KR102178350B1 - Bacillus safensis strain with antibiotic activity and antibiotic use thereof - Google Patents

Abstract

The present invention relates to a Bacillus safensis strain having an antimicrobial activity and an antibacterial use thereof, and more specifically, Bacillus safensis SR098 strain (KACC 92219P), a probiotic comprising the strain or a culture thereof, the strain Or it relates to an antibacterial composition comprising the culture, etc., a method of producing bacteriocin from the strain, and a method of inhibiting harmful bacteria using the strain.
The strain of the present invention shows excellent antibacterial activity against various food poisoning bacteria, and by using this, it is possible to reduce crop loss due to contamination in the production and distribution process of various agricultural and livestock products.

Description

Bacillus safensis strain with antibiotic activity and antibiotic use thereof {Bacillus safensis strain with antibiotic activity and antibiotic use thereof}

The present invention relates to a new strain for inhibiting the growth of harmful bacteria and its use, in particular, a novel strain having growth inhibitory activity against food poisoning bacteria such as Listeria monocytogenes, an antibacterial composition comprising the strain, and the like It relates to a method for producing a 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 that cause food poisoning.

In particular, Listeria monocytogenes contaminates most foods such as meat, fish and shellfish, cheese, ice cream, and frozen food, and is a deadly pathogen causing miscarriage, sepsis, meningitis, and food poisoning in pregnant women, newborns, and the elderly. Staphylococcus aureus is food poisoning. In addition, it is widely known as a bacterium that causes purulent diseases such as purulent, otitis media, and cystitis of the skin. These Listeria monocytogenes can proliferate by maintaining intracellular tension by accumulating osmolyte, an osmoprotective substance, in cells when subjected to osmotic stress such as high salt, high sugar, and drying, and even at low temperatures (4℃). Since it can proliferate and survive in the heat treatment process, attention is being focused on food hygiene.

On the other hand, chemical preparations are commercially used as preservatives to inhibit the growth of food poisoning microorganisms, but in recent years, due to the increase in consumer's health-oriented desire and stability problems, the avoidance of synthetic preservatives is remarkable. In order to cope with this problem, attention is focused on the development of an antibiotic substitute that is harmless to the human body. Such antibiotic substitutes include probiotics, bacterioncin, acidifers, immunomodulatory and enhancing substances, complex enzymes, and functional natural extracts, but among them, beneficial for host animals by improving the balance of intestinal microbes. Probiotics and bacteriocins that work are in the spotlight.

Probiotics are defined as microorganisms and components that improve the balance of intestinal microorganisms in humans or animals and have a beneficial effect on the host, and microorganisms used as probiotics include lactic acid bacteria, Bacillus bacillus, bacillus, and yeast. Conditions that must be provided as a probiotic in order to help the host include the ability to survive in the intestine by retaining the safety, acid resistance and bile acid resistance to the host, the ability to settle in the intestine, and to produce antibacterial substances to prevent pathogenic bacteria. The ability to inhibit, immune activity, easy mass production in the manufacturing process, and viability within the shelf life. In addition, in the case of livestock probiotics, it is easy to absorb nutrients due to improvement in growth rate and feed efficiency, digestion rate, and There is a demand for the effect of improving the odor of the livestock environment.

Bacteriocin is a protein or peptide-based antimicrobial substance secreted to the outside of cells, and is known to be a substance that kills or inhibits the growth of microorganisms closely related to the production strain. Currently, nisin, the only bacteriocin that has been put into practical use industrially, is used as a food preservative in processed cheese, vegetables, canned fruits, and fermented milk products in about 50 countries. As such bacteriocin is decomposed by various proteolytic enzymes in the digestive system, it has the advantage of being harmless and non-persistent to the human body, and according to the recognition that it is generally stable to the human body, various studies have recently been actively conducted to utilize it as a natural antibiotic or food preservative Has become.

Accordingly, Bacillus permilus HY1 strain having excellent antibacterial activity against Bacillus cereus and Listeria monocytogenes, and sulfectin produced therefrom (Patent Document 1), Bacillus bellengensis CJBV, and bacilomycin produced therefrom (Patent Document 2), Bacillus Te?quot;Nsys HD 15 (Patent Document 3) and Staphylococcus Pastry RSP-1 (Patent Document 4), and the like are known.

