KR101633888B1 - Composition Comprising of Pseudomonas aeruginosa Media Extract Having Antimicrobial and Antiseptic Activity and The Use Thereof - Google Patents

Abstract

The present invention provides an antimicrobial and antimicrobial composition comprising an extract of a culture solution of P. aeruginosa as an active ingredient. The present invention provides a composition for preventing or treating acne comprising an extract of a culture solution of P. aeruginosa as an active ingredient. The composition containing the extract of the cultured Pseudomonas aeruginosa extract of the present invention exhibits a broad spectrum of antimicrobial spectrum and a high antioxidative effect against various fungi. Such a composition can be applied to cosmetic compositions such as cosmetics and also can be applied to a variety of foods, medicines, It is useful for antibacterial, sterilization and preservation purposes in the field. The present invention identifies specific active ingredients causing the antimicrobial effect of the extract of the cultured Pseudomonas aeruginosa and provides the structure thereof.

Description

TECHNICAL FIELD The present invention relates to a composition comprising a Pseudomonas aeruginosa culture extract having antimicrobial and antiseptic activity and a use thereof.

The present invention relates to a composition comprising a Pseudomonas aeruginosa culture extract having antimicrobial and antiseptic activity and its use.

Parabens are commonly used as antiseptic preservatives in cosmetics, food, and pharmaceuticals to inhibit the growth of microorganisms and increase the shelf life. They are known to have no toxicity and have been used for a long time since they were first used in medicine in the mid-1920s. While the use of parabens is widespread and their use in various products is prolonged, studies on the estrogenicity of parabens have been reported, raising the question of the safety of parabens in humans.

 Studies on the acute, subacute and chronic toxicity of parabens have been conducted based on the results of various analyzes on experimental animals. The sensitization reactions of methylparaben such as skin inflammation, delayed, contact and hypersensitivity of methyl and propylparaben have been reported As regards the carcinogenicity of parabens, parabens have estrogenic properties, suggesting the association of parabens with breast cancer through cosmetic use. Anti-androgenicity of parabens is also known to interfere with the function of the male reproductive system, and the effects of parabens released by sewage on the ecosystem and the environment are also discussed. Despite its potential hazards, parabens are being used in a variety of areas because it is not as good as the chemical preservatives of natural ingredients. However, as the above-mentioned risk of parabens is reported, consumers express their refusal to use parabens preservatives and the demand for alternative natural preservatives is increasing.

Biosurfactant is a surfactant obtained from biomaterials such as microorganisms, animals and plants, and has been attracting attention because it is low in toxicity and easily decomposed by ecosystem. The advantages of biosurfactant for chemosynthetic surfactants are: first, it is non-toxic and easy to biodegrade; therefore, it is not a secondary pollutant when used; and secondly, it can be used for special purposes due to its complex chemical structure Third, it shows that the physical and chemical performance of surfactants, such as ability to degrade surface tension, stability to temperature, and pH, are comparable to those of conventional chemically synthesized surfactants.

Actual biosurfactants are used in most various industrial fields where chemical synthetic surfactants are used, such as pharmaceuticals, food, cosmetics, detergents, secondary recovery of crude oil, pulp and paper industry, oil pollution cleansing on land and sea, And also Pseudomonas sp. Researchers have also reported that the Ramsonite-biosurfactant produced from the Ramsey biosurfactant may be applied as a new field in biomedicine because it changes cell surface morphology and exerts its inhibitory effect against pathogens. Therefore, research on biosurfactants having such advantages has been actively conducted worldwide, and many types of biosurfactants have been reported.

Prior to the present invention, Pseudomonas sp. The inhibitory effect of various surfactants on the pathogenic bacteria was investigated by extracting the mixture of the surfactant with rhamnoside from the culture solution. As a result, compared with the conventional antibiotics , specific pathogens ( Propionibacterium acnes, Staphylococcus aureus ) And it has an excellent inhibitory effect. In addition, it has been confirmed that it has a sterilizing effect at a low concentration on various pathogens. Based on these results, it is possible to provide an environmentally friendly and biocompatible material which can be used as a cosmetic preservative, The present inventors have studied the development of a preservative and, at the same time, the development of raw materials capable of alleviating acne by inhibiting antimicrobial-resistant acne-causing bacteria, and the following experimental results can provide concrete details and basis for practicing the present invention.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to develop a composition for a naturally occurring substance which can effectively inhibit the growth of microorganisms and thereby increase the shelf life of cosmetics or foods. As a result, it was confirmed that the extract of Pseudomonas aeruginosa culture showed antibacterial effect on various bacteria, and it was confirmed that the extract of Pseudomonas aeruginosa culture liquid of the present invention could be used as a cosmetic preservative and food preservative instead of synthetic parabens cosmetic preservative Thereby completing the invention.

Accordingly, an object of the present invention is to provide a composition for ostomy containing an extract of the cultured Pseudomonas aeruginosa as an active ingredient.

Another object of the present invention is to provide an antibiotic composition comprising an extract of a culture solution of P. aeruginosa as an active ingredient.

Another object of the present invention is to provide a composition for preventing or treating acne, which comprises an extract of a culture solution of P. aeruginosa as an active ingredient.

It is another object of the present invention to disclose an effective ingredient which causes the antimicrobial effect of the extract of the cultured Pseudomonas aeruginosa.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention and claims.

According to one aspect of the present invention, there is provided an anticancer composition comprising an extract of a cultured broth of Pseudomonas aeruginosa as an active ingredient.

According to another aspect of the present invention, there is provided an antibiotic composition comprising, as an active ingredient, an extract of a cultured broth of Pseudomonas aeruginosa.

According to another aspect of the present invention, there is provided a composition for preventing or treating acne comprising an extract of a culture solution of P. aeruginosa as an active ingredient.

The present inventors have sought to develop a composition for a naturally occurring substance which can effectively inhibit the growth of microorganisms and thereby increase the shelf life of cosmetics or foods. As a result, it was found that the extract of Pseudomonas aeruginosa culture showed antimicrobial effect on various bacteria including skin onion and acne-causing bacteria, and that the extract of Pseudomonas aeruginosa culture liquid of the present invention replaces cosmetic preservatives of synthetic parabens, It can be used as a food preservative.

