KR101722230B1 - Composition for Preveting Biofilm Formation Containing Extract of Clove - Google Patents

Abstract

The present invention relates to a composition for inhibiting the formation of a biofilm containing an extract of cloves as an active ingredient, and more particularly to a composition for preventing Xanthomonas oryzae , Yersinia enterocolitica or Aeromonas a composition for preventing the formation of a biofilm containing an extract of cloves which inhibits the formation of a biofilm of hydrophila as an active ingredient, a composition for controlling dry leaf diseases of rice, and a composition for preventing or treating food poisoning, enteritis and the like.
The composition containing the clove extract according to the present invention contains Xanthomonas oryzae , Yersinia enterocolitica Or Aeromonas inhibit the formation of biofilm of hydrophila , and are effective in the prevention of dry leaf disease of rice and prevention and treatment of food poisoning or enteritis.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing biofilm formation,

The present invention relates to a composition for inhibiting the formation of a biofilm containing an extract of cloves as an active ingredient, and more particularly to a composition for preventing Xanthomonas oryzae , Yersinia enterocolitica or Aeromonas a composition for preventing the formation of a biofilm containing an extract of cloves which inhibits the formation of a biofilm of hydrophila as an active ingredient, a composition for controlling dry leaf diseases of rice, and a composition for preventing or treating food poisoning, enteritis and the like.

Biofilm is a membrane-like structure formed by microorganisms, and is a well-organized microbial system composed of microbial cell layers connected to the surface. Biomembranes have physicochemical gradients that affect the metabolism of microorganisms. Cells that form biofilms are more than 1000 times more resistant to antibiotics than floating cells, and biofilm formation plays an important role in the pathogenesis of many diseases. (Rasmussen and Givskov 2006).

On the other hand, enterohemorrhagic pathogenic Escherichia coli is a human pathogen that penetrates into the intestinal tract due to contamination of food or hemorrhagic colitis, that is, blood which can lead to a hemolytic uraemic reaction. Recently, it has become known that these pathogens can adhere to and survive on surfaces that are not normally suspended. Thus, biofilm produced by potential pathogens is considered to be a major cause of chronic and recurrent infections, and is believed to be due to the ability to form and sustain biofilms, especially on the surface of medical devices and in the human body. With the recent surge in biofilm research, most are aimed at prevention, control, and eradication of fundamental biofilms. Only a few studies have identified and characterized natural substances that inhibit biofilms formed by E. coli, including pathogenic E. coli strains. (5Z) -4-bromo-5- (bromomethylene) -3-butyl-2 (5H) -furanone produced from the seaweed Delisea pulchra and ursolic acid 3 produced from the ebony tree Dios pyros dendo of Gabon. Β-hydroxy-urs-12-en-28-oic acid, and a group capsule-type polysaccharide produced by the non-pathogenic Escherichia coli group significantly inhibited the formation of biofilm. However, the toxicity of these compounds and their poor water solubility limit their use in the food and pharmaceutical industries.

In addition, the biofilm produced by X. oryzae obstructs the passage of the conduit when it is infected with rice, so that the nutrients are not transferred smoothly and the water is not transmitted smoothly, so that the leaf starts to die and the rice is normally produced Which causes the blight of blight of the blight that can not be made.

Therefore, there is an urgent need to develop new natural substances which are stable without toxicity to human body, inhibit the growth of cells and exhibit drug resistance, and have excellent biofilm inhibiting effect.

Accordingly, the present inventors have made intensive efforts to develop a natural substance which is stable without toxicity on the human body, inhibits growth of cells and exhibits drug resistance without exhibiting drug resistance, and as a result, it has been found that clove extract is effective against Xanthomonas oryzae and Yersinia enterocolitica Wow Aeromonas the present inventors have confirmed that the present invention effectively inhibits the formation of biofilms of hydrophila .

An object of the present invention is to provide a method for effectively inhibiting Xanthomonas oryzae , Yersinia enterocolitica or Aeromonas a natural product extract capable of inhibiting the biofilm of hydrophila .

