KR20200006866A - Bacillus safensis strain with antibiotic activity and antibiotic use thereof - Google Patents

Abstract

The present invention relates to a Bacillus safensis strain having antibacterial activity and an antibacterial use thereof. More specifically, Bacillus safensis SR098 strain (KACC 92219P); probiotics comprising the strain, a culture thereof, etc.; an antibacterial composition comprising the strain a culture thereof, etc.; and a method for producing bacteriocin from the strain, and a method for inhibiting harmful bacteria using the strain. The strain of the present invention shows excellent antibacterial activity against various food poisoning bacteria. Crop loss due to contamination of production and distribution processes of various agricultural and stockbreeding products can be reduced by using the same.

Description

Bacillus safensis strain with antibacterial activity and its antibacterial use

The present invention relates to a novel strain (Strain) for the growth inhibition of harmful bacteria and its use, in particular a novel strain having a growth inhibitory activity against food poisoning bacteria such as Listeria monocytogenes, antimicrobial composition comprising the strain, the strain It relates to a method for producing bacteriocin from and a method for inhibiting harmful bacteria using the strain.

Pathogenic bacteria of the current I Staphylococcus aureus that is the problem in the world (Staphylococcus aureus) is widely recognized as a pathogen causing nosocomial infection with Gram-positive bacteria to be detected in about 30% of the population, and in addition to Salmonella spp. (Salmonella spp.), Cocos Staphylococcus aureus (Staphylococcus aureus), Chapter toxic E. coli (enterotoxigenic E. coli), Bacillus cereus (Bacillus cereus), Yersinia Enterococcus coli urticae (Yersinia enterocolitica) and Listeria monocytogenes jeneseu (Listeria monocytogenes ) are widely known as representative bacteria causing food poisoning.

In particular, Listeria monocytogenes pollutes most foods such as meat, fish, shellfish, cheese, ice cream, and frozen foods, and is a deadly pathogen that causes miscarriage, sepsis, meningitis, and food poisoning in pregnant women, newborns, and senior citizens, and Staphylococcus aureus is food poisoning. In addition, it is widely known as a bacterium that causes purulent diseases such as purulent otitis media, otitis media and cystitis. When Listeria monocytogenes is subjected to osmotic stress such as high salinity, high sugar content and drying, it can accumulate osmolyte, which is an osmoprotective substance, in the cell to maintain intracellular swelling and proliferate, even at low temperature (4 ° C). There is a growing interest in food hygiene because it can grow and survive in the heat treatment process.

On the other hand, chemical preservatives are used commercially as a preservative to inhibit the growth of food poisoning microorganisms, but the recent phenomenon of avoiding synthetic preservatives is prominent due to the increase of health-oriented needs and stability of consumers. In order to cope with these problems, attention is focused on the development of antibiotic substitutes that are harmless to the human body. Such antibiotic substitutes include probiotics, bacterioncins, acidifers, immunomodulatory and enhancers, complex enzymes, and functional natural extracts, which are beneficial to host animals by improving the balance of intestinal microorganisms. Probiotics and bacteriocins are in the spotlight.

Probiotics (probiotics) are defined as microorganisms and components that have a beneficial effect on the host by improving the balance of intestinal microorganisms in humans or animals, and microorganisms used as probiotics include lactic acid bacteria, Bacillus subtilis, Bacillus, yeast and the like. The conditions that must be provided as a probiotic to help the host include the safety of the host, acid resistance and bile acid resistance, the ability to survive in the intestine, the ability to settle in the intestine, and produce antibacterial substances to produce pathogenic bacteria. Ability to inhibit, immune activity, easy mass production and viability within the shelf life. In addition, livestock probiotics improve growth and feed efficiency, ease of absorption of nutrients due to improved digestibility, and Odor improvement effects of the livestock environment are required.

Bacteriocin is a protein or peptide-based antibacterial substance secreted to the outside of the cell is known to kill or inhibit the growth of microorganisms in close relation to the production strain. Currently, nisin, the only bacteriocin that is commercially available, has been used as a food preservative in processed cheeses, canned vegetables, fruits, and fermented milk products in about 50 countries. These bacteriocins are decomposed by various proteolytic enzymes of the digestive system, so they are harmless to the human body and have no residual properties. Therefore, in accordance with the recognition that they are generally stable to the human body, various studies for active use as natural antibiotics or food preservatives have been actively conducted. It is becoming.

Thus Bacillus permilus HY1 strain having excellent antimicrobial activity against Bacillus cereus and Listeria monocytogenes and sulfactin (Patent Document 1), Bacillus Belensis CJBV and Bacillomycin produced therefrom (Patent Document 2), Bacillus te quot; Ensis HD 15 (Patent Document 3), Staphylococcus pastry RSP-1 (Patent Document 4) and the like are known.

As such, attention is focused on the development of natural antimicrobial active substances that are harmless to the human body, and in particular, the need to effectively suppress antibiotic resistant harmful bacteria is very high.

