 5644) and Bacillus cereus (KCTC No. 13123). A pharmaceutical composition for preventing and treating vaginitis containing a combination consisting of one or more lactic acid bacteria selected from the group consisting of strains as an active ingredient is disclosed. However, these lactic acid bacteria compositions are not described in the strong antibacterial activity by the optimal combination of vaginal lactic acid bacteria disclosed in the present invention.

[Prior art literature]

[Patent Literature]

Korea Patent No. 2139687, Lactobacillus MG4288 for treatment or improvement of vaginitis having antibacterial effect on Gardnerella vaginalis and Candida albicans, registered on July 24, 2020.

Korea Patent No. 1717672, Lactobacillus sp. having activity to inhibit urinary tract infection and candida vaginitis. HY 7801 and products containing it as an active ingredient, registered on December 19, 2011.

Korean Patent Registration No. 1784847, composition containing lactic acid bacteria for preventing and treating vaginitis and its use, registered on September 28, 2017.

It is to provide a powdery feminine cleanser containing Lactobacillus rhamnosus and Lactobacillus acidophilus.

The powdered female cleanser of the present invention is Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 mixed lactic acid bacteria, rice starch, betulin, alpha-glucan oligosaccharide and a powdery feminine cleanser containing silica.

Lactobacillus rhamnosus and Lactobacillus acidophilus of the powdered feminine wash of the present invention are preferably Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14. .

The Lactobacillus rhamnosus HN001 ( Lactobacillus rhamnosus HN001) and Lactobacillus acidophilus La-14 ( Lactobacillus acidophilus La-14) mixed lactic acid bacteria, Lactobacillus rhamnosus HN001 and Bacillus acidophilus La-14 are 1: 2 It can be mixed in a ratio of ~6, preferably in a ratio of 1:4.

The Lactobacillus rhamnosus HN001 ( Lactobacillus rhamnosus HN001) and Lactobacillus acidophilus La-14 ( Lactobacillus acidophilus La-14) mixed strains may be included in an amount of 1 to 50% (hereinafter, weight %) based on the total weight of the composition. . Although not limited thereto, the mixed strain may not exhibit antifungal activity against Candida strains if it is less than 1% based on the total weight of the composition, and if it exceeds 50%, the effect of the concentration used may not be economical.

The powdery female cleanser is characterized in that it has at least one effect selected from the group consisting of removing odor of the vulva, relieving itching, improving skin elasticity, improving skin moisturization, improving skin radiance, improving skin clarity, and improving skin density. Odor or itching in the vulva can be caused by bacterial vaginosis, an increase in secretions with an unpleasant odor caused by yeast infections such as Candida, and odors caused by bacteria growing in synthetic fiber underwear. It is a common phenomenon experienced by most women. . The feminine cleanser composition of the present invention can remove the odor of the vulva by reducing the concentration of ammonia or trimethylamine, which is the cause of the vulvar odor.

The feminine cleanser composition of the present invention is characterized by inhibiting Candida albicans strains. By inhibiting the Candida albicans strain, it is possible to remove the vulvar odor caused by vaginitis caused by Candida and relieve itching. In particular, vaginitis caused by Candida is often accompanied by unbearable itching of the vulva, and the feminine cleanser composition of the present invention can alleviate itching by inhibiting Candida albicans strains.

Betulin included in the feminine cleanser composition of the present invention may be included in an amount of 0.01 to 20% (hereinafter referred to as % by weight) based on the total weight of the composition. Although not limited thereto, if betulin is less than 0.01% based on the total weight of the composition, the effect of increasing antifungal activity against Candida strains of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 mixed strain by betulin is increased. It may be insignificant, and if it exceeds 20%, the activity of the mixed strain of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 may decrease. Preferably, 0.01 to 10% may be included, and more preferably 0.2 to 2% may be included.

The alpha-glucan oligosaccharide contained in the feminine cleanser composition of the present invention may be included in an amount of 0.01 to 15% (hereinafter referred to as weight %) based on the total weight of the composition. If alpha-glucan oligosaccharide is less than 0.01% relative to the total weight of the composition, the effect of increasing antifungal activity against Candida strains of mixed strains of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 by betulin may be insignificant. If it is present and exceeds 15%, the activity of the mixed strain of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 may be reduced. Preferably, 0.01 to 10% may be included, and more preferably 0.15 to 1.5% may be included.

