KR102594523B1 - Powder-typed feminine cleanser comprising Lactobacillus rhamnosus and Lactobacillus acidophilus - Google Patents

Abstract

The present invention relates to a powdered feminine wash containing Lactobacillus rhamnosus and Lactobacillus acidophilus mixed in a certain ratio and containing rice starch, betulin, alpha-glucanoligosaccharide and silica, and Candida albi. By suppressing the Cans strain, it can contribute to improving women's health and quality of life by eliminating vulvar odor, relieving itching, and improving skin elasticity, moisture, gloss, clarity, and density.

Description

Powder-typed feminine cleanser comprising Lactobacillus rhamnosus and Lactobacillus acidophilus}

The present invention relates to a powdered feminine wash containing Lactobacillus rhamnosus and Lactobacillus acidophilus.

The microbial environment in a woman's vagina is very important for women's health. In particular, the female vulva is a very good environment for pathogens to grow because it is close to the anus and maintains a humid and warm environment through secretions. Lactic acid bacteria reside inside the vagina and excrete lactic acid and other antibacterial substances during the growth process, thereby maintaining the optimal vaginal acidity level below 4.5 and suppressing the growth of pathogenic strains. However, if these lactic acid bacteria lose their dominance due to various bacteria such as anaerobic bacteria or Candida, the balance of the vaginal microbial environment is disrupted and the likelihood of contracting vaginitis increases.

Vaginitis causes premature birth, low birth weight fetuses, and weakens immunity. Vaginitis in women is largely classified into three categories: bacterial vaginosis caused by bacterial infection, vaginitis caused by Candida fungus (Vulvovaginal candidiasis), and vaginitis caused by Trichomonas protozoan infection. Bacterial vaginosis is a disease in which the proliferation of specific bacteria in the female vagina increases vaginal acidity and changes the microbial environment. It is an inflammation that occurs mainly in women of childbearing age, and usually causes white or grayish-white vaginal discharge and a foul 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 must be taken because it affects the lactic acid flora in the vagina.

Candida vaginitis is a common disease that 75% of women experience at least once in their lives, and 45% of women experience it at least twice a year. Candida albicans, a fungus, is the causative agent. If antibiotics are used for a long time, pregnancy, diabetes, etc., the concentration of lactic acid bacteria and normal vaginal bacteria in the vagina decreases, causing overgrowth of the fungus and causing this disease. Symptoms of Candida vaginitis include itchiness of the vulva, foul-smelling vaginal discharge, pain during intercourse, and pain during urination. It is treated by taking antifungal medication or inserting vaginal tablets, but if left untreated, pelvic inflammatory disease and pregnancy can occur. It may cause complications such as complications. In addition, Candida vaginitis often recurs, in which case persistent vulvar itching occurs.

Recently, lactic acid bacteria administered orally are being used to prevent vaginitis and improve vaginal health. If you take lactic acid bacteria regularly, the lactic acid bacteria survive through the intestines and into the vagina, where they settle and proliferate into beneficial bacteria necessary for vaginal health. However, since the ingested lactic acid bacteria must pass through the digestive system, there is a problem that the number of guaranteed bacteria that settles in the vagina is small compared to the number of introduced bacteria.

In addition, in order to create a stable vaginal microbial environment and effectively sterilize pathogenic bacteria, the number of lactic acid bacteria species, dominant species, core species and their colony ratio in vaginal flora, and the lactic acid flora that are especially effective against vaginitis-causing bacteria such as Candida are determined. Research on characteristics is needed.

Meanwhile, Lactobacillus rhamnosus is a Gram-positive lactic acid bacterium most commonly found in the healthy female genital tract and urinary tract. It prevents the growth of pathogenic bacteria in the female genital tract, suppresses Candida, and maintains pH at an appropriate level. It is known as a strain that helps colon health and also contributes to basic colon health. However, Lactobacillus rhamnosus itself has a problem in that its antibacterial activity against vaginitis-causing bacteria such as Candida is limited.