As such, attention has been focused on the development of natural antibacterial 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. Korean Patent Registration No. 10-0851063 (2008.08.12) 2. Korean Patent Registration No. 10-1629551 (2016.06.03) 3. Korean Patent Registration No. 10-1655781 (2016.09.02) 4. Korean Patent Registration No. 10-1374696 (2014.03.10)

The technical task to be achieved by the present invention is a novel strain capable of inhibiting the growth of harmful bacteria, especially food poisoning bacteria, and furthermore, antibiotic-resistant harmful bacteria in an effective and environmentally friendly manner, and using these strains to inhibit the growth of harmful bacteria in agricultural and livestock products, food, etc. To provide a way.

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

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

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

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 antibacterial activity against the above strains.

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

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 antibacterial 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, Bacillus sapensis ( Bacillus safensis ) provides a bacteriocin production method comprising the step of culturing the SR098 strain (KACC 92219P).

According to an aspect of the present 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 include the step of concentrating the filtrate obtained in the step of filtering the supernatant.

According to one aspect of the present invention, Bacillus safensis SR098 strain (KACC 92219P), a culture of the strain and at least one selected from the bacteriocin produced by the strain is treated outside the cells, characterized in that the removal of harmful bacteria Or it provides 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 SR098 strain (KACC 92219P), a culture of the strain, and any one or more selected from the bacteriocin produced by the strain is administered to an animal other than humans. It provides a method of preventing, alleviating or treating harmful bacterial infections.

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 ) Can be applied to any one or more strains selected from the group consisting of.

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

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

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

The present invention provides Bacillus safensis SR098 strain (KACC 92219P). The present inventors selected a Bacillus safensis Bacillus safensis SR098 strain having antibacterial activity against various food poisoning bacteria such as Listeria monocytogenes and Yersinia enterocolitica , and confirmed a novel bacteriocin material generated from the strain and completed the present invention .

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

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

The present invention provides a composition for antibacterial 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.

In the present specification, “culture” is a result obtained by inoculating a strain in a medium and culturing it for a certain period of time, and the culture solution itself (crude culture) obtained by culturing the strain according to the present invention in a suitable liquid medium, the culture solution is filtered or centrifuged. The filtrate (filtrate or centrifuged supernatant) from which the strain was removed, cell lysate obtained by sonicating the culture solution or treating the culture solution with lysozyme, the culture solution, filtrate, extract obtained from the lysis solution, and the culture solution , A filtrate, a crushing solution, a concentrated solution obtained by concentrating an extract, and the like, but are not limited thereto.

In the present specification, the term "concentrate" means that the culture medium is concentrated.

In the present specification, the term "extract" refers to extraction from the culture solution or its concentrate, and as long as it is an extract capable of exhibiting the antibacterial effect of the present invention, it is not limited thereto, and the extract, the diluted solution or the concentrate of the extract, and the extract are dried The resulting dried product, or these crude or purified products, and fractions obtained by fractionating them may be included.

In the present specification, the term "dried product" refers to drying the culture, its concentrate, and its extract. The drying method may be ventilated drying, natural drying, spray drying, and freeze drying, but is not limited thereto.

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

In the present specification, the term “probiotic” refers to a living microbial preparation designed to improve human or animal health, and it may be independently ingested or administered or included in medicines, foods and feeds, or dietary additives.

In the present invention, the meaning of "antibacterial" means the ability to reduce, prevent, inhibit, or eliminate the growth or survival of microorganisms at a specific concentration. The antimicrobial composition may have the same meaning as an antibiotic, which is a generic term for an antimicrobial agent, and may have the same meaning as an antimicrobial agent, fungicide, preservative, preservative or disinfectant, preferably Gram-positive bacteria, Gram-negative bacteria, fungi (yeast and mold ) Refers to a substance capable of inhibiting or inhibiting the development and life function of one or more microorganisms selected from the group consisting of, that is, a substance having antibacterial and antifungal effects.

The antimicrobial composition of the present invention may be administered directly to a portion infected with harmful microorganisms or may be prepared and administered in an appropriate form. The dosage of the antimicrobial composition of the present invention refers to the amount required to exert the effect of killing harmful microorganisms in the subject to be administered, and the type of the dosage form and the age, weight, general health condition, sex and diet of the administration subject. , Administration route, administration time and duration. When used for livestock or fish, it can be formulated within the range permitted by veterinary medicine and the route of administration can be determined accordingly.

