KR101256182B1 - A manufacturing method of a yeast-statistic protein from Rahnella aquatilis and anti-yeast enhancing substance - Google Patents

Abstract

The present invention provides a method for preparing a extract of Ranella aquatilis AY2000 strain (Accession No. KACC 91164P) containing a proteinaceous antifungal substance, an extract prepared by this method, an antifungal composition comprising the extract and a fungus using the same. It relates to a method of inhibiting the growth of. The antifungal substance according to the present invention is a high molecular proteinaceous substance, which can effectively control fungi and has bacteriostatic effects, which can significantly reduce side effects compared to conventional low molecule antifungal agents. And synergistic effects with antifungal agents.

Description

A manufacturing method of a yeast-statistic protein from Rahnella aquatilis and anti-yeast enhancing substance}

The present invention relates to a method for preparing an antifungal extract in a Lanella aqua tilis AY2000 (Accession No. KACC 91164P) culture. More specifically, the present invention is an alcohol precipitation reaction to the culture of Ranella aquatylus AY2000 (Accession Number KACC 91164P), the addition of sugar hydrolyase to the precipitate and the high protein proteinaceous antifungal showing a high antifungal effect by filtration The present invention relates to a method for producing an extract including the substance, an extract prepared by the method, an antifungal composition comprising the extract as an active ingredient, and a method for inhibiting the growth of fungi using the extract or the antifungal composition.

Rahnella aquatilis Bacteria in the genus Gram-negative bacillus belonging to the Enterobacteriaceae family are widely present in nature. These strains generally exist mainly in water or soil, and the optimum growth temperature is 25 ℃, and cannot grow above 37 ℃. In addition, the bacteria of the genus R. aquatilis are Gram-negative bacteria, but there are no reports of pathogenicity.

A fungus is a microbial group composed of more than 72,000 species of fungi, including fungi, yeasts and mushrooms. It belongs to eukaryotic organisms with a nuclear membrane, and cell organelles such as mitochondria and endoplasmic reticulum are developed, and there are cell walls composed of chitin, glucan, and the like. Most of them form cellular mycelium to grow, develop, sexually and asexually reproduce, and spores as propagules, but some strains have unicellular proliferation. As a by-product bacteria, they are involved in organic decomposition in nature, but some of them are parasitic or symbiotic to animals and plants.

Generally, antibiotics include antibacterial, antifungal, and antiviral agents in a broad sense, but in a narrow sense, they mean substances that inhibit the growth of bacteria, and distinguish them from antifungal agents. Antifungal agents used today are more toxic to humans than antibacterial agents, so the number of drugs available is extremely limited. Therefore, a new concept of anti yeast is required in the constant development of new antifungal agents with fewer side effects. Antifungal agents used today are low-molecular organic compounds with a focus on stability.Among the major antifungal agents currently used are amphotericin B and the azole compounds ketoconazole, fluconazole and clotri. Clotrimazole and the like are known, all of which are manufactured by chemical synthesis, not materials extracted from natural products, and are evaluated to be not ideal for treating mycosis due to their various side effects. In particular, amphotericin B, known as the best antifungal agent, has a nickname 'amphoterrible' due to severe side effects such as high fever, lowering blood pressure, headache, vomiting, vasculitis, and kidney damage. The lack of selective toxicity to pathogens can cause many side effects in the host. In the case of ketoconazole, production has been stopped for three years.

A side effect of the fungal agent on a host is a problem due to the biological properties of the fungus that fungi also share many biochemical properties with host cells as eukaryotic organisms. Humans have been able to succeed in developing antibiotics because the target bacterial cells are prokaryotic organisms that are different from host cells, and because of their different biochemical properties, they have been able to apply active research to them.

