KR20150075447A - Cosmetic composition for prevention and improvement of vaginosis - Google Patents

Abstract

The present invention relates to a functional composition for preventing and alleviating cervical vaginosis. A method for producing the same comprises the steps of: selecting Lactobacillus plantarum MB01, Lactobacillus acidophilus MB07, and Lactobacillus crispatus MB08 from a specimen of a female, and evaluating biochemical properties of the selected lactobacillus; culturing the selected lactobacillus using a GPY culture medium, a GGPY culture medium, a P-GPY culture medium, and P-GGPY culture medium, confirming a growth speed and an acidity change, and evaluating an activity in inhibiting harmful microorganisms of the selected lactobacillus and an antibacterial effect of a supernatant in which the selected lactobacillus are cultured; and producing a functional composition for preventing and alleviating cervical vaginosis, wherein he functional composition contains the selected lactobacillus. The functional composition according to the present invention restores microorganism flora in the abnormal vagina to be normal flora, so that a woman can maintain her health and acidity in the vagina can be normally maintained. Accordingly, several sorts of disease can be prevented, and normal microorganism flora can be restored in a short time after menstruation.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a composition for prevention and improvement of vaginitis,

The present invention relates to a composition for preventing and improving female uterine vaginitis.

The quality of women is located at the entrance of the uterus and is directly affected by the menstrual cycle and is located close to the urethra and the anus and is highly likely to be contaminated by colon and microbial diseases (Cho, YH, 2006, Introduction to urinary tract infection, Kor. J. Urol., 47, 559-567). E. coli, Tricomonas vaginalis, Candida albicans, Chlamydia, Haemophilus vaginalis, Herpes viruses, and the like are examples of pathogenic microorganisms that cause infection in the female genital area. And Gardnerella sp., And vaginitis is a common infection of these strains, either alone or in combination. Bacterial vaginosis (BV) generally accounts for more than 50% of infections in the female reproductive system that represent symptoms of persistent chronic infections (Sobel, 1990). Especially, when women are sexually active, they can not maintain their normal state due to infiltration of pathogenic microorganisms. In addition to being able to suffer various forms of diseases such as local inflammation, harmful microorganisms penetrate into the pelvic girdle, Also.

In order to prevent the contamination of external microorganisms, there are two types of female hormones: female hormones (Lactobacilli, Peptosreptococcus, group β-hemolytic streptococcus, and coliform bacteria) Glycogen stored in vaginal epithelial cells under the influence of estrogen is used for lactic fermentation. The lactic acid fermentation is performed by the lactic acid bacteria present in the vagina, and the proper level of the lactic acid bacteria maintains the pH of the vagina at 4.5 to 5.5, thereby inhibiting the growth of pathogenic microorganisms invading from the outside.

On the other hand, lactic acid bacteria are widely used as probiotic bacteria. They are widely found in nature and are easily found in the intestines and fermented foods of humans and animals. Food and Drug Administration (Orrhge, K. et al., 2000, Bifidobacteria and lactobacilli in human health, Drugs Expt. Clin. Res., 26, 95-111). Lactic acid bacteria attach to the intestinal epithelial cells and become parasitic, thereby improving the properties of intestinal microflora. Thus, the intestinal microflora such as the stabilization of intestinal microflora, the reduction of the formation of decay products due to the inhibition of harmful bacteria and the prevention of disease, immune activation, It helps a lot. Lactic acid bacteria have several metabolic characteristics that inhibit the growth of various pathogenic microorganisms and pathogenic microorganisms because lactic acid bacteria produce organic acid, hydrogen peroxide, reuterin, diacetyl, acetaldehyde, bacteriocin, etc. as metabolic products (Fuller, K., 1989, Probiotics in man and animals, J. Appl. Bacteriol., 66, 365-378).

Bacterial vaginitis is treated clinically by oral administration of antibiotics or direct vaginal or vaginal administration (Blackwell et al. 1983), imidazole and nitroimidazole derivatives are often used to treat these symptoms, Other types of drugs that are used include nitrofurfuryl derivatives and various antibiotics. These active antibiotic components may be formulated for oral or topical administration, wherein metronidazole is often administered by the oral route, but mixed infections may be formulated for topical administration, particularly for pessary forms containing two or more active ingredients, Lt; / RTI > However, in the case of treatment with antibiotics, antibiotics affect the growth of normal bacterial flora, especially lactic acid bacteria, in the vagina as well as pathogenic microorganisms causing vaginitis, thereby reducing the number of bacterial flora in the vagina, Can be generated.