The composition of the present invention contains an extract of Pseudomonas aeruginosa culture medium as an active ingredient.

As used herein, the term " extract of Pseudomonas aeruginosa culture medium " means a product obtained by extracting from a culture solution in which Pseudomonas aeruginosa is cultured.

According to the present invention, the Pseudomonas aeruginosa culture extract of the present invention includes a biosurfactant, rhamnolipid.

According to one embodiment of the present invention, the rhamnoripid contained in the composition of the present invention is a di-rhamnolipid represented by the following formula (1) or a mono-rhamnolipid represented by the following formula (2)

 Formula 1

(2)

According to the present invention, the extract of the cultured Pseudomonas aeruginosa has a variety of structures of rhamnose ribides. Among them, the rhamnose ribide of the above formula (1) or (2) has a remarkably high antimicrobial activity as compared with other rhamniripids extracted from the extract of the cultured Pseudomonas aeruginosa.

According to one embodiment of the present invention, the Pseudomonas aeruginosa culture extract of the present invention is obtained by inoculating and cultivating Pseudomonas aeruginosa in a culture medium, centrifuging the culture to obtain a supernatant, adding a hydrochloric acid solution to the supernatant to precipitate, , Adding ethyl ether to the precipitate, separating and concentrating only the ethyl ether layer, and concentrating.

The extract of the present invention includes not only those obtained by using the above-mentioned solvents, but also those obtained by additionally applying a purification process thereto. For example, a fraction obtained by passing the above extract through an ultrafiltration membrane having a constant molecular weight cut-off value, and a separation by various chromatography (manufactured for separation according to size, charge, hydrophobicity or affinity) The fraction obtained by the purification method is also included in the extract of the present invention.

The extract of the present invention can be prepared in powder form by an additional process such as vacuum distillation and freeze-drying or spray-drying.

According to one embodiment of the present invention, the content of the extract of Pseudomonas aeruginosa culture in the composition of the present invention is 0.005-1.0 parts by weight based on 100 parts by weight of the total composition.

According to another embodiment of the present invention, the content of the Pseudomonas aeruginosa culture extract in the composition of the present invention is 0.01-0.5 parts by weight based on 100 parts by weight of the total composition.

According to a specific embodiment of the present invention, the content of the Pseudomonas aeruginosa culture extract in the composition of the present invention is 0.02-0.1 part by weight based on 100 parts by weight of the total composition.

According to one embodiment of the present invention, the composition of the present invention has antibacterial activity and / or antifungal activity. The composition of the present invention exhibits a wide range of antibiotic activity against various microorganisms, especially bacteria, fungi and yeast.

In further embodiments, the compositions of the present invention is Staphylococcus (Staphylococcus) genus Bacillus (Bacillus) genus Pseudomonas (Pseudomonas) genus Candida (Candida) in, propionyl sludge tumefaciens (Propionibacterium) genus Streptomyces Lactococcus (Streptococcus) genus Proteus (Proteus) genus Corynebacterium (Corynebacterium) genus Enterococcus (Enterococcus) genus, keulrep when Ella (Klebsiella) into and E. coli one or more bacteria selected from the group consisting of (Escherichia coli) Lt; / RTI >

According to another embodiment of the present invention, the composition of the present invention has a high antioxidative effect such as ascorbic acid (AA), which is a representative component having an antioxidative effect at a concentration of 0.5% or more.

The antiseptic / antimicrobial composition of the present invention having a wide spectrum of antibiotic spectrum and antioxidant effect as described above can be widely used for the purpose of antibacterial, sterilizing, disinfecting, and dissolving in foods, household products, agricultural chemicals, .

According to the present invention, the composition for the nasal according to the present invention can be prepared with a cosmetic composition. When the composition of the present invention is prepared with a cosmetic composition, the extract of the Pseudomonas aeruginosa culture solution can be used instead of a cosmetic preservative because it prevents the cosmetic composition from being deteriorated by bacteria. When the composition of the present invention is prepared with a cosmetic composition, the composition of the present invention may contain the above-mentioned effective components of the extract of P. aeruginosa culture, salts thereof, solvates thereof or hydrates thereof, as well as components conventionally used in cosmetic compositions Such as antioxidants, stabilizers, solubilizers, vitamins, customary adjuvants such as pigments and flavoring agents, and carriers.

According to the present invention, the composition for the nasal according to the present invention can be prepared with a food composition. When the composition of the present invention is prepared with a food composition, the extract of the cultured Pseudomonas aeruginosa can be used instead of a food preservative because it prevents the degradation of food by bacteria.

When the composition of the present invention is prepared as a food composition, it contains not only the extract of the cultured Pseudomonas aeruginosa as an active ingredient but also a component which is ordinarily added at the time of food production, for example, protein, carbohydrate, fat, nutrients, . Examples of the above-mentioned carbohydrates are monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavorings such as tau martin and stevia extract (e.g., rebaudioside A and glycyrrhizin) and synthetic flavorings (saccharine, aspartame, etc.) can be used as flavorings. For example, when the food composition of the present invention is prepared as a drink, it may further contain citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, mulberry extract, jujube extract, licorice extract, .

As used herein, the term " antibiotic composition " refers to a composition containing antibiotics that kill or inhibit growth of microorganisms including viruses, fungi, protozoa and bacteria. . Thus, the antibiotic has an antibiotic activity, that is, an antibacterial activity, an antifungal activity or an antiviral activity.

The composition of the present invention has a significant effect on prevention and treatment of skin infection or acne caused by the bacteria. As demonstrated in one embodiment below, the compositions of the present invention are useful for the treatment of acne-causing bacteria, And Gram-positive bacteria such as Staphylococcus aureus, Bacillus cereus, Gram-negative bacteria such as Pseudomonas aeroginosa, Candida albicans, and the like.

The composition for the prevention or treatment of acne of the present invention can be prepared by incorporating a cosmetically effective amount of the extract of the above-mentioned cultured Pseudomonas aeruginosa extract, which is an effective ingredient of the present invention, and a cosmetically acceptable carrier.

As used herein, the term " a cosmetically effective amount " means an amount sufficient to achieve the antibiotic efficacy against the microorganism of the composition of the present invention described above.