There is an urgent need for the development of new fungicides with fewer side effects that complement these weaknesses of antifungal agents, and as a result of this effort, the Applicant has sought to develop methods for producing new antifungal agents and substances that enhance the effectiveness of existing antifungal agents. Invented a method for preparing an extract comprising a high molecular proteinaceous material having antifungal activity effectively and an antifungal promoter using the same in Ranella Aquatilis AY2000 (Accession No. KACC 91164P).

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for producing an extract by performing a constant treatment in a culture medium of Ranella aquatilis AY2000 (Accession No. KACC 91164P) in order to develop a new fungicide with low side effects, and an extract prepared therefrom.

Still another object of the present invention is to provide an antifungal composition comprising the extract as an active ingredient and a known antifungal agent and an antifungal composition comprising the extract as an active ingredient. Another object of the present invention is to provide a method for inhibiting fungal growth using the extract or composition.

In order to achieve the above object, the present invention provides a step of alcohol precipitation in a culture culture of Ranella aquatilis AY2000 (Accession No. KACC 91164P), adding a hydrolytic enzyme to the precipitate, and filtering and filtering the additives. It provides a method of producing a Lanella aqua tilis AY2000 strain culture solution comprising a. Glycolytic enzymes used in the present invention are from the group consisting of beta-glucosidase, cellulose, cellulose, inulinase, invertase and pectinase. Can be selected. In addition, the extract may include an antifungal substance composed of a high molecular substance including two heterologous proteins.

In addition, the present invention precipitates the culture of ranella Aquatilis AY2000 strain (Accession No. KACC 91164P), and treats the hydrolase on the precipitate, and extracts produced by filtering the fractions of the ranella Aquatilis AY2000 strain culture solution And it provides an antifungal composition containing the extract as an active ingredient. Also preferably the composition may further comprise at least one selected from the above extracts and itraconazole and fluconazole.

In addition, the present invention provides a method for inhibiting the growth of fungi using the extract or composition, the growth inhibition method may be preferably to perform bacteriostatic action.

In the case of using the method for preparing extracts derived from the Ranella aquatilis AY2000 strain (Accession No. KACC 91164P) culture provided in the present invention, the extract prepared from this method and the antifungal composition containing the extract, fungi are effectively controlled. In addition, the anti-yeast substance produced by the aquatilis AY2000 strain is a high molecular substance, which has a low side effect on the human body, and if the anti-yeast substance is mixed with an existing antifungal agent to increase antifungal activity, Fungal agents can inhibit fungal growth even with small amounts, which can alleviate side effects on the human body.

1 (A) shows the result of gel filtration using Sephacryl S400HR after treatment with pectinase. (B) is the result of secondary gel filtration from pit 1 of (A) using Sephacryl S400HR.
FIG. 2 shows the results of none SDS-PAGE (A) and SDS-PAGE (B) on antifungal material after secondary gel filtration. FIG. Electrophoresis was performed using 8% separation gel and 4% stacking gel. P1 is the purified antifungal substance, M is the molecular marker of SDS-PAGE.
3 is a cell cycle when C. albicans treated with antifungal substances. Cell groups were analyzed at 0 hours (A), 2 hours (B), 4 hours (C) and 6 hours (D).
Figure 4 is a result showing the growth inhibitory effect when the anti-fungal treatment to C. alvicans cultured in YM broth.
5 is a result showing the antifungal activity of the combination of a commercially available antifungal agent and the antifungal substance of the present invention.

Hereinafter, the present invention will be described in more detail.

The present invention comprises the steps of: (a) alcohol precipitation of the culture culture of Ranella aquatilis AY2000 (Accession No. KACC 91164P);

(b) adding a sugar hydrolase to the precipitate; And

(C) comprising the step of fractionating the additives, provides a method for producing a Lanella aquatilis AY2000 strain culture extract comprising a substance having antifungal activity.

Ranella Aquatilis AY2000 strain (Accession No. KACC 91164P) has the property of being able to grow at a wide range of pH from acidic to weak alkali (pH4.5-8.0).