1. Korea Patent Registration No. 10-0851063 (2008.08.12) 2. Korea Patent Registration No. 10-1629551 (2016.06.03) 3. Korea Patent Registration No. 10-1655781 (2016.09.02) 4. Korea Patent Registration No. 10-1374696 (2014.03.10)

The technical problem to be achieved by the present invention is a novel strain capable of inhibiting the growth of harmful bacteria, particularly food poisoning bacteria, and further antibiotic resistant harmful bacteria, and using such strains to inhibit the growth of harmful bacteria in agricultural products, foods, etc. To provide a way.

In order to achieve the above object, the present invention provides a Bacillus safensis SR098 strain (KACC 92219P).

Bacillus safensis SR098 strain (KACC 92219P) according to the present invention may have a 16S rDNA of the nucleotide sequence represented by SEQ ID NO: 1.

According to an aspect of the present invention, there is provided a probiotic comprising Bacillus safensis SR098 strain (KACC 92219P), a culture of the strain, and any one or more of the bacteriocins produced by the strain as an active ingredient.

According to one aspect of the invention, the probiotic is L. monocytogenes jeneseu (Listeria monocytogenes), Staphylococcus aureus (Staphylococcus aureus), Bacillus turing Gen System (Bacillus thuringensis), Bacillus cereus (Bacillus cereus), for example reusi Any one selected from the group consisting of Yersinia enterocolitica , Shigella flexneri , Shigella boydii , Escherichia coli , and Cronobacter sakazakii . It may have an antimicrobial activity against the above strain.

According to an aspect of the present invention, there is provided an antimicrobial composition comprising any one or more of Bacillus safensis SR098 strain (KACC 92219P), a culture of the strain, and bacteriocin produced by the strain as an active ingredient. .

According to one aspect of the invention, the antimicrobial composition for L. monocytogenes jeneseu (Listeria monocytogenes), Staphylococcus aureus (Staphylococcus aureus), Bacillus turing Gen System (Bacillus thuringensis), Bacillus cereus (Bacillus cereus), Yersinia enterocolitica , Shigella flexneri , Shigella boydii , Escherichia coli and Cronobacter sakazakii It may have antimicrobial activity against any one or more strains.

According to one aspect of the present invention, the antimicrobial composition according to the present invention may be a pharmaceutical composition, a food composition, a food additive composition, a feed composition, a feed additive composition, a cosmetic composition or a quasi-drug composition.

According to one aspect of the present invention, there is provided a bacteriocin production method comprising culturing Bacillus safensis SR098 strain (KACC 92219P).

According to one aspect of the invention, the bacteriocin production method comprises the steps of centrifuging the culture obtained in the step of culturing the strain of the present invention; Filtering the supernatant obtained after the centrifugation; And it may further comprise the step of concentrating the filtrate obtained in the step of filtering the supernatant.

According to one embodiment of the present invention, Bacillus safensis ( Bacillus safensis ) SR098 strain (KACC 92219P), the culture of the strain and any one or more selected from the bacteriocin produced by the strain to remove the harmful bacteria, characterized in that the treatment Or a method of inhibiting activity.

According to one aspect of the invention, the harmful bacteria removal or inhibition method L. monocytogenes jeneseu (Listeria monocytogenes), Staphylococcus aureus (Staphylococcus aureus), Bacillus turing Gen System (Bacillus thuringensis), Bacillus cereus (Bacillus cereus ), Yersinia enterocolitica , Shigella flexneri , Shigella boydii , Escherichia coli and Cronobacter sakazakii It can be applied to any one or more strains selected from the group.

According to an aspect of the present invention, Bacillus safensis ( Bacillus safensis ) SR098 strain (KACC 92219P), the culture of the strain and any one or more selected from the bacteriocin produced by the strain is characterized in that the administration to animals other than humans It provides a method for preventing, alleviating or treating a harmful bacterial infection.

According to one aspect of the invention, the harmful bacteria infection prevention, mitigation or treatment method L. monocytogenes jeneseu (Listeria monocytogenes), Staphylococcus aureus (Staphylococcus aureus), Bacillus turing Gen System (Bacillus thuringensis), Bacillus celebrity Bacillus cereus , Yersinia enterocolitica , Shigella flexneri , Shigella boydii , Escherichia coli and Cronobacter sakazakii It can be applied to any one or more strains selected from the group consisting of).

The present invention provides a Bacillus safensis SR098 strain (KACC 92219P), a novel strain having excellent inhibitory activity against harmful bacteria such as food poisoning bacteria, especially Listeria monocytogenes, a culture of the strain and the bacteriocin produced by the strain. And it provides an antimicrobial composition and probiotic comprising a culture of the strain, and a method for inhibiting harmful bacteria using the same. By using the specific strain according to the present invention it is possible to reduce the loss of crops due to contamination of the production and distribution process of the agricultural products, it is possible to effectively control the food poisoning microorganisms.

1 is an experimental result evaluating the antimicrobial activity of Listeria monocytogenes of bacteriocin isolated from the strain of the present invention.