In order to achieve the above object, the present invention relates to a method for producing Xanthomonas oryzae , Yersinia enterocolitica And Aeromonas hydrophila . The present invention also provides a composition for preventing the formation of a biofilm of a microorganism.

The present invention also provides a composition for controlling dry leaf blight of rice, comprising clove extract as an active ingredient.

The present invention also relates to a method for the treatment of Yersinia enterocolitica selected from the group consisting of acute gastroenteritis, endophthalmitis, sepsis, tuberculous erythema, food poisoning and enteritis containing clove extracts as active ingredients Or a pharmaceutically acceptable salt thereof.

The present invention also relates to the use of clove extract as an active ingredient, Aeromonas hydrophila on And a method for preventing or treating food poisoning or enteritis.

The composition containing the clove extract according to the present invention contains Xanthomonas oryzae , Yersinia enterocolitica or Aeromonas inhibit the formation of biofilm of hydrophila , and are effective in the prevention of dry leaf disease of rice and prevention and treatment of food poisoning or enteritis.

Fig. 1 shows the results of X. oryzae The results are shown in FIG.
FIG. 2 shows the results of confirming the inhibitory effect of Y. enterocolitica on the biofilm formation by the clove extract.
Fig. 3 shows the results of confirming the inhibitory effect of A. hydrophila on the biofilm formation by the clove extract.

In one aspect, the present invention relates to a pharmaceutical composition comprising Xanthomonas oryzae, Yersinia enterocolitica and Aeromonas hydrophila . The present invention relates to a composition for preventing the formation of a biofilm of a microorganism.

In the present invention, the clove extract may be extracted with a solvent selected from the group consisting of water, alcohol, dichloromethane and ethyl acetate.

In one embodiment of the present invention, the clove extract was extracted with methanol and ethanol solvent. The solvents were divided and extracted four times, and all were mixed together. A water bath at 50 ° C was used for the extraction. After the extraction, the solid content was removed by using filter paper. The methanol extract and the ethanol extract were concentrated and pulverized using a rotary condenser.

In another embodiment of the present invention, the solvent fraction extract of the clove extract was first extracted with methanol and then divided into four portions using methanol, and then the whole was collected and mixed. A water bath at 50 ° C was used for the extraction. After extraction, the solid content was removed by using filter paper. The extract was concentrated and pulverized using a rotary evaporator. First, the powdered methanol extract was dissolved in water, and dichloromethane was added thereto. The mixture was allowed to stand for 1 hour and then fractionated. The process of adding dichloromethane to the fractionated water layer and allowing it to stand for 1 hour was repeated two more times. The collected fractions were filtered through a filter paper and then concentrated to a powder. The separated water layer was used to remove the remaining dichloromethane using a concentrator. This water layer was fractionated three times in the same manner using ethyl acetate. Thereafter, the remaining water layer was finally fractionated three times using butanol. All fractions, including the last remaining water layer, were concentrated and powdered. The four powders obtained by the fractionation were dissolved in 3 ml of water, and the supernatant, excluding the precipitate, was taken and used in the experiment by filtration using a filtration membrane having a pore size of 0.45 μm.

In another aspect, the clove extract water extract and a solvent extract fraction of the present invention at low concentrations, Xanthomonas oryzae , Yersinia enterocolitica And Effectively inhibited the biofilm formation of Aeromonas hydrophila .

In another aspect, the present invention relates to a composition for controlling dry leaf blight of rice, comprising an extract of cloves as an active ingredient.

Xanthomonas Rice blight caused by oryzae is a disease of rice causing the greatest economic loss in the production of rice. One of the mechanisms causing diseases is X. oryzae adsorbed on the conduit of rice to form a biofilm, blocking the passage of the conduit. As a result, nutrients are not transferred smoothly and water does not flow smoothly. Rice can not be produced normally. Therefore, inhibiting the plugging of a conduit to the paddy rice plants even if the X. oryzae infection to the normal growth if suppressing the formation of biofilm in the X. oryzae, while maintaining the yield of rice normally.