Probiotic agents for treating vaginitis are disclosed in Korean Patent No. 10-0400411, and a vaginal therapeutic composition is disclosed in Korean Patent No. 10-1092731. In addition, Lactobacillus valve L. var. Bulkuricus HW-7901 and a health functional food product containing it as an active ingredient are disclosed in Korean Patent No. 10-1084049. However, none of the literatures discloses a vaginitis prevention and improvement functional composition in the form of a single composition or a mixture of L. acidophilus, L. crispatus and L. plantarum.

Accordingly, an object of the present invention is to provide lactic acid bacteria such as L. acidophilus, L. crispatus, and L. plantarum which are effective for preventing and improving vaginitis.

Another object of the present invention is to provide a composition for preventing and / or improving vaginitis comprising L. acidophilus, L. crispatus and L. plantarum.

The above object of the present invention can be achieved by a method of selecting lactic acid bacteria, comprising the steps of: Lactobacillus plantarum MB01, Lactobacillus acidophilus MB07 and Lactobacillus crispatus MB08 of the above selected lactic acid bacteria, and evaluating their biochemical characteristics; Culturing said selected lactic acid bacterium using GPY medium, GGPY medium, P-GPY medium and P-GGPY medium to confirm growth rate and acidity change; Confirming the harmful microorganism inhibiting activity and the characteristic change of the selected lactic acid bacteria; Evaluating the antimicrobial effect of the above-mentioned lactic acid bacteria culture supernatant, and preparing a vaginitis prevention and improvement functional composition containing the selected lactic acid bacteria of the present invention.

The present invention has the effect of restoring the abnormal microorganism of female vagina to normal microorganism, thereby maintaining the health of female, maintaining the acidity of vagina in the normal state, preventing the various diseases and restoring normal microorganism microorganism within a short time after menstruation It has excellent effect.

FIG. 1 is a graph showing growth rate and acidity change of lactic acid bacteria Lactobacillus acidophilus in GPY medium according to the present invention. FIG.
2 is a graph showing the growth rates of C. ablicans and lactic acid bacteria in media containing povidone according to the present invention.
FIG. 3 is a graph showing changes in the number of live cells according to the mixed culture of the selected lactic acid bacteria and E. coli in the present invention.
FIG. 4 is a graph showing changes in the viable cell counts according to the mixed culture of the selected lactic acid bacteria and S. aureus in the present invention. FIG.
FIG. 5 is a graph showing changes in pH and acidity with time in the mixed culture of the selected lactic acid bacteria and E. coli according to the present invention. FIG.
FIG. 6 is a graph showing changes in pH and acidity with time in the mixed culture of the selected lactic acid bacteria and S. aureus according to the present invention. FIG.
FIG. 7 is a photograph showing the microbial growth inhibitory effect of the lactic acid bacteria culture supernatant selected according to the present invention. FIG.

In the present invention, female samples were obtained from the urology department of Seoul Hong-Tae, Daejeon Newborn Obstetrics and Gynecology and Inchon Maximus Obstetrics and Gynecology Department and the products of Difco (Becton, Dickinson and Co., Sparks, MD 21152, USA) were used.

In the present invention, the lactic acid bacteria may be in the form of a culture solution or a powder, and in the case of a powder, it may be prepared by any method capable of maintaining the activity of the microorganism, and most preferably, it is prepared in the form of a lyophilized powder.

The composition of the present invention may be prepared in the form of powders, gels, ointments, creams, solutions or solid preparations, and may contain conventional components incorporated into the composition for external application for skin such as antibiotics, binders, disintegrants, , A lubricant, a stabilizer, a preservative, a perfume or the like.

In addition, the composition of the present invention can be used according to a conventional method of use, and the number of times of use may be varied depending on the state of the user.

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are intended to illustrate the invention and are not intended to limit the scope of the invention.

Example 1: Selection of lactic acid bacteria useful in the present invention

Since May 1999, more than 200 female surveys have been obtained from obstetrics and gynecology clinics in Seoul, Daejeon and Incheon. Lactic acid bacteria were selected from MRS medium, which is a lactic acid selection medium, from the samples of patients having normal microorganism strains as much as possible in order to select lactic acid bacteria having strong lactic acid fermentation ability.

<Experimental Example 1> Evaluation of the characteristics of the selected lactic acid bacteria

Among the useful lactic acid bacteria biochemical characteristics selected in the present invention, the size was measured using a micrometer microscope, and the Gram negative / gram positive determination was determined by Gram stain of Difco. In the catalase test, the 1% H ₂ O ₂ solution was dropped on the colonies to determine whether it was foamed. The pH was measured using a pH meter (Orion Star A211, Thermo, Singapore) ml of phenolphthalein solution was added to 0.1N NaOH solution, and the amount of NaOH solution added until the color changed was multiplied by 0.059.