The present invention for achieving the above object Bacillus safensis ( Bacillus safensis ) SR098 strain (KACC 92219P), the antimicrobial composition comprising the strain or the culture thereof, the antimicrobial composition comprising the strain or its culture, etc. It is characterized in that the method for producing bacteriocin from the strain and a method for inhibiting harmful bacteria using the strain. Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

The present invention provides Bacillus safensis SR098 strain (KACC 92219P). The present inventors selected Bacillus sapensis Bacillus safensis SR098 strain having antimicrobial activity against various food poisoning bacteria such as Listeria monocytogenes and Yersinia enterocyticica , and identified novel bacteriocins generated from the strain and completed the present invention. .

More specifically, Bacillus safensis SR098 strain (KACC 92219P) according to the present invention may have a 16S rDNA of the nucleotide sequence represented by SEQ ID NO: 1.

The present invention provides a probiotic comprising Bacillus safensis SR098 strain (KACC 92219P), a culture of the strain, and at least one of the bacteriocins produced by the strain as an active ingredient.

The present invention provides an antimicrobial composition comprising any one or more of Bacillus safensis SR098 strain (KACC 92219P), a culture of the strain, and bacteriocin produced by the strain as an active ingredient.

As used herein, the term "culture" is a result obtained by inoculating a strain into a medium and culturing for a predetermined time. The culture solution itself (crude culture) obtained by culturing the strain according to the present invention in a suitable liquid medium, and filtering or centrifuging the culture solution. Filtrate from which strains were removed (filtrate or supernatant centrifuged), cell crushing solution obtained by sonicating the culture solution or lysozyme to the culture solution, extract of the culture solution, filtrate, and crushing solution, the culture solution , Concentrates including filtrates, crushed liquids, extracts, and the like and dried products thereof, but are not limited thereto.

As used herein, the term "concentrate" refers to the concentration of the culture solution.

As used herein, the term "extract" means extracted from the culture solution or its concentrate, and as long as the extract can exhibit the antimicrobial effect of the present invention, the present invention is not limited thereto, and the extract, the dilution or concentrate of the extract, and the extract are dried. It may include all of the dried product obtained, or these crude or purified product, fractions fractionated therefrom.

As used herein, the term "dry matter" means drying the culture, its concentrate, and its extract. Drying methods may be, but not limited to, ventilation drying, natural drying, spray drying and freeze drying.

Therefore, the probiotic or antimicrobial composition according to the present invention may contain the above-described strain itself or its culture, and may further include a suitable excipient or carrier.

As used herein, the term “probiotic” is a living microbial agent designed to promote the health of a human body or an animal, which may be ingested or administered alone or used in medicine, food and feed or dietary additives.

By “antibacterial” is meant herein the ability to reduce, prevent, inhibit, or eliminate the growth or survival of microorganisms at certain concentrations. The antimicrobial composition may mean the same as an antibiotic which is a generic term for an antimicrobial agent, and may mean the same as an antimicrobial agent, a fungicide, a preservative, a preservative or an antimicrobial agent, preferably Gram-positive bacteria, Gram-negative bacteria, fungi (yeast and mold) ) Means a substance capable of inhibiting or inhibiting the development and life function of one or more microorganisms selected from the group consisting of: antibacterial and antifungal effect.

The antimicrobial composition of the present invention may be directly administered to a part infected with a harmful microorganism or prepared and administered in a suitable form of the composition. Dosage of the antimicrobial composition of the present invention means the amount required to exert the effect of killing harmful microorganisms in the subject, the type of formulation and the age, weight, general state of health, sex and diet of the subject It may be adjusted according to various factors including the route of administration, time of administration and duration of administration. In the case of livestock or fish, the route of administration can be formulated within the range allowed by veterinary medicine and the route of administration can be determined accordingly.

The antimicrobial composition of the present invention may be prepared as a solution, powder, suspension, dispersion, emulsion, oily dispersion, paste, dust, propellant or granule, but is not limited thereto. According to a specific embodiment of the present invention, the antimicrobial composition according to the present invention can be widely used to achieve the purpose of antibacterial, sterilization, disinfection, antiseptic, etc. in pesticides, medicine, cosmetics, household goods, food. Specifically, for the purpose of antibacterial, disinfecting and disinfecting in agriculture, for antibiotics and antifouling agents in medicine, for antiseptic and antibacterial purposes in foods, for preventing dandruff in cosmetics and household goods, for preventing athlete's foot, for armpits. It may be used in products directly related to microorganisms such as anti-acne and anti acne or may be used for preservative, antibacterial or sterilizing purposes such as cleaning detergents or dish washing detergents, but is not limited thereto. It is not limited to this,

The pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the above-mentioned active ingredient, and the adjuvant may include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, Lubricants, flavors and the like can be used. The pharmaceutical composition may be preferably formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredient for administration.

Formulation forms of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, nontoxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, natural and synthetic gums such as starch, gelatin, glucose or beta-lactose, corn sweeteners, acacia, trackercance or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, 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 in compositions formulated in liquid solutions are sterile and biocompatible, which include saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

In addition, the composition of the present invention may also be a food composition, such a food composition may contain various flavors or natural carbohydrates as an additional component, as well as the conventional food composition, in addition to the above-described active ingredients. Examples of the food to which the present invention can be applied include, but are not particularly limited to, meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, Beverages, teas, drinks, alcoholic beverages and vitamin complexes, and the like, and includes all foods, in particular health foods in the conventional sense. The health food containing the composition according to the present invention is a folk object for the purpose of inhibiting the growth of harmful bacteria such as tea, jelly, juice, extract, beverage, etc. using the above strains, cultures thereof, or antibacterial substances isolated therefrom. Therapeutic agent is mentioned.

