JP2017128574A - Novel bacillus velezensis cjbv and antimicrobial composition containing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a newly isolated Bacillus velezensis strain CJBV having specific antibacterial activity against fungi and food poisoning-inducing bacteria.SOLUTION: According to the present invention, an antimicrobial composition comprises a Bacillomycin D analogue represented by formula (1) or an extract of a culture of Bacillus velezensis strain CJBV deposited as the accession number KCCM11528P, or a dried product thereof. Here, the extract or the dried product thereof includes a Bacillomycin D analogue.SELECTED DRAWING: None

Description

  The present invention relates to a novel Bacillus velezensis CJBV (Bacillus velezensis CJBV) strain having antibacterial activity specific to fungi and food poisoning-inducing bacteria, and an antibacterial composition using a basilomycin D analog isolated therefrom, and the same The present invention relates to a pharmaceutical composition for preventing or treating microbial infection diseases.

  When producing a fermented soybean food by a traditional method, since it is a food using microorganisms, it is exposed to the risk of food poisoning due to harmful bacteria contained in the microorganisms. However, harmful bacteria such as pathogenic fungi and food poisoning-inducing bacteria are not completely killed by general sterilization methods, but produce toxic substances to induce food poisoning.

  Therefore, chemical preservatives or preservatives are used to prevent deterioration and spoilage and maintain the quality of the food for a long period of time. However, the chemicals themselves act as environmental hormones or cause skin allergies. It tends to cause consumer resistance to chemical preservatives or preservatives.

  Therefore, interest in natural preservatives that are microorganisms or metabolites thereof and can inhibit the growth of spoiled microorganisms is rapidly increasing. As an example of natural antibacterial substances that can be used as preservatives, there is increasing interest in bacteriocin or bacteriocin-like substances. Bacteriocins are natural non-toxic preservatives and are antibacterial proteins consisting of proteins or protein-carbohydrate complexes produced by many gram positive and gram negative bacteria. In particular, since the US FDA approved the use of nisin among bacteriocins in 1998 for processed cheese spread foods, bacteriocin has been recognized as an antimicrobial preparation that can be used in the food industry.

  Research on the antibacterial substances produced by the genus Bacillus and lactic acid bacteria has been actively conducted, but unlike the lactic acid bacteria bacteriocin that can mainly inhibit the growth of Gram-positive bacteria, the bacteriocin produced by the Bacillus strain has its antibacterial properties. The active range is very wide and has various properties.

  Specifically, it is known that the genus Bacillus produces various antibacterial substances (Non-patent Document 1), and as reported antifungal substances, mycosubtilin, eumycin , Bacillomycin, surfactin, iturin, aminopolyol, lipoprotein, and other substances that have antibacterial and antifungal activities simultaneously. Bacilysin produced by Bacillus subtilis has been reported.

  As prior literature on this, Patent Document 1 describes that basilomycin D1, which is an antibacterial substance produced by Bacillus amyloliquefaciens strain (Bacillus amyloliquefaciens GYL4), has a wide range of activities against phytopathogenic fungi. Yes.

  Patent Document 2 discloses that as an antibacterial substance produced by Bacillus subtilis, UV254-B, which is a novel antibacterial substance completely different from an iturin substance containing basilomycin, is a food poisoning-inducing bacterium such as Bacillus cereus and Listeria monolith. It has been described that it exhibits antibacterial activity specifically only for cytogenes.

  However, the reported bacteriocin has only been reported to have antibacterial activity against specific microorganisms and is effective against various pathogenic bacteria that induce microbial infections including various crop diseases and food poisoning No bacteriocin has been reported. Furthermore, since Bacillus amyloliquefaciens described in the above document is a fermenting bacterium useful in fermented foods, even if the bacteriocin is applied to soybean fermented foods, food poisoning-inducing fungi In addition, since it is not considered to exhibit a specific activity only for bacteria, there is a problem that it is difficult to use it directly for fermented foods.

  Therefore, as a result of earnest research to develop a method that is harmless to the human body and suppresses various fungi and bacteria, the present inventors have newly isolated Bacillus bereshensis CJBV newly isolated from conventional miso onions. Not only has antibacterial activity against at least one kind of various fungi such as pathogenic fungi and yeast, but also has no antibacterial activity against fermented bacteria such as jean, and only the main harmful microorganisms of food such as food poisoning bacteria It was confirmed that the antibacterial activity was specifically suppressed, and the present invention was completed.

