KR20220048534A - Novel Strain of Penicillium bissettii with Anti-bacterial and Anti-fungal Activity and Uses thereof - Google Patents

Abstract

The present invention relates to a Penicillium bissettii strain having antibacterial and antifungal activities and producing penicillic acid and to uses thereof. The strain of the present invention and culture extract or fraction thereof exhibit excellent antibacterial and antifungal activities by directly affecting harmful microorganisms, thereby being usefully used for purposes such as a pharmaceutical composition, a cosmetic composition, and a preservative for food or feed preservation.

Description

A novel strain showing antibacterial and antifungal activity and uses thereof {Novel Strain of Penicillium bissettii with Anti-bacterial and Anti-fungal Activity and Uses thereof}

The present invention relates to a novel strain Penicillium viseti exhibiting antibacterial and antifungal activity and an antibacterial and antifungal composition containing the same as an active ingredient.

Some of the substances that play an important role in the process for maintaining homeostasis of living things are physiologically active substances derived from various living organisms. Until now, many studies have been conducted on numerous physiologically active substances, and among them, research on substances having antibacterial and antifungal activity is a very important area in life science and medicine.

It is known that all living things produce antibacterial substances for survival, and many studies are being done on microorganisms that produce antifungal substances. Research to develop has been attempted for a long time.

In addition, the strain isolated from the plant itself is known to produce useful secondary metabolites having anticancer or antibacterial activity, and the most representative example is Taxomyces andreanae isolated from the yew tree. It is a strain that produces paclitaxel, which is attracting attention as an anticancer drug. Due to the commercial success of these plant isolation strains, research and development efforts are continuously being made to find new plant-derived strains having novel physiologically active substances with various pharmacological effects.

On the other hand, Peucedanum insolens is a perennial plant of the umbel family of dicotyledonous plants, and it is a native plant known only in Korea, distributed mainly in forests or limestone areas of Gangwon-do and Gyeongsangbuk-do. Active studies have not yet been conducted.

Under this background, the inventors of the present inventors have made diligent efforts to find a new strain having excellent antibacterial activity. As a result, the Penicillium biseti strain isolated from the endemic plant of Korea, Deokwoo oil, effectively inhibits the growth of pathogenic bacteria and fungi, and the antibacterial agent And it was confirmed that it can be usefully used as an antifungal agent, and the present invention was completed.

Accordingly, the main object of the present invention is to provide a Penicillium viseti strain having antibacterial and antifungal activity.

Another object of the present invention is to provide an antibacterial and antifungal composition using the Penicillium viseti strain.

According to one aspect of the present invention, the present invention provides a Penicillium bissettii strain (Accession No.: KACC 83024BP) having antibacterial and antifungal activity.

The term " Penicillium bissettii " of the present invention is a novel Penicillium bissetti strain newly isolated and identified by the present inventors from the domestic native plant, Peucedanum insolens .

The present inventors have investigated and analyzed the antifungal and antibacterial effects of strains isolated from various domestically grown plants for the development of new antibacterial agents or antifungal agents for several years. Antifungal and antibacterial effects were verified (see FIGS. 2, 8, and 10).

The Penicillium bissettii strain (Accession No.: KACC 83024BP) of the present invention is characterized in that it produces penicillic acid.

As a result of comparative analysis of the gene sequence of the Penicillium viseti with the gene sequences of conventionally known strains, the present inventors confirmed that the Penicillium genus SK16-002 strain of the present invention is a new strain belonging to Penicillium viseti. Therefore, it was named Penicillium bissettii SK16-002 strain, deposited at the Agricultural Genetic Resources Center (KACC) of the Rural Development Administration on June 24, 2019, and was given an accession number KACC 83024BP.

In the present invention, " Penicillium bissettii strain (accession number: KACC 83024BP)", " Penicillium sp. SK16-002 strain", " Penicillium bissettii SK16 -002 strain" and "SK16-002 strain" are strains deposited with accession number KACC 83024BP and are used in the same meaning as each other, and may be used interchangeably in the present specification.

According to another aspect of the present invention, the present invention provides an antibacterial and antifungal pharmaceutical composition comprising a Penicillium bissettii strain (Accession No.: KACC 83024BP), its culture extract or fraction as an active ingredient. .

In the present invention, the Penicillium viseti strain (accession number: KACC 83024BP) or a culture solution thereof is characterized in that it exhibits antifungal and antibacterial activity by itself.