The composition of the present invention may also be a food additive composition. When the composition according to the present invention is used as a food additive, each of the above-mentioned active ingredients may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The food of the present invention may contain various flavors, sweeteners, natural carbohydrates, and the like as additional ingredients, like conventional foods. Natural carbohydrates described above include glucose, monosaccharides such as fructose, malsaccharides, disaccharides such as sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as tautin and stevia extract, synthetic sweeteners such as saccharin, aspartame, and the like can be used. Foods of the present invention other than the above include various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, And a carbonation agent used for the carbonated beverage.

The composition of the present invention may also be a feed additive composition. When the composition according to the present invention is used as a feed additive, each of the above-mentioned active ingredients may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. Or in animal feed. Feed additives of the present invention may be 20 to 90% high concentrate or in the form of powder or granules. The feed additives include organic acids such as citric acid, fumaric acid, adipic acid, lactic acid and malic acid, or phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate and polyphosphate (polyphosphate), polyphenols, catechins (catechin) and lees extracts ( rosemary extract), vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid and may further include any one or more of natural antioxidants. The feed additive of the present invention and the feed containing the same are supplementary ingredients such as amino acids, inorganic salts, vitamins, antibiotics, antimicrobials, antioxidants, antifungal enzymes, live microbial agents, etc. Wheat, oats, barley, corn and rice; Vegetable protein feed, such as rape, soybeans and sunflower; Animal protein feeds such as blood meal, meat meal, bone meal and fish meal; Sugar and dairy products, for example, a dry ingredient consisting of various powdered milk and whey powder, and a drying additive, all mixed together with a liquid component, and a component which becomes a liquid after heating, that is, a lipid, for example, animal liquefied by heating. In addition to the main components such as fats and vegetable fats can be used with substances such as nutritional supplements, digestive and absorption enhancers, growth promoters, disease prevention agents and the like. The feed additive may be administered alone in combination with other feed additives in an edible carrier. In addition, the feed additives can be easily administered as a top dressing or mixed directly into the feed or separately from the feed, in a separate oral formulation, by injection or transdermal or in combination with other ingredients.

In addition, the present invention may be a cosmetic composition. When used as a cosmetic composition, further substances may be added according to the formulation of the cosmetic. For example, but not limited to, when the formulation is a paste, cream or gel, the carrier component may be animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or Zinc oxide or the like may be used, and lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component when the formulation is a powder or a spray, and in particular in the case of a spray, additional chloro Propellants such as fluorohydrocarbons, propane / butane or dimethyl ether. In addition, when the formulation is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as the carrier component, preferably water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid ester of, 3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan, but is not limited thereto. If the dosage form is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystalline cellulose, Aluminum metahydroxide, bentonite, agar or tracant and the like can be used, and when the formulation is a surfactant-containing cleansing, as a carrier component, an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, isethionate, Imidazolinium derivatives, methyltaurate, sarcosinates, fatty acid amide ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters. Be But it is not limited thereto. The composition is used as a cosmetic composition, supple cosmetics, astringent cosmetics, nourishing cosmetics, nutrition cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, Body oil, body essence, makeup base, foundation, hair dye, shampoo, rinse or body cleansers, etc., but is not limited thereto.

The composition of the present invention may also be an external appearance composition. The "quasi drug" is a fiber, rubber product or the like used for the purpose of treating, alleviating, treating or preventing a disease of a human or animal, has a weak effect on the human body or does not directly act on the human body, and is not an apparatus or a machine. Similar to those used for sterilization, insecticidal and similar purposes for the prevention of infectious agents, for the purpose of diagnosing, treating, reducing, treating or preventing diseases of humans or animals. Non-apparatus, machinery, or device, and any item used for the purpose of pharmacologically affecting the structure and function of a person or animal, except for non-apparatus, machine, or device. In addition, the quasi-drug may include external skin preparations and personal hygiene products. Preferably, it may be, but is not limited to, a disinfectant cleaner, a shower foam, a gagreen, a wet tissue, a detergent soap, a hand wash, or an ointment.

According to one aspect of the invention, Bacillus safensis ( Bacillus safensis ) SR098 strain, its culture and antimicrobial material produced by the strain, and antimicrobial against various harmful bacteria of the probiotic or antimicrobial composition comprising any one or more of the above Serves the purpose. The antimicrobial use may be applied without limitation as long as the strain of the present invention is a harmful bacterium exhibiting antimicrobial activity.

The strain of the present invention, its culture and the antimicrobial material produced by the strain, and the probiotic or antimicrobial composition comprising any one or more of the above may have antimicrobial activity against harmful bacteria.