In one embodiment of the present invention, it has been confirmed that when methanol extract of clove is treated in a culture solution of X. oryzae , the formation of biofilm can be effectively inhibited.

In yet another aspect, the invention Yersinia selected from the group consisting of acute gastroenteritis, end ileitis, sepsis, tuberculosis rash, food poisoning and enteritis containing a clove extract as an active ingredient enterocolitica on To a composition for preventing or treating a disease caused by a disease.

Y. enterocolitica is a Gram-negative bacterium belonging to the intestinal bacteria and causing gastroenteritis (food poisoning, enteritis). It is known that it causes various diseases such as acute gastroenteritis, endocarditis, sepsis, tuberculous erythema and the like in humans. The bacterium grows over a very wide range of temperatures, and biofilms form resistance to external stimuli or antibiotic treatment, making it difficult to manage.

In one embodiment of the present invention, it has been confirmed that when the methanol extract of Clove and the ethanol extract are treated in a culture medium of Y. enterocolitica, the formation of biofilm can be effectively inhibited.

In another aspect, the present invention relates to a pharmaceutical composition comprising Aeromonas on hydrophila To a composition for preventing or treating food poisoning and enteritis.

A. hydrophila is a tuberous anaerobic Gram-negative bacterium that is distributed in fresh water such as rivers, rivers, ponds, etc. and is a bacterium that develops diseases by fixing in cold blooded and warm blooded animals. Because this bacterium is explosively increased in summer by drinking water, food, and meat products, infection rates between late spring and early autumn are high and all age groups can be infected. However, it usually occurs after 2 years of age. There are many cases.

In one embodiment of the present invention, it has been confirmed that when the methanol extract of clove and the dichloromethane solvent fraction extract are treated in the culture of A. hydrophila , the formation of biofilm can be effectively inhibited.

The carrier used in the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier, adjuvant and vehicle and is collectively referred to as a " pharmaceutically acceptable carrier ". Pharmaceutically acceptable carriers that can be used in the medicinal compositions of the present invention include, but are not limited to, ion exchange, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances Water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal silicas such as glycerin, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, , Magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substrate, polyethylene glycol, sodium carboxymethyl cellulose, polyarylate, wax, polyethylene-polyoxypropylene-barrier polymer, polyethylene glycol and wool.

The route of administration of the pharmaceutical composition according to the present invention may be, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, Sublingual or rectal.

Oral and parenteral administration is preferred. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

The pharmaceutical composition may be in the form of a sterile injectable preparation as a sterile injectable aqueous or oleaginous suspension. The suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (e. G., Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent (e.g., a solution in 1,3-butanediol). Vehicles and solvents that may be used tolerably include mannitol, water, a Ringgel solution and an isotonic sodium chloride solution. In addition, sterile, nonvolatile oils are conventionally employed as a solvent or suspending medium. For this purpose, any non-volatile oil with low irritation, including synthetic mono- or diglycerides, may be used. Fatty acids such as oleic acid and glyceride derivatives thereof are useful in injection formulations as well as pharmaceutically acceptable natural oils (e.g., olive oil or castor oil), especially those polyoxyethylated.

The pharmaceutical compositions of the present invention may be orally administered in any dosage form orally acceptable, including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of oral tablets, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When the aqueous suspension is orally administered, the active ingredient is combined with an emulsifying agent and a suspending agent. If desired, sweetening and / or flavoring agents and / or coloring agents may be added.

The pharmaceutical compositions of the present invention may also be administered in the form of suppositories for rectal administration. These compositions may be prepared by mixing the compounds of the invention with suitable non-polar excipients which are solid at room temperature but liquid at rectal temperature. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.

Oral administration of a pharmaceutical composition according to the present invention is particularly useful when the desired treatment is associated with a site or organ that is accessible by topical application. When topically applied to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active ingredient suspended or dissolved in the carrier. Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid paraffin, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax and water. Alternatively, the pharmaceutical composition may be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of the present invention may also be topically applied by rectal suppositories to the lower intestinal tract with a suitable enema. Topically applied transdermal patches are also included in the present invention.