Rice starch included in the feminine cleanser composition of the present invention may be included in 20 to 95% (hereinafter referred to as weight %) based on the total weight of the composition. Rice starch maintains the composition in a dry powder formulation to maintain smoothness during use, is harmless to the human body, and can improve the skin tone of the female vulva. If the content of the rice starch is less than 20%, it is not easy to maintain the powder formulation, and if it is more than 95%, the content of the mixed strain of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 may be lowered and the antifungal activity may be lowered .

Silica included in the feminine cleanser composition of the present invention may be included in an amount of 0.01 to 10% (hereinafter referred to as % by weight) based on the total weight of the composition. The silica may act as a carrier to maintain the composition in a dry powder formulation and maintain comfort during use. If the content of the silica is less than 0.01%, it is not easy to maintain the powder formulation, and if it is more than 10%, the content of the mixed strain of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 is relatively lowered, and antifungal activity may be lowered. have.

The feminine cleanser composition of the present invention may further include talc, aluminum hydroxide, calcium silicate, or polyamide powder in addition to silica as a carrier component.

Therefore, the present invention is Lactobacillus rhamnosus HN001 ( Lactobacillus rhamnosus HN001) and Lactobacillus acidophilus La-14 ( Lactobacillus acidophilus La-14) mixed lactic acid bacteria 1 to 50% by weight, rice starch 20 to 95% by weight, betulin 0.01 ~ 20% by weight, 0.01-15% by weight of alpha-glucan oligosaccharide and 0.01-10% by weight of silica are provided.

The powdery feminine cleanser of the present invention can be used by spraying on the human body, underwear, hygiene products, etc. in powder form. The powdered feminine cleanser of the present invention may contain a moisturizer, conditioning agent, thickener, neutralizer, colorant, fragrance, emollient, thickener, nutrient, foaming agent, organic disinfectant, etc., or may further contain a surfactant having cleansing power. .

The feminine cleanser composition of the present invention is preferably in powder form, and may be used by mixing with purified water.

The present invention relates to a powdered female cleanser, wherein Lactobacillus rhamnosus and Lactobacillus acidophilus mixed in a certain ratio can remove vulvar odor and alleviate itching by inhibiting Candida albicans strains, rice starch, It is a powder type containing betulin, alpha-glucan oligosaccharide and silica, which can improve skin elasticity, moisturization, gloss, clarity and density of the female vulva, helping to improve women's quality of life and health. have.

1 is a graph showing the inhibitory action against Candida albicans according to the mixing ratio of Lactobacillus rhamnosus and Lactobacillus acidophilus .

2 is a view showing the effect of relieving vulvar odor and itching after using a feminine cleanser containing Lactobacillus rhamnosus and Lactobacillus acidophilus in a ratio of 1:4 according to the present invention.

Figure 3 is a graph showing the skin elasticity improvement rate (%) after using a powdered feminine cleanser according to the present invention.

Figure 4 is a graph showing the skin moisturizing improvement rate (%) after using a powdered feminine cleanser according to the present invention.

5 is a graph showing the skin gloss improvement rate (%) after using the powdered feminine cleanser according to the present invention.

6 is a graph showing the improvement rate (%) of clear skin after using the powdered feminine cleanser according to the present invention.

7 is a graph showing the skin density improvement rate (%) after using a powdered feminine cleanser according to the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the disclosure herein is provided so that it will be thorough and complete, and will fully convey the spirit of the invention to those skilled in the art.

<Example 1. Lactobacillus rhamnosus and Lactobacillus acidophilus inhibiting performance of the causative agent of vaginitis>

1.1. Lactobacillus rhamnosus and Lactobacillus acidophilus mixture composition test for inhibition of vaginitis causative bacteria

Lactobacillus rhamnosus and Lactobacillus acidophilus are lactic acid bacteria found in the vagina of healthy women. These affect the secretion of lactic acid and the vaginal pH environment, respectively, and are known to exhibit a certain inhibitory effect on the growth of Candida albicans , which is known as a causative agent of vaginitis. However, while the present inventors were studying the relationship between the characteristics of the microbial flora in the vagina and vaginal health, they showed a great difference in the bactericidal effect on the causative agent of vaginitis according to the mixing ratio of the lactic acid bacteria strain, and in some cases, the fact that the growth of the causative agent of vaginitis is promoted found In particular, when mixing Lactobacillus rhamnosus and Lactobacillus acidophilus at a certain ratio among these mixed strains, it was confirmed that the growth inhibitory effect on Candida albicans was remarkably increased, and the present invention was completed.