The antibacterial composition of the present invention may be prepared as a solution, powder, suspension, dispersion, emulsion, oil 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 in pesticides, medicines, cosmetics, household goods, foods, etc. to achieve the purpose of antibacterial, sterilization, disinfection, and antiseptic. Specifically, for the purpose of antibacterial, sterilization, and disinfection in agriculture, for the same purpose as antibiotics or antifouling agents in medicine, for preservative or antibacterial purposes in food, for preventing dandruff, preventing athlete's foot in cosmetics and household goods, underarms It can be used for products directly related to microorganisms, such as for suppressing collection and anti-acne, or for antiseptic, antibacterial, or sterilizing purposes, such as cleaning detergents or dishwashing detergents, but is not limited to this purpose. Although 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-described active ingredients, and the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, expanding agents, lubricants, It is possible to use a lubricant or flavoring agent. For administration, the pharmaceutical composition may contain one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients, and may be preferably formulated into a pharmaceutical composition.

The formulation form 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, non-toxic 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 are, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, lacquercanth 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. As acceptable pharmaceutical carriers for compositions formulated as liquid solutions, sterilization and biocompatible, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, 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. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

In addition, the composition of the present invention may also be a food composition, and such a food composition may contain various flavoring agents or natural carbohydrates, etc. as an additional component, as in a conventional food composition in addition to containing the above-described active ingredient. Examples of the food to which the present invention can be applied are not particularly limited, and dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, Beverages, teas, drinks, alcoholic beverages, and vitamin complexes are included, and foods in the usual sense, especially health foods, are included. As a health food containing the composition according to the present invention, a private sector for the purpose of inhibiting the growth of harmful bacteria such as tea, jelly, juice, extract, beverage, etc. using the strain, its culture, or an antibacterial substance isolated therefrom as an active ingredient Therapeutic agents are mentioned.

The composition of the present invention may also be a food additive composition. When using the composition according to the present invention as a food additive, each of the above-described active ingredients may be added as it is or may be used 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 flavoring agents, sweeteners, natural carbohydrates, and the like as an additional component, like ordinary foods. The above-described natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame can be used. In the food of the present invention other than the above, various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, Carbonating agents used in carbonated beverages may be contained.

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-described active ingredients may be added as it is or may be used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The feed additive of the present invention is a plant. Or it can be added to animal feed. The feed additive of the present invention may be a high concentration of 20 to 90% or a powder or granular form. The feed additives include organic acids such as citric acid, humic acid, adipic acid, lactic acid, and malic acid, or phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, and polyphosphate (polyphosphate), polyphenols, catechins, and extracts of lee ( rosemary extract), vitamin C, green tea extract, licorice extract, chitosan, tannic acid, and natural antioxidants such as phytic acid. The feed additive of the present invention and the feed containing the same include various supplements such as amino acids, inorganic salts, vitamins, antibiotics, antibacterial substances, antioxidants, anti-fungal enzymes, and live microorganism preparations as auxiliary ingredients. Wheat, oats, barley, corn and rice; Vegetable protein feeds, such as those based on rapeseed, soybeans and sunflowers; Animal protein feeds such as blood meal, meat meal, bone meal and fish meal; After mixing all of the dry ingredients and dry additives consisting of sugar and dairy products, such as various powdered milk and whey powder, the liquid ingredient and the ingredient that becomes liquid after heating, i.e., a lipid, for example, an animal that is liquefied arbitrarily by heating In addition to the main ingredients such as fat and vegetable fat, it may be used with substances such as nutritional supplements, digestion and absorption enhancers, growth promoting agents, disease preventing 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 additive may be easily administered as a top dressing or directly mixed with the feed or separately from the feed, in a separate oral formulation, by injection or transdermally, or in combination with other ingredients.

In addition, the present invention may be a cosmetic composition. When used as a cosmetic composition, additional substances can be added according to the formulation of the cosmetic. For example, but not limited thereto, for example, when the formulation is a paste, cream or gel, 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 when the formulation is powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of spray, additional chloro Propellants such as fluorohydrocarbons, propane/butane or dimethyl ether. In addition, when the formulation is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, preferably water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan, but is not limited thereto. When the formulation is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, Aluminum metahydroxide, bentonite, agar, or tracant may be used, and when the formulation is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, Imidazolinium derivatives, methyltaurates, 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 are used. May be, but is not limited thereto. The composition is used as a cosmetic composition and is used as a softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, It may be formulated as a body oil, body essence, makeup base, foundation, hair dye, shampoo, conditioner, or body cleaner, but is not limited thereto.