The pharmaceutical composition of the present invention can be administered by inhalation aerosol or by inhalation. Such compositions may be prepared according to techniques well known in the art of pharmacy and may be prepared using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art, Solution.

 The specific effective amount for a particular patient will depend on a variety of factors including the activity of the particular compound employed, age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease It will be appreciated that it can vary. The pharmaceutical composition according to the present invention can be formulated into pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.

When the medicinal composition according to the present invention is injected into subcutaneous cells of a fish, it can be administered to a dacryocyst or digestive tract. Injection can be injected into muscle cells or other cells within the muscle tissue and injected into the intestinal cells in the abdominal cavity.

In a preferred embodiment, the pharmaceutical compositions for oral administration can be prepared by mixing the active ingredients together with solid excipients and may be prepared in the form of granules for preparation in the form of tablets or dragees. Suitable excipients include sugars such as lactose, sucrose, mannitol and sorbitol or starches from corn, wheat flour, rice, potato or other plants, cellulose such as methylcellulose, hydrocyclopropylmethylcellulose or sodium carboxymethylcellulose , Arabic gum, carbohydrates such as gum including Tagakans gum, or protein fillers such as gelatin and collagen. If necessary, disintegrating or solubilizing agents in the form of respective salts such as cross-linked polyvinylpyrrolidone, agar and alginic acid or sodium alginic acid may be added.

In a preferred embodiment, for parenteral administration, the pharmaceutical composition of the present invention can be prepared as a water-soluble solution. Preferably, physically suitable buffer solutions such as Hank's solution, Ringer's solution or physically buffered saline can be used. Water-soluble injection suspensions may contain a substrate capable of increasing the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. In addition, suspensions of the active ingredient may be prepared in suitable oily injection suspensions. Suitable lipophilic solvents or carriers include fatty acids such as sesame oil or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Polycationic amino polymers can also be used as carriers. Optionally, the suspension may employ a suitable stabilizing agent or agent to increase the solubility of the compound and to produce a high concentration of solution.

 The formulation of the present invention can be prepared by adding a stabilizer, a buffer or an anti-adsorption agent as described above. In particular, when used as an injection for medical use or an animal medicine, an osmotic pressure ratio of 1 to 2 is acceptable as an osmotic pressure ratio. The osmotic pressure ratio can be adjusted by increasing or decreasing sodium chloride during the chemical reduction.

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 illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example  One: cloves  Preparation of extract

Cling (origin: China) was purchased from Kyungdong market and crushed to a size of 1 mm or less. The ground cloves were extracted with methanol and ethanol solvent. The clove used was 200 g per each solvent. At the time of extraction, 2,300 ml of the solvent was divided into four portions in 800 ml, 500 ml, 500 ml and 500 ml, and they were mixed together. A water bath at 50 ° C was used for the extraction. After extraction, the solid content was removed by using filter paper.

The methanol extracts were concentrated and powdered using a rotary evaporator. The amount of the powdered extract was 59.02 g. The powdered methanol extract (0.54 g) was dissolved in 10 ml of ethanol and dissolved. The supernatant, excluding the insoluble matter, was taken and filtered using a filtration membrane having a pore size of 0.45 μm. The solids content in the solution was 54.01 g / l. The degree of inhibition of biofilm formation was tested using the solid content.

The ethanol extracts were concentrated and powdered using a rotary evaporator. The amount of the powdered extract was 58.23 g. 0.47 g of powdered ethanol extract was dissolved in 10 ml of ethanol and dissolved. Then, the supernatant liquid excluding the insoluble matter was taken and filtered through a filtration membrane having a pore size of 0.45 μm. The solids content in the solution was 25.40 g / l. The degree of inhibition of biofilm formation was tested using the solid content.