In the present invention, the culture of Ranella aquatilis AY2000 strain can be used as long as the ranella aquatilis AY2000 strain is obtained by the culture medium, but in the present invention, the strain was inoculated using MYCS medium and cultured with shaking. .

In the present invention, it is preferable to use alcohol having 1 to 6 carbon atoms for alcohol precipitation, and isopropanol, ethanol, methanol and the like can be used, and ethanol is most preferred.

In the present invention, the sugar hydrolase performs a function of degrading sugars that may be present in the culture by using water molecules, and any enzyme capable of hydrolyzing sugars is possible, but preferably beta-glucosidase ( β-glucosidase, cellulase (cellulose), inulinase (inulinase), invertase (invertase) and pectinase (pectinase) is selected from the group consisting of, most preferably pectinase.

In the present invention, fractionation is for purification of the active substance, and it is possible to use a filtration method, a chromatographic method, and the like that are obvious to those skilled in the art, and the gel filtration used by the inventors in the examples is most preferred.

In the present invention, the "antifungal substance" means a substance that inhibits fungal growth, and the "fungal" is a microbial group composed of more than 72,000 species including fungi, yeasts and mushrooms. It does not limit. In particular, in one embodiment of the present invention Saccharomyces is a non-pathogenic yeast The minimum inhibitory concentration was confirmed in cerevisiae and the pathogen Candida The effect of cell cycle on albocans was analyzed, which indicates that the extract of the present invention has a growth inhibitory effect on both pathogens and non-pathogens.

Further, in the present invention, the substance having antifungal activity is preferably a high molecular proteinaceous substance containing two heterologous proteins.

In addition, the present invention precipitates the culture of ranella Aquatilis AY2000 strain (Accession No. KACC 91164P), and treated with the hydrolase on the precipitate, and the filtered production of ranella Aquatilis AY2000 strain culture solution Provide an extract.

In addition, the present invention precipitates the culture of ranella Aquatilis AY2000 strain (Accession No. KACC 91164P), and treated with the hydrolase on the precipitate, and the filtered production of ranella Aquatilis AY2000 strain culture solution It provides an antifungal composition containing the extract as an active ingredient.

The antifungal composition according to the present invention can be used for pharmaceutical compositions, cosmetic compositions, agrochemical compositions, food additive compositions and the like.

The pharmaceutical composition may be administered orally in the form of a solid, semi-solid or liquid by adding a commercially available inorganic or organic carrier, or be administered parenterally, rectally, topically, transdermally, intravenously, intramuscularly, intraperitoneally, subcutaneously, or the like. Can be.

The preparation for oral administration includes tablets, pills, granules, soft and hard capsules, powders, fine granules, powders, emulsions, syrups, pellets and beverages. Etc. can be mentioned. In addition, preparations for parenteral administration include injections, drops, ointments, lotions, sprays, suspensions, emulsions, suppositories, and the like.

In order to formulate the active ingredient of the present invention, it can be easily formulated according to a conventional method, and surfactants, excipients, coloring agents, spices, preservatives, stabilizers, buffers, suspensions, and other commonly used auxiliaries can be used.

In addition, the dosage of the active ingredient will vary depending on the age, sex and weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the route of administration and the judgment of the prescriber. Dosage determination based on these factors is within the level of those skilled in the art, and generally the dosage may range from 0.001 mg / kg / day to approximately 1000 mg / kg / day, although any method is intended to limit the scope of the invention. no.

The cosmetic composition according to the present invention is not particularly limited to the formulation, softening cream, astringent makeup, nourishing cream, nourishing cream, massage cream, essence, eye cream, eye essence, lotion, lipstick, powder, baby powder, body lotion, Cosmetics such as body cream, body oil or body essence; Human body cleaners such as soap, cleansing cream, cleansing lotion, cleansing foam, cleansing water or body cleanser; Hair composition such as hair nourishing cosmetics, hair essence, hair serum scalp treatment, hair treatment, hair conditioner, hair shampoo or hair lotion; Or tablets and the like. The external preparation composition for each of these formulations may include various components formulated into conventional human cleansing compositions according to the various formulations described above or as appropriate for the end purpose, and the type and amount of these components may be easily selected by those skilled in the art. have.