The Penicillium viseti strain (Accession No.: KACC 83024BP) or a culture solution thereof has antibacterial activity against various fungi. The strain or its culture medium of the present invention has antibacterial activity against Gram-positive bacteria, Gram-negative bacteria and antibiotic-resistant strains, and specifically, Gram-negative Escherichia genus, Klepsiella genus Antibacterial activity against genus strains and, more specifically, E. coli ( Escherichia coli ), and Klepsiella pneumoniae ), but has antibacterial activity against pathogenic bacteria such as, but is not limited thereto. In addition, specifically Gram-positive Staphylococcus genus, Streptococcus genus, and Enterococcus genus It has bacterial activity against strains, more specifically Staphylococcus aureus ( Staphylococcus aureus ), Streptococcus pseudopneumoniae ), and Enterococcus faecalis ) It has antibacterial activity against pathogenic bacteria such as, but is not limited thereto.

In addition, the Penicillium viseti strain (Accession No.: KACC 83024BP) or its culture medium has antifungal activity against various fungi, specifically, has antifungal activity against fungi of the genus Candida, more specifically Candida albicans ( Candida albicans ) has antifungal activity, but is not limited thereto.

The term "extract" used in the present invention is an extract obtained according to the extraction treatment of the Penicillium viseti strain (accession number: KACC 83024BP) culture solution, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, and the extract of the extract. It includes extracts of all formulations that can be formed using the extract itself and the extract, such as a crude product, a purified product, or a mixture thereof. The extract of the present invention may be prepared and used in the form of a dry powder after extraction.

In the extraction of the culture medium extract of the Penicillium viseti strain (Accession No.: KACC 83024BP) of the present invention, the method of extracting the extract is not particularly limited, and can be extracted according to a method commonly used in the art. there is. Non-limiting examples of the extraction method include hot water extraction, ultrasonic extraction, filtration, reflux extraction, and the like, and these may be performed alone or in combination of two or more extraction methods.

In the present invention, the type of extraction solvent used for extracting the culture medium extract of the Penicillium viseti strain (accession number: KACC 83024BP) is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent include water; C1-C4 lower alcohols, such as methanol, ethanol, propyl alcohol, and butyl alcohol; polyhydric alcohols such as glycerin, butylene glycol and propylene glycol; and hydrocarbon solvents such as methyl acetate, ethyl acetate, acetone, benzene, hexane, diethyl ether, and dichloromethane; Or a mixture thereof may be used. Preferably, water, lower alcohol, 1,3-butylene glycol, and ethyl acetate may be used alone or in a mixture of two or more, and most preferably, ethyl acetate is used as the extraction solvent. can be used as

In one embodiment of the present invention, it was confirmed that the culture extract of the Penicillium viseti strain (Accession No.: KACC 83024BP) according to the present invention has a growth inhibitory effect against various pathogenic fungi and pathogenic bacteria.

Penicillium bissettii ( Penicillium bissettii ) strain (Accession No.: KACC 83024BP) of the present invention, its culture extract or fractions exhibit excellent antibacterial and antifungal activity through direct influence on harmful microorganisms, so that antibacterial and antifungal pharmaceuticals It can be usefully used as a composition.

The pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, silicic acid. calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. it is not The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, or the like.

A suitable dosage of the pharmaceutical composition of the present invention is variously prescribed depending on factors such as formulation method, administration method, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity of the patient. can be The daily dose of the pharmaceutical composition of the present invention is, for example, 0.001-100 mg/kg.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. or it may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, syrup, or emulsion in oil or an aqueous medium, or may be in the form of an extract, powder, powder, granule, tablet or capsule, and may additionally include a dispersant or stabilizer.

According to another aspect of the present invention, the present invention provides a cosmetic composition for antibacterial and antifungal containing a Penicillium bissettii strain (Accession No.: KACC 83024BP), its culture extract or fraction as an active ingredient.

The Penicillium bissettii strain (Accession No.: KACC 83024BP) of the present invention, its culture extract or fraction, exhibits excellent antibacterial and antifungal activity through direct influence on harmful microorganisms. Therefore, antibacterial and antifungal cosmetic composition can be usefully used as

The cosmetic composition of the present invention includes, as an active ingredient, components commonly used in cosmetic compositions in addition to the Penicillium bissettii strain (Accession No.: KACC 83024BP) or its culture medium, for example, antioxidants, stabilizers, conventional adjuvants such as solubilizers, vitamins, pigments and fragrances, and carriers.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , oil, powder foundation, emulsion foundation, wax foundation, spray, etc., but is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion (skin), a nourishing lotion (milk lotion), a nourishing 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, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide, etc. are used as carrier components. can be

When 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, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propane /may contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and microcrystals Adult cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide as carrier components Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester and the like may be used.

According to another aspect of the present invention, the present invention provides an antibacterial and antifungal feed additive composition comprising a Penicillium bissettii strain (accession number: KACC 83024BP), its culture extract or fraction as an active ingredient. .