The above-described synergistic effect on Candida albicans inhibitory activity is very useful industrially as it can effectively prevent vaginitis even with a small total bacterial count when applied daily as well as a quick therapeutic effect when vaginitis occurs.

The inventors of the present invention extensively screened lactic acid bacteria distributed in the vagina of healthy women, Lactobacillus rhamnosus and Lactobacillus acidophilus, which show a certain inhibitory effect on Candida albicans even when used alone. was selected, and the bacteria used in the present invention are shown in Table 1 below.

number	scientific name	strain
No. One	Lactobacillus rhamnosus	HN001
No. 2	Lactobacillus acidophilus	La-14
No. 3	Candida albicans	ATCC 10231

First, Candida albicans ( Candida albicans , ATCC 10231) was inoculated in a Potato Dextrose agar (PDA) medium, pre-cultured at 25° C. for 2 days, and then 10 ⁶ CFU/ml bacterial solution was prepared using sterile physiological saline.

strain	sample
	Con.	One	2	3	4	5	6	7	8	9	10	11
HN001	-	One	-	One	One	One	One	One	2	4	6	8
La-14	-	-	One	One	2	4	6	8	One	One	One	One

According to the ratio, as shown in Table 2, Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 were homogenized by adding distilled water to a final concentration of 2×10 ⁶ CFU/ml to prepare test solutions. Thereafter, 0.1 ml of Candida albicans bacterial solution was added to the test solution, mixed, cultured at 25° C. for 24 hours, and the number of viable cells was measured by a dilution culture method. The survival rate of Candida albicans strains is shown in FIG. 1 . At this time, the group in which the Candida albicans strain was added to sterile physiological saline was used as a control group (Con.). The survival rate of the Candida albicans strain was 100% immediately after inoculation, and the number of bacteria was shown after culturing for 24 hours. As shown in FIG. 1 , Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 independently exhibited inhibitory effects against Candida albicans strains. In particular, it was confirmed that when Lactobacillus rhamnosus HN001 was used alone, it was effective in killing about 41.2% of Candida bacteria compared to the control group.

Lactobacillus rhamnosus strain is a lactic acid bacterium found in the vagina of healthy women, and is a strain used for vaginitis. However, since the use of lactic acid bacteria for vaginitis is known to be helpful in preventing recurrence after the use of antibiotics, it is difficult to expect the removal of pathogenic bacteria that cause vaginitis only with the lactic acid bacteria. In addition, it is advantageous to utilize complex lactic acid bacteria to stably establish a healthy vaginal flora after using antibiotics. However, even beneficial lactic acid bacteria show a great difference in their effects depending on the condition of the bacteria mixture. Accordingly, the present inventors increased the inhibitory activity of the causative agent of vaginitis by mixing with Lactobacillus rhamnosus , searched for lactic acid bacteria species capable of establishing a healthy vaginal bacterial flora, and confirmed the mixing ratio.

As shown in Figure 1 , Lactobacillus acidophilus La-14 exhibited an effect of killing Candida bacteria by about 8.2% compared to the control group. Lactobacillus acidophilus La-14 is a Gram-positive bacterium of the genus Lactobacillus, which is found in the gastrointestinal tract and mouth of humans and animals, and is commercially used in many dairy products together with Streptococcus thermophilus and Lactobacillus delbrueckii subsp. According to FIG. 1 , when Lactobacillus acidophilus La-14 was mixed with Lactobacillus rhamnosus HN001, the inhibitory effect on Candida greatly varied depending on the mixing ratio. That is, when Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 were mixed at a ratio of 1:1, the inhibitory effect of Candida strains was superior to that of the control group, and the effect was superior to that of Lactobacillus acidophilus La-14 strain alone, but Lactobacillus rhamnosus HN001 treatment alone did not show the same inhibitory effect on Candida strains.

In addition, when Lactobacillus rhamnosus HN001, which has a relatively excellent inhibitory effect on Candida strains, was mixed at a higher ratio (2-8:1) than Lactobacillus acidophilus La-14, the survival rate of Candida strains increased rather than the control group. . These results can be interpreted as creating an environment favorable to the survival of Candida strains under certain conditions while growing in a mixture of various types of lactic acid bacteria.