Korean Published Patent No. 10-2013-0008673 Korean Published Patent No. 10-2013-0025453

Sholberg et al., 1995 Can. J. Microbio. 41, 247-252

  The main object of the present invention is to provide a newly isolated Bacillus beresensis CJBV strain having antibacterial activity specific to fungi and food poisoning-inducing bacteria.

  Another object of the present invention is to provide a basilomycin D analog produced from the Bacillus berescensis CJBV strain.

  Furthermore, an object of the present invention is to provide an antibacterial composition using the Bacillus berescensis CJBV strain and the basilomycin D analog.

  Furthermore, this invention aims at providing the foodstuff composition containing the said antimicrobial composition.

  Furthermore, this invention aims at providing the feed composition containing the said antimicrobial composition.

  Furthermore, this invention aims at providing the antibacterial agent for plant crops containing the said antibacterial composition.

  Furthermore, an object of the present invention is to provide a method for controlling phytopathogenic fungi, which comprises a step of treating the antibacterial agent with a plant or soil.

  Furthermore, an object of the present invention is to provide a pharmaceutical composition for preventing or treating a microbial infection disease comprising the antibacterial composition.

  Furthermore, an object of the present invention is to provide a health functional food for preventing or ameliorating a microbial infection disease containing the antibacterial composition.

  Furthermore, an object of the present invention is to provide a quasi-drug containing the antibacterial composition.

  One aspect of the present invention for achieving the above object provides a newly isolated Bacillus velezensis CJBV strain having the accession number KCCM11528P having specific antibacterial activity against fungi and food poisoning-inducing bacteria.

  The term “Bacillus” in the present invention means an aerobic or facultative anaerobic Gram-positive Bacillus genus widely distributed in nature. Examples of microorganisms belonging to the genus Bacillus include Bacillus berescensis. The present inventors have elucidated a novel strain belonging to Bacillus berencensis, which is Bacillus having specific antibacterial activity against fungi and food poisoning-inducing bacteria, as follows.

  First, to isolate Bacillus strains from conventional miso onion, to test pathogenic fungi, fungi such as yeast and food poisoning bacteria, to select strains with excellent antibacterial activity, and to identify the selected strains 16S rDNA and gyrA gene base sequence analysis was performed. The 16S rDNA gene base sequence of the selected strain is SEQ ID NO: 1, which shows 99% homology with Bacillus berensis. Moreover, although the gyrA gene base sequence of the selected strain is SEQ ID NO: 2, this shows 100% homology with Bacillus berescensis (Example 1). Therefore, the strain was identified as Bacillus velezensis, named Bacillus velezensis CJBV, and deposited on April 17, 2014 at the Korean Microbiology Conservation Center (KCCM), an international depositary organization under the Budapest Treaty. And the accession number KCCM11528P was assigned (Example 1).

  Therefore, the Bacillus berescensis CJBV has a 16s rDNA base sequence represented by SEQ ID NO: 1.

  According to one aspect of the present invention, the newly isolated Bacillus beresensis CJBV not only has excellent antibacterial activity against fungi such as phytopathogenic fungi and yeast, but also suppresses food poisoning-inducing bacteria, and various fungi And antibacterial activity capable of simultaneously acting on food poisoning-inducing bacteria (Example 3, FIG. 3 and FIG. 4).

  In one embodiment, the fungus is selected from the group consisting of Colletotrichum orbiculare, Fusarium oxysporum, Candida albicans, and Zygosaccharomyces bailii. It may be a pathogenic fungus, but is not limited thereto.

  The term “choletoricum orbiclare” in the present invention means a fungus that causes anthracnose in cucumber, cucumber, watermelon and other vegetable crops of the family Cucurbitaceae.

  The term “fusarium oxysporum” in the present invention means a pathogenic fungus that causes Fusarium wilt in vegetable crops of the Cucurbitaceae family such as cucumber, cucumber, and watermelon.

  The inventors of the present invention have confirmed that newly isolated Bacillus berescensis CJBV has an antibacterial effect on the above-mentioned Coretotricum orbiclare and Fusarium oxysporum. Therefore, the novel strain can suppress plant infectious diseases such as anthracnose and vine split disease in cucumber vegetable crops such as cucumber, cucumber and watermelon, and therefore can be used as a microbial preparation for controlling plant diseases. .