Korean Patent No. 2139687 has Gardnerella vaginalis ( Gardnerella Vaginalis) vaginalis ) and Candida albicans ( Lactobacillus rhamnosus ) MG4288 strain (accession number KCTC13823BP), which has antibacterial activity against Candida albicans, the strain culture medium, and the cell-free supernatant of the strain are described. In addition, Korean Patent No. 1098241 discloses Lactobacillus sp, which has the activity of suppressing urinary tract infections and candidal vaginitis. HY 7801 and products containing it as an active ingredient are described, and Korean Patent No. 1784847 relates to a composition containing salt, sugar, and lactic acid bacteria, including salt selected from molten salt, refined salt, or sea salt; (2) Sugars such as xylose and arabinose; and (3) Lactobacillus acidophilus ( Lactobacillus acidophilus ; KCTC No.3164), Bifidobacterium longum subspecies longum ( Bifidobacterium longum subsp. longum; KCTC No. 3128), Streptococcus sp; KCTC No. 5644) and Bacillus cereus (KCTC No. 13123). Prevention of vaginitis containing as an active ingredient a combination of one or more lactic acid bacteria selected from the group consisting of and Pharmaceutical compositions for therapeutic use are described. However, these lactic acid bacteria compositions do not have strong antibacterial activity due to the optimal combination of vaginal lactic acid bacteria disclosed in the present invention.

Korean Patent No. 2139687, Lactobacillus MG4288 for treating or improving vaginitis with antibacterial effect on Gardnerella vaginalis and Candida albicans, registered on July 24, 2020. Korean Patent No. 1717672, Lactobacillus sp., which has the activity of suppressing urinary tract infections and candidal vaginitis. HY 7801 and products containing it as an active ingredient, registered on December 19, 2011. Korean Patent No. 1784847, Composition containing lactic acid bacteria for preventing and treating vaginitis and its uses, registered on September 28, 2017.

The aim is to provide a powder-type feminine wash containing Lactobacillus rhamnosus and Lactobacillus acidophilus.

The powdered feminine wash of the present invention is a mixture of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 ( Lactobacillus acidophilus La-14), rice starch, betulin, and alpha-glucan oligosaccharide. and a powder-type feminine wash containing silica.

Lactobacillus rhamnosus and Lactobacillus acidophilus of the powdered feminine wash of the present invention are preferably Lactobacillus rhamnosus HN001 ( Lactobacillus rhamnosus HN001) and Lactobacillus acidophilus La-14 ( 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 at a ratio of ~6, preferably at a ratio of 1:4.

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

The powdered feminine wash is characterized by having one or more effects selected from the group consisting of removing vulvar odor, relieving itching, improving skin elasticity, improving skin moisturization, improving skin gloss, improving skin clarity, and improving skin density. Bad odor or itchiness of the vulva can be caused by bacterial vaginosis, increased discharge of unpleasant odor due to vaginitis caused by fungi such as Candida, or odor caused by bacteria growing on synthetic fiber underwear, and is a common phenomenon experienced by most women. . The feminine wash composition of the present invention can specifically remove vulvar odor by reducing the concentration of ammonia or trimethylamine, which causes vulvar odor.

The feminine wash composition of the present invention is characterized by inhibiting Candida albicans strains. By suppressing Candida albicans strains, vulvar odor caused by vaginitis caused by Candida bacteria can be removed and itching can be alleviated. In particular, vaginitis caused by Candida bacteria is often accompanied by unbearable itching of the vulva, and the feminine wash composition of the present invention can relieve 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 weight percent) based on the total weight of the composition. Although it is not limited to this, if betulin is less than 0.01% of the total weight of the composition, the effect of increasing the antifungal activity of the mixed strain of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 by betulin against Candida strains It may be insignificant, and if it exceeds 20%, the activity of the Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 mixed strain itself may decrease. Preferably, it may contain 0.01 to 10%, and more preferably, it may contain 0.2 to 2%.

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 percent) based on the total weight of the composition. If alpha-glucanoligosaccharide is less than 0.01% of the total weight of the composition, the effect of betulin on increasing the antifungal activity of the mixed strain of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 against Candida strains may be minimal. And, if it exceeds 15%, the activity of the Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 mixed strain itself may be reduced. Preferably, it may contain 0.01 to 10%, and more preferably, it may contain 0.15 to 1.5%.

Rice starch contained in the feminine cleanser composition of the present invention may be included in an amount of 20 to 95% (hereinafter referred to as weight percent) based on the total weight of the composition. Rice starch maintains the composition in a dry powder form so that it remains soft and dry when used, is harmless to the human body, and can improve the skin tone of the female vulva. If the rice starch content is less than 20%, it is not easy to maintain the powder formulation, and if it is more than 95%, the content of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 mixed strains may decrease, resulting in reduced antifungal activity. .