However, when Lactobacillus acidophilus La-14 was mixed at a higher ratio (1:2~8) than Lactobacillus rhamnosus HN001 , it showed higher Candida strain inhibitory activity compared to the control group. In the case of 2 to 6, Candida inhibitory activity was very high. In particular, when the mixing ratio was 1:4, the survival rate of Candida strain was significantly reduced compared to the case of single treatment with Lactobacillus rhamnosus HN001. This suggests that growth conditions composed of a certain mixing ratio of beneficial lactic acid bacteria can provide an environment capable of synergistically suppressing the activity of Candida strains, one of the causative bacteria of vaginitis.

1.2. Lactobacillus rhamnosus, Lactobacillus acidophilus and betulin mixture composition test for suppression of vaginitis causative bacteria

As shown in Example 1.1, the antifungal effect against Candida strains can be maximized while directly applying Lactobacillus rhamnosus and Lactobacillus acidophilus mixed at a ratio of 1:4 with the highest inhibitory activity of Candida strains to the female vulva. Possible compositions were screened.

As a result of mixing and applying polysaccharides as food for lactic acid bacteria such as Lactobacillus rhamnosus or Lactobacillus acidophilus, samples for skin protection and regeneration, anti-inflammatory, antibacterial natural products, skin tone improving cosmetics, etc. with the strain mixture, betulin and alpha - It was confirmed that the glucan oligosaccharide maintained a stable liquid or powder formulation without inhibiting the growth of a 1:4 mixed strain of Lactobacillus rhamnosus and Lactobacillus acidophilus. Thereafter, the antifungal activity of the mixed strain of Example 1.1 and the mixture of betulin and alpha-glucan oligosaccharide against Candida albicans was confirmed.

Betulin is a triterpene derived from birch trees and is known to lower cholesterol levels, help prevent obesity, and improve insulin sensitivity. In addition, it has been confirmed that skin wrinkles are improved through the effect of promoting cell regeneration and collagen production by inhibiting collagenase activity. However, when betulin was used with the mixed strain of Lactobacillus rhamnosus and Lactobacillus acidophilus of the present invention, it was confirmed that the antifungal activity against the Candida strain was increased.

As in Example 1.1, Candida albicans (ATCC 10231) was inoculated in Potato Dextrose agar (PDA) medium, pre-cultured at 25 ° C for 2 days, and then 10 ⁶ CFU / ml Candida using sterile physiological saline albicans bacterial solution was prepared. In addition, distilled water was added to obtain Lactobacillus rhamnosus HN001 4×10 ⁴ CFU/ml and Lactobacillus acidophilus La-14 1.6×10 ⁵ CFU/ml, and homogenized 1:4 strain mixture 2×10 ⁵ CFU/ml test solution was prepared, Samples A, B, C and D were prepared by mixing betulin and alpha-glucan oligosaccharide as shown in Table 3 below.

	HN001 (cfu/ml)	La-14 (cfu/ml)	betulin	Alpha- Glucan oligosaccharides	survival rate (%)
control group	-	-	-	-	92.86
A	4×10 4	1.6×10 5	-	-	76.28
B	4×10 4	1.6×10 5	2 %	-	48.65
C	4×10 4	1.6×10 5	-	1.5%	60.27
D	4×10 4	1.6×10 5	2 %	1.5%	45.67

Thereafter, 0.1 ml of Candida albicans bacterial solution was added to the samples A, B, C and D, mixed, incubated at 25 ° C for 24 hours, and the number of viable cells was measured by a dilution culture method, and the number of Candida albicans strains was calculated based on the initial number of bacteria. The survival rate (%) was converted and shown in Table 3 and FIG. 2 . At this time, the group in which the Candida albicans strain was added to sterile physiological saline was used as a control (Con.). As shown in Table 4 and FIG. 2 , Lactobacillus rhamnosus and Lactobacillus acidophilus mixed at a ratio of 1:4 2×10 ⁵ CFU/ml of the mixed strain inhibited the number of Candida albicans bacteria by about 25%. In the case of sample B in which 2% (w/v) of betulin was added, the antifungal effect of the mixed strains of Lactobacillus rhamnosus and Lactobacillus acidophilus against Candida strains was increased by 36% or more.