  Therefore, using the newly isolated Bacillus bereshensis CJBV as an environmentally friendly non-polluting biological pesticide is useful for solving ecosystem destruction and toxicity problems due to organic synthetic pesticides, and reducing the financial burden on each farmer. it can. Moreover, since it is sustainable due to the characteristics of microorganisms, long-term benefits are expected in the future.

  The biological pesticide is defined as a product obtained by commercializing microorganisms separated and collected from the natural environment to control phytopathogenic fungi in crops.

  The term “Candida albicans” in the present invention is a genus name of a fungus belonging to imperfect fungi, and causes dermatomycosis, oral candidiasis, intestinal disease, vaginitis (Candida vaginitis) and the like. In particular, Candida vaginitis is a common disease in which about 75% of women suffer from Candida vaginitis of the vagina and vulva more than once in their lifetime, and 45% of women recur more than twice a year. Is caused by Candida albicans, but it can be transmitted when Candida albicans is normally present in the oral cavity, intestines, vagina, etc. of healthy humans when female hormones become abnormal or when immunity is extremely reduced. .

  The present inventors have confirmed that the newly isolated Bacillus berescensis CJBV has an antibacterial effect on the Candida albicans. Thus, the novel strain can effectively prevent or treat dermatomycosis, Candida vaginitis, oral microbial infection, and bowel disease.

  The term “Digosaccharomyces baili” in the present invention is a yeast of the genus Saccharomyces. Further, it is a spoilage yeast, and in beverage products and processed starch products, it produces a large amount of carbon dioxide gas together with alcoholic fermentation to lower the quality of food.

  The inventors of the present invention have confirmed that the newly isolated Bacillus berescensis CJBV has an antibacterial effect against the above-mentioned Digosaccharomyces baili. Thus, the novel strain can improve the preservability of food products such as beverage products and processed starch products, and can prevent or treat microbial infection diseases such as food poisoning that can occur from them.

  As another specific example, the food poisoning-inducing bacterium may be, but is not limited to, Bacillus cereus or Listeria monocytogenes.

  The term “Bacillus cereus” in the present invention is a Gram-positive bacterium that forms heat-resistant spores, but when infected causes diarrhea and vomiting. Aerobic bacteria, widely distributed in nature such as soil and cereals. Since the spores of this strain are superior in surface adhesion ability compared to the spores of other strains, they are characterized by difficulty in cleaning and disinfection. In addition, the Bacillus cereus may grow by germination of spores in meat, vegetables, rice, noodles, etc. that are left at room temperature for several hours after cooking, and may germinate in milk, cheese, etc. after pasteurization. Since it produces heat-resistant toxin and shows resistance to heat, its suppression is not easy.

  The present inventors have confirmed that newly isolated Bacillus berescensis CJBV has an antibacterial effect on the Bacillus cereus. Therefore, food poisoning, diarrhea, and vomiting due to infection can be prevented or treated by the novel strain.

  The term “Listeria monocytogenes” in the present invention is a zoonotic pathogen, which can be transmitted to humans through livestock foods such as milk and cheese, and has recently attracted worldwide attention. One of the pathogenic food poisoning bacteria. When listeriosis is caused by infection with the bacteria, the mortality rate is as high as about 30%. In particular, in the case of human infection, a weakly immune person or an immunocompromised person such as an infant, an organ transplant patient, a lymphoma, or an AIDS patient is likely to be infected and may cause sepsis, encephalitis and meningitis. Unlike other pathogenic bacteria, the pathogenic bacteria have the property that they can grow even at low temperatures, and it is known that the pathogenicity of bacteria grown at 4 ° C is stronger than that grown at 37 ° C. . Due to the characteristics of these bacteria, food poisoning due to Listeria monocytogenes can occur in foods that are considered to be safe at home, foods that are refrigerated, refrigerated foods, and preserved foods.

  The present inventors have confirmed that the newly isolated Bacillus bereshensis CJBV has an antibacterial effect on the Listeria monocytogenes. Thus, the novel strain can prevent or treat microbial infection diseases such as food poisoning and listeriosis.

  In particular, the present inventors not only have the effect that the newly isolated Bacillus berecensis CJBV of the present invention can simultaneously suppress the Bacillus cereus and Listeria monocytogenes, It was confirmed that there was no antibacterial activity against Bacillus subtilis and Bacillus amyloliquefaciens, which are useful fermented foods in fermented foods (Example 3 and FIG. 4).