Silica contained in the feminine cleanser composition of the present invention may be included in an amount of 0.01 to 10% (hereinafter referred to as weight percent) based on the total weight of the composition. The silica acts as a carrier to maintain the composition in a dry powder formulation and can maintain comfort during use. If the silica content 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 strains of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 may be relatively low, resulting in reduced antifungal activity. there is.

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 1~50% by weight, rice starch 20~95% by weight, betulin 0.01~20% by weight, alpha-glucan oligosaccharide 0.01 Provided is a powdered feminine wash comprising ~15% by weight and 0.01~10% by weight of silica.

The powdered feminine cleanser of the present invention can be used in powder form by spraying it on the human body, underwear, sanitary products, etc. The powdered feminine wash of the present invention may contain moisturizers, conditioning agents, thickeners, neutralizers, pigments, fragrances, emollients, thickeners, nutrients, foaming agents, organic disinfectants, etc., or may further include surfactants with cleansing properties. .

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

The present invention relates to a powdered feminine wash. Lactobacillus rhamnosus and Lactobacillus acidophilus mixed in a certain ratio can eliminate vulvar odor and relieve itching by suppressing Candida albicans strains. Rice starch, It is a powder type containing betulin, alpha-glucan oligosaccharide and silica and can improve the skin elasticity, moisturizing, gloss, clarity and density of the female vulva, which can be helpful in improving the quality of life and health of women. there is.

Figure 1 is a graph showing the inhibitory effect on Candida albicans according to the mixing ratio of Lactobacillus rhamnosus and Lactobacillus acidophilus .
Figure 2 is a diagram showing the effect on alleviating vulvar odor and itching after using a feminine wash 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 the powdered feminine wash according to the present invention.
Figure 4 is a graph showing the improvement rate (%) in skin moisturization after using the powdered feminine cleanser according to the present invention.
Figure 5 is a graph showing the skin gloss improvement rate (%) after using the powdered feminine cleanser according to the present invention.
Figure 6 is a graph showing the improvement rate (%) in skin clarity after using the powdered feminine wash according to the present invention.
Figure 7 is a graph showing the improvement rate (%) in skin density after using the powdered feminine wash 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 content introduced herein is provided to be thorough and complete, and to fully convey the spirit of the present invention to those skilled in the art.

< Example One. Lactobacillus rhamnosus and Lactobacillus of acidophilus Vaginitis-causing bacteria inhibition performance test>

1.1. Lactobacillus rhamnosus and Lactobacillus Acidophilus Testing the performance of the mixed composition to inhibit vaginitis-causing bacteria

Lactobacillus rhamnosus and Lactobacillus acidophilus are lactic acid bacteria found in the vagina of healthy women. They each affect the secretion of lactic acid and the vaginal pH environment, and are known to have a certain inhibitory effect on the growth of Candida albicans , known as the causative agent of vaginitis. However, while researching the relationship between the characteristics of the vaginal microbial flora and vaginal health, the present inventor found a significant difference in the sterilization effect on vaginitis-causing bacteria depending on the mixing ratio of lactic acid bacteria strains, and in some cases, promotes the growth of vaginitis-causing bacteria. was discovered. In particular, among these mixed strains, it was confirmed that the growth inhibitory effect on Candida albicans was significantly increased when Lactobacillus rhamnosus and Lactobacillus acidophilus were mixed at a certain ratio, and the present invention was completed.

The synergistic effect on the Candida albicans inhibition ability as described above is very useful industrially as it not only provides a quick treatment effect when vaginitis occurs, but also effectively prevents vaginitis with a small total number of bacteria when applied daily.

The inventor of the present invention extensively screened the lactic acid bacteria distributed in the vagina of healthy women and identified Lactobacillus rhamnosus and Lactobacillus acidophilus , which have a certain inhibitory effect on Candida albicans even when used alone. were 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 albicans , ATCC 10231) was inoculated on Potato Dextrose agar (PDA) medium and pre-cultured at 25°C for 2 days, and then 10 ⁶ CFU/ml bacterial solution was prepared using sterilized saline solution.