More specifically, the harmful bacteria is Listeria monocytogenes jeneseu (Listeria monocytogenes), Staphylococcus aureus (Staphylococcus aureus), Bacillus turing Gen System (Bacillus thuringensis), Bacillus cereus (Bacillus cereus), Yersinia Enterococcus coli urticae ( Yersinia enterocolitica ), Shigella flexneri , Shigella boydii , Escherichia coli , Cronobacter sakazakii , but are not limited thereto.

More specifically, the harmful bacterium may be any one or more selected from Listeria monocytogenes , Staphylococcus aureus , Bacillus thuringensis and Bacillus cereus . .

In addition, the strain of the present invention, its culture and the antimicrobial material produced by the strain, and the probiotic or antimicrobial composition comprising any one or more of the above may have an antimicrobial activity against antibiotic resistant strains.

The antibiotic resistant strains are vancomycin (Vancomycin), penicillin (Penicillin), methicillin (Methicillin), cephalothin (Eethromycin), nofloxacin (Norfloxacin), oxacillin (Oxacillin), gentamicin It may be a strain that is resistant to antibiotics such as, but not limited to, Gentamicin, Chloramphenicol, Sulfonamides, Streptomycin, Streptomycin, Carbapenem, and Tetracyclin .

According to one aspect of the present invention, there is provided a bacteriocin production method comprising culturing Bacillus safensis SR098 strain (KACC 92219P).

The medium used in the step of culturing the strain of the present invention can be used without limitation as long as it can help the growth of the strain and maintain a high antimicrobial activity. Therefore, it may be cultured in one or more media selected from the group consisting of LB medium, NB medium, BHI medium, KB (King's B) or TSB medium, but is not limited thereto. The strain may preferably be cultured in TSB medium, and more preferably the TSB medium may contain pancreatic digest of casein, enzymatic digest of soya been, sodium chloride. It may include di-potassium hydrogen phosphate and glucose.

Incubation temperature and incubation time of the strain according to the present invention can be carried out without limitation within the temperature and time to help the growth of the strain and maintain high antibacterial activity. Therefore, the culture may be incubated for 2 to 48 hours at pH 5.0 to 7.0, more preferably pH 5.7 to 6.8, 32 hours, but can be cultured divided into two times of 16 hours.

The bacteriocin production method of the present invention may further comprise the step of treating the culture obtained in the step of culturing the strain of the present invention by any one or more of filtration, concentration, extraction and drying process. . For example, centrifuging the culture; Filtering the supernatant obtained above; And it may further comprise the step of concentrating the filtrate obtained above.

The filtration process, the concentration process, the extraction process, and the drying process may be applied to all conventional methods that can be used by those skilled in the art for microbial culture. Therefore, commonly used filter filtration, membrane filtration, ultrafiltration, centrifugal separation, heat concentration, lyophilization, crushing, grinding, solvent extraction and hot water extraction may be used alone or in combination.

The filtration or extraction of the method for producing bacteriocin may be a process of removing impurities from the culture and further separating the antimicrobial active material. As used herein, 'separation' refers to a process comprising a purification step of obtaining a sufficiently purified compound of interest.

According to one aspect of the invention, Bacillus safensis ( Bacillus safensis ) SR098 strain (KACC 92219P), the culture of the strain and provides a method for inhibiting harmful bacteria characterized in that the treatment of the bacteriocin produced by the strain.

Specifically, the harmful bacteria is Listeria monocytogenes jeneseu (Listeria monocytogenes), Staphylococcus aureus (Staphylococcus aureus), Bacillus turing Gen System (Bacillus thuringensis), Bacillus cereus (Bacillus cereus), Yersinia Enterococcus coli urticae ( Yersinia enterocolitica ), Shigella flexneri , Shigella boydii , Escherichia coli , Cronobacter sakazakii , but are not limited thereto.

More specifically, the harmful bacterium may be any one or more selected from Listeria monocytogenes , Staphylococcus aureus , Bacillus thuringensis and Bacillus cereus . .

Specifically, the present invention may be subjected to extracellular treatment of any one or more of Bacillus safensis SR098 strain (KACC 92219P), the culture of the strain and the bacteriocin produced by the strain.

Specifically, the harmful bacterium removal or activity inhibition method may be applied to antibiotic resistant strains.

The antibiotic resistant strains are vancomycin (Vancomycin), penicillin (Penicillin), methicillin (Methicillin), cephalothin (Eethromycin), nofloxacin (Norfloxacin), oxacillin (Oxacillin), gentamicin It may be a strain that is resistant to antibiotics such as, but not limited to, Gentamicin, Chloramphenicol, Sulfonamides, Streptomycin, Streptomycin, Carbapenem, and Tetracyclin .

According to one aspect of the invention, Bacillus safensis ( Bacillus safensis ) SR098 strain (KACC 92219P), a harmful bacterium infection characterized in that the subject is administered any one or more selected from the culture of the strain and the bacteriocin produced by the strain. Provided are methods for preventing, alleviating, or treating a disease.

Specifically, the present invention may be administered to an animal other than human, any one or more selected from Bacillus safensis SR098 strain (KACC 92219P), the culture of the strain, and the bacteriocin produced by the strain.

Specifically, the method for preventing, alleviating, or treating a harmful bacterial infection disease may be applied to an antibiotic resistant strain infection disease.