  As another specific example, the Bacillus berescensis CJBV is an antibiotic Bacillomycin D, and a basilomycin D analog (analog) represented by Chemical Formula 1, that is, C14-Basilomycin D, C15-Basilomycin D, And at least one basilomycin D analog selected from the group consisting of C16-bacilomycin D can be produced and secreted.

  In the above chemical formula, R is CH3 in the case of C14-bacilomycin D, CH2-CH3 in the case of C15-bacilomycin D, and CH2-CH2-CH3 in the case of C16-bacilomycin D.

  The term “basilomycin D” in the present invention is a substance belonging to the Iturin system, which is one of the cyclic peptide groups, and includes seven amino acids (2Asn, Glu, Tyr, Pro, Ser, Thr) and one It has a cyclic structure consisting of β-amino acids and has antibiotic activity.

  As a result of separating the antibacterial substance produced and secreted by the newly isolated Bacillus bereshensis CJBV of the present invention and measuring the UV spectrum and the molecular weight, the present inventors found that the separated substance is the above three basilomycin D analogs. This was confirmed (Example 2, FIGS. 1 and 2).

  Another aspect of the present invention to achieve the above object provides a basilomycin D analog produced from the novel strain of Bacillus beresensis CJBV.

  The basilomycin D analog is represented by the following chemical formula 1.

  In the chemical formula, R is CH 3, CH 2 -CH 3, CH 2 -CH 2 -CH 3, the case of CH 3 is named C14-bacilomycin D, the case of CH 2 -CH 3 is named C 15 -bacilomycin D, and CH 2- The case of CH2-CH3 was named C16-bacilomycin D.

  As a result of separating the antibacterial substance produced and secreted by the newly isolated Bacillus bereshensis CJBV of the present invention and measuring the UV spectrum and the molecular weight, the present inventors found that the separated substance is the above three basilomycin D analogs. This was confirmed (Example 2, FIGS. 1 and 2).

  A basilomycin D analog produced from the novel strain of Bacillus berescensis CJBV is converted into a phytopathogenic fungus that contains anthracnose causing cholectricum orbiclare, Fusarium oxysporum causing vine split disease, dermatomycosis and Candida Candida albicans that can cause vaginitis, enteritis, oral inflammation, etc., Digosaccharomyces baili, a degrading yeast, Bacillus cereus, a food poisoning bacterium, and Listeria monocytogenes. The antibacterial effect was confirmed (FIGS. 3 and 4). Therefore, the basilomycin D analog produced from the novel Bacillus bereshensis CJBV strain can be used as an antibacterial composition, and this can be used as a feed, pharmaceutical composition, health function for prevention, improvement or treatment of microbial infection diseases. It can be effectively used in foods, quasi-drug compositions, foods having antibacterial activity, antibacterial agents for plant crops, and control methods using the same.

  As yet another aspect for achieving the above object, the present invention provides the newly isolated Bacillus berescensis CJBV strain, the spores of the strain, the culture of the strain, the concentrate, the extract thereof, and the drying thereof. An antibacterial composition comprising at least one selected from the group consisting of substances as an active ingredient is provided. Although the state of a strain is a liquid state or a dry state, it is not limited to these.

  The term “spore” in the present invention refers to a germ cell such as a bacterium, and for the purpose of the present invention, the spore means a germ cell of Bacillus beresensis CJBV. In the present invention, the spore of Bacillus beresensis CJBV exhibits antibacterial activity against fungi and food poisoning bacteria, so it can be used in microbial preparations for controlling plant diseases, foods with antibacterial activity, feeds, pharmaceuticals, quasi drugs, and health functional foods. It can be used by containing.

  The term “culture” in the present invention means a product obtained by culturing the above strain, and may be a culture stock solution containing cells, or cells from which the culture strain and culture supernatant have been removed. Or concentrated bacterial cells. The composition of the culture may include not only components necessary for normal Bacillus culture, but also components that act synergistically with the growth of Bacillus, and those compositions are easy for those having ordinary knowledge in the art. Can be selected.

  The term “concentrate” in the present invention means a concentrate of the culture.