The composition for external application for skin of the present invention can be prepared in any formulations conventionally produced in the art and can be used in the form of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, But are not limited to, cleansing, oils, powder foundations, emulsion foundations, wax foundations and sprays. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. Especially, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane or dimethyl ether.

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters.

The ingredients contained in the composition for external application for skin of the present invention include the ingredients commonly used in the composition for external application for skin in addition to the above-mentioned effective ingredient and carrier component, and include conventional ingredients such as antioxidants, stabilizers, solubilizers, vitamins, Phosphorous assistant.

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides an antimicrobial and antimicrobial composition comprising an extract of a culture solution of P. aeruginosa as an active ingredient.

(Ii) The present invention provides a composition for preventing or treating acne comprising an extract of a culture solution of P. aeruginosa as an active ingredient.

(Iii) The composition containing the extract of the Pseudomonas aeruginosa culture liquid of the present invention exhibits a wide spectrum of antimicrobial spectrum and high antioxidative effect against various fungi. Such a composition can be applied to cosmetic compositions such as cosmetics, It can be usefully used for antibacterial, sterilizing, and preservative purposes in various fields such as the field of medicine.

(Iv) The active ingredient causing the antimicrobial effect of the Pseudomonas aeruginosa culture extract of the present invention was confirmed to be a rhamnolipid of a specific structure (Rha C10 C10, Rha Rha C10 C10) among various rhamnolipids, To improve the efficiency of industrial applications.

FIG. 1 shows the results of confirming the antimicrobial effect of the extract of the cultured broth of Pseudomonas aeruginosa (R1) by performing a paper disk diffusion test.
2 is a graph showing cfu as a result of a challenge test for a product of a toner formulation containing a solution of P. aeruginosa culture.
Fig. 3 is a photograph showing a state after plate-smearing in a buoyancy test for a product of a toner formulation containing an extract of the cultured Pseudomonas aeruginosa.
FIG. 4 shows the result of measuring the improvement rate of acne related trouble according to the concentration of the extract of P. aeruginosa cultures (the red bars indicate nightly formulations and the purple bars indicate cream formulations).
FIG. 5 is a photograph showing the improvement of the acne-related trouble after applying the composition of the present invention containing the extract of P. aeruginosa culture to the skin of the subjects.
6 is a view showing the structure of rhamnose and rhamnolipid.
FIG. 7 shows the results of analysis of rhamnoside through 1D 1H-NMR spectrum.
Fig. 8 shows the result of lambda proton analysis.
9 shows the results of 2D-NMR analysis.
10 shows the result of 2D NMR ROESY experiment.
Fig. 11 shows the result of measuring the relative density of rhamnose in a sample through 1D < 1 > H-NMR spectral preliminary analysis.
Fig. 12 shows the results of the antioxidant activity test of the extract of the culture broth of Pseudomonas aeruginosa .
Figure 13 shows the results of testing the antimicrobial activity against S. aureus of fractions separated from R1 and R1.
Figure 14 shows the LC analysis results of fractions separated from R1 and R1.
Fig. 15 is a structural analysis result of active ingredient peaks 1 and 2 using LC-MS and LC-MS / MS.
16 shows the LC analysis result of R1.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and methods

1. Production of biological surfactants

The strain (CJM01) was selected prior to the experiment. As a result, the strain was found to be Pseudomonas aeruginosa and the strain was cultured in M9 medium (CaCl ₂ 0.015 g / L, Na ₂ HPO ₄ 6 g / L, KH ₂ PO ₄ 3 g / L, NaCl 0.5 g / L, NH ₄ Cl 1 g / L, MgSO ₄ 0.0625 g / L, glucose 20 g / L) and cultured at 37 ° C for 72 hours. After the culture, the culture was centrifuged to obtain a culture solution in which cells were completely removed. The cell-free medium was lowered to pH 2 using HCL and then precipitated at 4 ° C for one day. After precipitation, the supernatant was removed by centrifugation again and only the precipitate was obtained. The precipitate was extracted with ethyl ether, and the ethyl ether layer was separated. The concentrate was concentrated on a rotary evaporator to evaporate all the ethyl ether. The extract (biosurfactant crude) was named R1.

2. Antimicrobial activity of extracted biosurfactant TEST (paper disk diffusion test)

The biological surfactant in the form of mixture extracted from the culture broth of P. aeruginosa was designated as R1 and subjected to a paper-disk diffusion method for Staphylococcus aureus , Bacillus cereus , and Pseudomonas aeroginosa ( Escherichia coli ) (paper disk diffusion test).

S. aureus, B. cereus, P. aeroginosa and E. coli were inoculated into LB (Luria-Bertani) medium (10 g / L of tryptone and 10 g / L of NaCl and 5 g / L of yeast extract) Liquid culture was carried out under aerobic conditions for 18 hours. After culturing, 200 μl of each bacterial culture was applied to an LB agar plate. R1 extracted from the culture medium of P. aeruginosa was dissolved in methanol at a concentration of 2 mg / ml. Methyl Paraben, Phenoxy Ethanol, Naturotics, And dissolved in methanol at a concentration of 10 mg / ml. 20 μl of the dissolved R1, methylparaben, phenoxyethanol, and Necturotics solution was placed on a paper disk (diameter: 6 mm), and the paper disk was sufficiently dried. The paper discs were then placed on stained agar plates of S. aureus, B. cereus, P. aeroginosa and E. coli . Each agar plate was incubated for 18 hours under aerobic conditions at 37 占 폚. After 18 hours, the clear zone generated around the paper disk was observed.

3. The challenge test of the extracted biosurfactants

Positive pathogen gram in the toner formulation and the lotion formulation in order to compare methylparaben to determine the potential as the R1 preservatives (Staphylococcus aureus, Bacillus cereus), gram-negative pathogenic bacteria (Pseudomonas aeroginosa), the sterilizing power of the fungi (Candida albicans) Buoyancy test was performed. The buoyancy test was conducted on the basis of the American Cosmetics Association (CTFA), the criteria for which a specific substance killed 99.9% of the bacteria inoculated within 7 days of toner and lotion formulation It has potential as a preservative. Prior to the experiment, toner and lotion formulations were prepared and their compositions are shown in Tables 1 and 2.

