KR101818859B1 - Pseudomonas azotoformans strain KACC 92125P and composition for comprising the same - Google Patents

Abstract

The Pseudomonas azo topomancer strain KACC 92125P of the present invention exhibits antibacterial activity against Staphylococcus aureus and Aspergillus flavus , which are food-harmful microorganisms, and has an antibacterial activity against environmental microorganisms such as Staphylococcus aureus and Aspergillus flavus , The composition containing the strain can be used as a biological agent and widely applied to foods, agriculture, environmental industries, and the like.

Description

Pseudomonas azo topomancer strain KACC 92125P and a composition comprising it Pseudomonas azotoformans strain KACC 92125P and composition for comprising the same,

The present invention relates to a novel Pseudomonas azotopneumans strain KACC 92125P exhibiting an antimicrobial activity against Staphylococcus aureus and Aspergillus flavus , a composition comprising the same, and a pathogenic microorganism using the same Lt; / RTI >

Staphylococcus aureus is a gram-positive anaerobic bacterium that is present on the skin and nasal surface of healthy people or livestock and can cause inflammation in the skin, respiratory tract, sinus, etc. of humans and animals. If the food is contaminated by the bacteria, the bacteria may proliferate in the food to produce enterotoxin, which may cause food poisoning if the food containing the food is ingested. In addition, this bacterium secretes toxins (lucocidin), hemolysin, coagulase, etc. which kill phagocytes, causing the infectious disease to escape from the resistance of infected host cells. In addition, it also proliferates in high salt concentration, and it is resistant especially in dry state, so it can survive for a long time (several months) in food or scurf to induce food poisoning.

Staphylococcal food poisoning is one of the most commonly reported food poisoning in the world and has been associated with the ingestion of foods containing staphylococcal enterotoxins, mainly produced by Staphylococcus aureus (Non-Patent Document 1). According to the Statistical Information of the Food and Drug Administration, 46 cases of food poisoning accidents occurred in Korea due to Staphylococcus aureus for the last 5 years, and the total number of patients was 807. The major symptoms of food poisoning caused by Staphylococcus aureus are nausea, vomiting, and cramps.

Aspergillus flavus , on the other hand, is a fungal toxin and can contaminate food by producing aflatoxin, known as a carcinogen. The aflatoxins produced by Aspergillus flavus have been shown to be less resistant to infection by inhibiting immune function when exposed to humans and animals, causing growth retardation, mortality, reduced feed efficiency and increased infection rates by disease (Non-Patent Document 2). Animals vary in susceptibility to toxicity by species, and there are significant differences in the same species depending on their age, sex, weight, diet, exposure to infectious agents, and other fungal toxins or drugs. In particular, mycotoxins are a serious source of damage to farms that raise livestock.

Staphylococcus aureus, which is resistant to stresses such as the drying environment, is often found in food manufacturing facilities and on environmental surfaces, which cause contamination or recontamination of food, and cross-contamination Lt; / RTI > Therefore, effective sterilization methods must be developed to prevent cross-contamination of harmful microorganisms in various environments.

Chemical fungicides such as hypochlorite, chlorine dioxide, ethanol, and hydrogen peroxide have been developed to inhibit harmful microbes contaminated on the environment surface, and the sterilizing power of these fungicides Although verified, these chemical disinfectants can cause side effects such as skin irritation and accumulation of toxic substances in workers. Therefore, there is a need to develop a method that can inhibit the use of a natural product formulation on a food contact surface rather than a chemical composition.

On the other hand, biofilm is a binding layer of polysaccharide (EPS) secreted by microorganisms and microorganisms attached to biological or nonbiological surfaces. When microorganisms form a biofilm on the food contact surface, they are highly resistant to environmental stresses such as drying. If biofilms are formed on various environmental surfaces using antimicrobial antimicrobials against Staphylococcus aureus, the surface may exhibit a persistent antimicrobial activity against Staphylococcus aureus.

To date, there have been few new microorganisms that can inhibit foodborne pathogens contaminated on the surface of the environment. In particular, there have been few studies to improve the resistance of environmental microorganisms to environmental stress by inducing biofilm formation. Therefore, the discovery of new microorganisms capable of inhibiting microorganisms from various environmental surfaces must be continuously carried out, and methods for applying the newly discovered useful microorganisms should also be developed.

