KR101819344B1 - Bacillus safensis strain KACC 92124P and composition for comprising the same - Google Patents

Abstract

The Bacillus subtilis strain KACC 92124P of the present invention exhibits antimicrobial activity against food-borne microorganisms Staphylococcus aureus and Escherichia coli O157: H7, The composition containing the strain can be used as a biological agent and can be widely applied to foods, agriculture, environmental industries, and the like.

Description

Bacillus sapensis strain KACC 92124P and a composition comprising it {Bacillus safensis strain KACC 92124P and composition for comprising the same}

The present invention relates to a novel Bacillus sabensis strain KACC 92124P exhibiting antimicrobial activity against Staphylococcus aureus and Escherichia coli O157: H7, a composition comprising the same, and inhibiting the activity of a pathogenic microorganism using the same ≪ / RTI >

Staphylococcus aureus is a gram-positive anaerobic bacterium that is present on the skin and nasal surface of healthy people and 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 is proliferating even in high salt concentration, and it is resistant especially in dry condition, 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.

On the other hand, Escherichia Coli O157: H7) was first discovered in the United States in 1982 and is a pathogenic Escherichia coli that causes diarrhea and abdominal pain. It is a colitis hemorrhagic Escherichia coli and usually causes blood diarrhea. The intestinal hemorrhagic Escherichia coli is very infectious, and once it enters the human body, it causes abdominal pain, diarrhea and blood stains. It produces toxic protein synthetics called 'vero toxin' and destroys red blood cells , Resulting in hemolytic uremic toxicity that causes the kidney to lose its function by intensively attacking the kidney (Non-Patent Document 2). When the uraemic disease occurs, it is secondarily reported that the disorder occurs in the nervous system, the respiratory system, the circulatory system, and finally it leads to death, and the mortality rate reaches 5 to 10%, and it is reported that infection occurs in all ages. ≪ / RTI >

The intestinal hemorrhagic Escherichia coli is infected when it is consumed at an incubation temperature of less than 65 ° C or when drinking potable water contaminated on the animal's side and is also infected through a patient infected with intestinal hemorrhagic Escherichia coli, Recently, eating out is becoming commonplace, and food poisoning is occurring mainly in restaurants and group foodservice stations. Therefore, it is necessary to prevent and treat food poisoning against intestinal hemorrhagic Escherichia coli.

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, it is necessary to develop a method for inhibiting microorganisms from harmful substances by using natural products rather than chemical compounds on the food contact surface.

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 antimicrobial agents against Staphylococcus aureus, the surface may exhibit persistent antimicrobial activity against Staphylococcus aureus.

Bacillus spp. Is a potent anaerobic gram-positive bacterium that forms spores. This bacterium tends to form a chain arrangement, and the presence or absence of the cavernous fascia and flagella varies depending on the species. Most are non-pathogenic and widely distributed in the natural world such as soil and water. Bacillus spp. Is a microorganism which is excellent in preservation because it forms strong apophysis that can withstand various conditions and is easy to use because it can be mixed with chemical pesticides and can be used together. The antimicrobial effect of Bacillus sp. Bacillus has been known for a long time, but unlike chemical products, it is characterized by antimicrobial activity by antagonism by microorganisms.

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. World Health Organization 2011, PUBLIC HEALTH REVIEW OF THE ENTEROHAEMORRHAGIC ESCHERICHIA COLI OUTBREAK IN GERMANY

The object of the present invention is to provide a method for the treatment of Staphylococcus aureus and Escherichia coli coli O157: H7), a composition comprising the same, and a method for inhibiting the activity of a pathogenic microorganism using the same.

Accordingly, the present inventors have isolated about 2,100 microorganisms from the soil, food, and food contact surfaces. Among these microorganisms, Staphylococcus aureus and Escherichia coli coli O157: H7). As a result, a novel Bacillus sp. strain Bacillus sampensis strain KACC 92124P was isolated and identified. As a result, the present invention was completed.

The Bacillus safensis strain KACC 92124P according to the present invention is a novel strain of Bacillus sabensis derived from soil. Although the Bacillus subtilis strain of the present invention is isolated and identified from the soil, the route of intake is not limited thereto.