As shown in Table 1, MB01, MB07 and MB08 were selected as the final lactic acid bacteria in consideration of the final acidity after lactic acid fermentation, tolerance to povidone, and survival rate in mixed culture with Staphylococci and Escherichia coli, Were selected. The three selected lactic acid bacteria were identified as Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus crispatus in Inha University professor Han Hong 's lab and Seoul National University, respectively. Lactobacillus plantarum MB01, Lactobacillus acidophilus MB07 and Lactobacillus crispatus MB08 were named according to the strain classification criteria of the inventors.

The useful lactic acid bacteria biochemical characteristics selected in the present invention Colony Characteristics of Colonies Size of bacterium Gram Staining Catalase Test CPR test final pH total Acidity (%) Microscopic Morphology MB01 white, entier, flat 1.5m + - + 3.82 1.50 rod MB07 white, entier, flat 2.5 + - + 3.80 1.48 rod MB1 white, entire, convex 0.1 + - + 4.29 1.48 rod MBa2-2 white, irregular, flat 1.0 + - + 3.97 1.86 coccobacillus MB08 white, entier, flat 2.0 + - + 3.84 2.19 rod MBb white, convex, entire 3.0 + + - 6.06 0.28 yeast MBd white, convex, entire 1.5 + ++ - 6.56 0.22 yeast (slim) MBe white, convex, irregular 0.5 + - + 4.65 1.03 coccobacillus MBh pale yellow 0.6 + + + 4.80 0.86 coccus MB1-1 white, convex, entire 1.0 + + - 4.57 1.10 staphylococcus MB1-2 white convex, entire 1.0 + + + 4.93 0.73 staphylococcus MB1-3 white, convex, entire 0.8 + + + 4.85 0.79 staphylococcus MBi2 white, convex, entire 0.5 + - + 5.00 0.63 streptococcus

Example 2 Culture of Lactic Acid Bacteria Sorted According to the Present Invention Using GPY Medium and GGPY Medium

The above three lactic acid bacteria were cultured using a medium having the composition shown in Table 2 below. Each lactic acid bacterium was previously cultured in MRS medium for 48 hours, inoculated into GPY and GGPY medium at 1% (v / v), and cultured at 37 ° C for 72 hours.

The composition ratio of the GPY and GGPY liquid media of the present invention badge GPY GGPY ingredient Composition ratio (%) Composition ratio (%) Glucose 2.0 2.0 Glycerin - 10.0 Peptone 0.1 0.1 Yeast Extract 0.1 0.1

<Experimental Example 2> Growth and acidity change of lactic acid bacteria of the present invention using GPY medium and GGPY medium

The growth of lactic acid bacteria was measured by counting the number of microbial cells inoculated with the lactic acid bacteria in the MRS liquid medium and the acidity was measured in the same manner as in Experimental Example 1 above. The growth rate and acidity of Lactobacillus crispatus MB08, which is considered to be superior to lactic acid fermentation ability, are shown in FIG. 1.

As shown in FIG. 1, the growth of the lactic acid bacteria in the GPY liquid medium having a low viscosity was remarkably increased, but the survival rate of the useful lactic acid bacteria at the end of the 72 hour culture was not significantly different . In acidity, it was confirmed that the acidity increase in GPY liquid medium increased with time. In the GGPY medium, the acidity did not increase significantly after 24 h, but survived to 1.0 x 10 ⁵ pfc / ml after 72 h.

Example 3 Culture of the Present Invention Lactic Acid Bacteria Using P-GPY Medium and P-GGPY Medium

(P-GPY and P-GGPY) to which povidone was added were difficult to inhibit with candida bacteria only by lactic acid fermentation of normal lactic acid bacteria. The above three kinds of lactic acid bacteria were cultured using a medium having the composition shown in [Table 3]. Each of the lactic acid bacteria was pre-cultured in MRS medium for 48 hours, inoculated 1% (v / v) in P-GPY and P-GPY medium, and cultured at 37 ° C for 72 hours.

The composition ratio of the P-GPY and P-GGPY liquid media of the present invention badge P-GPY P-GGPY ingredient Composition ratio (%) Composition ratio (%) Povidone 0.09 0.09 Glucose 2.0 2.0 Glycerin - 10.0 Peptone 0.1 0.1 Yeast Extract 0.1 0.1

<Experimental Example 3> Growth and acidity change of lactic acid bacteria according to the present invention using povidone-added medium

Lactic acid bacteria were cultured in MRS medium for 48 hours and then inoculated at 1% (v / v). C. albicans was cultured for 24 hours in YM medium, . The degree of growth of the microorganisms was measured by absorbance at 530 nm using a colorimetric system at the time points of 24 hours and 48 hours. Lactobacillus crispatus MB08 and Lactobacillus plantarum MB01, which were considered to be excellent in lactic acid fermentation ability, were cultured in a medium supplemented with Candida albicans and povidone and a culture medium containing povidone at a proper concentration.