Seo, S., Bohac, G. A., 2013. Staphylococcus aureus. In: Doyle, M. P., Buchanan, R. L. (Eds.), Food Microbiology: Fundamentals and Frontiers. ASM Press, Washington D.C., pp. 547-573. Nam, K., H., Korean Journal of Poultry Science (1994) 21 (2), 113-117

It is an object of the present invention to provide a novel strain Pseudomonas azotopneumans strain KACC 92125P which shows antimicrobial activity against Staphylococcus aureus and Aspergillus flavus , a composition comprising the same, And to provide a method for inhibiting the activity of pathogenic microorganisms.

Accordingly, the present inventors have isolated about 2,100 microorganisms from the soil, food, and food contact surfaces, and have obtained excellent antimicrobial activity against Staphylococcus aureus and Aspergillus flavus among the microorganisms As a result, it was found that Pseudomonas azo topomancer strain KACC 92125P, a novel Pseudomonas sp. Strain, was isolated and identified to complete the present invention.

The Pseudomonas azotoformans strain KACC 92125P according to the present invention is a novel strain of Pseudomonas azo topomans derived from food. Although the Pseudomonas aeruginosa strains of the present invention are isolated and identified from foods, the route of intake is not limited thereto.

As a result of 16S rRNA base sequence analysis for identification and classification of microorganisms showing excellent antimicrobial effect on Staphylococcus aureus and Aspergillus flavus isolated through the examples of the present invention, Respectively. As a result of the analysis, the strain showed the highest molecular phylogenetic relationship with Pseudomonas azotoformans strain NBRC 12693, as can be seen from the tree of FIG.

Therefore, the microorganism of the present invention having the 16S rRNA nucleotide sequence shown in Fig. 3 can be used as Pseudomonas sp. azotoformans lettuce, and deposited with the National Academy of Sciences on Mar. 30, 2016 (Accession No. KACC 92125P). In the present invention, the above-mentioned Pseudomonas aeruginosa azotoformans lettuce) is only Pseudomonas azo Topo's KACC 92125P strain (Pseudomonas azotoformans strain KACC 92125P).

The Pseudomonas aeruginosa strain KACC 92125P of the present invention exhibits antimicrobial activity against Staphylococcus aureus and Aspergillus flavus at the same time, so that the composition comprising the strain of the present invention It can be used as a biological agent and widely applied to food, agriculture, and environmental industries.

In the following examples, it was confirmed that the Pseudomonas azotopneumans strain KACC 92125P of the present invention exhibited antibacterial activity simultaneously against Staphylococcus aureus and Aspergillus flavus. In addition, the suspension of Pseudomonas azotopneumans strain KACC 92125P was spray-dried on stainless steel, and the number of the strains was measured on the cultured surface.

Therefore, the Pseudomonas azo topomancer strain KACC 92125P according to the present invention is an antimicrobial substance against Staphylococcus aureus and Aspergillus flavus, and can be variously used for the inhibition of the activity of pathogenic microorganisms in livestock, .

Thus, in one embodiment of the present invention, there is provided a composition comprising Pseudomonas azotopneumans strain KACC 92125P, a culture thereof, an extract thereof or a suspension thereof.

The Pseudomonas azotopneumans strain KACC 92125P contained in the composition according to the present invention may exist as a living cell or dead cell, and may also exist in a form of dried or lyophilized. The forms and formulations of the strains suitable for inclusion in the various compositions are well known to those skilled in the art. But are not limited to, for example, in the form of an extract, a culture, a concentrate, a suspension, and the like.

In one embodiment of the present invention, there is provided an antimicrobial composition comprising Pseudomonas azotopneumans strain KACC 92125P, a culture thereof, an extract thereof or a suspension thereof.

The antimicrobial composition according to the present invention exhibits antibacterial activity against Staphylococcus aureus and Aspergillus flavus .

Staphylococcus aureus can cause mastitis in cows and chlorines, can cause edematous dermatitis and arthritis in chickens, and can cause infectious diseases such as pneumonia, diabetes, osteomyelitis, endocarditis, and bacteremia sepsis. In addition, aspergillus flavasus can cause ear infections in corn, and in particular, aspergillus flavas can form a fungal toxin called aflatoxin in cereal grains, , It may cause symptoms or diseases such as edema, indigestion, liver disease, cancer, abortion, malformations, pancreatic ulcers, and facial nerve paralysis. In addition, Staphylococcus aureus and Aspergillus flavus are known as typical food poisoning bacteria, and can cause food poisoning and enteritis in livestock.