As a result of 16S rRNA sequence analysis for identification and classification of microorganisms showing excellent antimicrobial activity against Staphylococcus aureus and enterohemorrhagic Escherichia coli isolated through the examples of the present invention, . As a result of the analysis, the strain showed the highest molecular phylogenetic relationship with Bacillus safensis strain FO-36b as shown in the tree of FIG.

Therefore, the microorganism of the present invention having the 16S rRNA nucleotide sequence of FIG. 3 was designated as Bacillus safensis oak forest, and deposited on March 30, 2016 (Accession No. KACC 92124P) . In the present invention, the Bacillus sapensis oak forest ( Bacillus safensis oak forest) can be used interchangeably with Bacillus safensis strain KACC 92124P.

The Bacillus sabensis strain KACC 92124P of the present invention is effective against Staphylococcus aureus and Escherichia coli coli O157: H7), the composition containing the strain of the present invention can be used as a biological agent and can be widely applied to foods, agriculture, and the environment industry.

In the following examples, it was confirmed that the bacteriocin susceptible strain KACC 92124P of the present invention simultaneously exhibited antibacterial activity against Staphylococcus aureus and enterohemorrhagic Escherichia coli. In addition, the suspension of the Bacillus subtilis strain KACC 92124P was spray-dried on stainless steel to measure the number of the strains on the cultured surface, and it was confirmed that the biofilm-forming ability was excellent.

Therefore, the Bacillus subtilis strain KACC 92124P according to the present invention is an antimicrobial substance against Staphylococcus aureus and enterohemorrhagic Escherichia coli, and can be variously used for suppressing the activity of the pathogenic microorganism in livestock, housing, and the like.

Thus, in one embodiment of the present invention, there is provided a composition comprising a Bacillus subsp. Strain KACC 92124P, a culture thereof, an extract thereof or a suspension thereof.

The Bacillus subtilis strain KACC 92124P contained in the composition according to the present invention may exist as living cells or dead cells, and may also be present 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 Bacillus subtilis strain KACC 92124P, a culture thereof, an extract thereof or a suspension thereof.

The antimicrobial composition according to the present invention can be used for the treatment of Staphylococcus aureus and Escherichia coli coli O157: H7).

Staphylococcus aureus and intestinal hemorrhagic Escherichia coli can cause mastitis in cows and chlorines and can cause edematous dermatitis and arthritis in chickens and cause pneumonia, diabetes, osteomyelitis, endocarditis, bacteremia, sepsis, hemorrhagic colitis, hemolytic Urinary tract syndrome, thrombotic thrombocytopenic purpura, and the like. In addition, Staphylococcus aureus and enterohemorrhagic Escherichia coli are known as typical food poisoning bacteria, and can cause food poisoning and enteritis in livestock.

Therefore, the composition can be used for prevention, treatment, and improvement of diseases caused by infection with pathogenic microorganisms in an animal, and preferably the animal includes cattle such as cattle, horses, and pigs.

The present invention also provides a feed or feed additive composition comprising Bacillus subtilis strain KACC 92124P, 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.

The present invention also provides a biological control agent comprising Bacillus subtilis strain KACC 92124P, a culture thereof, an extract thereof or a suspension thereof.

In the present invention, the bacillus sabensis strain KACC 92124P 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 from about 0.1 to 99% by weight of the Bacillus subtilis strain KACC 92124P, 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.

The controlling agent according to the present invention can be used in combination with or without being mixed with pesticides, nematicides, fungicides, fungicides, bactericides, herbicides, plant growth regulators, electrodeposits, fertilizers, microbial materials, have.

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 is selected from the group consisting of Staphylococcus aureus and Escherichia coli coli O157: H7).

As described above, by inhibiting the activity of the pathogenic microorganism, it is possible to prevent food poisoning, enteritis, mastitis, edematous dermatitis, arthritis, pneumonia, diabetes, osteomyelitis, heart disease caused by Staphylococcus aureus induced in animals such as poultry and livestock Endocarditis, bacteremia, sepsis and hemorrhagic colitis caused by intestinal hemorrhagic Escherichia coli, hemolytic urethral syndrome, and thrombotic thrombocytopenic purpura can be prevented.