Food or feed preservatives are additives used to prevent deterioration, spoilage, discoloration and chemical changes of food or feed, and include sterilizers and antioxidants, and inhibit the growth of microorganisms such as bacteria, mold, yeast, etc. , functional antibacterial agents such as inhibiting the growth of decaying microorganisms or sterilizing in cosmetics and pharmaceuticals. Ideal conditions for these preservatives in food or feed should be non-toxic and effective even in small amounts. Penicillium bissettii ( Penicillium bissettii ) strain (Accession No.: KACC 83024BP) of the present invention, its culture extract or fraction directly affects harmful microorganisms, thereby exhibiting excellent antibacterial and antifungal activity. It can be widely used as preservatives, cosmetic preservatives and pharmaceutical preservatives.

According to another aspect of the present invention, the present invention kills pathogenic bacteria by administering the Penicillium bissettii strain (Accession No.: KACC 83024BP), its culture extract or fraction to an individual in need of treatment It provides a method for treating bacterial or fungal infections in animals other than humans, comprising the step of:

Although the treatment method according to the present invention is a method of treating animals other than humans, it does not mean that the treatment method is ineffective in humans. In addition, considering that humans have bacterial or fungal infectious diseases whose symptoms can be improved by administration of the therapeutic composition according to the present invention, it can be sufficiently used for the treatment of bacterial or fungal infectious diseases in humans.

In the present invention, the term "animals other than humans" means horses, sheep, pigs, goats, camels, except for humans, who have diseases caused by bacterial or fungal infection whose symptoms can be improved by administration of the therapeutic composition according to the present invention. , antelope, means animals such as dogs. By administering the therapeutic composition according to the present invention to animals other than humans, it is possible to effectively prevent and treat diseases caused by bacterial and fungal infections.

In the present invention, the term "administration" means introducing a predetermined substance to an animal by any suitable method, and the administration route of the therapeutic composition according to the present invention is oral or through any general route as long as it can reach the target tissue. It may be administered parenterally. In addition, the therapeutic composition according to the present invention may be administered by any device capable of moving the active ingredient to the target cell.

A preferred dosage of the therapeutic composition according to the present invention varies depending on the condition and weight of the animal to be treated, the degree of disease, the drug form, the route of administration, and the duration, but may be appropriately selected by those skilled in the art. However, for a desirable effect, the pharmaceutical composition of the present invention is 0.001 to 10 mg/kg, preferably 0.01 to 1 mg/kg, and may be administered 1 to 3 times a day.

According to another aspect of the present invention, the present invention comprises the steps of culturing a Penicillium bissettii strain (accession number: KACC 83024BP); obtaining a culture solution produced by culturing the strain; and separating penicillic acid from the obtained culture medium. In an example of the present invention, the HPLC peak area of the ethyl acetate extract (PBME) of the Penicillium bissettii strain (accession number: KACC 83024BP) is commercially sold HPLC peak area of penicillic acid As a result of comparison, it was analyzed that about 80.44% of the ethyl acetate extract of the culture medium of the Penicillium viseti strain was penicillic acid, and through this, penicillic acid of high purity can be produced through the culture of the Penicillium viseti strain of the present invention. was able to confirm

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

(1) The present invention provides an antibacterial and antifungal composition comprising, as an active ingredient, a Penicillium bissettii strain, a culture solution extract or fraction thereof, derived from Duckweed.

(2) Penicillium bissettii ( Penicillium bissettii ) strain of the present invention, a composition comprising a culture extract or fraction thereof exhibits a wide antibacterial and antifungal spectrum against various bacteria, and the composition of the present invention is It can be applied to various bacterial infections that occur.

(3) In addition, the composition of the present invention can be usefully used for antibacterial, antifungal and antiseptic purposes in various fields such as cosmetic compositions, additive compositions for food or feed preservation.