Accordingly, the composition can be used for prevention, treatment, and improvement of diseases caused by infection with pathogenic microorganisms of animals or plants. Preferably, the animal is a livestock such as cattle, horses, , Rye, rice and the like.

The present invention also provides a feed or feed additive composition comprising Pseudomonas azo topomancer strain KACC 92125P, a culture thereof, an extract thereof or a suspension thereof.

When used as feedstuffs, the compositions may comprise any protein-containing organic kernel meal commonly used to meet dietary needs of the animal. These protein-containing flours usually consist mainly of corn, soy flour, or corn / soy flour mix.

When used as a feed additive, the composition may be 20 to 90% high concentrate or may be prepared in powder or granular form. The feed additive may be selected from the group consisting of organic acids such as citric acid, fumaric acid, adipic acid, lactic acid and malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polymerized phosphate), polyphenol, catechin, alpha-tocopherol, Extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid, and the like. When used as a feed, the composition may be formulated in conventional feed form and may contain conventional feed ingredients.

The feed additives and feeds may be selected from the group consisting of cereals, such as ground or crushed wheat, oats, barley, corn and rice; Feeds based on vegetable protein such as rapeseed, soybeans and sunflower; Animal protein feeds such as blood, meat, bone meal and fish meal; A sugar or a milk product, for example, a dry component comprising various powdered milk and whey powder, and may further include nutritional supplements, digestion and absorption enhancers, growth promoters, and the like.

The feed additive may be administered to animals singly or in combination with other feed additives in edible carriers. The feed additives can also be administered to the animal either as a top dressing or they can be mixed directly with the animal feed or in a separate oral form separate from the feed. When the feed additive is administered separately from an animal feed, it can be prepared in an immediate release or sustained release formulation, in combination with a pharmaceutically acceptable edible carrier as is well known in the art. Such edible carriers may be solid or liquid, such as corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the feed additive can be a tablet, capsule, powder, troche or emulsion or top-dressing in finely divided form. When a liquid carrier is used, the feed additive can be a gelatin soft capsule, or a syrup or suspension, emulsion, or solution formulation.

The feed and feed additives may also contain adjuvants such as preservatives, stabilizers, wetting or emulsifying agents, solution promoters, and the like. The feed additive may be used by adding to the animal's feed by pouring, spraying or mixing.

The feed or feed additive of the present invention is applicable to a large number of animal diets including mammals and poultry.

The above mammals can be used for pets (eg, dogs, cats) as well as pigs, cows, sheep, goats, laboratory animals and laboratory animals as well as poultry such as chickens, turkeys, ducks, Quail, and the like, but the present invention is not limited thereto.

In addition, the present invention provides a biological control agent comprising Pseudomonas azo topomancer strain KACC 92125P, a culture thereof, an extract thereof or a suspension thereof.

In the present invention, Pseudomonas azo topomancer strain KACC 92125P used in the above-mentioned controlling agent can be used as it is without adding any other ingredients. If necessary, it may be prepared by adding a composition prepared by combining with various carriers such as a solid carrier or a liquid carrier or a preparation auxiliary agent such as an additive to prepare a microorganism or a microorganism in the form of a wettable powder, a solution, a suspension, a granule, The formulations obtained by formulating them into formulations can be used. The formulation may comprise about 0.1 to 99% by weight of Pseudomonas azotopneumans strain KACC 92125P, and may include about 10 ³ to 10 ¹⁰ CFU of the strain per g of the formulation, although not limited thereto.

Examples of the solid carrier used in the formulation include mineral powders (e.g., kaolin clay, bentonite, diatomaceous earth, synthetic silicon oxide, talc, quartz, vermiculite, pearlite and the like), inorganic salts (e.g., ammonium sulfate, ammonium phosphate, (Wheat flour, soybean powder, wheat bran, chitin, rice bran, skim milk powder and whole milk powder), activated carbon and calcium carbonate, and the like. Examples of the liquid carrier include water, glycerol, vegetable oils such as soybean oil and rapeseed oil, liquid animal oils such as fish oil, ethylene glycol, poly (ethylene glycol), propylene glycol and poly (propylene glycol) .

Examples of the adjuvant for the preparation include additives such as casein, gelatin, polysaccharides such as starch powder, gum arabic, cellulose derivatives and alginic acid, lignin derivatives, bentonites, sugars, vegetable oils, synthetic oils, synthetic water- ), Poly (acrylic acid), etc.), propylene glycol, ethylene glycol and the like; Defoaming agents such as silicone compounds; Thickeners such as natural polysaccharides (e.g., xanthan gum), inorganic substances (e.g., aluminum and bentonite), and synthetic water-soluble polymers (e.g., poly (acrylic acid) and the like).