In the case of adding 1.5% (w/v) of alpha-glucan oligosaccharide that can be provided as food for lactic acid bacteria such as Lactobacillus rhamnosus or Lactobacillus acidophilus (C), Lactobacillus rhamnosus and Lactobacillus acidophilus The antifungal effect of the phyllus mixed strain against the Candida strain was increased by more than 20%. In addition, when adding betulin 2% (w / v) and alpha-glucan oligosaccharide 1.5% (w / v) to the Lactobacillus rhamnosus and Lactobacillus acidophilus mixed strains mixed together at a ratio of 1:4 (D), there was no significant difference from the betulin additionally administered group (B), but there was a tendency to increase the antifungal effect against Candida strains than the betulin additionally administered group (B).

Through the above results, the Lactobacillus rhamnosus and Lactobacillus acidophilus mixed strains mixed at a ratio of 1:4 of the present invention showed a significant increase in antifungal activity against Candida strains at a specific mixing ratio, and betulin and / Or it was confirmed that the antifungal effect of the mixed strains of Lactobacillus rhamnosus and Lactobacillus acidophilus mixed at a ratio of 1:4 was increased by adding alpha-glucan oligosaccharide.

<Example 2. Deodorization performance test of mixed composition of Lactobacillus rhamnosus and Lactobacillus acidophilus>

Vaginitis, such as bacterial vaginosis and candida vaginitis, is particularly stressful for women with an unpleasant smell of vaginal discharge. The present inventors tested the deodorizing performance of a 1:4 mixture of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14, which had the most excellent Candida strain inhibitory effect, and a sample to which betulin was added.

The deodorization performance was confirmed according to the measurement of ammonia or trimethylamine concentration reduction rate in the EL608 deodorant test method, which is an environmental label certification standard of the Ministry of Environment. After adjusting the concentration of ammonia or trimethylamine diluted with acetone in the test vessel to the vicinity of the initial concentration (C _b,i ) according to the certification standard, mix Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 (1:4 mixing ratio) strain 2 ×10 ⁵ CFU/ml or Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 (1:4 mixing ratio) mixed strain 2×10 ⁵ CFU/ml with 2% (w/v) betulin added to the sample in the test container Injection was injected through the nozzle. After 6 hours had elapsed after the injection of each sample, the gas in the test vessel was collected with a gas sampler specified in SPS-KCLI2218-6218, and the concentration (C _t,i ) of ammonia or trimethylamine as an odor component was measured. At this time, the gas sampler was used by inserting a direct reading detector tube corresponding to the odor component to be measured and the expected concentration range.

Deodorization performance (odor component concentration reduction rate) was calculated according to the following equation, and the results are shown in Table 4 .

Odor component concentration reduction rate (%) =

Test Items	(Concentration reduction rate %)		note
	HN001+La-14	HN001+La-14+betulin
ammonia	60.0	54.8	(21.3±0.6)℃ (50.1±0.6)% RH
trimethylamine	62.5	57.3

As shown in Table 4 , it was confirmed that the 1:4 mixed strain of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14, which had the highest Candida strain inhibitory effect, showed an effect of reducing both ammonia and trimethylamine concentrations by more than 60%. . In addition, when betulin was added to the 1:4 mixed strain of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14, it was confirmed that the deodorizing effect increased. As shown in the above results, the present invention Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus Compared to the mixture of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 at different ratios, the 1:2~6 mixture of La-14 not only has an excellent inhibitory effect on Candida albicans, a causative agent of vaginitis, but also directly reduces the concentration of ammonia and trimethylamine. It was confirmed that the antifungal effect and deodorizing effect of Candida albicans were further increased when betulin was added thereto.

<Example 3. Confirmation of the effect of a powdered feminine wash containing a mixture of Lactobacillus rhamnosus and Lactobacillus acidophilus>

3.1. Manufacture of powder type feminine cleanser

A powder type feminine cleanser containing a 1:4 mixture of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14, which has the most excellent Candida strain inhibitory effect, was prepared.

A powder type feminine cleanser was prepared as shown in Table 5 below, including a mixture of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 in a 1:4 ratio. Powdery feminine cleanser is a mixture of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 in a ratio of 1:4, and betulin, which increases antifungal activity against Candida strains, is added, and rice starch and Silica was added. In addition, alpha-glucan oligosaccharide, which increases the role of a lactic acid bacteria medium and antifungal activity against mixed strains, was added.