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 above, a test solution was prepared by homogenizing Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 by adding distilled water to a final concentration of 2 × 10 ⁶ CFU/ml. Afterwards, Candida albicans was added to the test solution. After adding and mixing 0.1 ml of the bacterial solution, cultured at 25°C for 24 hours, measured the number of viable bacteria using a dilution culture method, and Candida albicans The survival rate of the strains is shown in Figure 1 . At this time, the group in which Candida albicans strain was added to sterilized saline solution was used as the control group (Con.). Candida albicans The survival rate of the strain is Candida albicans immediately after inoculation. The number of bacteria was shown after culturing for 24 hours, assuming that the strain was 100%.

As shown in Figure 1 , Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 each individually exhibited an effect on inhibiting 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 is a lactic acid bacteria found in the vagina of healthy women and is used for vaginitis. However, since the use of lactic acid bacteria for vaginitis is known to help prevent recurrence after the use of antibiotics, it is difficult to expect the removal of pathogenic bacteria that cause vaginitis with the lactic acid bacteria alone. In addition, the use of complex lactic acid bacteria is advantageous for stably establishing healthy vaginal flora after antibiotic use. However, even beneficial lactic acid bacteria show significant differences in their effectiveness depending on the state of the bacteria mixture. Accordingly, the present inventor searched for lactic acid bacteria species that can increase the inhibitory activity of the causative bacteria of vaginitis and establish healthy vaginal flora by mixing with Lactobacillus rhamnosus and confirmed the mixing ratio.

As shown in Figure 1 , Lactobacillus acidophilus La-14 showed a Candida killing effect of about 8.2% compared to the control group. Lactobacillus acidophilus La-14 is a Gram-positive bacterium of the Lactobacillus genus, a lactic acid bacterium found in the gastrointestinal tract and mouth of humans and animals, and is commercially used in many dairy products along with Streptococcus thermophilus and Lactobacillus delbrueckii subsp. According to Figure 1 , when Lactobacillus acidophilus La-14 was mixed with Lactobacillus rhamnosus HN001, the inhibitory effect on Candida bacteria varied greatly depending on the mixing ratio. In other words, when Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 were mixed in a 1:1 ratio, the inhibitory effect on Candida strains was superior to that of the control group, and the effect was superior to that of Lactobacillus acidophilus La-14 strain alone; however, Lactobacillus rhamnosus It did not show the same inhibitory effect on Candida strains as when treated with HN001 alone.

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

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

1.2. Lactobacillus rhamnosus , Lactobacillus Acidophilus and Betulin Testing the performance of the mixed composition to inhibit vaginitis-causing bacteria

As seen in Example 1.1 above, Lactobacillus rhamnosus and Lactobacillus acidophilus mixed at a ratio of 1:4, which have the highest inhibitory activity against Candida strains, can be applied directly to the female vulva to maximize the antifungal effect against Candida strains. Compositions that could be used were screened.

As a result of mixing polysaccharides as food for lactic acid bacteria such as Lactobacillus rhamnosus or Lactobacillus acidophilus, samples for skin protection and regeneration, anti-inflammatory and antibacterial natural products, skin tone improving cosmetics, etc., with the above strain mixture, betulin and alpha -It was confirmed that glucan oligosaccharide maintained a stable liquid or powder formulation without inhibiting the growth of Lactobacillus rhamnosus and Lactobacillus acidophilus 1:4 mixed strains. Afterwards, the antifungal activity of the mixed strain of Example 1.1 and the mixture of betulin and alpha-glucanoligosaccharide 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. It has also been confirmed to improve skin wrinkles by inhibiting collagenase activity and promoting cell regeneration and collagen production. However, when betulin was used together with the mixed strain of Lactobacillus rhamnosus and Lactobacillus acidophilus of the present invention, it was confirmed that the antifungal activity against Candida strains increased.

As in Example 1.1, Candida albicans ( Candida albicans , ATCC 10231) was inoculated onto Potato Dextrose agar (PDA) medium and pre-cultured at 25°C for 2 days. Then, 10 ⁶ CFU/ml Candida albicans was inoculated using sterile saline solution. A bacterial solution was prepared. Then, a 1:4 strain mixture homogenized by adding distilled water to Lactobacillus rhamnosus HN001 4×10 ⁴ CFU/ml and Lactobacillus acidophilus La-14 1.6×10 ⁵ CFU/ml was prepared to prepare a test solution of 2×10 ⁵ CFU/ml, 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 oligosaccharide 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, Candida in samples A, B, C and D albicans After adding and mixing 0.1 ml of the bacterial solution, cultured at 25°C for 24 hours, measured the number of viable bacteria using a dilution culture method, and Candida albicans The number of strains was converted into a survival rate (%) relative to the initial number of bacteria and is shown in Table 3 and Figure 2 . At this time , Candida in sterile saline solution albicans The group to which the strain was added was used as the control group (Con.).