As used herein, the term "prevention" may refer to any action of inhibiting or delaying the onset of a harmful bacterial infection disease by administering to a subject a composition for preventing or treating a microbial infection disease according to the present invention.

As used herein, the term "treatment" may refer to any action of administering the composition of the present invention to an individual suspected of developing a microbial infection disease so as to improve or benefit from the symptoms of the microbial infection disease.

As used herein, the term "relaxation" may mean any action that at least reduces the parameters associated with the condition being treated, for example, the degree of symptoms.

As used herein, the term "individual" may mean any animal, including humans, who have or are likely to develop a microbial infectious disease.

Any one or more of the strain of the present invention, its culture and the bacteriocin produced by the strain can be directly treated or administered to the infected part of the harmful bacterium, or can be prepared or treated with a suitable probiotic or antimicrobial composition of the above-mentioned forms.

The harmful bacterial infection diseases include food poisoning, dermatitis, vaginitis, diarrhea, vomiting, enteritis, gastroenteritis, constipation, abdominal pain, abdominal distension, cholera, listeriosis, cellulitis, urinary tract infection, meningitis, peritonitis, cystitis, lymphadenitis, sensitis , Respiratory disease, pneumonia, purulent inflammation, or sepsis.

Dosage of the antimicrobial composition of the present invention means the amount required to exert the effect of killing harmful microorganisms in the subject, the type of formulation and the age, weight, general state of health, sex and diet of the subject It may be adjusted according to various factors including the route of administration, time of administration and duration of administration. In the case of livestock or fish, the route of administration can be formulated to the extent allowed by veterinary medicine.

According to one aspect of the present invention, there is provided a use of any one or more of Bacillus safensis SR098 strain (KACC 92219P), a culture of the strain, and the bacteriocin produced by the strain in the preparation of the antimicrobial composition. .

Specifically, Bacillus safensis ( Bacillus safensis ) SR098 strain (KACC 92219P), the culture of the strain, and any one or more of the bacteriocin produced by the strain can be prepared as a probiotic or antimicrobial composition. The probiotic and antimicrobial compositions are as described above.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these are only intended to describe the present invention in more detail, but the scope of the present invention is not limited thereto.

Example 1 Screening and Identification of Microorganisms

Strains showing excellent antimicrobial activity were selected by comparing antimicrobial activity against representative food poisoning bacteria against various microorganisms collected in soil.

The experimental results, the screening strain is Bacillus cereus (Bacillus cereus), Bacillus turing Gen System (Bacillus thuringensis), L. monocytogenes jeneseu (Listeria monocytogenes), and Staphylococcus brother-less with respect to Gram-positive sikjungdokgyun such as (Staphylococcus aureus) It was confirmed that it has excellent antimicrobial activity.

Molecular phenotypic and other biochemical analyzes based on the 16S rDNA sequence of the selected strains confirmed that the strain was Bacillus safensis , and the strain was named Bacillus safensis SR098. On February 7, 2018, he was deposited with the National Academy of Agricultural Science and was assigned accession number KACC 92219P.

Example 2 Cultivation and Bacteriocin Isolation of Strains of the Invention

The culture supernatant of the Bacillus safensis SR098 strain selected in Example 1 was separated using an ultrafiltration membrane (3,000 MWCO PES, Vivaspin 20, Sartorius, Germany) to obtain a fraction by molecular weight.

Specific culture method is as follows. 3 ml of Tryptic soy broth (TSB) was put into a 50 ml conical tube, and one colony of Bacillus safensis SR098 of the present invention was taken and inoculated for 16 hours. The culture obtained above was centrifuged at 10,000 g for 20 minutes to remove the cells, and then filtered through a 0.22 μm filter. The filtered supernatant was concentrated to a final 20% by 3 kDa ultrafiltration (4,000 rpm).

Specific bacteriocin separation method is as follows. 30% precipitate of ammonium sulfate was removed by centrifugation and precipitated up to 60%, then 60% precipitate was removed by centrifugation, and the supernatant was again precipitated to 80%, and the precipitate was suspended in 20 mM Tris-Cl buffer at pH 8.0. Heat-treated at 80 ° C. for 30 minutes. Through heat treatment, a large amount of protein was denatured and precipitated. The precipitate was removed by centrifugation, and finally, the remaining supernatant was separated and concentrated to a 3 kDa pore size membrane.

Experimental Example 1 Investigation of Antimicrobial Spectrum of Selected Microorganisms

The antimicrobial activity against various food poisoning bacteria was confirmed for the fractions of 3 kDa or more in the fractions according to the present invention obtained in Example 2.

Specifically, the indicators for measuring antimicrobial activity were inoculated in TSA medium (Tryptic soy liquid medium), respectively, and incubated overnight at 37 ° C. 1% of each indicator culture medium was inoculated into soft agar medium (0.8% agar) cooled to 45 ° C. The soft agar medium inoculated with the indicator bacteria was poured into a disposable medium dish and cooled to prepare a plate medium. A paper disk was placed on the prepared flat medium, and 10 µl each of the strain fractions of the present invention prepared on the paper disk was incubated, and then incubated at an optimal growth temperature of each indicator for 24 hours. After incubation, the antimicrobial activity of each strain was measured by measuring the diameter of the growth zone (Clear zone) for each indicator. By comparing the diameters of growth inhibition rings, strains with high antimicrobial activity were selected for each strain. It was evaluated as-(not inhibiting growth), + (low-ring formation) depending on the occurrence of a low-ring ring showing the antimicrobial activity of the selected strains. The experimental results are shown in Tables 1 and 2.