The antifungal composition according to the present invention can be used for the therapeutic or prophylactic control of phytopathogenic fungi. Thus, the invention can also use the compositions according to the invention on seeds, plants or plant parts, fruits or soil on which the plants are growing.

The composition according to the invention for controlling phytopathogenic fungi in crop protection comprises an active compound according to the invention in an amount that is effective but not phytotoxic. "Effective but not phytotoxic amount" means on the one hand the amount of the composition according to the invention sufficient to satisfactorily or completely eliminate the fungal disease of the plant, without causing any significant symptoms of phytotoxicity to the crop. do. In general, such application rates can vary over a relatively wide range. This amount depends on a number of factors such as, for example, the fungus to be controlled, the plant, the climatic conditions and the components of the composition according to the invention.

All plants and plant parts can be treated according to the invention. Here, a plant should be understood to mean all plants and plant populations, such as wild plants or crops (including naturally occurring crops) that they want or do not want. Crops include plant varieties and transgenic plants that may or may not be protected by the sovereignty of plant breeders, by conventional plant cultivation and optimization methods, by biotechnology or genetic engineering, or by these methods. It may be a plant obtainable in combination. Some of the plants are to be understood as meaning all the above-ground and underground parts and organs of the plant, for example, shoots, leaves, flowers and roots, examples of which are leaves, needles, stems, trunks. ), Flowers, fruits, fruits, seeds, roots, tubers and rhizomes may be mentioned. Harvesting materials, and nutritional and reproductive materials, such as seedlings, tubers, rhizomes, cuttings and seeds, are also included in plant parts.

Treatment of plants and plant parts with the compositions according to the invention is carried out by conventional treatment methods, for example by dipping, spraying, spraying, irrigation, evaporation, dusting, spraying, spraying, forming, applying, spraying, spraying (de (Wrenching), by trickle irrigation, and in the case of propagation materials, especially seeds, also by dry seed treatment powder, wet seed treatment solution, slurry treatment water soluble powder, by shell formation, by work or multi-coating applications, etc. By acting directly or on its surroundings, habitat or storage space. It is also possible to apply the active compounds in microvolumes or to inject the active compound preparations or the active compounds themselves into the soil.

The composition for food additives according to the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the composition of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. There is no particular limitation on the kind of the food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, and the like and include all foods in a conventional sense.

The present invention also provides an antifungal composition further comprising at least one selected from itraconazole and fluconazole in the extract.

The term "itraconazole" used in the present invention is an oral azole antifungal drug and, together with its metabolite, hydroxyitraconazole, is a drug useful for systemic infection by fungi, and was approved by the US FDA in 1992. .

As used herein, the term “fluconazole” is a triazole antifungal agent used for the treatment and prevention of systemic infections or surface diseases caused by fungi.

The weight ratio of extract to itraconazole or fluconazole is preferably 500: 1 to 1: 500.

In another aspect, the present invention provides a method for inhibiting the growth of fungi using the extract or composition, preferably performing fungal fungal action.

Hereinafter, the present invention will be described in more detail with reference to Examples. The embodiments are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

< Example 1  >

1-1 : Strain culture and preparation of antifungal substance

R. aquatilis AY2000 strain (Accession No. KACC 91164P) was first inoculated with AY2000 strain in MYCS medium, followed by shaking culture (25 ° C, 24hr), and then the precipitate formed by adding 2 times the amount of ethanol to the culture supernatant from which the cells were removed. Immediately collected by centrifugation (12000rpm, 10min, 4 ℃). This precipitate was dissolved in pre-cooled distilled water (4 ° C.) and then dialyzed (SPECTRA / POR; MW6,000-8,000, 4 ° C.) to remove ethanol. The dialyzed solution was vacuum frozen and dried (-20 ° C.) and used as a sample for purification.