As a result, FIG. 2A shows the growth of Lactobacillus and Candida in the P-GGPY medium supplemented with povidone, and FIG. 2B shows the growth in the same medium without povidone. Lactobacillus plantarum MB01 showed approximately 1.0 level of OD ₅₃₀ in the culture medium without povidone at 24 hours and 48 hours, but it was 0.81 ~ 0.93 in the medium supplemented with povidone Giving. On the other hand, in the case of C. albicans, the OD ₅₃₀ value was about 1.3 when povidone was absent, and it was confirmed that when povidone was added, it dropped to the level of 0.2 to 0.8.

&Lt; Experimental Example 4 > Effect of the present invention on inhibition of harmful microorganisms and characterization of lactic acid bacteria

Escherichia coli and Staphylococcus aureus, which are gram-negative bacteria and Gram-positive bacteria, were selected as harmful microorganisms and mixed with the above three lactic acid bacteria. Three kinds of lactic acid bacteria of the present invention were cultured separately in MRS medium for 18 hours. E. aureus was cultivated in TSB (Tryptic soy broth) for 18 hours, and each of them was diluted 1% (v / v) Liquid medium, and then incubated at 37 ° C to measure viable cell count, acidity and pH. L. plantarum MB01 and L. acidophilus MB07 were grown on MRS media, L. crispatus MB08 on TSA (Tryptic soy agar) medium, E. coli and S. aureus on EMB (Eosin Methylene Blue) medium And then diluted.

As shown in FIGS. 3 and 4, E. coli and S. aureus were killed at 9 and 24 hours after incubation, respectively.

As shown in FIGS. 5 and 6, when the pH of the medium was changed, E. coli and S. aureus, which are typical microorganisms capable of causing vaginitis, were cultured in the presence of lactic acid bacteria, 5.0) and the pH value at 24 hours (not more than 4.0), it was confirmed that the increase of the acidity inhibits the survival of the microorganism causing the vaginitis.

&Lt; Experimental Example 5 > Antibacterial effect of the lactic acid bacteria culture supernatant of the present invention

Three lactic acid bacteria selected in Example 1 were separately cultured in MRS medium for 18 hours and the culture supernatant was obtained. The culture supernatant of L. plantarum MB01 was used as it was, and the culture supernatant of L. acidophilus MB7 and L. crispatus MB08 was concentrated 10 times and used in the experiment. The culture supernatant (300 μL) of each of the above strains was added to the wells punched using a cork boiler at the center of the TSA medium coated with E. coli, S. aureus, and C. albicans, and cultured at 37 ° C. for 24 hours. The size of the inhibitory ring was confirmed.

As a result, as shown in FIG. 7, the culture supernatants of the three lactic acid bacteria showed a marked inhibitory effect on E. coli, but the inhibitory effect on S. aureus was not as clear as that of E. coli. Another reason is that S. aureus is more resistant to changes in pH than E. coli. Another reason is that lactic acid bacteria are incubated for only 18 hours and then the supernatant is obtained . The lactic acid bacteria showed sufficient efficacy when cultured for 72 hours.

<Example 4> Preparation of composition for prevention and treatment of vaginitis containing the selected lactic acid bacterium

0.1 to 10% by weight based on the total weight% of the above-mentioned selected lactic acid bacterium was added to the sweet potato starch as a supplementary raw material. Examples of the additives include talc, potato starch, corn starch, silica powder, and the like. In the present invention, the lactic acid bacteria in the composition for prevention and treatment of vaginitis containing the produced lactic acid bacterium exhibit insufficient effect of the pathogenic microorganism of the present invention in an amount of less than 0.1% by weight based on the total weight%, and the microorganism inhibition in the case of 0.1 to 10% Since the effect is sufficiently exhibited, 0.1 to 10% by weight is most preferable.

INDUSTRIAL APPLICABILITY As described above, the present invention is an industrially advantageous invention because it has an excellent effect of providing vaginal lactobacillus L. acidophilus, L. crispatus, L. plantarum, and a composition for prevention and treatment of vaginitis containing the lactic acid bacteria culture liquid and the active ingredient thereof .

Korea Biotechnology Research Institute KCTC11877BP 20110223

Claims (4)

Lactobacillus plantarum MB01 strain (KCTC11877BP), which is characteristic of restoring vaginal environment to normal state.
Lactobacillus plantarum is a Lactobacillus plantarum MB01 microbial agent characterized by containing as an active ingredient that MB01 lyophilized strain (KCTC11877BP).
Wherein the Lactobacillus plantarum MB01 microbial agent is present in an amount of 0.1 to 10% by weight based on the total composition.
The cosmetic composition according to claim 3, wherein the cosmetic composition is a powder selected from the group consisting of a water-soluble liquid, lotion, cream, gel, pack, essence, O / W, W / O or W / Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt;

Images