Toner composition R1-0% R1-0.01% R1-0.05% R1 or methyl paraben-0.1% R1 or methyl paraben 0.5% R1 or methyl paraben-1% MeoH Hyaluronic acid 2 g 2 g 2 g 2 g 2 g 2 g 2 g glycerin 3 g 3 g 3 g 3 g 3 g 3 g 3 g Aloewater 40 g 40 g 40 g 40 g 40 g 40 g 40 g Marigold extract 2.8 g 2.8 g 2.8 g 2.8 g 2.8 g 2.8 g 2.8 g Distilled water 52.2 g 52.18 g 52.1 g 52 g 51.2 g 50.2 g 50.2 g R1 or methyl paraben (500 mg / ml MeOH) 0 g 20 μl 100 μl 200 μl 1 ml 2 ml 0 MeOH 0 0 0 0 0 0 2 ml gun 100 g 100 g 100 g 100 g 100 g 100 g 100 g

Lotion composition R1 or methyl paraben-0% R1 or methyl paraben 0.5% MeOH Aloe number 82.5 g 81.5 g 81.5 g glycerin 5 g 5 g 5 g Sorbitan olivate 1.5 g 1.5 g 1.5 g Olive oil 7 g 7 g 7 g Hyaluronic acid 2 g 2 g 2 g Marigold extract 2 g 2 g 2 g R1 or methyl paraben (500 mg / ml MeOH) 0 1 ml 0 MeOH 0 0 1 ml gun 100 g 100 g 100 g

4. Experiments on Gram-positive pathogenic bacteria ( Staphylococcus aureus , Bacillus cereus ) in toner formulations

R1 dissolved in methanol was added to the prepared toner formulation so that the final concentrations of R1 were 0%, 0.01%, 0.05%, and 0.1%, and S. aureus and B. cereus were inoculated into each container. In addition, methylparaben dissolved in methanol was added to the toner formulation so that the final concentration of methylparaben was 0%, 0.1%, 0.5%, and 1%, and each of the containers was inoculated with S. aureus and B. cereus . The number of inocula was 10 ⁶ -10 ⁷ cfu (colony forming unit) / ml. After storing the inoculated toner at room temperature, each toner was diluted immediately after inoculation, 1 day, 3 days, 5 days, and 7 days, and 200 μl was plated on an LB agar plate to calculate the number of cells. To determine whether or not it was dead. Methanol alone was added to the toner formulation as a control, followed by repeated inoculation with the culture medium.

5. Experiments on Gram-negative pathogenic bacteria ( Pseudomonas aeroginosa ) and fungi ( Candida albicans ) in toner formulations

R1 and methylparaben dissolved in methanol were added to the prepared toner formulation so that the final concentrations were 0%, 0.1%, 0.5%, and 1%, respectively. In each container, P. aeruginosa and C. albicans were inoculated. The number of inocula was 10 ⁶ -10 ⁷ cfu (colony forming unit) / ml. After the inoculated toner was stored at room temperature, each toner was diluted immediately after inoculation, 1 day, 3 days, 5 days, and 7 days, and the P. aeruginosa inoculated toner was inoculated onto the LB agar plate and C. albicans was inoculated The amount of the toner was determined by counting 200 μl of YPD agar plate and counting the number of cells in comparison with the number of the inoculated cells. Methanol alone was added to the toner formulation as a control, followed by repeated inoculation with the culture medium.

YPD agar (5 g / L of peptone, 5 g / L of yeast extract, 5 g / L of dextrose, 15 g / L of agar)

6. Experiments on Staphylococcus aureus , Bacillus cereus , Gram negative pathogens ( Pseudomonas aeroginosa ) and fungi ( Candida albicans ) in lotion formulations

To the prepared lotion formulation, R1 and methylparaben dissolved in methanol were added respectively so that the final concentrations were 0% and 0.5%. The culture medium of S. aureus, B. cereus, P. aeruginosa and C. albicans was inoculated into each container Respectively. The number of inocula was 10 ⁶ -10 ⁷ cfu (colony forming unit) / ml. The lotion, which was inoculated with S. aureus, B. cereus, and P. aeruginosa, was diluted immediately after inoculation, 1 day, 3 days, 5 days, and 7 days after storage at room temperature. C. albicans was inoculated with the YPD The cells were counted by staining 200 μl on agar plates and compared with the number of cells inoculated. Methanol alone was added to the lotion formulation as a control, and the bacterial culture was inoculated and repeatedly tested.

7. Improvement of acne-related problems in products containing extracted biosurfactants

7-1. Type of product, amount of product and formulation

Natural cosmetics containing biosurfactant R1 extracted from the culture of P. aeruginosa were applied to subjects with acne-related problems (purulence, wheat gluten, acne scar, pimples, etc.) (5 g / / 2 weeks).

Type of product - Kinds One Cream containing 0% microbial culture extract 2 Cream containing 0.02% of microbial culture extract 3 Cream containing 0.1% of microbial culture extract 4 Night containing 0% microbial culture extract 5 Night containing 0.02% of microbial culture extract 6 A night containing 0.1% of the microbial culture extract 7 Night containing 0.5% of microbial culture extract

Composition of a nightly product containing 0.5% R1 mixture ingredient g % Beads wax 7 23.33333 Tocopheryl acetate 0.3 One Karen dura extract 0.1 0.333333 Tea tree E.O 0.3 One Sunflower seed oil 0.15 0.5 Rosemary leaf extract Tocopherol 0.15 0.5 Grape E.O 0.2 0.666667 Large evening primrose oil 5 16.66667 Jojoba oil 3 10 Organic Tamanu Oil 13.65 45.5 R1 (mixture) 0.15 0.5 gun 30 100

Composition of a nightly product containing 0.1% R1 mixture ingredient g % Beads wax 7 23.33333 Tocopheryl acetate 0.3 One Karen dura extract 0.1 0.333333 Tea tree E.O 0.3 One Sunflower seed oil 0.15 0.5 Rosemary leaf extract Tocopherol 0.15 0.5 Grape E.O 0.2 0.666667 Large evening primrose oil 5 16.66667 Jojoba oil 3 10 Organic Tamanu Oil 13.77 45.9 R1 (mixture) 0.03 0.1 gun 30 100