The composition of the present invention may also be a quasi-drug composition. The above "quasi-drug" refers to fibers, rubber products, or the like, which are used for the purpose of treating, alleviating, treating or preventing diseases of humans or animals, weakly acting on the human body or not directly acting on the human body, and are not instruments or machines. It is one of the products used for sterilization, insecticide, and similar purposes for the prevention of infectious types, and among the products used for the purpose of diagnosing, treating, alleviating, treating or preventing diseases of humans or animals. It may mean an article other than an appliance, machine, or device among articles that are not instruments, machines, or devices, and articles used for the purpose of having a pharmacological effect on the structure and function of humans or animals. In addition, the quasi-drug may include skin external preparations and personal hygiene products. Preferably, it may be a disinfectant cleaner, shower foam, gagrin, wet tissue, detergent soap, hand wash, or ointment, but is not limited thereto.

According to an aspect of the present invention, antibacterial against various harmful bacteria of a Bacillus safensis SR098 strain, a culture thereof and an antibacterial substance produced by the strain, and a probiotic or antibacterial composition comprising any one or more of the above Provides a use. The antimicrobial use may be applied without limitation if the strain of the present invention is a harmful bacteria exhibiting antibacterial activity.

The strain of the present invention, its culture, and the antimicrobial material produced by the strain, and a probiotic or antibacterial composition comprising any one or more of the above may have antibacterial 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 flexneri ), Shigella boydii ( Shigella boydii ), Escherichia coli ( Escherichia coli ), Cronobacter sakazakii ( Cronobacter sakazakii ), but is not limited thereto.

More specifically, the harmful bacteria 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 a probiotic or antibacterial composition comprising any one or more of the above may have antibacterial activity against antibiotic resistant strains.

The antibiotic-resistant strains include Vancomycin, Penicillin, Methicillin, Cephalosin, Erythromycin, Norfloxacin, Oxacillin, and Gentamicin. Gentamicin), chloramphenicol, sulfonamides, streptomycin, carbapenem, and tetracycline may be a strain having resistance to antibiotics such as, but is not limited thereto. .

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

The medium used in the step of culturing the strain of the present invention may be used without limitation as long as it is a medium capable of supporting the growth of the strain and maintaining high antibacterial activity. Therefore, it may be cultured in one or more medium 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 be preferably cultured in TSB medium, and more preferably, the TSB medium is a pancreatic digest of casein, enzymatic digest of soya been, sodium chloride , Di-potassium hydrogen phosphate and glucose.

The cultivation temperature and cultivation time of the strain according to the present invention may be performed without limitation within a temperature and time capable of helping the growth of the strain and maintaining high antibacterial activity. Therefore, the cultivation may be performed for 2 to 48 hours at pH 5.0 to 7.0, more preferably at pH 5.7 to 6.8, for 32 hours, but may be cultivated by dividing in two times each for 16 hours.

The bacteriocin production method of the present invention may further include a step of treating the culture obtained in the step of culturing the strain of the present invention through any one or more of a filtration process, a concentration process, an extraction process, and a drying process. . In one example, centrifuging the culture; Filtering the supernatant obtained above; And it may further include 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 a person skilled in the art can use for microbial culture. Therefore, commonly used filter filtration, membrane filtration, ultrafiltration, centrifugation, heat concentration, freeze drying, crushing, pulverization, solvent extraction, hot water extraction, and the like may be used alone or in combination.

The filtration or extraction process of the method for producing bacteriocin may be a process of removing impurities from the culture and further separating an antimicrobial active substance. In the above, "separation" refers to a process including a purification step of obtaining a sufficiently purified corresponding compound.

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

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 , and Cronobacter sakazakii , but are not limited thereto.

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

Specifically, the present invention is 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 can be treated extracellularly.

Specifically, the method for removing harmful bacteria or inhibiting activity may be applied to antibiotic-resistant strains.