1 is a photograph taken by collecting a sample of deokki oil vegetable.
Figure 2 is a drawing showing the creation of a biological phylogeny of Penicillium bissettii ( Penicillium bissettii ) SK16-002 strain (accession number: KACC 83024BP).
Figure 3 is Penicillium bissettii ( Penicillium bissettii ) SK16-002 strain (accession number: KACC 83024BP) strain CYA (Czapek Yeast Autolysate agar), YES (Yeast extract sucrose agar), MEA (Malt extract agar), and DG18 This picture was taken after culturing for 7 days at 23°C using 4 types of medium (Dichloran 18% Glycerol agar).
Figure 4 is a micrograph of the Penicillium bissettii ( Penicillium bissettii ) SK16-002 strain (Accession No.: KACC 83024BP) strain.
5 is a photograph showing the results of measuring the antibacterial activity of the ethyl acetate extract of the Penicillium bissettii SK16-002 strain (Accession No.: KACC 83024BP) culture using the disk diffusion method using the disk diffusion method.
6 is a graph showing the results of HPLC analysis of the ethyl acetate extract of the Penicillium bissettii SK16-002 strain (Accession No.: KACC 83024BP).
Figure 7 shows the antibacterial activity of the HPLC first fraction, the second fraction, and the third fraction of the ethyl acetate extract of the Penicillium bissettii SK16-002 strain (accession number: KACC 83024BP) using a disk diffusion method. This is a picture showing the measurement result.
8 is a graph showing the results of prep-LC chromatography of the ethyl acetate extract of the Penicillium bissettii SK16-002 strain (Accession No.: KACC 83024BP) culture.
9 is a graph showing the results of MS analysis performed to analyze the active ingredients present in the ethyl acetate extract of the Penicillium bissettii SK16-002 strain (accession number: KACC 83024BP).
10 is a graph showing the results of HR-MS analysis performed to analyze the active ingredients present in the ethyl acetate extract of the Penicillium bissettii SK16-002 strain (accession number: KACC 83024BP).
11 is a graph showing the results of NMR spectrum analysis performed to analyze the active ingredients present in the ethyl acetate extract of the Penicillium bissettii SK16-002 strain (accession number: KACC 83024BP).
12 is a photograph showing the results of measuring the antimicrobial activity of Penicillium bissettii SK16-002 strain (accession number: KACC 83024BP) culture medium ethyl acetate extract, its fractions, and penicillic acid using a disc diffusion method; am.
FIG. 13 is a graph of a standard curve ( FIG. 13b ) created by integrating the results of HPLC quantitative analysis for each concentration of penicillic acid ( FIG. 13a ) and the peak area of penicillic acid shown in about 5 minutes based on the results ( FIG. 13b ).
Figure 14 is Penicillium bissettii ( Penicillium bissettii ) SK16-002 strain (accession number: KACC 83024BP) culture medium ethyl acetate extract (PBME treatment group) and its second fraction (PBME-fraction treatment group), and in the same strain It is a graph showing the results of performing quantitative analysis based on MTT assay to analyze the antibacterial activity of the isolated penicillic acid.
Figure 15 is Penicillium bissettii ( Penicillium bissettii ) SK16-002 strain (accession number: KACC 83024BP) culture medium ethyl acetate extract (PBME treatment group) and its second fraction (PBME-fraction treatment group), and in the same strain It is a graph showing the cytotoxicity of the isolated penicillic acid.
Figure 16 is Penicillium bissettii ( Penicillium bissettii ) SK16-002 strain (Accession No.: KACC 83024BP) in the culture medium ethyl acetate extract (PBME treatment group) and its second fraction (PBME-fraction treatment group), and in the same strain It is a graph showing the antioxidant capacity of the isolated penicillic acid.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and therefore, the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Experimental Materials and Methods

1-1. strain isolation

The plant endogenous bacteria Penicillium bissettii SK16-002 strain was collected from the roots of deokki oil plants collected in September 2016 in Bonghwa-gun, Gyeongsangbuk-do, Republic of Korea [37°05' 08.0" north latitude, 129°08' 26.7" east longitude]. separated (Fig. 1).

The collected plant samples were used to isolate plant endophytes after washing the soil dust attached to the surface with running tap water and wiping it dry. After slicing the root tissue into small pieces of 0.5 cm in size, sterilize in 2% sodium hypochlorite solution for 1 minute, surface sterilize in 70% ethanol for 1 minute, wash in sterile distilled water for 1 minute, remove moisture, and strain It was placed on the separation medium.

The strain separation medium was prepared by adding 50 ppm Rose Bengal, 50 ppm Kanamycin, and 50 ppm Streptomycin to MEA (malt extract agar, Difco) medium. After the sterilized root tissue was placed on the prepared medium, it was cultured for 5-7 days in an incubator at 22°C.

A method of transferring a young mycelium from the edge of a flora growing from a plant tissue to a new PDA (potato dextrose agar) medium and culturing, germinating single conidia in WA (water agar) for 20 hours, and then transferring a single spore to a new PDA medium. was separated into pure water.

1-2. Extraction of strain culture

The pure isolated strain was cultured in PDA medium at room temperature for 7 days to prepare an inoculum. The strain was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of PDB (potato dextrose broth), and cultured with shaking at 23° C. and 120 rpm for 3 weeks. The mycelia were filtered with miracloth, the same amount of ethyl acetate (EtOAc) was added to the collected culture filtrate for extraction, and the extract was concentrated under reduced pressure and dried to prepare a crude extract.

1-3. Species identification of the endogenous bacteria Penicillium viseti SK16-002 strain

Species identification of the endogenous plant, Penicillium viseti SK16-002 strain, was performed by combining molecular identification using DNA sequencing, culturing in the medium, and branching shape of the sporangia. First, for molecular identification, DNA isolated from hyphae cultured in a liquid medium was used for three marker sites required for species identification of Penicillium sp. ) was obtained (Visage and Houbraken, 2014; Visage et al., 2016).