Composition of a nightly product containing 0.02% R1 mixture ingredient g % Beads wax 7 23.33333 Tocopheryl acetate 0.3 One Karen dura extract 0.1 0.333333 Tea tree E.O 0.3 One Sunflower seed oil 0.15 0.5 Rosemary leaf extract Tocopherol 0.15 0.5 Grape E.O 0.2 0.666667 Large evening primrose oil 5 16.66667 Jojoba oil 3 10 Organic Tamanu Oil 13.77 45.9 R1 (mixture) 0.03 0.1 gun 30 100

Composition of a nightly formulated product containing 0% R1 mixture ingredient g % Beads wax 7 23.33333 Tocopheryl acetate 0.3 One Karen dura extract 0.1 0.333333 Tea tree E.O 0.3 One Sunflower seed oil 0.15 0.5 Rosemary leaf extract Tocopherol 0.15 0.5 Grape E.O 0.2 0.666667 Large evening primrose oil 5 16.66667 Jojoba oil 3 10 Organic Tamanu Oil 13.8 46 R1 (mixture) 0 0 gun 30 100

Composition of a cream-formulated product containing 0% R1 mixture material g % Location Huzel Can 18.5 37 Golden jojoba oil 3 6 Evening Flower Oil 2.5 5 Olive oil wax 2.5 5 Aloe vera leaf extract 18 36 glycerin Glyceryl acrylate / acrylic acid copolymer Propylene glycol Centipede extract 3 6 Tocopheryl acetate One 2 Tea tree essential oil 0.5 One Grapefruit essential oil 0.5 One Natural herbal preservative 0.5 One R1 (mixture) 0 0 gun 50 100

Composition of cream formulated product containing 0.02% R1 mixture material g % Location Huzel Can 18.49 36.98 Golden jojoba oil 3 6 Evening Flower Oil 2.5 5 Olive oil wax 2.5 5 Aloe vera leaf extract 18 36 glycerin Glyceryl acrylate / acrylic acid copolymer Propylene glycol Centipede extract 3 6 Tocopheryl acetate One 2 Tea tree essential oil 0.5 One Grapefruit essential oil 0.5 One Natural herbal preservative 0.5 One R1 (mixture) 0.01 0.02 gun 50 100

Composition of a cream formulation containing 0.1% R1 mixture material g % Location Huzel Can 18.45 36.9 Golden jojoba oil 3 6 Evening Flower Oil 2.5 5 Olive oil wax 2.5 5 Aloe vera leaf extract 18 36 glycerin Glyceryl acrylate / acrylic acid copolymer Propylene glycol Centipede extract 3 6 Tocopheryl acetate One 2 Tea tree essential oil 0.5 One Grapefruit essential oil 0.5 One Natural herbal preservative 0.5 One R1 (mixture) 0.05 0.1 gun 50 100

7-2. Subject

The purpose of this study was to evaluate the efficacy and safety of acne - related problems such as acne - related problems when applied to subjects with acne - related problems.

The primary end point was the symptom improvement rate of the acne-related trouble 2 weeks after use of the product, and whether the symptom improvement rate of the acne-related trouble before and after the test was different was evaluated. Based on the clinical trials performed in the past, the total number of subjects was reduced to 30, taking into account the dropout rate. Three individuals who did not sign the clinical trial agreement for personal reasons did not participate in the clinical trial. 27, and the dropout rate was 0%, and the final evaluation was 27 subjects.

7-3. Test Methods

Use 5 g of the test product every two weeks and apply cream or chestnut product to the skin lesion area designated by the tester for 2 weeks (morning, evening, always) more than once a day.

Inha University and Incheon University students were recruited to participate in clinical trials for evaluating the effectiveness of acne-related trouble cosmetics. Clinical studies were conducted for volunteers who were voluntarily applied and who did not meet the exclusion criteria. All subjects were selected through screening. A total of 27 subjects agreed to take the test. There were 16 men and 11 women, and the subjects were between 20 and 28 years of age. The number of subjects who dropped out and dropped out was 27.

In order to evaluate the improvement effect of acne related problems, each visit was carried out with a visual evaluation and an image taking, and a questionnaire survey was conducted with subjective opinions of the subjects directly checking their degree of change (degree of change and presence of an adverse reaction) Respectively. The visual acuity of the lesion was determined by the number of acne in the lesion area of the lesion determined by the examiner before application of the product at the 0th day of the clinical trial. The images of the lesion sites of the subjects participating in the clinical study were photographed before the test and during the clinical test visit. In addition, subjects' subjective opinions were surveyed.

7-4. Evaluation Criteria, Evaluation Method, and Analysis Method

The subjects who used the subjects who were fit for the selection criteria and those who used the products for 2 weeks were selected as the subjects to be evaluated, and a gross evaluation was conducted for the improvement of acne related troubles such as identification of the number of pimples and image shooting in progress at 0 week, 1 week and 2 weeks And questionnaires were administered to the subjects. The results of the tests were analyzed by statistical estimation.

8. NMR analysis

It contains 90% of R2, R3 and Rumorrifice (one kind of biological surfactant) obtained by changing the culture and extraction method against P. aeruginosa such as the biological surfactant R1 extracted from the culture of the microorganism ( P. aeruginosa ) The R90 purchased from SIGMA-ALDRICH was analyzed by NMR.

The structure of the ranorifide consists of a lipid moiety comprising one or two rhamnose (sugar) moieties (Figure 6, left) and two aliphatic chains (Figure 7, right). The diversity of the rhamnoglyphide structure is due to the number of one or two Ramanose rings and the structure (length and saturation) of the two aliphatic chains of the lipid moiety. Four different biosurfactant extracts (Samples 0, 1, 2 and 3) and commercially available (Sigma) rhamnolipid (Sample 90) were dissolved in a mixture of chlorophyll-methanol (2: 1) Respectively.

9. Antioxidative effect of Pseudomonas aeruginosa culture extract

The antioxidative effect of Pseudomonas aeruginosa extract as an antiseptic was tested.

DPPH radicals are purple, and when they react with antioxidants they become yellow. The more the DPPH radical is reacted with the sample with the excellent antioxidant ability, the clearer the color change becomes. R1 and ascorbic acid (AA) and methylparaben (MTP) were dissolved in methanol at different concentrations, respectively. 100 μl of each of the thus obtained samples was mixed with 100 μl of a DPPH radical solution, which was dissolved in methanol at a concentration of 0.006%, to make a total of 200 μl, followed by reaction for 30 minutes in a light-shielded space. After completion of the reaction, the absorbance of each mixture was measured at 517 nm, and the antioxidant activity of each sample was measured.