Rice starch is used as an absorbent and bulking agent, so that the composition can be maintained in a powder form and bulked up, and silica is a powder type and acts as an anti-caking agent.

ingredient	Content (% by weight)
rice starch	85.5
HN001+ La-14 (1:4)	10.0
betulin	2.0
alpha-glucan oligosaccharide	1.5
silica	1.0

3.2. Confirmation of the effect of a powder-type feminine cleanser on vulvar itching and odor The effect of this powder-type feminine cleanser on vulvar itching and odor was requested by the Korea Research Institute of Dermatology, and 21 adult women aged 25 years or older were surveyed from September 2020 to April 4. Weekly test subjects were applied to the vulva and changes were observed.

During the test period of 4 weeks, the test substance was sprayed in a small amount of lukewarm water 5 times in the shower once a day, applied to the vulva, gently massaged, and washed off with lukewarm water. Before using the test substance and after using the test substance for 2 and 4 weeks, a survey was conducted on the subjects, and the results are shown in Tables 6 and 7 and FIG. 3 .

Before using test substance
question		frequency	percentage
No discomfort due to itching of the vulva	Not at all	4	19.0
	Not like that	16	76.2
	is average	One	4.8
	Yes	0	0.0
	it really is	0	0.0
There is no smell in the vulva	Not at all	4	19.0
	Not like that	16	76.2
	is average	One	4.8
	Yes	0	0.0
	it really is	0	0.0
Sum		21	100.0

period of use question		2 weeks later		4 weeks later
		frequency	percentage	frequency	percentage
The vulvar itching was more relieved	Not at all	0	0.0	0	0.0
	Not like that	0	0.0	0	0.
	is average	4	19.0	One	4.8
	Yes	16	76.2	13	61.9
	it really is	One	4.8	7	33.3
The vulvar odor was more mitigated	Not at all	0	0.0	0	0.0
	Not like that	0	0.0	0	0.0
	is average	3	14.3	0	0.0
	Yes	16	76.2	14	66.7
	it really is	2	9.5	7	33.3
Sum		21	100.0	21	100.0

As shown in Table 6 , most of the female subjects suffered from itching or odor of the vulva. This can be said to indicate that vaginitis is a common disease in women enough to be called 'a woman's cold', and many women suffer from discomfort caused by it. As shown in Table 7 and FIG. 3 , when the 1:4 mixture of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 of the present invention, which has the most excellent Candida strain inhibitory effect, is directly applied to the female vulva, 80.1% after 2 weeks, After 4 weeks, 95.2% of the subjects responded that the itching in the vulva was alleviated. In the item related to the smell of the vulva, 85.7% of the subjects after 2 weeks of use and all the subjects who participated in the experiment after 4 weeks of use responded that the smell of the vulva was alleviated.

In addition, it was confirmed that the satisfaction of this feminine cleanser was high, as the subject felt that the skin radiance, the clearness of the skin color, and the degree of skin density were all improved.

3.2 Confirmation of the effect of powder type feminine cleanser on vulvar skin

Twenty-one adult women over the age of 25 were used on the subject's vulva for 4 weeks. During the test period of 4 weeks, the test subject sprayed the test substance 5 times in a small amount of lukewarm water in the shower once a day, applied it to the vulva, massaged it gently, and washed it off with lukewarm water. Elasticity, moisture, gloss, clarity, and density were measured before, after 2 weeks of use, and after 4 weeks of use of the test substance, and the results are shown in Tables 8 and 9.

Before using test substance
question		frequency	percentage	question		frequency	percentage
The skin of the vulva is not dry.	Not at all	6	28.6	The skin color of the vulva is not dull	Not at all	6	28.6
	Not like that	15	76.2		Not like that	15	71.4
	is average	0	0.0		is average	0	0.0
	Yes	0	0.0		Yes	0	0.0
	it really is	0	0.0		it really is	0	0.0
The skin of the vulva is elastic	Not at all	5	23.8	The skin of the vulva is dense and firm	Not at all	6	28.6
	Not like that	16	76.2		Not like that	15	71.4
	is average	0	0.0		is average	0	0.0
	Yes	0	0.0		Yes	0	0.0
	it really is	0	0.0		it really is	0	0.0
The skin of the vulva becomes shiny	Not at all	7	33.3
	Not like that	14	66.7
	is average	0	0.0
	Yes	0	0.0
	it really is	0	0.0
Sum		21	100	Sum		21	100