In the present invention, the controlling agent may be applied as an antibacterial agent, a house disinfectant, a negative additive, or the like.

In addition, the controlling agent according to the present invention can be used in combination with or without mixing with an insecticide, a nematicide, a fungicide, a fungicide, a bactericide, a herbicide, a plant growth regulator, an electrodeposit agent, a fertilizer, a microbial material, It can also be used.

The present invention also provides a method for inhibiting the activity of a pathogenic microorganism, comprising the step of treating the biological control agent to a livestock, a house or the vicinity thereof.

The method for inhibiting the activity of a pathogenic microorganism according to the present invention may include, but is not limited to, a step of directly treating a livestock, a house or the vicinity thereof, or a step of forming a biofilm. For example, there is a method for inhibiting the activity of a pathogenic microorganism capable of infecting livestock by adding the biological control agent to a feed and feeding it to a livestock, or to a farm or a vicinity thereof, And a method of imparting antimicrobial properties to pathogens by inducing biofilm formation or the like.

In the present invention, the pathogenic microorganism may be Staphylococcus aureus and Aspergillus flavus .

As described above, by inhibiting the activity of the pathogenic microorganisms, it is possible to prevent the pathogenic microorganisms from causing diseases such as food poisoning, enteritis, mastitis, edematous dermatitis, arthritis, pneumonia, diabetes, osteomyelitis, endocarditis, Bacteremia, septicemia, and infectious diseases caused by pathogenic microorganisms such as hard ulcers and liver cancer caused by Aspergillus flavus can be prevented.

The present invention provides an anti-icarus agent for Aspergillus flavus , comprising Pseudomonas azo topomancer strain KACC 92125P, a culture thereof, an extract thereof or a suspension thereof.

The present invention also provides a method of inhibiting the activity of Aspergillus flavus comprising the step of treating the plant with the controlling agent.

The method for inhibiting the activity of Aspergillus flavus according to the present invention can inhibit the activity of Aspergillus flavus , which is capable of infecting plants by spraying the plant with the above-mentioned controlling agent.

As described above, by inhibiting the activity of Aspergillus flavus, it is possible to prevent ear blight in cereals such as wheat, barley, oats, rye and rice. It can also prevent the fungal toxins of aflatoxin formed by Aspergillus flavus. The aflatoxin can cause facial nerve paralysis, palpable ulcer, liver cancer, and the like.

The present invention provides a pharmaceutical composition for preventing or treating infectious diseases caused by a pathogenic microorganism comprising Pseudomonas aeruginosa topomancer strain KACC 92125P, a culture thereof, an extract thereof or a suspension thereof.

In the present invention, the pathogenic microorganisms include Staphylococcus aureus and Aspergillus flavus .

The composition according to the present invention is useful for the treatment of infectious diseases such as food poisoning, enteritis, mastitis, edema dermatitis, arthritis, pneumonia, diabetes, osteomyelitis, endocarditis, bacteremia and sepsis caused by Staphylococcus aureus in animals such as poultry and livestock Infection or toxic diseases caused by Aspergillus flavus caused by pathogenic microorganisms such as acute ulcer, liver cancer, or mycotoxins can be prevented or treated.

When the composition of the present invention is used as a pharmaceutical composition, the pharmaceutical composition of the present invention may be prepared by using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned active ingredients. Examples of the adjuvants include excipients, Release agents, sweeteners, binders, coating agents, swelling agents, lubricants, lubricants or flavors.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.

For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, natural and synthetic gums such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, Sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

Further, it can be suitably formulated according to each disease or ingredient by using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

The present invention also provides a method for treating an antimicrobial surface of a Pseudomonas aeruginosa strain KACC 92125P, a culture thereof, an extract thereof, or a suspension thereof, on a surface thereof.

 In the present invention, the step of treating the surface may include spraying a suspension of the strain, the culture of the strain, the extract of the strain, or the strain on the surface and drying the surface. Further, after the spray-drying, the step of culturing in an incubator for 3 to 120 hours, preferably 3 to 72 hours, more preferably 6 to 48 hours in a temperature range of 20 to 30 DEG C is further carried out .

In the present invention, the surface may be stainless steel, glass, wood, vinyl, plastic, rubber or tile, but the surface is not limited thereto.