The present invention provides a pharmaceutical composition for the prophylaxis or treatment of an infectious disease caused by a pathogenic microorganism comprising a culture of Bacillus subtilis strain KACC 92124P, an extract thereof or a suspension thereof.

In the present invention, the pathogenic microorganism is selected from the group consisting of Staphylococcus aureus and Escherichia coli coli O157: H7).

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 Infectious diseases such as hemorrhagic colitis caused by enterohemorrhagic Escherichia coli, hemolytic urethral syndrome, thrombotic thrombocytopenic purpura 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.

In addition, the present invention provides a method for treating an antimicrobial surface comprising the step of treating the surface of a Bacillus subtilis strain KACC 92124P, a culture thereof, an extract thereof or a suspension 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 the Bacillus subsp. Strain KACC 92124P was sprayed onto a stainless steel coupon, dried and then cultured at 25 DEG C for 120 hours. As a result, the number of the strains was about 1,000 times or more And that the strains were retained thereafter, it was confirmed that the strain could form a biofilm in a stainless steel coupon and exhibit antimicrobial activity.

Therefore, the strain according to the present invention can exhibit antibacterial activity by forming a biofilm on a surface of stainless steel, glass, wood, vinyl, plastic, etc. by forming a biofilm on a surface in a non-selective manner, ( Staphylococcus aureus ) and enterohemorrhagic Escherichia coli coli O157: H7). < / RTI >

The bacillus sampinesys strain KACC 92124P of the present invention exhibits antimicrobial activity against Staphylococcus aureus and Escherichia coli O157: H7, which are food-harmful microorganisms, and exhibits antibacterial activity against Bacillus sabensis strain KACC Since 92124P is well-viable, the composition containing the strain can be used as a biological agent and widely applied to foods, agriculture, environmental industries, and the like.

FIG. 1 shows the antimicrobial activity against the strain Staphylococcus aureus according to the present invention through the formation of an inhibitory ring.
Fig. 2 is a graph showing the results of measurement of the concentration of Escherichia coli coli O157: H7) through the formation of inhibitory rings.
3 shows the nucleotide sequence of SEQ ID NO: 1 of Bacillus subsp. Strain KACC 92124P of the present invention.
Figure 4 is a tree showing the molecular phylogenetic relationship of the Bacillus sampinesis strain KACC 92124P of the present invention.
FIG. 5 is a graph showing a growth curve of the biofilm of the Bacillus subtilis strain KACC 92124P of the present invention.
FIG. 6 shows the results of confirming the resistance of the biofilm formed from the Bacillus subtilis strain KACC 92124P of the present invention to the stress in the drying environment.
FIG. 7 shows the results of confirming the antimicrobial activity against Staphylococcus aureus of a biofilm formed from Bacillus sampense strain KACC 92124P 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 ℃ 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 inoculation were inoculated for 24 hours after inoculation, 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 inhibition ring formed on the agar plate was measured, and it was confirmed that the diameter of the inhibition ring by the antagonistic microorganism (oak forest 5-25) isolated from the soil was the largest at 3.7 cm.

[ Example  2] from soil, food, food contact surface Intestinal hemorrhage E. coli ( Escherichia  coli  O157: H7) of the antagonistic microorganisms

Antibacterial agents were Escherichia coli coli O157: H7) was used as the starting material. 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 1.6 cm was formed by the antagonistic microorganism (oak forest 5-25) isolated from the soil.

[Example 3] Identification of antagonistic microorganisms

An antagonistic microorganism (oak forest 5-25) showing antibacterial activity against Staphylococcus aureus and Escherichia coli O157: H7 was streaked on Bennet's agar and cultured for 2 days at 25 ° C. And then subjected to 16S rRNA gene sequencing 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). As a result of the identification, it was confirmed that the microorganism was 99% likely to be of the genus Bacillus spp. The nucleic acid sequence of SEQ ID NO: 1 is shown in Fig.

In order to identify the genus Bacillus spp., A phylogenetic tree was first drawn and analyzed. As can be seen in the tree of FIG. 4 showing the 16S rRNA gene sequence information analyzed by the neighbor-joining method of the Mega software (version 5.05), the Bacillus safensis strain FO-36b, and showed the highest molecular phylogenetic relationship with Bacillus subtilis. Therefore, the antagonistic microorganism (oak forest 5-25) isolated in Examples 1 and 2 was named Bacillus safensis oak forest and deposited with the National Academy of Sciences on Mar. 30, 2016, No. KACC 92124P.