As shown in Table 4 and Figure 2 , the mixed 1:4 mixed strain of Lactobacillus rhamnosus and Lactobacillus acidophilus is 2×10 ⁵ CFU/ml. Candida albicans bacterial count was inhibited by about 25%. In the case of Sample B, in which 2% (w/v) of betulin was added, the antifungal effect against Candida strains of Lactobacillus rhamnosus and Lactobacillus acidophilus mixed strains increased by more than 36%.

When 1.5% (w/v) of alpha-glucanoligosaccharide, which can be provided as food for lactic acid bacteria such as Lactobacillus rhamnosus or Lactobacillus acidophilus, is added (C), Lactobacillus rhamnosus and Lactobacillus acido The antifungal effect of the Pillus mixed strain against Candida strains increased by more than 20%. In addition, when adding 2% (w/v) of betulin and 1.5% (w/v) of alpha-glucanoligosaccharide to the mixed strains of Lactobacillus rhamnosus and Lactobacillus acidophilus mixed 1:4. (D), there was no significant difference from the additional betulin administration group (B), but there was a tendency to increase the antifungal effect against Candida strains compared to the additional betulin administration group (B).

Through the above results, the 1:4 mixed Lactobacillus rhamnosus and Lactobacillus acidophilus mixed strain of the present invention significantly increased antifungal activity against Candida strains at a specific mixing ratio, including betulin and /Or, it was confirmed that the addition of alpha-glucanoligosaccharide increased the antifungal effect of the Lactobacillus rhamnosus and Lactobacillus acidophilus mixed strains mixed at a ratio of 1:4.

< Example 2. Lactobacillus rhamnosus and Lactobacillus Acidophilus Deodorizing performance test of mixed composition>

Vaginitis such as bacterial vaginosis and candida vaginitis cause great stress to women due to the unpleasant odor of vaginal discharge. The present inventor tested the deodorizing performance of a 1:4 mixture of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14, which had the best effect on inhibiting Candida strains, and a sample to which betulin was added.

Deodorization performance was confirmed by measuring the reduction rate of ammonia or trimethylamine concentration among the EL608 deodorant test methods in the Ministry of Environment's environmental label certification standard. Adjust the concentration of ammonia or trimethylamine diluted with acetone in the test container to around the initial concentration (C _b,i ) according to the certification standard, and then mix Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 (1:4 mixing ratio) strain 2. ×10 ⁵ CFU/ml or mixed strain of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 (1:4 mixing ratio) 2×10 ⁵ CFU/ml with 2% (w/v) of betulin added to the sample in the test container. It was sprayed and injected through the injection port. At 6 hours after injection of each sample, the gas in the test container was collected using a gas sampler specified in SPS-KCLI2218-6218, and the concentration (C _t,i ) of ammonia or trimethylamine, which is an odor component, was measured. At this time, a direct-reading detection tube corresponding to the odor component to be measured and the expected concentration range was inserted into the gas sampler.

Deodorization performance (reduction rate of odor component concentration) 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 above, it was confirmed that the 1:4 mixed strain of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14, which had the best effect in suppressing Candida strains, reduced the concentrations of ammonia and trimethylamine by more than 60%. . Also Lactobacillus When betulin was added to a 1:4 mixed strain of rhamnosus HN001 and Lactobacillus acidophilus La-14, it was confirmed that the deodorizing effect increased.

As seen from the above results, the 1:2 to 6 mixture of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 of the present invention is more effective in reducing Candida albicans, a vaginitis-causing bacteria, compared to the mixture of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 at other ratios. Not only is the inhibitory effect excellent, but it also has the effect of directly reducing the concentration of ammonia and trimethylamine, and when betulin was added, it was confirmed that the Candida albicans antifungal effect and deodorizing effect were further increased.