In the following examples, the suspension of Pseudomonas azotopneumans strain KACC 92125P was sprayed onto a stainless steel coupon, dried and then cultured at a temperature of 25 캜 for 120 hours. As a result, the number of the strains was about 100 Fold and the number of the population was maintained after that, it was confirmed that the strain could form a biofilm in a stainless steel coupon and exhibit antibacterial activity.

Therefore, the strain according to the present invention can exhibit antibacterial activity by forming a biofilm on the surface of stainless steel, glass, wood, vinyl, plastic, rubber, Can exhibit excellent antimicrobial activity against Staphylococcus aureus and Aspergillus flavus .

The Pseudomonas azo topomancer strain KACC 92125P of the present invention exhibits antimicrobial activity against Staphylococcus aureus and Aspergillus flavus , which are food-harmful microorganisms, and exhibits antimicrobial activity against Pseudomonas azotopoulum Since the strain KACC 92125P is well-viable, the composition containing the strain can be used as a biological agent and widely applicable to foods, agriculture, and the environment industry.

FIG. 1 shows the antimicrobial activity against the strain Staphylococcus aureus according to the present invention through the formation of an inhibitory ring.
Figure 2 shows the results of the antimicrobial activity of the strain according to the present invention against Aspergillus flavus through the formation of inhibitory rings.
Fig. 3 shows the nucleotide sequence of SEQ ID NO: 1 of Pseudomonas azo topomancer strain KACC 92125P of the present invention.
4 is a tree showing the molecular phylogenetic relationship of the Pseudomonas azo topomancer strain KACC 92125P of the present invention.
FIG. 5 is a graph showing a biofilm growth curve of the Pseudomonas aeruginosa topomancer strain KACC 92125P of the present invention.
FIG. 6 shows the result of SEM observation of the morphology of the biofilm formed by Pseudomonas aeruginosa strain KACC 92125P of the present invention.
FIG. 7 shows the results of confirming the resistance of the biofilm formed from Pseudomonas aeruginosa strain KACC 92125P of the present invention to the stress of the drying environment.
FIG. 8 shows the results of confirming the antimicrobial activity against Staphylococcus aureus of the biofilm formed from the Pseudomonas aeruginosa strains KACC 92125P of the present invention.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

[ Example  1] from soil, food, food contact surface Staphylococcus Aureus ( Staphylococcus aureus ) For the detection of antagonistic microorganisms

Soil samples were collected by collecting soil from various mountains, lakes and dams in Korea. The soil dug a depth of about 10 cm and collected the soil inside. 1 g of this soil was suspended in 10 ml of sterilized water and incubated with shaking at 25 캜 for 1 to 3 hours at 200 rpm. After incubation, if the soil sinks in the culture solution, 0.1 ml of the supernatant is taken on humic acid vitamin agar and cultured at 28 ° C for 3 to 14 days. The grown microorganism colonies are taken in an inoculation loop and placed in 20% glycerol, And stored frozen at 80 ° C.

A single colony of the stored soil microorganism was plated on Bennet 's agar using platinum at 25 ℃ for 6 days. At this time, after culturing for 3 days, it was confirmed that the strain was well grown, and the strain that did not grow well was stained with a sterilized cotton swab and cultured.

After 6 days, if colonies grew, about 1 to 5 colonies were inoculated into 10 ml of TSB containing small-sized beads and cultured with shaking at about 200 rpm at 25 DEG C for 3 days.

After culturing for 3 days, the soil microorganisms were spotted in 10 μl of TSA, dried in a laminar flow biosafety cabinet hood for 15 to 30 minutes, and cultured in a 25 ° C. incubator for 24 hours .

In order to isolate microorganisms from food, eight randomly selected shops in traditional markets in Seoul were selected for fresh vegetables (lettuce, ice lettuce, cabbage, red cabbage, cabbage, sesame leaf, chinese cabbage, chicory and mustard leaves) Aquatic products (fish [mackerel, reference], shellfish [shrimp] and shellfish [mussels, shellfish] were purchased.

10 g of fresh vegetables or aquatic products were placed in a 400 ml polyolefin container with 100 ml of sterilized 0.1% peptone water (PW) and homogenized for 2 minutes using a homogenizer. The homogenized samples were each diluted with 0.1% PW and plated on TSA (tryptic soy agar) medium supplemented with 0.1% (w / v) sodium pyruvate, and cultured at 25 ° C for 48 hours Respectively. After culturing, colonies with different shapes and colors were selected, plated on TSA medium with platinum powder, cultured at 25 ° C for 24 hours, and stored at 4 ° C.