Indicator bacteria-Gram-positive bacteria Bacillus cereus ATCC14579 Bacillus thuringensis ATCC29730 Listeria monocytogens ATCC15313 Listeria monocytogenes EDGe Staphylococcus aureus D-1 Staphylococcus aureus D-26
+ + + + - +

Indicator bacteria-Gram negative bacteria Cronobacter sakazakii ATCC29544 Eschericchia coli O157: H7
ATCC35150 Salmonella enterica subsp
arizonae ATCC13314 Salmonella enterica subsp enterica
serovar Typhimurium ATCC14028 Shigella boydii ATCC8700 Shigella flexneri ATCC12022 Yersinia entertocolitica subsp Enterocolitica ATCC55075 Yersinia entertocolitica subsp Enterocolitica ATCC9610 - - - - - - - -

Like the table results, the present invention is 3 kDa or more fractions isolated from the culture supernatant of the strain Bacillus cereus (Bacillus cereus), Bacillus turing Gen System (Bacillus thuringensis), L. monocytogenes jeneseu (Listeria monocytogenes), and Staphylococcus brother-less It was confirmed that it possesses excellent antimicrobial activity against Gram-positive bacteria such as ( Staphylococcus aureus ).

Experimental Example 2 Characterization of Bacteriocin According to the Present Invention

For the fractions (fractions) of 3 kDa or more obtained in Example 2, the experiments were performed under various temperature conditions, pH conditions, and various enzyme conditions, and the experimental results are shown in Table 3.

Experimental treatment stability No treatment (control) + Temperature conditions 60 ℃, 30 min + 80 ℃, 30 min - 100 ℃, 30 min - pH condition 2.0 + 4.0 + 6.0 + 8.0 + 10.0 + Enzyme treatment α-chymotrypsin - pronase E - proteinase K - trypsin - amylase + DNase + RNase +

As a result, it was confirmed that stability was maintained at various temperature and pH conditions, and it was found that the bacteriocin, a protein substance, was sensitive to proteolytic enzymes.

Experimental Example 3 Growth Inhibition of Bacteriocin Against Listeria

The bacteriocin obtained in Example 2 was confirmed to inhibit the growth of Listeria monocytogenes.

Specifically, in order to confirm the growth inhibitory effect of the bacteriocin produced by the strain of the present invention, the growth inhibitory ability of the target test bacteria was observed using a microplate reader (sense Hidex, Finland). Bacteriocin 1AU (minimum volume of soil supernatant supernatant to identify growth inhibition of 1 AU = 1 cm), 2 AU and 4 AU, respectively, at 10 ³ CFU / mL Listeria monocytogenes ( L. monocytogenes) ) ATCC 15313 was treated and incubated at 28 ° C. and 120 rpm for 24 hours, and the growth of the test strain was observed by measuring the OD value in units of 1 hour.

As a result of the experiment, it was confirmed that the logarithm of Listeria monocytogenes was delayed by 4 hours when the bacteriocin obtained in Example 2 was treated with 1AU, and the growth of Listeria monocytogenes was inhibited by 4 hours when treated with 4AU (Fig. 1).

In the above, the applicant has described the preferred embodiments of the present invention, these embodiments are only one embodiment for implementing the technical spirit of the present invention, any modification or modification of the present invention as long as the technical spirit of the present invention is implemented. Should be interpreted as being within the scope.