10. Search for active ingredients causing antimicrobial effect of Pseudomonas aeruginosa culture extract

Pseudomonas aeruginosa culture solution is a mixture of several components rather than a single component. In order to investigate the effect of various components of Pseudomonas aeruginosa extract on the antimicrobial effect,

Isolation of R1 in mixture state extracted from microorganism (P. aeruginosa) culture

1 mg of R1 in the form of mixture extracted from the culture medium of P. aeruginosa was dissolved in methanol and injected into sep-Pak vac 1cc tc18 cartridge column. The column was then washed with solvent A and solvent B, a developing solvent, was introduced. The concentration of Solvent B was injected into the column differently, and the fraction passed through the column was collected and named as fraction 1, fraction 2, fraction 3, fraction 4, fraction 5, and fraction 6, respectively. The concentrations of solvent B were 50%, 55%, 60%, 65%, 70% and 75%, respectively. The composition of solvent A and solvent B is shown in Table 11.

Composition of Solvent A and Solvent B Solvent A 0.1% Formic acid in water Solvent B 0.1% Formic acid in ACN

Analysis of antimicrobial activity of fractions isolated from R1

Each fraction obtained was dried and dissolved in methanol at a concentration of 10 mg / ml. 20 μl of each fraction dissolved in methanol was dropped on agar plate coated with S. aureus culture, incubated at 37 ° C for 18 hours, and observed clear zone formed on agar plate after 18 hours.

Analysis of fractions separated from R1 by LC / MS

LCMS performed for the structural analysis of the separated fractions consisted of SB-Aq RRHD (analytical column) of 1.8 μm 2.1 * 100 mm and Zorbax SB-C8 (guard column) of 2.1 * 5 mm 1.8 μm in negative mode ionic polarity . The separated fractions were dissolved in methanol to prepare a sample. 20 μl of the sample was injected into the column-mounted HPLC and analyzed by connecting it to a mass spectrometer. In the HPLC, a mixture of solvent A and solvent B was used as a developing solvent, and the mixing ratio of the solvent A and solvent B at the initial stage was 50%. 100% Solvent B (19 min), 100% Solvent B (20.5 min), 50% Solvent B (21 min), 50% Solvent B (25 min), HPLC flow rate: 0.15 ml / min

The scanning mass range was 75 ~ 1000 m / z. MS / MS was carried out for specific components causing antimicrobial effect.

Experiment result

1. Antimicrobial activity test of extracted biological surfactant

Bacterial surfactant in the form of mixture extracted from the culture medium of P. aeruginosa was designated as R1 and tested for antimicrobial activity against Staphylococcus aureus , Bacillus cereus , and Gram negative aeruginosa ( Escherichia coli ) Respectively. As a result of paper disk diffusion test, R1 produced significantly larger clear zones at concentrations five times lower than that of methylparaben, phenoxyethanol, and nectirox, which have been used as conventional cosmetic preservatives (FIG. 1).

2. Flotation test of extracted biological surfactant

As a result of the buoyancy test, in the toner formulation, R1 killed 99.9% of the inoculated bacteria within 7 days for all the inoculated bacteria, especially R1 at 0.01%, which is 10 times lower than methylparaben, against Staphylococcus aureus, Bacillus cereus was killed in 99.9% of the inoculation days in one day. For Gram-negative pathogens ( Pseudomonas aeroginosa ) and fungi ( Candida albicans ), 99.9% of the inoculum was killed in 5 days at the same concentration of methylparaben (Fig. 2-3). For all strains inoculated from the rosin formulation, R1 killed 99.9% of the bacteria inoculated within 7 days at the same concentration as methylparaben (Table 12). These results show that R1 has a potential as a preservative.

Flotation test of R1 in lotion formulation (% - Survival rate of inoculum) 0 days 1 day 3 days 5 days 7 days Staphylococcusaureus 0% 100% 6.74% 0.06% 30% 78% MeOH 100% 15.70% 4.27% 0.04% 0.04% R10.5% 100% 0% 0% 0% 0% MTP 0.5% 100% 0% 0% 0% 0% Bacillus
cereus 0% 100% 0.09% 0.23% 35% 54% MeOH 100% 0.17% 0.02% 0.02% 0.02% R10.5% 100% 0.01% 0% 0% 0% MTP 0.5% 100% 0.12% 0.06% 0.02% 0.01% Pseudomonasaeroginosa 0% 100% 0.30% 0.60% 0.02% 0.02% MeOH 100% 7.30% 0.80% 0.03% 0.02% R10.5% 100% 7.30% 0.60% 0.006% 0% MTP 0.5% 100% 0% 0% 0% 0% Candida
albicans 0% 100% 37.50% One% 0.5% 0.5% MeOH 100% 27.50% 1.80% 0.4% 0.3% R10.5% 100% 16.30% 0.10% 0% 0% MTP 0.5% 100% 0% 0% 0% 0%

3. Improvement of acne-related troubles of products containing extracted biosurfactants

Natural cosmetics containing biosurfactant R1 extracted from microbial ( P. aeruginosa ) culture medium were applied to subjects with acne related trouble symptoms. As a result, it was found that subjects using products containing R1 And skin irritation (itching, burning, red spot, swelling, swelling, stinging, etc.) were not observed in all subjects.