[ Example  4] Bacillus Sapensis  Strain KACC  Confirm the ability of 92124P to form biofilm

A single colony of Bacillus subsp. Strain KACC 92124P isolated from food through the above example was inoculated into 10 ml of Bennet's broth and subcultured three times at 48 hours intervals in an incubator at 25 ° C.

The culture solution of Bacillus subsp. Strain KACC 92124P, which had been cultured in 10 ml of Bennet's broth for 48 hours, was centrifuged at 2,002 rcf. Then, 1 ml of the suspension was diluted 100 times with 99 ml of PBS (phosphate buffer saline) Finally, a suspension of 5 log CFU / ml bacillus sampinesys strain KACC 92124P 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 Bennet's broth was supplied to coupons attached to the cells of Bacillus subsp. Strain KACC 92124P and stored for up to 120 hours in a 25 ° C incubator. After confirming the number of Bacillus subsp. Strain KACC 92124P every 24 hours, Respectively. At this time, it was judged that the biofilm was formed when the population of Bacillus subsp. Strain KACC 92124P was significantly increased on the surface of stainless steel. FIG. 5 shows a growth curve in which a biofilm is formed.

As shown in FIG. 5, the initial population of Bacillus subtilis strain KACC 92124P attached to stainless steel was 2.6 log CFU / coupon, which was significantly increased after 24 hours of storage, and 8.2 log CFU / coupon , But the population remained after that. These results indicate that Bacillus sabensis strain KACC 92124P can form a biofilm on stainless steel surfaces during storage at 25 ° C after supplying Bennet's broth.

[ Example  5] Bacillus Sapensis  Strain KACC  Identification of 92124P resistance to stress in dry environment

In order to attach the Bacillus subtilis strain KACC 92124P to the stainless steel surface, 8 log CFU / ml of Bacillus subtilis strain KACC 92124P cultured in 10 ml of Bennet's broth for 120 hours at a temperature of 25 ° C was sprayed with sterilized spray And allowed to attach for 1 hour while drying.

To form a biofilm of Bacillus subtilis strain KACC 92124P on a stainless steel surface, 5 log CFU / ml of Bacillus sabensis strain KACC 92124P cultivated in 10 ml of Bennet's broth for 120 hours at 25 ° C was sprayed with sterile spray After drying for 1 hour, 4 ml of Bennet's broth was supplied and stored at 25 ° C for 5 days.

Stainless steel coupons with biofilm prepared with Bacillus subtilis strain KACC 92124P washed or sterilized distilled water were stored for 5 days at 25 ° C and 43% relative humidity and stored for 24 hours at the storage temperature of Bacillus sapensis strain KACC 92124P Were identified. The results are shown graphically in Fig.

As a result, when the Bacillus subtilis strain KACC 92124P was simply adhered, the population decreased by more than 5.0 log CFU / coupon after 24 hours of storage but decreased by only 2.5 log CFU / coupon when the biofilm was formed. These results indicate that when the tested Bacillus subsp. Strain KACC 92124P forms a biofilm on a stainless steel surface, the resistance to the drying environment is increased.

[ Example  6] Bacillus Sapensis  Strain KACC  92124P Staphylococcus Aureus  Check for reduction effect

(About 4 log CFU / coupon) of a suspension of Staphylococcus aureus was prepared by preparing a stainless steel coupon having a biofilm of Bacillus subsp. Strain KACC 92124P prepared in the same manner as in Example 5, Spot-inoculation of 18 ± 2 drops on stainless steel coupons with or without biofilms of sis strain. 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, Staphylococcus aureus inoculated with a stainless steel coupon containing a biofilm formed by Bacillus subsp. Strain KACC 92124P was found to die faster. That is, the number of Staphylococcus aureus exposed to the biofilm formed by Bacillus subtilis strain KACC 92124P decreased significantly during the storage time and decreased to 1.8 log CFU / coupon after 48 hours of storage. As a result, the bactericidal activity against Staphylococcus aureus of Bacillus subsp. Strain KACC 92124P was confirmed.