  The term “extract” in the present invention means an extract extracted from the culture or a concentrate thereof, and any extract that exhibits a specific antibacterial effect on the fungi and food poisoning-inducing bacteria of the present invention may be used. It is not limited, and all of extract, diluted solution or concentrate of the extract, dried product obtained by drying the extract, or their preparation or purified product, and fractions thereof are included.

  The term “dried product” in the present invention means a product obtained by drying the culture, its concentrate, its extract, and its fraction. Ventilation drying, natural drying, spray drying and freeze drying can be used as the drying method, but the drying method is not limited thereto.

  The newly isolated Bacillus bereshensis CJBV of the present invention can be cultured by a normal method for culturing Bacillus strains, but is not limited thereto. As the medium, a natural medium or a synthetic medium can be used. As the carbon source of the medium, for example, glucose, sucrose, dextrin, glycerin, starch and the like can be used, and as the nitrogen source, peptone, meat extract, yeast extract, dry yeast, soybean, ammonium salt, nitrate And other organic or inorganic nitrogen-containing compounds can be used, but are not limited to these components. As the inorganic salt contained in the medium, magnesium, manganese, calcium, iron, phosphorus and the like can be used, but are not limited thereto. In addition to the components of the carbon source, nitrogen source and inorganic salt, amino acids, vitamins, nucleic acids and related compounds may be added to the medium.

  The antibacterial composition may include other by-products produced in the process of culturing Bacillus bereshensis CJBV.

  As yet another aspect of the present invention for achieving the above object, the present invention provides a food composition comprising the antibacterial composition.

  The type of the food is not particularly limited, and includes all foods in a non-limiting sense. Examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice creams, various soups, Examples include soft drinks, tea, drinks, alcoholic beverages, and vitamin complexes. When using the said composition as a food additive, the said composition may be added as it is, may be used with another foodstuff or a food component, and may be used suitably by a normal method.

  The food composition may include a pharmaceutically acceptable carrier.

  In particular, the present inventors have the effect that the newly isolated Bacillus beresensis CJBV of the present invention can simultaneously suppress the degrading yeast Digosaccharomyces baili, the food poisoning bacteria Bacillus cereus, and Listeria monocytogenes. In addition, there is no antibacterial activity against Bacillus subtilis and Bacillus amyloliquefaciens, which are useful fermented foods in fermented foods (Example 3 and FIG. 4). Therefore, the antibacterial composition can be directly used for any food including fermented food.

  As still another aspect of the present invention for achieving the above object, the present invention provides a feed composition comprising the antibacterial composition.

  The term “feed” in the context of the present invention means any natural or artificial diet, a meal etc., or a component of said meal for an animal to eat, ingest and digest, or suitable for it.

  The kind of the said feed is not specifically limited, The feed normally used in the said technical field can be used. The feed composition may include a feed additive. The feed additive of the present invention corresponds to an auxiliary feed in the feed management method, and may contain a live fungicide. Examples of the feed include vegetable feeds such as cereals, nuts, food processing by-products, algae, fiber, pharmaceutical by-products, fats and oils, starches, cucurbitaceae plants or cereal by-products, Examples thereof include animal feeds such as proteins, inorganic substances, fats and oils, minerals, single-cell proteins, zooplanktons, and foods and drinks. These may be used alone or in combination of two or more.

  Here, the feed composition containing the antibacterial composition of the present invention may further contain an excipient, a diluent and an additive.

  The newly isolated Bacillus beresensis CJBV of the present invention was confirmed to have the effect of simultaneously suppressing the degrading yeast Digosaccharomyces baili, the food poisoning bacteria Bacillus cereus, and Listeria monocytogenes. . Therefore, the present invention can prevent microbial infection diseases including food poisoning of livestock by providing a feed composition having antibacterial and antifungal activities.

  As still another aspect of the present invention for achieving the above object, the present invention provides an antibacterial agent for plant crops comprising the antibacterial composition.

  The present inventors have shown that the newly isolated Bacillus beresensis CJBV of the present invention has an inhibitory effect on phytopathogenic fungi including, for example, Colletotricum orbiclare causing anthrax and Fusarium oxysporum causing vine split disease. Confirmed (FIG. 3). Therefore, the antibacterial composition of the present invention is used as an environmentally friendly non-polluting biological pesticide, is useful for solving ecosystem destruction and toxicity problems with organic synthetic pesticides, and can reduce the financial burden on each farmer. . In addition, long-term benefits are expected because it is sustainable due to the characteristics of microorganisms.