Acne related trouble improvement rate Subject number gender Extract content and product formulation Improvement rate One female 0.5% / night 57% 2 south 0.1% / night 57% 3 south 0.02% / cream 75% 4 south 0.02% / night 75% 5 south 0% / cream 60% 6 female 0% / night 47% 7 female 0.1% / night 43% 8 female 0.5% / night 50% 9 female 0% / cream 20% 10 female 0.1% / night 100% 11 female 0.02% / night 50% 12 female 0% / cream 38% 13 south 0.1% / cream 54% 14 south 0.02% / night 100% 15 south 0.1% / cream 80% 16 female 0.02% / cream 88% 17 female 0% / cream 31% 18 female 0.02% / night 50% 19 south 0.1% / night 64% 20 south 0.1% / night 60% 21 south 0.02% / cream 63% 22 south 0.02% / night 57% 23 south 0.1% / night 60% 24 south 0.5% / night 60% 25 south 0.5% / night 65% 26 south 0.5% / night 100% 27 south 0.02% / cream 100%

At the beginning of clinical trial, the number of progressive acne in the lesion area determined by the examiner before application of the product was set as the starting point, and the degree of improvement after 2 weeks of the test was quantified. A higher improvement was observed in subjects using products containing microbial culture extracts compared to products not containing culture extracts. In particular, the highest rate of improvement was found in subjects using a cream-form product containing 0.02% of the microbial culture extract, followed by a higher rate of improvement in subjects using a cream-type product containing 0.1% of the microbial culture extract 4). Based on this, natural cosmetics containing the microbial culture extract prepared by the present inventors can be considered to help improve acne. The results of image capturing by the subjects are shown in Figs. 5A to 5I.

Overall validation Subject number evaluation Subject number evaluation One 4 15 4 2 3 16 4 3 4 17 3 4 5 18 4 5 4 19 4 6 3 20 4 7 3 21 3 8 3 22 3 9 3 23 4 10 5 24 4 11 3 25 4 12 3 26 5 13 4 27 4 14 4 Average 3.7

5: no symptoms, 4: overall symptom improved clearly

3: overall symptom improved slightly, 2: no change in symptoms before the test

1: Symptoms are worse than before

Satisfaction rating of products after use Subject number evaluation Subject number evaluation One 3 15 5 2 4 16 3 3 4 17 4 4 5 18 5 5 4 19 4 6 4 20 4 7 4 21 4 8 3 22 4 9 3 23 4 10 5 24 4 11 2 25 3 12 4.5 26 5 13 4 27 4 14 4 Average 3.9

1: Not sure, 2: Not satisfied, 3: Normal, 4: Satisfactory, 5: Very satisfied

Overall satisfaction with the improvement of the subjects' acne related trouble after 2 weeks of clinical trial was highly evaluated. The most prominent changes in questionnaire evaluation and visual evaluation were skin problems such as purulent acne and temporary rashes. On the other hand, there was relatively little change in the improvement of acne scars and acne scars. It was also found that the subjects who used cream formulations had higher improvement and satisfaction than those who did not use night formulations.

As a result of 2 weeks clinical trials, it was found that natural cosmetic products containing microbial culture extract prepared by the present inventors through visual evaluation, questionnaire evaluation and image photographing are products for improving acne related trouble.

4. NMR analysis

General observation

All samples contained a similar set of rhamnoside feeds consisting of four major components (two types of di-rhamnolipid and two types of mono-rhamnolipid). The content of the A-fraction of the di-rhamnoside in each sample was different (sample 35 contained up to 35% and samples 2 and 3 contained more than 65%) (Table 17). Samples 0, 1, 2, and 3 also contained aromatic components that were expected to affect antimicrobial activity.

1D-NMR analysis

The portion corresponding to ranoriphipide in the 1D1H-NMR spectrum for Sample 1 is shown in Fig. The signal strengths (integrals) are shown below the spectrum. All signal intensities were normalized to methyl peaks at 0.82 ppm, and the peaks correspond to 6 protons (2 methyl groups). Therefore, a single proton peak appears to have a signal intensity of about 100 units. For example, the peaks of the two groups at 1.4-1.5 ppm and 2.4-2.6 ppm exhibit signal intensities of ~ 400 and correspond to the C ₂ H ₂ / C ₅ H ₂ and C ₇ H ₂ / C ₁₀ H ₂ groups It coincides with the four proton signals that appear. The mass ratio of mono and di-rhamnoside can be measured by comparing the intensity of signals obtained from lipid and rhamnose groups.

If the sample contains only mono-rhamnolipids, then only one proton from lambos will match one proton in the lipid moiety. Also, if the sample contains only a di-rhamnolipid, it will match "2 -to-1 ". As evidenced in FIG. 8, the H1 and H4 lipoproteins (dark orange box) represent the same type of signal, with a sum of intensities of approximately 100, indicating that each Rumannipid molecule in the mixture has exactly this type of proton Quot; has one < / RTI >

As a result, the sum of the signal intensities obtained from H1 '/ 1 ", H2' / 2", H3 '/ 3 ", H5' / 5" rhamnose protons (blue box in FIG. 8) Is higher than expected. One H1 'proton per molecule in the mono-rhamnolipid mixture should have a signal with a value of 100. The protons of the three H2 'H3', H5 'should have a signal with a value of 300. However, these signal intensities in sample 1 were measured at about 150-480. From this signal strength value it can be inferred that about 60% of the di-rhamnorefeeds are present in sample 1. Samples 0, 2, 3 and 90 were run on 1D < 1 > H-NMR spectra with similar analysis.

2D-NMR analysis

The presence of the dia-rhamnorefide in the samples was confirmed by the 2D 1H-13C-HSQC experiment. Formation of β-L-Rha p (1 '' → 2 ') - β-L-Rha p bond resulted in strong NMR signal transfer of C2' and C1 'atoms of di-rhamnose connected via oxygen 6). The C ^{1 '/ 1 "} H and C ^{2' / 2"} H zones of the HSQC spectrum are shown in FIG. The cross-peaks ( ¹³ C scale, red box) appearing at 102 ppm correspond to the correlation coefficient of H1 "/ C1" in the di-ranoripalipide. While the cross-peaks appearing at 95-96 ppm are consistent with the correlation coefficient of H1 '/ C1' of mono-rhamnolipid (structure of FIG. 6). Two cross-peaks (Figure 9 blue box) appearing at ~ 79 ppm correspond to two C2 'atoms of the di-rhamnolipid moiety.