period of use question		2 weeks later		4 weeks later
		frequency	percentage	frequency	percentage
The skin of the vulva seems to be more moist.	Not at all	0	0.0	0	0.0
	Not like that	0	0.0	0	0.0
	is average	One	4.8	0	0.0
	Yes	19	90.5	18	85.7
	it really is	One	4.8	3	14.3
The skin of the vulva appears more elastic	Not at all	0	0.0	0	0.0
	Not like that	0	0.0	0	0.0
	is average	3	14.3	One	4.8
	Yes	17	81.0	19	91.5
	it really is	One	4.8	One	4.8
The skin of the vulva appears shiny	Not at all	0	0.0	0	0.0
	Not like that	0	0.0	0	0.0
	is average	7	33.3	5	23.8
	Yes	13	61.9	15	71.4
	it really is	One	4.8	One	4.8
The skin color of the vulva seems to have become clearer	Not at all	0	0.0	0	0.0
	Not like that	0	0.0	0	0.0
	is average	5	23.8	2	9.5
	Yes	15	71.4	18	85.7
	it really is	One	4.8	One	4.8
It seems that the skin of the vulva has become dense and firm	Not at all	0	0.0	0	0.0
	Not like that	0	0.0	0	0.0
	is average	4	19.0	3	14.3
	Yes	16	76.2	16	76.2
	it really is	One	4.8	2	9.5
Sum		21	100.0	21	100.0

As shown in Tables 8 and 9 , the subjects showed higher satisfaction in proportion to the period of use in all of the skin moisturization, elasticity, gloss, clarity, and density of the vulva compared to before the use of the test substance. In particular, the response that skin moisturization and density were greatly improved was high, and 76.2 to 100% of the subjects thought that skin elasticity, gloss, and clarity were all improved.

3.3. Check the skin elasticity improvement effect

A ballistometer (Ballistometer BLS780, Dia-Stron Ltd., UK) was applied to evaluate skin elasticity improvement of the composition, and the left armpit area of all subjects was measured, and the results are shown in FIG. 4 . The ballistometer calculates skin elasticity by digitizing the vibration energy when the 2 mm diameter arm mounted on the probe contacts the skin surface, and MApp, a dedicated analysis program for the ballistometer, is used for analysis. CoR value (Coefficient of Restitution), which is a coefficient of recovery, was used as a value to measure skin elasticity, which represents the ratio of impact speed to the rebound speed of the arm. The average value of CoR for the first three bounces was calculated, and the maximum value was 1.0, which means that skin elasticity improved as the measured value increased compared to before using the test substance, and the skin elasticity improvement rate (%) was calculated. Instrumental measurements were made before, after 2 weeks of use, and after 4 weeks of use of the test substance.

As shown in FIG. 4 , when the powdered feminine cleanser of the present invention was used, skin elasticity increased in proportion to the period of use, and after 4 weeks of use, skin elasticity was improved by 2.46% compared to before use.

3.4. Check the effect of improving skin hydration

Epsilon E100 (Biox Systems Ltd., UK) was applied to evaluate skin moisturizing improvement of the powdery feminine cleanser of the present invention. Epsilon E100 calculates the moisture of the skin surface in contact with the sensor as an Epsilon (ε) value. The same person in charge of the test measured the left armpit area of all test subjects. For this analysis, the change in skin moisture was analyzed using Epsilon E100 Software V3.1, an analysis program dedicated to Epsilon E100, and the skin moisturizing improvement rate compared to before using the test substance. (%) was calculated and shown in FIG. 5 .

As shown in FIG. 5 , when the powdered feminine cleanser of the present invention was used, the skin moisturizing rate was improved according to the period of use, and after 4 weeks of use, the skin moisturizing rate was improved by 22.48% compared to before use.

3.5. Confirmation of skin radiance improvement effect

A gloss meter (Multi Gloss 268 PLUS, Konica Minolta, Japan) was applied to evaluate skin gloss improvement of the powdery feminine cleanser of the present invention. The gloss meter measured the left armpit of all test subjects three times in succession, calculated the average value, and calculated the skin gloss improvement rate (%) compared to before using the test substance, and is shown in FIG. 6 . The measurement value of the gloss meter consists of three measurement values of 20˚, 60˚, and 85˚, and 20˚, 60˚, and 85˚ represent the measured ratio of reflected light projected at each angle. The value at 60 ° was used as a value for measuring the skin gloss of the composition.

As shown in FIG. 6 , when the powdered feminine cleanser of the present invention was used, skin gloss was improved according to the period of use, and after 4 weeks of use, skin gloss was improved by 16.13% compared to before use.