For microbial isolates from food and food contact surfaces, single colonies were inoculated into 10 ml TSB and subcultured three times at 24 h intervals at 25 ° C incubator.

10 μl of each TSB microorganism was inoculated into 10 ml of TSA, and then dried in a laminar flow biosafety cabinet hood for 15 to 30 minutes. The cells were cultured in a 25 ° C. incubator for 24 hours Respectively.

In the case of microorganisms isolated from the soil, the microorganisms isolated from the food after 3 days of spot inoculation were cultured for 24 hours in the inoculated culture, and then 99 ml of TSA maintained at 45 ° C and 1 ml of Staphylococcus aureus 10 ⁷ CFU / ml to prepare 100 ml of agar containing 10 ⁵ CFU / ml of bacteria. 10 ml of agar mixed with Staphylococcus aureus was added to the strain grown TSA.

The agar plate was dried in a laminar flow biosafety cabinet hood for 30 minutes and then incubated at 37 ° C for 24 hours to confirm the size of the inhibitory ring. The results are shown in Fig.

As shown in FIG. 1, the diameter of the inhibitory ring formed on the agar plate was measured, and it was confirmed that the diameter of the inhibitory ring by the antagonistic microorganism (Lettuce-9) isolated from the food was the largest at 3.9 cm.

[ Example  2] from soil, food, food contact surface Aspergillus Flavors Aspergillus flavus )on  Of antagonistic microorganisms

The size of the inhibitory ring was confirmed in the same manner as in Example 1, except that Aspergillus flavus was used as an antimicrobial target. FIG. 2 shows the size of the inhibition ring.

As shown in FIG. 2, the diameter of the inhibitory ring formed on the agar plate was measured. As a result, it was confirmed that the inhibitory ring having a diameter of 1.1 cm was formed by the antagonistic microorganism (Lettuce-9) isolated from the food.

[ Example  3] Identification of antagonistic microorganisms

Staphylococcus aureus and Aspergillus flavus were streaked onto TSA medium and cultured for 2 days at 25 ° C. The 16S rRNA gene sequence analysis As a result, it was found to have the nucleic acid sequence of Fig. 16S rRNA gene sequence information was identified by blast analysis of NCBI (www.ncbi.nlm.nih.gov). The results identified microorganism Pseudomonas species (Pseudomonas spp.) was found to be 99%. The nucleic acid sequence of SEQ ID NO: 1 is shown in Fig.

This Pseudomonas To identify the species names of the spp., phylogenetic trees were first drawn and analyzed. As can be seen in the tree of FIG. 4 showing the 16S rRNA sequence information analyzed by the neighbor-joining method of the Mega software (version 5.05), Pseudomonas azotoformans strain NBRC 12693, and showed the highest molecular phylogenetic relationship with Pseudomonas azo topomans. Therefore, the antagonistic microorganism (Lettuce-9) isolated in Examples 1 and 2 was treated with Pseudomonas aeruginosa azotoformans lettuce), and deposited with the National Academy of Sciences on Mar. 30, 2016, and granted accession number KACC 92125P.

[ Example  4] Pseudomonas Azotopomans  Strain KACC  Confirm the ability of 92125P to form biofilm

A single colony of Pseudomonas azo topomancer strain KACC 92125P isolated from food through the above example was inoculated into 10 ml of TSB medium and subcultured three times at 48 간격 intervals in a 25 째 C incubator.

The culture solution of Pseudomonas azo topomancer strain KACC 92125P, which had been cultured in 10 ml TSB for 48 hours, was centrifuged at 2,002 rcf, and 1 ml of the suspension was diluted 100 times with 99 ml of PBS (phosphate buffer saline) Once more, a suspension of 5 log CFU / ml Pseudomonas azotopneumans strain KACC 92125P was prepared.

The suspension thus prepared was sprayed onto a stainless steel coupon using a sterilized sprayer and then dried in a laminar flow biosafety hood for 1 hour to simply attach the suspension to the coupon surface.

4 ml of TSB was supplied to the coupon with Pseudomonas azo topomancer KACC 92125P cells and stored for up to 120 hours in an incubator at 25 ° C and the number of Pseudomonas azotopneumans KACC 92125P was confirmed every 24 hours, The film forming ability was confirmed. At this time, it was judged that a biofilm was formed when the population of Pseudomonas aeruginosa strain KACC 92125P was significantly increased on the surface of stainless steel. FIG. 5 shows a growth curve in which a biofilm is formed.