The obtained nucleotide sequence was secured and assembled using a sequencing program (Sequencher) to determine a consensus sequence, and BLAST search was performed on the nucleotide database of NCBI. Multiple alignment and phylogenetic relationship analysis were performed using the MEGA program.

As a result of the NCBI Blast search performed based on ITS sequence information, the Penicillium viseti SK16-002 strain was found to have high sequence homology with the strains belonging to the Lanata-Divaricata section of the Penicillium genus.

Accordingly, after multiple alignment of the marker sequences with respect to the standard strains of species belonging to the section Lanata-Divaricata of the genus Penicillium, phylogenetic relationships were analyzed by parallel mixing. As a result, the SK16-002 strain was located in the same branch as Penicillium viseti (FIG. 2).

In addition, the SK16-002 strain was cultured at 23 °C using 4 types of medium: CYA (Czapek Yeast Autolysate agar), YES (Yeast extract sucrose agar), MEA (Malt extract agar), and DG18 (Dichloran 18% Glycerol agar). When cultured for 7 days under the condition, it grew with a light grayish-green color (line A in Fig. 3), and showed a pink pigment in addition to the dark brown pigment on the back side (line B in Fig. 3). The conidia showed a broom-shaped phialide characteristic of the Penicillium sp. strain (FIG. 4). Moreover, it did not grow at 37 degreeC. By combining these results, the SK16-002 strain was identified as a Penicillium bissettii species.

The identified strain was named Penicillium viseti strain (accession number: KACC 83024BP), and the National Institute of Biological Resources' Wildlife Genetic Resources Bank (NIBRGR 0000118808) and the National Academy of Agricultural Sciences Agricultural Genetic Resources Information Center with deposit number KACC 83024BP. deposited.

1-4. pathogenic microorganisms

The pathogenic microorganisms used in the experiment were E. coli ( Escherichia coli , K12, Gram-negative), Klepsiella pneumoniae (KCTC2208, Gram-negative), Staphylococcus aureus , KCTC1928, acquired from KCTC. Gram-positive), Streptococcus pseudopneumoniae ( Streptococcus pseudopneumoniae , KCTC 5764, Gram-positive), Enterococcus faecalis ( Enterococcus faecalis , KCTC2011, Gram-positive), and Candida albicans ( Candida albicans 17485), KCTC 17485 6 strains were used, and culture conditions for each pathogen were LB (Luria-Bertani broth) medium, E. coli NB (Nutrient Broth) medium, Klebsiella pneumoniae, Staphylococcus aureus, And Streptococcus pseudopneumoniae BHI (Brain Heart Infusion) medium, Enterococcus faecalis and Candida albicans GPY (Glucose Peptone Yeast Extract) medium was used. Candida albicans strains were cultured at 30°C, and the remaining strains were cultured at 7°C. In common, after inoculating the appropriate bacteria for each medium, the species was cultured for 16 hours at 200 rpm in a shaking incubator at the appropriate temperature, followed by main culture for 4 to 6 hours.

Example 2. Investigation of antibacterial activity based on the disc diffusion method

The antibacterial activity against pathogenic microorganisms was investigated by using a disc diffusion method using a total of 96 unknown sample extracts isolated from the roots of the deuciferous oil plants of Example 1-1.

The disc diffusion method was used as an experimental method for the investigation of antibacterial activity. By measuring the absorbance of the cultured cells at 600 nm and quantifying the number of cells, the concentration of cells applied on an agar plate was fixed at a concentration of 1.5×10 ⁸ cfu in all experiments, and the experiment was carried out.

In order to confirm the antibacterial activity of the sample, a 6mm paper disc was placed on the parafilm, and 20 μl of the extract (10mg/mL) estimated to have antibacterial ability was placed on the disc and dried for 30 minutes. In the case of four bacterial strains of E. coli, Klebsiella pneumoniae, Staphylococcus aureus, and Streptococcus pseudopneumoniae, 2 μl gentamicin was used as a positive control, and Enterococcus fae In the case of the callis strain, 2 μl of ampicillin was used, and in the case of the Candida albicans strain, 2 μl of fluconazole was used, and H ₂ O, and DMSO were used as negative controls.

After culturing the strains in each medium condition for 24 hours, the culture medium was applied to the agar plate with a cotton swab, and a dried paper disc was placed on the agar. Incubated for 18 hours at a temperature suitable for each bacteria in an incubator, and the experimental results were photographed with a digital camera (EOS M3, Canon), and then the diameter of the inhibition zone was measured using ImageJ software. did

As a result of the experiment, as a result of confirming the antibacterial activity three times against the six pathogenic microorganisms prepared in Examples 1-3 using the disc diffusion method, the culture solution extract of the Penicillium viseti strain, which is one of 96 unknown sample extracts ( Penicillium bissettii medium ethyl acetate extract, PBME) was found to form an inhibitory ring in all six pathogenic microorganisms, and it was confirmed that it had antibacterial activity against Gram-negative, Gram-positive and fungi (see FIG. 5).