3.6. Confirmation of skin clearing improvement effect

SkinColorCatch (Delfin Technologies Ltd., FinLand) was applied to evaluate the skin clearness improvement of the powdered feminine cleanser of the present invention. SkinColorCatch was measured by contacting the probe vertically to the left armpit of all test subjects, and white LED light was irradiated at 45° within the probe, and the light reflected on the skin was detected by the RGB color sensor, and compared with before using the test substance. The skin clarity improvement rate (%) was calculated and shown in FIG. 7 . The measurement value of SkinColorCatch consists of five factors: L ^* , a ^* , b ^* , melanin index, and erythema index. L ^* value is bright-ness, a ^* value is redness, and b ^* The value is yellowness, the melanin index indicates the amount of melanin present in the skin, and the erythema index indicates the amount of skin erythema. As a value for measuring the improvement in skin clarity of the composition, L ^* value representing the brightness of the skin was used for analysis.

As shown in FIG. 7 , when the powdered feminine cleanser of the present invention was used, skin clearness was improved according to the period of use, and after 4 weeks of use, skin clearness was improved by 1.04% compared to before use.

3.7. Check the effect of improving skin density

In order to evaluate the skin density improvement of the powdered female cleanser of the present invention, a DUB-Skin Scanner (Taberna pro medicum, Luneburg, Germany), a device for imaging high-resolution ultrasound waves, was applied. Distilled water was injected into the water chamber of the probe equipped with a scanning head so that it was perpendicular to the skin, and then the skin density was measured by pressing the left armpit of all subjects with the same pressure. As a value for measuring the improvement in skin density of the composition, the Density value representing skin density was analyzed, and the analysis range corresponds to the upper part of the subcutaneous fat layer right below the epidermis. The degree of denseness was analyzed in stages of 0 (sparse) to 255 (dense), and the measurement unit % value was used. The skin density improvement rate (%) was calculated compared to before the test substance was used, and is shown in FIG. 8 .

As shown in FIG. 8 , when the powdered feminine cleanser of the present invention was used, skin density was improved according to the period of use, and after 4 weeks of use, skin density was improved by 3.69% compared to before use.

As can be seen from the above results, the 1:4 mixed strain of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 and the mixed composition of betulin and alpha-glucan oligosaccharide of the present invention have excellent inhibitory effects on Candida albicans, the causative agent of vaginitis. In addition, by directly reducing the concentration of ammonia and trimethylamine, when applied to the vulva by an actual user, it is effective in removing itching and odor in the vulva, and it is made of a powder-type feminine cleanser including rice starch and silica, resulting in skin elasticity. , it was confirmed that there is an improvement effect in moisturizing, gloss, clarity and density.

Claims (6)

1. Lactobacillus rhamnosus HN001 ( Lactobacillus rhamnosus HN001) and Lactobacillus acidophilus La-14 ( Lactobacillus acidophilus La-14) mixed lactic acid bacteria, rice starch, betulin, alpha-glucan at a ratio of 1:2 to 6 A powder type feminine cleanser containing oligosaccharides and silica.
2. According to claim 1,

   The powdery female cleanser contains 1 to 50% by weight of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 mixed lactic acid bacteria, 20 to 95% by weight of rice starch, A powdery feminine cleanser comprising 0.01 to 20% by weight of thuline, 0.01 to 15% by weight of alpha-glucan oligosaccharide, and 0.01 to 10% by weight of silica.
3. According to claim 1 or 2,

   The Lactobacillus rhamnosus HN001 ( Lactobacillus rhamnosus HN001) and Lactobacillus acidophilus La-14 ( Lactobacillus acidophilus La-14) mixed lactic acid bacteria, Lactobacillus rhamnosus HN001 and Bacillus acidophilus La-14 are 1: 4 Powdery feminine cleanser, characterized in that mixed in a ratio of.
4. According to claim 1,

   The powdery female cleanser has at least one effect selected from the group consisting of removing odor of the vulva, relieving itching, improving skin elasticity, improving skin moisturization, improving skin radiance, improving skin clarity, and improving skin density Powder type, characterized in that it has an effect feminine cleanser.
5. According to claim 4,

   The odor removal of the vulva is a powder-type feminine cleanser, characterized in that to reduce the concentration of ammonia or trimethylamine.
6. According to claim 1 or 2,

   The feminine cleanser composition is a powder-type feminine cleanser, characterized in that for inhibiting Candida albicans strains.

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