After storage at 25 ° C for 120 hours, the characteristics of the bacteria were determined by scanning electron microscopy (SEM) to identify biofilms formed by Pseudomonas aeruginosa strains KACC 92125P in stainless steel. A biofilm formed by SEM is shown in Fig.

As shown in FIG. 5, the initial population of Pseudomonas aeruginosa strains KACC 92125P attached to stainless steel was 3.3 log CFU / coupon, which was significantly increased after 24 hours of storage and 8.5 log CFU / coupon, but the population remained after that. These results indicate that Pseudomonas azotopneumans strain KACC 92125P can form a biofilm on stainless steel surfaces during storage at 25 ° C after the supply of TSB.

In addition, the appearance of the Pseudomonas aeruginosa topomancer strain KACC 92125P formed on the stainless steel surface using a SEM (FIG. 6), and the Pseudomonas aeruginosa strains KACC 92125P Were found to have many clusters, and strains such as fibers connecting the surface of the stainless steel and bacteria or different bacteria were found.

[ Example  5] Pseudomonas Azotopomans  Strain KACC  Identification of 92125P resistance to stress in dry environment

To attach the Pseudomonas aeruginosa topomancer strain KACC 92125P to the stainless steel surface, 9 log CFU / ml of Pseudomonas aeruginosa strain KACC 92125P cultivated in 10 ml of TSB medium for 48 hours at a temperature of 25 ° C was added to the sterilized spray , And adhered while drying for 1 hour.

To form a biofilm of Pseudomonas aeruginosa topomancer strain KACC 92125P on a stainless steel surface, 5 log CFU / ml of Pseudomonas azotopneumans strain KACC 92125P cultured in 10 ml of TSB medium for 48 hours at a temperature of 25 ° C After spraying with a sterile spray and drying for 1 hour, 4 ml of TSB medium was supplied and stored at 25 ° C for 5 days.

Pseudomonas azo topomancer strain KACC 92125P-adhered or biofilm-formed stainless steel coupons were stored for 5 days at 25 ° C and 43% relative humidity, and every 24 hours of storage, the population of Pseudomonas azotopneumans strain KACC 92125P Respectively. The results are shown graphically in Fig.

As a result, when the Pseudomonas aeruginosa topomancer strain KACC 92125P was simply adhered, it showed a decrease of more than 3.4 log CFU / coupon after 24 hours of storage, but only 0.8 log CFU / coupon of biofilm formation . These results indicate that the resistance to the drying environment is increased when the tested Pseudomonas aeruginosa topomancer strain KACC 92125P forms a biofilm on a stainless steel surface.

[ Example  6] Pseudomonas Azotopomans  Strain KACC  92125P Staphylococcus Aureus ( Staphylococcus aureus ) For Reduction  Check the effect

A stainless steel coupon having a biofilm of Pseudomonas aeruginosa strains KACC 92125P was prepared in the same manner as in Example 5, and 0.1 ml (about 4 log CFU / coupon) of a suspension of Staphylococcus aureus was taken Spot-inoculation on 18 ± 2 drops onto a stainless steel coupon with or without a biofilm of Pseudomonas aeruginosa topomancer strain KACC 92125P. Thereafter, the cells were stored for 48 hours at a relative humidity of 43% and a temperature of 25 ° C, and the number of Staphylococcus aureus was determined at 0, 3, 6, 12, 24 and 48 hours after storage. The results are shown graphically in Fig.

The initial population of inoculated Staphylococcus aureus was 4.2 log CFU / coupon. The number of Staphylococcus aureus present on the surface of the stainless steel coupon without biofilm remained 3.0 log CFU / coupon after 48 hours of storage. On the other hand, it was confirmed that Staphylococcus aureus inoculated with a stainless steel coupon containing a biofilm formed by Pseudomonas aeruginosa topomancer strain KACC 92125P died more rapidly. That is, the number of Staphylococcus aureus exposed to the biofilm formed by Pseudomonas aeruginosa topomancer strain KACC 92125P decreased significantly during the storage time and decreased to 1.9 log CFU / coupon after 48 hours of storage. As a result, strong antimicrobial activity against Staphylococcus aureus of Pseudomonas azo topomancer strain KACC 92125P was confirmed.