[Table 1]

(+: less than 3mm, ++: less than 3mm to 6mm, +++: less than 6mm)

Example 3. Analysis of culture medium components of the Penicillium viseti strain

3-1. HPLC fractionation

The culture medium ethyl acetate extract (Penicillium bissettii medium ethyl acetate extract, PBME) of the Penicillium bissetti strain was fractionated by HPLC (shimadzu LC-20A). HPLC fractionation conditions were maintained at a flow rate of 1 mL/min using a SUPELCOSIL ^TM C-18 (25 cm x 4.5 mm, 5 μm) column. As the HPLC developing solvents, A (10% ACN), and B (ACN) were used. After setting the development ratio of the solvent B to 20%, the development was started, and the ratio of the solvent B in the development time of 13 minutes was set to 100%.

The solvent development ratio was maintained until 19 minutes, and the development ratio of solvent B at 20 minutes was set to be 20%, and then HPLC fractionation was performed by maintaining the solvent development ratio until 23 minutes.

Retention Time 1st fraction (①) from 1 to 3.5 minutes, the second fraction containing the main peak from 4.5 to 5.5 minutes (②), and the third fraction from 7 to 10 minutes ( ③) were recovered, respectively (see FIG. 6).

In order to confirm the antibacterial activity of the first to third fractions, an experiment was conducted in the same manner as in the disk diffusion method of Example 2.

Experimental results, referring to Figure 7, Staphylococcus aureus ( Staphylococcus aureus , KCTC1928, Gram-positive), Streptococcus pseudopneumoniae in the HPLC second fraction of the ethyl acetate extract of the culture medium of the Penicillium viseti strain ( Streptococcus pseudopneumoniae , KCTC 5764, gram-positive), Enterococcus faecalis ( Enterococcus faecalis , KCTC2011, gram-positive), and Candida albicans ( Candida albicans , KCTC 17485, fungus) against four strains forming an inhibitory ring could confirm that

3-2. Prep-LC fractionation

The culture medium ethyl acetate extract (Penicillium bissettii medium ethyl acetate extract, PBME) of the Penicillium bissetti strain was fractionated with Prep-LC (shimadzu LC-8A). For prep-LC fractionation conditions, 120 μl of a 10 mg/mL sample was injected for 40 minutes at a flow rate of 8 mL/min using a Kromasil 100-5-C18 column. Prep-LC solvents A (0.05% TFA in water) and B (ACN) were used. After setting the development ratio of solvent B to 20%, the development was started, and the ratio of solvent B in 15 minutes of development time was set to 80%. Then, after setting the development rate of solvent B for 15.5 minutes to 100%, the solvent development rate was maintained until 20 minutes.

The development rate of solvent B in 20.5 minutes was lowered to 20, and this was maintained until the development time of 30 minutes to proceed with prep-LC. Retention Time A fraction (referred to as a PBME fraction) containing a main peak from approximately 12 to 13 minutes was recovered (see FIG. 8 ).

3-3. MS, HR-MS, and NMR analysis

MS, HR-MS, and NMR analysis were performed to identify substances present in the PBME fraction obtained by the Prep-LC fractionation method of Example 3-2.

The MS (Mass Spectroscopy) result for the PBME fraction is as shown in FIG. 9, the HR-MS (High Resolution - Mass Spectroscopy) result is shown as in FIG. 10, and the NMR result is summarized in FIG.

Combining the MS, HR-MS, and NMR results, the compound present in the PBME fraction (Retention Time 12 to 13 minutes) obtained by fractionating the ethyl acetate extract of the culture medium of the Penicillium viseti strain by Prep-LC is penicillic acid ( penicillic acid).

3-4. Analysis of antibacterial activity of PBME fraction and penicillic acid

In order to confirm the antibacterial activity of the first to third fractions obtained in Example 3-1, an experiment was performed in the same manner as in Example 2 above. The experiment was carried out by dividing the culture medium ethyl acetate extract treatment group (PBME treatment group) of the Penicillium viseti strain and the second fraction treatment group (PBME-fraction treatment group) of the culture medium ethyl acetate extract of the Penicillium viseti strain, The same method as in Example 2 was used, except that penicillic acid (catalog number 11440) purchased from Cayman was used as a positive control group.

As a result of the experiment, referring to FIG. 12 , the culture medium ethyl acetate extract treatment group (PBME treatment group) of the Penicillium viseti strain and its second fraction treatment group, like the penicillic acid treatment group as a control group, for all 6 pathogenic strains It was confirmed that it exhibited excellent antibacterial activity.