< Example 3. Lactobacillus rhamnosus and Lactobacillus Acidophilus Powder type containing mixture feminine cleanser Check the effect>

3.1. Manufacturing of powdered feminine cleanser

A powder-type feminine wash containing a 1:4 mixture of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14, which has the most effective effect in suppressing Candida strains, was prepared.

Powdered feminine wash 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. Powdered feminine wash is a mixture of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 in a 1:4 ratio, and betulin, which increases antifungal activity against Candida strains, is added, and rice starch and rice starch are added to powder the composition. Silica was added. In addition, alpha-glucan oligosaccharide was added, which acts as a medium for lactic acid bacteria and increases antifungal activity against mixed strains.

Rice starch is used as an absorbent and bulking agent to maintain the composition in powder form and increase its volume, while 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-glucanoligosaccharide 1.5 silica 1.0

3.2. Verification of the effect of powdered feminine cleanser on vulvar itching and odor

To determine the effect of this powder-type feminine cleanser on vulvar itching and odor, the Korea Dermatological Research Institute was asked to apply it to the vulva of 21 adult women over 25 years of age for 4 weeks from September 2020 and observe changes.

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

Before using test substance question frequency percentage No discomfort due to itchiness 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 Vulvar itching was more alleviated 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 has become more relieved. 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 were experiencing discomfort due to vulvar itching or odor. This can be said to indicate that vaginitis is a common disease among women, so much so that it is called the 'women's cold', and that many women are experiencing discomfort due to it.

As shown in Table 7 and Figure 3 , when the 1:4 mixture of Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14 of the present invention, which has the best effect in suppressing Candida strains, is applied directly to the female vulva, after 2 weeks, 80.1%, After 4 weeks, 95.2% of subjects responded that vulvar itching was relieved. In the items related to vulvar odor, 85.7% of the subjects responded that the vulvar odor was alleviated after 2 weeks of use, and after 4 weeks of use, all subjects who participated in the experiment responded that the vulvar odor was alleviated.

In addition, the subjects felt that their skin glow, clear skin color, and skin density had all improved, confirming that they were highly satisfied with this feminine wash.

3.2 Confirmation of the effect of powdered feminine cleanser on vulvar skin

It was used on the vulvas of 21 adult women over 25 years of age 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 when showering once a day, applied it to the vulva, gently massaged it, and then washed it off with lukewarm water. Elasticity, moisture, shine, clarity, and density were measured before using the test substance, after 2 weeks of use, and after 4 weeks of use, 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 vulvar skin color 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 vulvar skin is elastic Not at all 5 23.8 The vulvar skin 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 vulvar skin seems to have become 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 vulvar skin seems to have become 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 to be 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 The vulvar skin seems to have become denser and firmer. 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 Table 8 and Table 9 , the test subjects showed higher levels of satisfaction in proportion to the period of use in terms of moisturizing, elasticity, gloss, clarity, and density of the skin of the vulva compared to before using the test substance. In particular, there was a high response that skin moisturization and density were greatly improved, and 76.2 to 100% of subjects thought that skin elasticity, gloss, and clarity were all improved.

3.3. Confirm the effect of improving skin elasticity

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

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

3.3. Confirm the effect of improving skin moisturization

Epsilon E100 (Biox Systems Ltd., UK) was applied to evaluate the skin moisturizing improvement of the powdered feminine cleanser of the present invention. Epsilon E100 calculates the moisture on the skin surface in contact with the sensor as an Epsilon (ε) value. As the skin moisture content increases, the brightness of the image increases, making the blue part appear closer to white. The same tester measured the left armpit area of all test subjects, and the change in skin moisture was analyzed using Epsilon E100 Software V3.1, an analysis program dedicated to Epsilon E100, and the improvement in skin moisturization compared to before using the test substance. (%) was calculated and shown in Figure 5 .

As shown in Figure 5 , when using the powdered feminine wash of the present invention, the skin moisturizing rate improved depending on the period of use, and after 4 weeks of use, the skin moisturizing rate improved by 22.48% compared to before use.

3.4. Confirm the effect of improving skin radiance

A gloss meter (Multi Gloss 268 PLUS, Konica Minolta, Japan) was applied to evaluate the skin gloss improvement of the powdered feminine cleanser of the present invention. The gloss meter measured the left armpit area 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, which is shown in Figure 6 . The measurement value of the gloss meter consists of three measurement values: 20˚, 60˚, and 85˚. 20˚, 60˚, and 85˚ represent the ratio of light emitted at each angle to be reflected and measured. The value at 60° was used to measure the skin gloss of the composition.