Agricultural Biotechnology Research Institute KACC92219P 20180207

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Bacillus Pumilus strain with antibiotic activity and antibiotic          use <130> P18-015 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1485 <212> DNA <213> Bacillus pumilus <220> <221> rRNA (222) (1) .. (1485) <223> 16S rDNA of Bacillus safensis SR098 <400> 1 gctcaggacg aacgctggcg gcgtgcctaa tacatgcaag tcgagcggac agaagggagc 60 ttgctcccgg atgttagcgg cggacgggtg agtaacacgt gggtaacctg cctgtaagac 120 tgggataact ccgggaaacc ggagctaata ccggatagtt ccttgaaccg catggttcaa 180 ggatgaaaga cggtttcggc tgtcacttac agatggaccc gcggcgcatt agctagttgg 240 tggggtaatg gctcaccaag gcgacgatgc gtagccgacc tgagagggtg atcggccaca 300 ctgggactga gacacggccc agactcctac gggaggcagc agtagggaat cttccgcaat 360 ggacgaaagt ctgacggagc aacgccgcgt gagtgatgaa ggttttcgga tcgtaaagct 420 ctgttgttag ggaagaacaa gtgcgagagt aactgctcgc accttgacgg tacctaacca 480 gaaagccacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc 540 cggaattatt gggcgtaaag ggctcgcagg cggtttctta agtctgatgt gaaagccccc 600 ggctcaaccg gggagggtca ttggaaactg ggaaacttga gtgcagaaga ggagagtgga 660 attccacgtg tagcggtgaa atgcgtagag atgtggagga acaccagtgg cgaaggcgac 720 tctctggtct gtaactgacg ctgaggagcg aaagcgtggg gagcgaacag gattagatac 780 cctggtagtc cacgccgtaa acgatgagtg ctaagtgtta gggggtttcc gccccttagt 840 gctgcagcta acgcattaag cactccgcct ggggagtacg gtcgcaagac tgaaactcaa 900 aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga 960 agaaccttac caggtcttga catcctctga caaccctaga gatagggctt tcccttcggg 1020 gacagagtga caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag 1080 tcccgcaacg agcgcaaccc ttgatcttag ttgccagcat tcagttgggc actctaaggt 1140 gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg 1200 acctgggcta cacacgtgct acaatggaca gaacaaaggg ctgcaagacc gcaaggttta 1260 gccaatccca taaatctgtt ctcagttcgg atcgcagtct gcaactcgac tgcgtgaagc 1320 tggaatcgct agtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac 1380 acaccgcccg tcacaccacg agagtttgca acacccgaag tcggtgaggt aacctttatg 1440 gagccagccg ccgaaggtgg ggcagatgat tggggtgaag tcgta 1485

Claims (13)

Bacillus safensis SR098 strain (KACC 92219P).
The method of claim 1,
The strain is Bacillus safensis SR098 strain (KACC 92219P), characterized in that it has 16S rDNA of the nucleotide sequence represented by SEQ ID NO: 1.
Probiotics comprising any one or more of the strain according to claim 1, the culture of the strain and the bacteriocin produced by the strain as an active ingredient.
The method of claim 3,
The probiotics are L. monocytogenes jeneseu (Listeria monocytogenes), Staphylococcus aureus (Staphylococcus aureus), Bacillus turing Gen System (Bacillus thuringensis), Bacillus cereus (Bacillus cereus), Yersinia Enterococcus coli urticae (Yersinia enterocolitica) , Shigella flexneri , Shigella boydii , Escherichia coli and Cronobacter sakazakii have antimicrobial activity against any one or more strains selected from the group consisting of. Probiotic, characterized in that.
Antimicrobial composition comprising any one or more of the strain according to claim 1, the culture of the strain and the bacteriocin produced by the strain as an active ingredient.
The method of claim 5,
For the antimicrobial composition L. monocytogenes jeneseu (Listeria monocytogenes), Staphylococcus aureus (Staphylococcus aureus), Bacillus turing Gen System (Bacillus thuringensis), Bacillus cereus (Bacillus cereus), Yersinia Enterococcus coli urticae (Yersinia antimicrobial activity against any one or more strains selected from the group consisting of enterocolitica , Shigella flexneri , Shigella boydii , Escherichia coli and Cronobacter sakazakii Antimicrobial composition comprising a.
The antimicrobial composition according to claim 5, wherein the antimicrobial composition is a pharmaceutical composition, a food composition, a food additive composition, a feed composition, a feed additive composition, a cosmetic composition or a quasi-drug composition.
Bacteriocin production method comprising the step of culturing Bacillus safensis SR098 strain (KACC 92219P).
The method of claim 8,
Centrifuging the culture obtained in the step of culturing the strain; Filtering the supernatant obtained after the centrifugation; And concentrating the filtrate obtained in the step of filtering the supernatant.
Bacillus safensis ( Bacillus safensis ) SR098 strain (KACC 92219P), a method for removing or inhibiting harmful bacteria characterized in that any one or more selected from the culture of the strain and the bacteriocin produced by the strain extracellularly treated.
The method of claim 10,
The harmful bacteria is Listeria monocytogenes jeneseu (Listeria monocytogenes), Staphylococcus aureus (Staphylococcus aureus), Bacillus turing Gen System (Bacillus thuringensis), Bacillus cereus (Bacillus cereus), Yersinia Enterococcus coli urticae (Yersinia enterocolitica) , Shigella flexneri , Shigella boydii , Shigella boydii , Escherichia coli , Escherichia coli and Cronobacter sakazakii ( Cronobacter sakazakii ) characterized in that any one or more selected from the group consisting of Activity Inhibition Method.
Preventing, alleviating, or preventing a harmful bacterial infection disease characterized by administering at least one selected from Bacillus safensis SR098 strain (KACC 92219P), the culture of the strain, and the bacteriocin produced by the strain to animals other than humans. Method of treatment.
The method of claim 12,
The harmful bacteria is Listeria monocytogenes jeneseu (Listeria monocytogenes), Staphylococcus aureus (Staphylococcus aureus), Bacillus turing Gen System (Bacillus thuringensis), Bacillus cereus (Bacillus cereus), Yersinia Enterococcus coli urticae (Yersinia enterocolitica) , Shigella flexneri , Shigella boydii , Shigella boydii , Escherichia coli and Escherichia coli ( Cronobacter sakazakii ) characterized in that any one or more selected from the group consisting of How to prevent, alleviate or treat.

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