The antibiotic-resistant strains include Vancomycin, Penicillin, Methicillin, Cephalosin, Erythromycin, Norfloxacin, Oxacillin, and Gentamicin. Gentamicin), chloramphenicol, sulfonamides, streptomycin, carbapenem, and tetracycline may be a strain having resistance to antibiotics such as, but is not limited thereto. .

According to an aspect of the present invention, Bacillus safensis SR098 strain (KACC 92219P), a culture of the strain, and a harmful bacteria infection, characterized in that at least one selected from the bacteriocin produced by the strain is administered to an individual. Provides a method of preventing, alleviating or treating disease.

Specifically, 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 can be administered to animals other than humans.

Specifically, the method of preventing, alleviating, or treating harmful bacterial infections may be applied to antibiotic-resistant strain infections.

In the present specification, the term "prevention" may mean any action of inhibiting or delaying the onset of harmful bacterial infectious diseases by administering the composition for preventing or treating microbial infectious diseases according to the present invention to an individual.

In the present specification, the term "treatment" may refer to any action in which the composition of the present invention is administered to an individual suspected of developing a microbial infection to improve or benefit the symptoms of a microbial infection.

In the present specification, the term "relaxation" may refer to any action that at least reduces the severity of a parameter related to the condition being treated, for example, symptoms.

In the present specification, the term "individual" may mean all animals including humans who have developed or are likely to develop a microbial infection.

Any one or more of the strain of the present invention, its culture, and the bacteriocin produced by the strain may be directly treated or administered to a portion infected with harmful bacteria, or may be treated or administered by preparing the probiotic or antibacterial composition of the appropriate form described above.

Food poisoning, skin mycosis, vaginitis, diarrhea, vomiting, enteritis, gastroenteritis, constipation, abdominal pain, abdominal distention, cholera, listeriosis, cellulitis, urinary tract infection, meningitis, peritonitis, cystitis, lymphangitis, superficial, otitis media , Respiratory disease, pneumonia, purulent inflammation, or sepsis.

The dosage of the antimicrobial composition of the present invention refers to the amount required to exert the effect of killing harmful microorganisms in the subject to be administered, and the type of the dosage form and the age, weight, general health condition, sex and diet of the administration subject. , Administration route, administration time and duration. When used for livestock or fish, it can be formulated within the range permitted by veterinary medicine and the route of administration can be determined accordingly.

According to one aspect of the present invention, Bacillus safensis SR098 strain (KACC 92219P), a culture of the strain, and a bacteriocin produced by the strain provide a use of any one or more of the production of an 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 used as an active ingredient to prepare a probiotic or antibacterial composition. The probiotic and antibacterial composition are as described above.

Hereinafter, the present invention will be described in more detail through examples. However, these are only for describing the present invention in more detail, and the scope of the present invention is not limited thereto.

[Example 1] Selection and identification of microorganisms

By comparing the antimicrobial activity of representative food poisoning bacteria with respect to various microorganisms collected from the soil, strains showing excellent antibacterial activity were selected.

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 antibacterial activity.

It was confirmed that the strain was Bacillus safensis through molecular phylogenetic analysis based on the 16S rDNA sequence of the selected strain, and other biochemical analysis, and this strain was named Bacillus safensis SR098. , As of February 7, 2018, it was deposited with the National Academy of Agricultural Sciences and was granted the deposit number KACC 92219P.

[Example 2] Culture of the strain of the present invention and isolation of bacteriocin

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

The specific culture method is as follows. Tryptic soy broth (TSB) 3 ml was added to a 50 ml conical tube, and one colony of Bacillus safensis SR098 of the present invention was taken and inoculated and incubated 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 subjected to 3 kDa ultrafiltration (4,000 rpm) and concentrated to a final 20%.

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

[Experimental Example 1] Investigation of the antibacterial spectrum of the selected microorganism

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

Specifically, indicator bacteria for measuring antimicrobial activity were each inoculated into TSA medium (Tryptic soy liquid medium) and cultured at 37° C. overnight. 1% of each indicator bacteria culture was inoculated into a soft agar medium (0.8% agar) that was sterilized and 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 flat medium. A paper disk was placed on the prepared plate medium, and 10 μl of the prepared strain fractions of the present invention were instilled on the paper disk, followed by incubation for 24 hours at the optimum growth temperature of each indicator. After the culture was completed, the antibacterial activity of each strain was measured by measuring the diameter of the growth inhibition ring (Clear zone) for each indicator. By comparing the diameter of the growth inhibition ring, strains with high antibacterial activity were selected for each strain. According to the occurrence of low-lying rings indicating antimicrobial activity of the selected strains,-(cannot inhibit growth) and + (low-lipid ring formation) were evaluated. The experimental results are shown in Tables 1 and 2.