Example  2: After methanol extraction, by solvent fraction Clove extract  Produce

Clays were purchased from Kyungdong market and crushed to a size of 1 mm or less. The ground cloves were extracted with methanol. The clove used was 200 g. When extracting, 2,300 ml of methanol was divided into four portions of 800 ml, 500 ml, 500 ml and 500 ml, and they were mixed together. A water bath at 50 ° C was used for the extraction. After extraction, the solid content was removed by using filter paper. The extract was concentrated and pulverized using a rotary evaporator. At this time, 59.02 g of powder was obtained, and 35.04 g of the powder was used in the experiment.

First, the powdered methanol extract was dissolved in 150 ml of water, and 150 ml of dichloromethane was added thereto. The mixture was allowed to stand for 1 hour and then fractionated. 150 ml of dichloromethane was again added to the separated water layer and the mixture was allowed to stand for one hour. This procedure was repeated two more times (using a total of 450 ml of dichloromethane). Collected dichloromethane was filtered through filter paper and concentrated to powder. The separated water layer was used to remove the remaining dichloromethane using a concentrator. This water layer was fractionated three times with ethyl acetate (150 ml) in the same manner. The remaining water layer was then fractionated three times with 150 ml of butanol in the same manner. All fractions, including the last remaining water layer, were concentrated and powdered. The obtained powder was 23.09 g in the dichloromethane layer, 4.69 g in the ethyl ether layer, 2.82 g in the butanol layer and 2.72 g in the water layer. The four powders obtained by the fractionation were dissolved in 3 ml of water, and the supernatant, excluding the precipitate, was taken and used in the experiment by filtration using a filtration membrane having a pore size of 0.45 μm. The solid content was 27.53 g / l in the dichloromethane layer, 1.80 g / l in the ethyl ether layer, 21.19 g / l in the butanol layer and 21.07 g / l in the water layer.

Example 3: Effect of clove extract Xanthomonas Analysis of inhibition effect of oryzae on biofilm formation

X. oryzae (KACC 10331) was distributed at the Agricultural Genetic Resources Information Center of RDA.

The X. oryzae strain stored at -80 ° C was plated in a solid culture medium, YGC medium, and cultured at 28 ° C for 2 days, and then cultured as an active growth strain. Table 1 shows the composition of the medium of the YGC medium, which is a solid medium using agar.

Composition of YGC medium (100 ml) ingredient content Glucose 5 g Yeast extracts 0.5 g CaCO ₃ 1.25 g Agar 1.5 g Water Fit total volume to 100ml

X. oryzae cultured in YGC medium The strain was inoculated into 5 ml of 210 medium, which was a liquid medium, and then cultured at 28 DEG C and 250 rpm for 2 days. The composition of the 210 medium used for the cultivation is shown in Table 2.

210 Medium composition ingredient content Sucrose 0.5 g Casein hydrolysates 0.8 g Yeast extract 0.4 g K ₂ HPO ₄ 0.3 g MgSO ₄ .7H ₂ O 30 mg Water Fit total volume to 100ml

X. oryzae cultured on 210 medium XOM2 medium, a biofilm formation medium, was used in order to confirm that the clove extract inhibited the formation of the biofilm of the strain.

The composition of the XOM2 medium is shown in Table 3.

The pH of the XOM2 medium (pH 6.5) ingredient content L-methionine 670 μM Na-glutamate · H ₂ O 10 mM KH ₂ PO ₄ 14.7 mM MnSO ₄ 40 μM Fe (III) -EDTA 240 μM MgCl ₂ 5mM D-xylose 0.18%

100 [mu] l of XOM2 medium was dispensed into 96-well PVC microplates, and 5 [mu] l of the clove extract (water extract and solvent fraction extract) prepared in Example 1 and Example 3 were further added. To each of the divided wells was added X. oryzae The strain was inoculated to a cell concentration Abs600 = 0.05 and cultured at 28 DEG C for 24 hours.

After removing the culture medium, the cells were washed three times with water, and 100 μl of 1% crystal violet solution was added to each well, and allowed to stand at room temperature for 15 minutes for reaction. The reaction solution was removed, washed gently three times, 100 .mu.l of 95% ethanol was placed in each well, allowed to stand for 15 minutes, and the degree of formation of the biofilm was measured with Abs595 using a microplate reader.