As shown in Figure 6 , when using the powdered feminine wash of the present invention, skin gloss improved depending on the period of use, and after 4 weeks of use, skin gloss improved by 16.13% compared to before use.

3.5. Confirm the effect of improving skin clarity

SkinColorCatch (Delfin Technologies Ltd., FinLand) was applied to evaluate the improvement in skin clarity of the powdered feminine wash of the present invention. SkinColorCatch was measured by contacting the probe perpendicularly to the left armpit area of all test subjects. White LED light was illuminated at 45° within the probe, and the light reflected on the skin was detected with an RGB color sensor, and compared with before using the test substance. The skin clearness improvement rate (%) was calculated and shown in Figure 7 . The measurement value of SkinColorCatch consists of five factors: L ^* , a ^* , b ^* , melanin index, and erythema index. The L ^* value is brightness, a ^* value is redness, and b ^* The value represents yellowness, the melanin index represents the amount of melanin present in the skin, and the erythema index represents the amount of skin erythema. The L ^* value, which indicates the brightness of the skin, was used in the analysis as a value to measure the composition's improvement in skin clarity.

As shown in Figure 7 , when using the powdered feminine wash of the present invention, skin clarity improved depending on the period of use, and after 4 weeks of use, skin clarity improved by 1.04% compared to before use.

3.6 Confirmation of skin density improvement effect

To evaluate the improvement in skin density of the powdered feminine wash of the present invention, the DUB-Skin Scanner (Taberna pro medicum, Luneburg, Germany), a high-resolution ultrasound imaging device, was applied. Distilled water was injected into the water chamber of the probe equipped with a scanning head so that it was at a right angle to the skin, and skin density was measured by pressing the left armpit area of all test subjects with the same pressure. Density value, which represents skin density, was analyzed as a measure of the composition's improvement in skin density, and the analysis range is from just below the epidermis to the upper part of the subcutaneous fat layer. The degree of compactness was analyzed in stages from 0 (sparseness) to 255 (denseness), and the measurement unit % was used. The skin density improvement rate (%) was calculated compared to before using the test substance, and is shown in Figure 8 .

As shown in Figure 8 , when using the powdered feminine wash of the present invention, skin density improved depending on the period of use, and after 4 weeks of use, skin density 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, betulin, and alpha-glucan oligosaccharide mixed composition of the present invention has an excellent inhibitory effect on Candida albicans, a vaginitis-causing bacteria. In addition, by directly reducing the concentration of ammonia and trimethylamine, it is effective in removing vulvar itching and bad odor when applied to the vulva by actual users, and by manufacturing it as a powdered feminine wash containing rice starch and silica, it provides skin elasticity. , it was confirmed that there was an improvement effect in moisturizing, gloss, clarity, and density.

Claims (6)

Lactobacillus rhamnosus HN001 ( Lactobacillus rhamnosus HN001) and Lactobacillus acidophilus La-14 ( Lactobacillus acidophilus La-14) mixed in a ratio of 1:2 to 6, lactic acid bacteria, rice starch, betulin, and alpha-glucan Powdered feminine wash containing oligosaccharide and silica. According to paragraph 1,
The powdered feminine wash contains 1 to 50% by weight of mixed lactic acid bacteria Lactobacillus rhamnosus HN001 and Lactobacillus acidophilus La-14, 20 to 95% by weight of rice starch, and 20 to 95% by weight of rice starch. A powdery feminine wash comprising 0.01 to 20% by weight of thulin, 0.01 to 15% by weight of alpha-glucan oligosaccharide, and 0.01 to 10% by weight of silica. According to claim 1 or 2,
The 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 Powdered feminine wash, characterized in that it is mixed in a ratio of. According to paragraph 1,
The powdered feminine wash is a powder type characterized in that it has one or more effects selected from the group consisting of removing vulvar odor, relieving itching, improving skin elasticity, improving skin moisturization, improving skin gloss, improving skin clarity, and improving skin density. Feminine cleanser. According to paragraph 4,
A powder-type feminine cleanser that removes odor from the vulva by reducing the concentration of ammonia or trimethylamine. According to claim 1 or 2,
The feminine wash is a powder-type feminine wash, characterized in that it inhibits Candida albicans strains.