3-5. Analysis of production of penicillic acid

In order to verify the productivity of penicillic acid present in the extract isolated from the Penicillium viseti strain, HPLC quantitative analysis was performed using commercially available penicillic acid (Cayman, catalog number 11440) as a control, and a standard curve (Standard curve) was made and the productivity of the Penicillium viseti strain extract was comparatively analyzed.

Referring to FIG. 13 , a standard curve ( FIG. 13b ) was obtained by integrating the peak area of penicillic acid shown in about 5 minutes based on the HPLC quantitative analysis result for each concentration of penicillic acid ( FIG. 13a ), and then, penicillic acid As a result of comparison with the peak area of the culture medium ethyl acetate extract (PBME) of the L. viseti strain, it was evaluated that about 80.44% of the culture medium ethyl acetate extract of the Penicillium viseti strain was penicillic acid.

Through the above results, it was confirmed that high purity penicillic acid can be produced through the culture of the Penicillium viseti strain.

3-6. Analysis of antibacterial activity of penicillic acid secreted by Penicillium viseti strain

MTT tetrazolium-based quantitative analysis was performed to analyze the antibacterial activity of penicillic acid secreted by the Penicillium viseti strain. The tetrazolium salt is reduced to formazan crystal by a reductase of living cells.

By measuring the absorbance of the cultured strain at 600nm to quantify the strain, the concentration of the bacteria was fixed to 1.0 × 10 ⁸ cfu in all experiments, and the experiment was carried out. The bacteria were settled by centrifugation (3000 rpm, 300 seconds), the supernatant was discarded, and the supernatant was resuspended in PBS (0.2% Glucose) in the same volume as the supernatant. After transferring 180 μl of the bacterial suspension to a 1.5 mL centrifuge tube at 37° C., put it on to confirm the quantitative antibacterial activity of the sample, and serially dilute the extract estimated to have antibacterial ability 10 times from 10 mg/mL to 20 μl After each treatment, incubated with shaking at 37° C. for 2 hours. Then, 20 μl of MTT reagent was added to a 1.5 ml centrifuge tube, the tube was shaken to mix, and a reduction reaction was initiated, followed by incubation with shaking by keeping it dark using a 37° C. foil for 30 minutes. After dissolving cells and formazan crystals by administering 500 μl of DMSO to a 1.5 mL centrifuge tube, the dissolution process was accelerated at 37° C. for 30 min and a shaking incubator at 200 rpm. After putting 200 μl of the formazan crystal solution in a 1.5 mL centrifuge tube into a 96 well plate, the absorbance was measured at 560 nm through a plate reader immediately.

4 bacterial strains of E. coli, Klebsiella pneumoniae, Staphylococcus aureus, and Streptococcus pseudopneumoniae were used as positive controls 2㎍ of gentamicin, Enterococcus fae In the case of the Callis strain, 2 μg of ampicillin was used as a positive control, and in the case of the Candida albicans strain, 2 μg of fluconazole was used as a positive control.

Since the formazan crystal exhibits maximum absorbance at 560 nm, quantitative antibacterial activity was evaluated using it (see FIG. 14 ), and IC ₅₀ value, which is a measure of quantitative antibacterial activity, was derived. The IC ₅₀ value is the half maximal inhibitory concentration, and is the concentration of the sample that can inhibit the antibacterial activity by half. That is, it is determined that the smaller the value, the better the antibacterial activity. All raw data obtained in the experiment were graphed using Origin software (version 9.0), and IC ₅₀ values were calculated using Graphpad prism software (version 4.0) and summarized in Table 2 below.

[Table 2]

Referring to [Fig. 14] and [Table 2], the culture medium ethyl acetate extract (PBME-treated group) of the Penicillium viseti strain and its second fraction (PBME-fraction-treated group), and penicillic acid isolated from the same strain As compared to the control group treated with gentamicin, ampicillin, and fluconazole, it was confirmed that the antibacterial activity was equal to or greater than that of the control group, thereby exhibiting excellent antibacterial activity.

Example 4. Cytotoxicity

To measure the cytotoxicity of the ethyl acetate extract (PBME-treated group) and its second fraction (PBME-fraction-treated group) of the culture medium of the Penicillium viseti strain of the present invention, and penicillic acid isolated from the same strain, MTT ( 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed.

First, HaCaT cells, a human skin keratinocyte line, were cultured in a complete medium [10% FBS, 1% Antibiotics in DMEM (Dulbecco's Modified Eagle Medium)] in a petri dish in a 5% carbon dioxide incubator at 37°C. Samples were prepared by diluting 10% DMSO with distilled water at a concentration of 10mg/mL, diluted by 1/10 with distilled water, and adding 1/10 to complete medium so that the final DMSO concentration was 0.1%. Cisplatin, which is used as an anticancer drug, was used as a control group for the experiment. MTT reagent was prepared by filtering using a 0.25um syringe filter that was dissolved in PBS at a concentration of 5mg/mL. When HaCaT cells showed growth of about 70 to 80% relative to the total volume of the Petri dish, subculture was performed and used in the experiment.