As a result, as shown in Figure 1, the solvent fraction of the clove extract at low concentration X. oryzae It was confirmed that the formation of the biofilm of the strain was inhibited.

Example 4: Preparation of clove extract Yersinia Analysis of inhibition effect of enterocolitica on biofilm formation

The Y. enterocolitica (KCCM 41657) was used at the Korea Microorganism Conservation Center.

The Y. enterocolitica The strain was plated on LB solid medium and cultured at 26 ° C for 2 days to make the strain active. The cultured strains were inoculated in 5 ml of TYE liquid medium and cultured in a test tube at 26 ° C and 250 rpm for 1 day.

The composition of the TYE medium is shown in Table 4.

Composition of TYE medium (100 ml) ingredient content Tryptone 1 g Yeast extracts 0.5 g Water Fit total volume to 100ml

Y cultured in TYE medium . enterocolitica In order to confirm that the clove extract inhibited the formation of the biofilm of the strain, M63 medium for biofilm formation was used.

The composition of the M63 medium is shown in Table 5.

Composition of M63 medium ingredient content 5x M63 medium (available destination base) 80 ml 20% Glucose 1 ml 20% MgSO ₄ 0.1 ml Water Fit total volume to 100ml

100 μl of M63 medium was dispensed into a 96-well PVC microplate, and the clove extracts (water extract and solvent fraction extract) prepared in Example 1 and Example 3 were further divided into 5 μl portions. The Y. enterocolitica strain cultured in TYE medium was inoculated into each of the divided wells at a cell concentration Abs600 = 0.05 and cultured at 26 DEG C for 24 hours.

After removing the culture medium, the cells were washed three times with water, and 100 μl of 1% crystal violet solution was added to each well, and allowed to stand at room temperature for 15 minutes for reaction. The reaction solution was removed, washed gently three times, 100 .mu.l of 95% ethanol was placed in each well, allowed to stand for 15 minutes, and the degree of formation of the biofilm was measured with Abs595 using a microplate reader.

As a result, as shown in Fig. 2, in the methanol extract and ethanol extract of clove, all of Y. enterocolitica It was confirmed that the formation of the biofilm of the strain was inhibited.

Example 5: Effect of clove extract Aeromonas Analysis of inhibition effect of hydrophila on biofilm formation

In the present embodiment, Aeromonas hydrophila Was used by LG Life Science Research Institute.

A. hydrophila stored at -80 ° C The strain was plated on LB solid medium and incubated at 30 ° C for 1 day to make the strain vigorous. The cultured strains were inoculated into 5 ml of LB liquid medium and then incubated in a test tube at 30 ° C for 1 day.

A cultured in LB medium. hydrophila TSBY medium, which is a biofilm formation medium, was used to confirm that the clove extract inhibited the formation of the biofilm of the strain.

The composition of the TSBY medium is shown in Table 9.

Composition of TSBY medium ingredient content Tryptic Soy Broth 3 g Yeast extract 1.6 g Water Fit total volume to 100ml

100 μl of TSBY medium was dispensed into a 96-well PVC microplate, and 5 μl of the clove extract (water extract and solvent fraction extract) prepared in Example 1 and Example 3 was further added. To each of the divided wells was added A. hydrophila The strain was inoculated at a cell concentration Abs600 = 0.05 and cultured at 30 DEG C for 24 hours.

After removing the culture medium, the cells were washed three times with water, and 100 μl of 1% crystal violet solution was added to each well, and allowed to stand at room temperature for 15 minutes for reaction. The reaction solution was removed, washed gently three times, 100 .mu.l of 95% ethanol was placed in each well, allowed to stand for 15 minutes, and the degree of formation of the biofilm was measured with Abs595 using a microplate reader.

As a result, as shown in Fig. 3, it was confirmed that the formation of the biofilm of the A. hydrophila strain was inhibited in both the methanol extract of clove and the dichloromethane fraction extract.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (4)

A composition for preventing the formation of biofilm of Aeromonas hydrophila containing methanol extract of cloves of 0.033 g / L to 0.067 g / L.

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