National Academy of Agricultural Sciences (Domestic) KACC92125P 20160330

<110> KOREAN UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> Pseudomonas azotoformans strain KACC 92125P and composition for          comprising the same <130> P16U13C0801 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1481 <212> RNA <213> Pseudomonas azotoformans strain KACC 92125P <400> 1 tcagattaac gctggcggca ggcctaacac atgcaagtcg agcggtagag agaagcttgc 60 ttctcttgag agcggcggac gggtgagtaa tgcctaggaa tctgcctggt agtgggggat 120 aacgttcgga aacggacgct aataccgcat acgtcctacg ggagaaagca ggggaccttc 180 gggccttgcg ctatcagatg agcctaggtc ggattagcta gttggtgggg taatggctca 240 ccaaggcgac gatccgtaac tggtctgaga ggatgatcag tcacactgga actgagacac 300 ggtccagact cctacgggag gcagcagtgg ggaatattgg acaatgggcg aaagcctgat 360 ccagccatgc cgcgtgtgtg aagaaggtct tcggattgta aagcacttta agttgggagg 420 aagggttgta gattaatact ctgcaatttt gacgttaccg acagaataag caccggctaa 480 ctctgtgcca gcagccgcgg taatacagag ggtgcaagcg ttaatcggaa ttactgggcg 540 taaagcgcgc gtaggtggtt tgttaagttg gatgtgaaat ccccgggctc aacctgggaa 600 ctgcattcaa aactgactga ctagagtatg gtagagggtg gtggaatttc ctgtgtagcg 660 gtgaaatgcg tagatatagg aaggaacacc agtggcgaag gcgaccacct ggactaatac 720 tgacactgag gtgcgaaagc gtggggagca aacaggatta gataccctgg tagtccacgc 780 cgtaaacgat gtcaactagc cgttggaagc cttgagcttt tagtggcgca gctaacgcat 840 taagttgacc gcctggggag tacggccgca aggttaaaac tcaaatgaat tgacgggggc 900 ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggcc 960 ttgacatcca atgaactttc cagagatgga ttggtgcctt cgggaacatt gagacaggtg 1020 ctgcatggct gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgt aacgagcgca 1080 acccttgtcc ttagttacca gcacgtcatg gtgggcactc taaggagact gccggtgaca 1140 aaccggagga aggtggggat gacgtcaagt catcatggcc cttacggcct gggctacaca 1200 cgtgctacaa tggtcggtac agagggttgc caagccgcga ggtggagcta atcccataaa 1260 accgatcgta gtccggatcg cagtctgcaa ctcgactgcg tgaagtcgga atcgctagta 1320 atcgcgaatc agaatgtcgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac 1380 accatgggag tgggttgcac cagaagtagc tagtctaacc ttcgggagga cggttaccac 1440 ggtgtgattc atgactgggg tgaagtcgta caggggaacc c 1481

Claims (14)

Pseudomonas azotoformans strain of Accession No. KACC 92125P. The present invention relates to a method for the antibacterial use of Staphylococcus aureus and Aspergillus flavus containing Pseudomonas aeruginosa topomancer strain KACC 92125P, a culture thereof, an extract thereof or a suspension thereof as an active ingredient, Composition. A feed or feed additive composition comprising Pseudomonas aeruginosa topomancer strain KACC 92125P, a culture thereof, an extract thereof or a suspension thereof. A biological control agent for Staphylococcus aureus and Aspergillus flavus , comprising Pseudomonas aeruginosa topomancer strain KACC 92125P, a culture thereof, an extract thereof or a suspension thereof. 4. A method for inhibiting the activity of Staphylococcus aureus and Aspergillus flavus , comprising the step of treating the control agent of claim 4 at, or near, a livestock house or a house. Pseudomonas azo topomancer strain KACC 92125P, a culture thereof, an extract thereof, or a suspension thereof, aspergillus flavus . A method for inhibiting the activity of Aspergillus flavus , comprising treating the plant with the controlling agent of claim 6. delete delete The surface of Staphylococcus aureus and Aspergillus flavus containing Pseudomonas azo topomancer strain KACC 92125P, a culture thereof, an extract thereof or a suspension thereof, Lt; / RTI &gt; 11. The method of claim 10,
Wherein the step of treating the surface further comprises the step of spraying and culturing the Pseudomonas aeruginosa topomancer strain KACC 92125P, a culture thereof, an extract thereof or a suspension thereof on the surface. 12. The method of claim 11,
And culturing at 20 to 30 캜 for 6 to 120 hours after spraying. 11. The method of claim 10,
Wherein the surface is at least one selected from the group consisting of stainless steel, glass, wood, vinyl, plastic, rubber and tiles. delete

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