1-2 : Purification Method of Sample

First, the samples were pretreated with sugar hydrolyzate. That is, the sample (80 mg) was dissolved in 4 ml of 10 mM acetate buffer (pH4.1) and then centrifuged (14,000 rpm, 20 min, 4 ° C.) to remove insoluble substances and measure the sugar content of the dissolved substance. The dissolved substance was added to 1 unit of sugar to 100 units of sugar hydrolyzate, followed by enzymatic reaction (50 ℃, 20min), followed by immediate cooling (4 ℃), and immediately Sephacryl S400HR column (Φ1.6x60cm, Pharmacia) was subjected to gel filtration (Flow rate 44ml / hr). At this time, fractions (5ml / tube) were measured on protein absorbance (A280), collected based on the divided peaks and lyophilized, and then measured antifungal activity on each peak.

1-3 : Electrophoresis

Electrophoresis of the purified antifungal material was performed by SDS-PAGE (12) and none SDS-PAGE containing 8% separation gel. In addition, ProSieve color protein marker (MW10-176kDa, Lonza Rockland Inc. Cat. No. 50550) was used for the molecular weight measurement in SDS-PAGE.

1-4 : Antifungal  Activity measurement

Antifungal activity measurements were made according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS). In other words, the medium used was pre-sterilized YM medium (two-fold concentrated medium) containing 5 µg / ml of tetracycline (Tc), and the antifungal substance solution was prepared in two steps in a 96-well microplate. Diluted twice. Also. This microplate was pre-cultured (28 ° C, overnight) yeast ( S. cerevisiae) ATCC2120; 10 ⁵ cells / ml) were inoculated in 10 μl and cultured again (28 ° C., 24 hr). The degree of fungal growth was measured by absorbance (A600 nm). The antifungal activity of the antifungal substance was represented by a minimal inhibitory concentration (MIC) where no fungal growth occurred above 95%, and amphotericin B was used as a control. All activity measurements were expressed as the average value of MIC obtained through 2 replicates.

1-5 : Sugar and Protein Assay

The total content of sugars was determined by the phenol-sulfuric acid method and expressed on the basis of glucose. In addition, the protein content of the lowry method was used based on bovine serum albumin (BSA).

1-6 : existing With antifungal agents  Combined Effect Measurement Method

Experiments using the purified antifungal substance in combination with existing antifungal agents to measure the effect were carried out by the method described below. Conventional antifungal agents (amphotericin B, nystatin, itraconazole, fluconazole) were combined with purified antifungal substances in various ratios in S. cerevisiae in 96-well microplates containing YM broth (pH 5.5). (10 ⁵ cell / ml) was inoculated and incubated (28 ° C., 24 hr), and MIC for each antifungal agent alone and MIC for each mixture of antifungal and antifungal substances were measured. From these MIC values, the fractional inhibitory concentrations (FICs) for each antifungal agent were then calculated, followed by the FIC index using ΣFIC = FIC _A + FIC _B (A and B are the two antifungal agents in the well). The analysis was carried out and the MIC values of the antifungal substance were expressed as protein content.