National Academy of Agricultural Sciences (Domestic) KACC92124P 20160330

<110> KOREAN UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> Bacillus safensis strain KACC 92124P and composition for          comprising the same <130> P16U13C0800 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1499 <212> RNA <213> Bacillus safensis strain KACC 92124P <400> 1 aggacgaacg ctggcggcgt gcctaataca tgcaagtcga gcggacagaa gggagcttgc 60 tcccggatgt tagcggcgga cgggtgagta acacgtgggt aacctgcctg taagactggg 120 ataactccgg gaaaccggag ctaataccgg atagttcctt gaaccgcatg gttcaaggat 180 gaaagacggt ttcggctgtc acttacagat ggacccgcgg cgcattagct agttggtggg 240 gtaatggctc accaaggcga cgatgcgtag ccgacctgag agggtgatcg gccacactgg 300 gactgagaca cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac 360 gaaagtctga cggagcaacg ccgcgtgagt gatgaaggtt ttcggatcgt aaagctctgt 420 tgttagggaa gaacaagtgc gagagtaact gctcgcacct tgacggtacc taaccagaaa 480 gccacggcta actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga 540 attattgggc gtaaagggct cgcaggcggt ttcttaagtc tgatgtgaaa gcccccggct 600 caaccgggga gggtcattgg aaactgggaa acttgagtgc agaagaggag agtggaattc 660 cacgtgtagc ggtgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcgactctc 720 tggtctgtaa ctgacgctga ggagcgaaag cgtggggagc gaacaggatt agataccctg 780 gtagtccacg ccgtaaacga tgagtgctaa gtgttagggg gtttccgccc cttagtgctg 840 cagctaacgc attaagcact ccgcctgggg agtacggtcg caagactgaa actcaaagga 900 attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa 960 ccttaccagg tcttgacatc ctctgacaac cctagagata gggctttccc ttcggggaca 1020 gagtgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1080 gcaacgagcg caacccttga tcttagttgc cagcattcag ttgggcactc taaggtgact 1140 gccggtgaca aaccggagga aggtggggat gacgtcaaat catcatgccc cttatgacct 1200 gggctacaca cgtgctacaa tggacagaac aaagggctgc aagaccgcaa ggtttagcca 1260 atcccataaa tctgttctca gttcggatcg cagtctgcaa ctcgactgcg tgaagctgga 1320 atcgctagta atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac 1380 cgcccgtcac accacgagag tttgcaacac ccgaagtcgg tgaggtaacc tttatggagc 1440 cagccgccga aggtggggca gatgattggg gtgaagtcgt acagggaaaa ccgggtaaa 1499

Claims (12)

Bacillus safensis strain of Accession No. KACC 92124P. A composition for antibacterial activity against Staphylococcus aureus and Escherichia coli O157: H7, which comprises a culture of Bacillus subtilis strain KACC 92124P, a culture thereof, an extract thereof or a suspension thereof as an active ingredient . A feed or feed additive composition comprising a Bacillus subtilis strain KACC 92124P, a culture thereof, an extract thereof or a suspension thereof. A biological control agent for Staphylococcus aureus and Escherichia coli O157: H7, comprising a Bacillus subsp. Strain KACC 92124P, a culture thereof, an extract thereof or a suspension thereof. A method for inhibiting the activity of Staphylococcus aureus and enterohemorrhagic Escherichia coli O157: H7, comprising the step of treating the control agent of claim 4 at, or near, a livestock house or a house. delete delete ( Staphylococcus aureus and Escherichia coli O157: H7), which comprises treating the surface of a Bacillus subtilis strain KACC 92124P, a culture thereof, an extract thereof or a suspension thereof, to a surface of Staphylococcus aureus and Escherichia coli O157: Antibacterial treatment method for. 9. The method of claim 8,
Wherein the step of treating the surface further comprises a step of spraying a culture of Bacillus subtilis strain KACC 92124P, a culture thereof, an extract thereof or a suspension thereof onto a surface and culturing the surface. 10. The method of claim 9,
And culturing at 20 to 30 캜 for 6 to 120 hours after spraying. 9. The method of claim 8,
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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