Dispense HaCaT cells to 10,000 cells per well in a 96-well plate, add 100 μl of complete medium to each well, incubate in an incubator at 37° C. and 5% carbon dioxide environment for 24 hours, and then complete medium in each well was removed, replaced with a medium containing the sample, and cultured for 48 hours in an incubator at 37°C and 5% carbon dioxide environment. Thereafter, 10 μl of MTT reagent was treated in each well and further cultured in a 5% carbon dioxide incubator at 37° C. for 4 hours. Then, using a multi-pipette, carefully remove the medium from the 96-well plate, add 200 μl of DMSO solvent to each well, and react in a shaking incubator at 37° C. Thus, the absorbance at 560 nm was measured. When measuring the absorbance, it was diluted and measured so that the maximum value was 1.000.

As a result of the experiment, the IC ₅₀ value of the culture medium ethyl acetate extract (PBME-treated group) of the Penicillium viseti strain was measured to be 46.918 μg/mL, and the second fraction (PBME- fraction treated group) was measured to have an IC ₅₀ value of 84.838 μg/mL, and the IC ₅₀ value of penicillic acid isolated from the same strain was measured to be 69.386 μg/mL, which was similar to that of cisplatin used as a positive control. It was evaluated to show cytotoxicity (see FIG. 15 ).

Example 4. Antioxidant activity

2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation scavenging ability is obtained by the reaction of diammonium salt with potassium persulfide to generate ABTS radicals, giving it a characteristic blue-green color. This is a method of measuring decolorization to light green as a sample exhibiting an antioxidant effect is added.

Antioxidant activity measurement test using ABTS radicals was conducted by mixing 7 mM 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and 2.4 mM potassium persulfide, blocking light for 16 hours before use, and leaving it at room temperature. reacted.

After the reaction, in 1ml of ABTS (in PBS, pH 7.4), the ethyl acetate extract (PBME-treated group), the second fraction thereof (PBME-fraction-treated group), and penicillic acid isolated from the same strain in 1ml (in PBS, pH 7.4) of the Viseti strain were each 0.5 After the reaction was added by increasing the concentration by 10 times from ng/mL to 0.5 mg/mL (in PBS, pH 7.4), centrifuged after 10 minutes, and only the supernatant was obtained and absorbance was measured at 734 nm [Fig. 16] arranged in For comparative experiments, gallic acid was used as a positive control.

As a result of the experiment, it was confirmed that the ethyl acetate extract (PBME-treated group), the second fraction thereof (PBME-fraction treatment group), and penicillic acid isolated from the same strain of the Viseti strain had an antioxidant activity equal to or higher than that of gallic acid. .

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Agricultural Biotechnology Research Institute KACC83024 20190624

Claims (10)

Penicillium bissettii having antibacterial and antifungal activity ( Penicillium bissettii ) strain (accession number: KACC 83024BP). According to claim 1,
The strain is Penicillium bissettii ( Penicillium bissettii ) strain (accession number: KACC 83024BP), characterized in that it produces penicillic acid. An antibacterial and antifungal pharmaceutical composition comprising the strain of claim 1, an extract or fraction thereof as an active ingredient. 4. The method of claim 3,
The composition is Escherichia coli , Klepsiella pneumoniae , Staphylococcus aureus , Streptococcus pseudopneumoniae , and Enterococcus faecalis. ( Enterococcus faecalis ) Antibacterial and antifungal pharmaceutical composition, characterized in that it has antibacterial activity against pathogenic bacteria composed of. 4. The method of claim 3,
The composition is Candida albicans ( Candida albicans ) Antibacterial and antifungal pharmaceutical composition, characterized in that it has antifungal activity. 4. The method of claim 3,
The culture medium extract is an antibacterial and antifungal pharmaceutical composition, characterized in that it is an ethyl acetate extract of the Penicillium bissettii strain (Accession No.: KACC 83024BP). An antibacterial and antifungal cosmetic composition comprising the strain of claim 1, an extract or fraction thereof as an active ingredient. A preservative composition for feed or food containing the strain of claim 1, an extract or fraction thereof as an active ingredient. The method of treating a bacterial or fungal infection of an animal other than humans, comprising the step of administering the strain of claim 1, a culture solution extract or fraction thereof to an individual in need of treatment to kill pathogenic bacteria or fungi. Culturing the strain of claim 1;
obtaining a culture solution produced by culturing the strain; and
Separating penicillic acid from the obtained culture medium; including,
Method for producing penicillic acid.

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