Assign spin system

To determine the relative densities and numbers of other rhamnolipids in the sample, spin systems were separated using 2D NMR spectra (HSQC, TOCSY, COZY-DQF), which contained the rhamnose ring structure of the rhamnose- Structure. We were also able to prove the six unknown systems of the laminocyte portion (Systems 1-6) and the eight systems of fatty acid tail (Systems 7-14). The systems have been pre-assigned as a type of rhamnose moiety (the first (') or second (") single ring structure of the di-rhamnose) and the position of the hydroxyl fatty acid (directly or indirectly, 6) is based on known chemical shifts (Part 3, 2D-NMR analysis, the example of FIG. 9). Systems 1 and 2 were assigned to the second Ramsey ring structure of Dai-Lamboth, while systems 3 and 4 were assigned to the first Ramsey ring structure of Dai-Lamboth. Systems 5 and 6 are consistent with the mono-rhamnolipid Laminocyte structure. Systems 7 and 8, as well as their minimal components 9 and 10, were assigned to a second fatty acid in lipids (not directly linked to the rhamnose part). As a result, the system was assigned to the first 11-14 fatty acid chain and was directly linked to the rhamnose portion.

Spin system connection

After assigning the spin system for the structural part of the rhamnorefide molecule, we performed several possible ways to connect them to the Ramanoride feed. 2D NMR ROESY experiments were used to detect dipole-dipole interactions through the surface of proton and fatty acid proton present in the laminocyte structure. As expected, cross-peaks of the H1 protons of the Laminos system 3-6 and the H1 protons of the fatty acid systems 11-14 were detected (FIG. 10). This experiment makes it possible to link the spin system assigned to the first ring structure or a single structure to the spin system assigned to the fatty acid.

No cross-peaks were observed between the second ring structure in dia-laminase and the H1 protons in systems 1 and 2 assigned to fatty acid protons. Finally, the spin system was organized into four rhamnolipid molecules I-IV (Table 16).

Relative density of I-IV molecules in NMR sample

A preliminary analysis of 1D < 1 > H-NMR spectra clearly shows the relative density of rhamnose in the samples (Fig. 11). The same results were also observed in the fatty acid system. The relative intensities of I-IV molecules were estimated using signal strength. The signal intensities were estimated using 1D 1HNMR to obtain well separated signals assigned to H1 / H1 "and H2 / H2" lambda proton, H1 and H4 fatty acid proton Lt; / RTI > A summary of the analysis is presented in Table 16.

R90 obtained from SIGMA-ALDRICH containing R2, R3 and 90% of rhamnolipid obtained by changing the culture and extraction method against P. aeruginosa such as the biological surfactant R1 extracted from the culture of microorganism ( P. aeruginosa ) NMR analysis revealed that both the extract and purchased R90 contained rhamnolipid. This proves that R1 extracted from the culture medium of P. aeruginosa contains a rhamnolipid series biological surfactant (Fig. 11) (Table 16). Further, through NMR analysis, five samples contained rhamnolipid, and the content thereof was found to be different. And ranorifide may be associated with other biological studies.

5. Antioxidative effect of Pseudomonas aeruginosa culture extract

In order to investigate the antioxidant effect of R1 extract of Pseudomonas aeruginosa , the DPPH scavenging activity test showed that R1 had the same level of antioxidative activity as ascorbic acid (AA) . Methylparaben (MTP), on the other hand, showed significantly lower antioxidative effect at the same concentration as R1. Based on these results, it can be considered that the extract of cultured Pseudomonas aeruginosa (R1) having antioxidant effect can be applied as a preservative (Fig. 12).

6. Detection of active ingredients causing antimicrobial effect of Pseudomonas aeruginosa culture extract

The R1 in the mixture state extracted from the culture of the microorganism ( P. aeruginosa ) was separated to obtain Fraction 1, Fraction 2, Fraction 3, Fraction 4, Fraction 5 and Fraction 6. The antimicrobial activity against S. aureus was tested in each of the obtained samples, and fraction 1, fraction 2 and fraction 3 showed antimicrobial activity corresponding to R1, and fractions 4 and 5 showed relatively weaker antibacterial activity than R1 Fraction 6 showed no antimicrobial activity (Fig. 13). LC analysis was carried out on all the samples. As a result, two peaks were found to be common in Fraction 1, Fraction 2 and Fraction 3, which were gradually decreased in Fraction 4 and Fraction 5, 2 (Fig. 14). The presence of Peak 1 and Peak 2 was judged to determine antimicrobial activity when coupled with the antimicrobial activity test. As a result of the MS 1 and Peak 2 MS and MS / MS tests, peak 1 had a molecular weight of 650 and Rha-Rha (Di-rhamnolipid) having a structure of -C10-C10, Peak 2 was analyzed with a mono-rhamnolipid having a molecular weight of 504, a structure of Rha-C10-C10 (Fig. 15A-D). R1 has rhamnoside having various structures (Fig. 16 and Table 18). Among them, Rham-rifide having the structure of Rha-Rha-C10-C10 and Rha-C10-C10 is an effective one having antibacterial activity Respectively. In addition, the antimicrobial activity of S. aureus of Rhamnolipid (R90, R95) extracted from P. aeruginosa culture purchased from Sigma-Aldrich Korea was significantly lower than that of R1 (Fig. 17).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

Staphylococcus aureus , Bacillus cereus , Pseudomonas aeroginasa , Escherichia coli , and Candida albicans , which contain an extract of Pseudomonas aeruginosa CJM01 as an active ingredient, Wherein the antimicrobial agent has antimicrobial activity against all pathogens.
Staphylococcus aureus , Bacillus cereus , Pseudomonas aeroginasa , Escherichia coli , and Candida albicans , which contain an extract of Pseudomonas aeruginosa CJM01 as an active ingredient, Wherein the antibiotic composition has an antimicrobial activity against all pathogens.
delete The composition according to any one of claims 1 to 2, wherein the extract of Pseudomonas aeruginosa culture is obtained by adding a hydrochloric acid solution to a culture solution of Pseudomonas aeruginosa to precipitate and then adding ethyl ether to the precipitate.
The composition according to any one of claims 1 to 2, wherein the Pseudomonas aeruginosa culture extract comprises Rhamnolipid.
6. The composition according to claim 5, wherein the ranoriphipide is a di-rhamnolipid represented by the following formula (1) or a mono-rhamnolipid represented by the formula (2)
Formula 1

(2)

The composition according to any one of claims 1 to 2, wherein the composition comprises 0.005-1.0 parts by weight of a Pseudomonas aeruginosa culture extract based on 100 parts by weight of the total composition.
delete The composition of claim 1, wherein the composition is a cosmetic composition.
delete delete delete

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