1-7 : C. albicans Cell cycle assay

The effect of antifungal substances on the cell cycle of C. albicans was performed according to the method described below. C. albicans from the logarithmic phase grown in YM medium (28 ℃, overnight) was added to eppendorf tube to 1x10 ⁸ cell / ml, and fresh medium (1ml) containing antifungal substance (20㎍ protein) was added. Mix well. Antifungal substance treatment cells were incubated (28 ° C.) and centrifuged (2,000 rpm, 5 min, 4 ° C.) at 200 μl in tubes at 2 hour intervals. PBS (1 ml) was used to completely remove the antifungal substances. ) Was added to wash the cells, and then 70% ethanol (1 ml) stored at -20 ° C in advance was added, mixed well, and fixed. After that, the cells were left at 4 ° C. overnight, the supernatant was removed, and suspended in 50 mM Na-citrate (0.5 ml) containing RNase A (100 μg) to react (37 ° C., 2 hr). Subsequently, 50 mM Na-citrate (0.5 ml) containing propidium iodide (50 μg) was added to fluoresce the cells (4 ° C., 1 hr). The stained cells were washed twice with PBS to form cell suspensions, passed through a nylon mesh (pore size 35 μm), single cells isolated and DNA flow cytometry (Becton Dickinson, San Jose, Calif., USA). The fluorescence reaction was analyzed by ModiFit LT (Beckton Dickinson) program. The excitation was performed at 488 nm and emission at 630 nm.

< Example 2 > Antifungal  refine

A freeze-dried sample (antifungal material) was used by dialysis of the precipitate formed by adding 2 times the amount of ethanol to the culture medium of R. aqualitis .

Glycolytic hydrolysis of beta-glucosidase, cellulose, inulinase, invertase and pectinase was performed on the freeze-dried antifungal substance. Enzymes were used to remove sugars not related to antifungal activity.

Among pectinase treatments, some of these enzymes could be observed on silica gel TLC while maintaining antifungal activity. Thereafter, the experiment proceeded to the treatment with pectinase. First, antifungal sample was treated with pectinase, followed by primary gel filtration (Sephacryl S400HR). As a result, three peaks appeared (Fig. 1 (A)), but among them, there was antifungal activity at peak 1 (fraction no. 12-18). Gel filtration was carried out to obtain a purified antifungal substance (Fig. 1 (B)) . The MIC of the purified antifungal substance against S. cerevisiae was determined to be 39 to 78 µg / ml in dry weight, 2.5 to 5.2 µg / ml in protein amount and 38 to 76 µg / ml in sugar content. Was calculated. As a result of measuring the ratio of sugar and protein of the purified antifungal substance, sugar / protein = 15.2 / 1 to 14.6 / 1, which was about 15 times higher than that of protein. Purification yield of the antifungal material was about 2.1% based on the total amount of protein in the R. aquatilis AY2000 strain culture. In addition, antifungal substances were treated with proteinase K (20unit) (37 ℃, 1hr, 10mM Tris-HCl buffer; pH7.5). It showed that the more than ㎍ / ㎖, the substance directly related to the activity of the antifungal substance was confirmed that the protein. The purified antifungal substance was first subjected to PAGE without SDS, and thus had no molecular weight and moved from the front line (FIG. 2 (A)). However, SDS-PAGE showed two polypeptides of 43 kDa and 150 kDa. (Fig. 2 (B)). Therefore, the purified antifungal substance was determined to be a high molecular proteinaceous substance containing two heterologous proteins.

< Example 3 > Antifungal and Antifungal  Combined effect

The antifungal activity against S. cerevisiae was investigated in combination with four commercially available antifungal agents to examine the effects of the antifungal substance of the present invention in combination with the existing antifungal agents. In experiments with two drugs, if the FIC index is less than 0.50, the two drugs are synergistic.If greater than 0.50 and less than 4.0, the two drugs are indifferent. Drugs are antagonistic with each other. As a result of the mixed experiment, the MIC of the single drug in the combination experiment of the antifungal substance and amphotericin B of the present invention was 5.0 μg / ml and 2.5 μg / ml, respectively, but the MIC of these mixtures was the antifungal substance (2.5 μg / ml) and amphotericin B (1.25 μg / ml). Therefore, the FIC index was calculated as 1.0 and these two drugs did not show synergistic or inhibitory effects. In addition, in the combined test of the antifungal substance and nystatin of the present invention, the MIC of the single agent was 5 μg / ml for each of the two drugs, but the mixed solution thereof was the antifungal substance (5 μg / ml) and nystatin ( 0.18 μg / ml). As a result, the FIC index was calculated to be 1.036. These two drugs also had no synergistic or inhibitory effect. On the other hand, the FIC index was measured based on the MIC measured in the mixing of itraconazole and the antifungal substance of the present invention or the mixing experiment of fluconazole and the antifungal substance of the present invention, and the results were 0.394 and 0.500, respectively. Therefore, itraconazole or fluconazole were further increased antifungal activity when used in combination with antifungal substances than used alone (Fig. 5). The antifungal agents used were all 20 μg / ml (w / v) and the antifungal substance of the present invention used 40 μg / ml (protein conc.).