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

Indicator-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 ( Staphylococcus aureus ) It was confirmed that it has excellent antibacterial activity against Gram-positive bacteria such as D-26.

[Experimental Example 2] Evaluation of the properties of bacteriocin according to the present invention

Table 3 shows the results of the stability experiments for the fraction (fraction) of 3 kDa or more obtained in Example 2 under various temperature conditions, pH conditions, and various enzyme conditions.

Experiment treatment stability No treatment (control) + Temperature condition 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 of the experiment, it was confirmed that stability was maintained under various temperature and pH conditions, and it was found that it is a protein substance, bacteriocin, because it is sensitive to proteolytic enzymes.

[Experimental Example 3] Growth inhibitory ability of the bacteriocin according to the present invention against Listeria bacteria

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

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 test bacteria was observed using a microplate reader (sense Hidex, Finland). Bacteriocin 1AU (arbitrary unit, 1 AU = the minimum volume of the soil isolate culture supernatant to confirm growth inhibition of 1 cm), 2 AU and 4 AU each of 10 ³ CFU/ml Listeria monocytogenes ( L. monocytogenes) ) Treated with ATCC 15313 and cultured at 28° C. and 120 rpm for 24 hours, and the OD value was measured every hour to observe the growth of the test strain.

As a result of the experiment, it was confirmed that the logarithmic period of Listeria monocytogenes was delayed by about 4 hours when the bacteriocin obtained in Example 2 was treated with 1AU, and that the growth of Listeria monocytogenes was inhibited until 24 hours when 4AU was treated (Fig. 1).

In the above, the applicant has described the preferred embodiments of the present invention, but such embodiments are only one embodiment embodying the technical idea of the present invention, and any change or modification of the present invention as long as the technical idea of the present invention is implemented. Should be construed as falling within the scope

Institute of Agricultural Biotechnology KACC92219P 20180207

<110> REPUBLIC OF KOREA(MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Bacillus Pumilus strain with antibiotic activity and antibiotic use thereof <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 (14)

Bacillus safensis SR098 strain (KACC 92219P).
The method of claim 1,
The strain is Bacillus sapensis ( Bacillus safensis ) SR098 strain (KACC 92219P), characterized in that it has a 16S rDNA of the base sequence shown in SEQ ID NO: 1.
A probiotic 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 probiotic is any one or more selected from the group consisting of Listeria monocytogenes , Staphylococcus aureus D-26, Bacillus thuringensis , and Bacillus cereus . A probiotic, characterized in that it has antibacterial activity against the strain.
The antibacterial 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,
The antimicrobial composition is any selected from the group consisting of Listeria monocytogenes , Staphylococcus aureus D-26, Bacillus thuringensis , and Bacillus cereus Antibacterial composition, characterized in that it has antibacterial activity against one or more strains.
The antibacterial composition of claim 5, wherein the antibacterial composition is a food composition, a food additive composition, a feed composition, a feed additive composition, or a cosmetic composition.
Bacillus sapensis ( Bacillus safensis ) SR098 strain (KACC 92219P) bacteriocin production method comprising the step of culturing.
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. A method for producing bacteriocin, characterized in that it further comprises.
Bacillus sapensis ( Bacillus safensis ) SR098 strain (KACC 92219P), in the method for removing harmful bacteria or inhibiting activity, characterized in that at least one selected from the culture of the strain and the bacteriocin produced by the strain is treated outside the cell, The harmful bacteria are Listeria monocytogenes , Staphylococcus aureus D-26, Bacillus thuringensis , and Bacillus cereus , any one or more selected from the group consisting of Characterized in that, the method.
delete 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 administered to animals other than humans to prevent, alleviate, or In the treatment method, the harmful bacteria are Listeria monocytogenes , Staphylococcus aureus D-26, Bacillus thuringensis , and Bacillus cereus . Any one or more selected from the method, characterized in that.
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