< Example  4> Cell cycle analysis by antifungal substance

It is not clear whether the antifungal action of the antifungal substance of the present invention is bactericidal or bacteriostatic. Therefore, the antifungal action of the antifungal substance against the growth of C. albicans and the growth period of the C. albicans The purpose of this study was to analyze the growth inhibition of fungal substances in association with the cell cycle.

Investigation of the effects of antifungal agents on the cell cycle of C. albicans revealed that the first cell cycle stage was the antifungal substance treated cells (G1; 51.63%, S; 16.12%, G2 / M; 31.76%) or untreated cells ( G1; 65.61%, S; 13.84%, G2 / M; 20.33%) were all in order of cells belonging to G1> G2 / M> S phase (Fig. 3 (A)). In non-fungal cells, G1 (67.54%) and S phase cells (17.99%) increased slightly over 2 hours, but G2 / M cells (12.73%) decreased (Figure 3 B)). After 4 hours, the number of G1 cells (36.67%) decreased and S cells (17.35%) were almost identical, but the number of cells belonging to G2 / M groups (44.54%) increased rather (Fig. 3 ( C)), After 6 hours, the distribution of cells in each cell cycle was G1 (63.24%), S (17.55%), G2 / M (17.70%) and G1> G2 / M = S It was shown that the cell cycle in progress in the order of (Fig. 3 (D)). On the other hand, in the antifungal substance treated cells, the distribution of cells belonging to each cell cycle after 2 hours was G1 (52.07%), S (15.67%), G2 / M (31.76%), and G1 phase (50.14%) after 4 hours. , S (16.93%), G2 / M (31.32%), and after 6 hours, G1 (49.28%), S (17.37%), G2 / M (31.79%) Figure 1 remained at the initial cell cycle stage with little change in cell cycle (Figures 3B-D). This phenomenon indicates that the cell cycle was stopped because the antifungal substance affected C. albicans , and the cell growth was stopped when the antifungal substance was added to the logarithmic growth stage in which C. albicans growth was progressed (Fig. 4). This is supported by this. In addition, all cells treated with antifungal substances in the course of the cell cycle of C. albicans were stopped.However, after 6 hours of treatment with antifungal substances, the number of dead cells (sub G1 phase) was changed (1.19 ~ 2.19). %) Was extremely minimal (FIG. 3 AD). Accordingly, antifungal substances were considered to have a function of inhibiting or retarding their growth rather than killing C. albicans directly.

Agricultural Biotechnology Research Institute KACC91164 20050531

Claims (9)

delete delete delete delete delete Ranella Aquatilis AY2000 strain (Accession No. KACC 91164P) was precipitated, and the precipitate was treated with Pectinase, a hydrolase, and the treated product was filtered and fractionated to prepare the Lanella Aquatilis AY2000 strain culture extract. An antifungal composition against Candida albicans containing as an active ingredient. 7. The antifungal composition for Candida albicans according to claim 6, further comprising at least one selected from itraconazole and fluconazole. A method for inhibiting growth of Candida albicans using the composition of any one of claims 6 to 7. The method of claim 8, wherein the growth inhibition method is a method of inhibiting the growth of Candida albicans, characterized in that the bacteriostatic action against Candida albicans.

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