  As yet another aspect of the present invention for achieving the above object, the present invention provides a method for controlling phytopathogenic fungi, comprising a step of treating the plant crop antibacterial agent on a plant body or soil.

  The phytopathogenic fungi are preferably, but are not limited to, Choletotricum orbiclare that causes anthrax and Fusarium oxysporum that causes vine split disease.

  The microbial preparation is prepared by foliar treatment on the ground part of the plant body and irrigation on the root part of the plant body after 15 days of planting, or in the rainy season and immediately before the onset, when the soil is opened before planting the crop. Although it is preferable to spray, it is not limited to these.

  As still another aspect for achieving the above object, the present invention provides a pharmaceutical composition for preventing or treating a microbial infection disease comprising the antimicrobial composition.

  The term “prevention” in the present invention means any action that suppresses or delays the onset of a microbial infection disease by administering to the individual the composition for the prevention or treatment of the microbial infection disease according to the present invention.

  The term “treatment” in the present invention means any action that improves or suitably changes the symptoms of a microbial infection disease by administering the composition of the present invention to an individual suspected of having a microbial infection disease.

  The term “amelioration” in the context of the present invention means any act that at least reduces a parameter relating to the condition to be treated, eg the degree of symptoms.

  The term “individual” in the present invention means any animal including a human who is likely to develop or may develop a microbial infection disease.

  Here, the microbial infection disease is a disease caused by infection with microorganisms, and is preferably a disease caused by infection with fungi or food poisoning-inducing bacteria.

  By way of example, the fungi may include Coretotricum orbiclare, Fusarium oxysporum f. sp. It may be selected from the group consisting of Leyco Persicium JCM12575, Candida albicans, and Gigosaccharomyces baili.

  As another example, the food poisoning-inducing bacterium may be Bacillus cereus or Listeria monocytogenes.

  As yet another example, the microbial infection diseases include food poisoning, dermatomycosis, vaginitis, diarrhea, vomiting, enteritis, gastroenteritis, stool, abdominal pain, abdominal distension, cholera, listeriosis, cellulitis, urinary tract It may be an infection, meningitis, peritonitis, cystitis, lymphangitis, itching, otitis media, respiratory disease, pneumonia, purulent inflammation, or sepsis.

  Cellulitis refers to acute suppurative inflammation that appears in the dermis and subcutaneous tissue, with suppurative inflammation occurring in the tissue and strong neutrophil infiltration extending into the interstitial space chronically and extensively. Yes, with hyperemia and edema.

  By felon is meant acute suppuration of the fingers or toes.

  The present inventors are Candida albicans, a degrading yeast, which can cause dermatomycosis, candida vaginitis, enteritis, oral inflammation, etc. when present in the oral cavity, intestine, and vagina of animals including humans and is infected. This can cause food poisoning, vomiting, diarrhea, fever, etc., infection can cause food poisoning, diarrhea, vomiting, Bacillus cereus, infection can cause food poisoning, listeriosis, sepsis, encephalitis, meningitis, etc. The excellent inhibitory effect of the strain of the present invention against Listeria monocytogenes was confirmed (FIGS. 3 and 4). Therefore, the antibacterial composition of the present invention can be effectively used as a pharmaceutical composition for preventing or treating microbial infection diseases.

  The composition for preventing or treating microbial infection disease according to the present invention may further contain a pharmaceutically acceptable carrier, and is formulated with the carrier to prepare a food, health functional food, pharmaceutical, quasi drug, feed additive. And as a drinking water additive. The term “pharmaceutically acceptable carrier” in the present invention means a carrier or diluent that does not irritate the organism and does not inhibit the biological activity and properties of the administered compound.

  The type of the carrier used in the present invention is not particularly limited, and any carrier can be used as long as it is a pharmaceutically acceptable carrier usually used in the art. Examples of the carrier include saline, sterilized water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerin, ethanol and the like. These may be used alone or in combination of two or more.

  If necessary, other usual additives such as antioxidants, buffers and / or bacteriostatic agents may be added and used as diluents, dispersants, surfactants, binders, lubricants. Further, an agent or the like may be added to prepare an injection dosage form such as an aqueous solution, suspension or emulsion, a pill, a capsule, a granule or a tablet.

  The administration method of the composition for preventing or treating microbial infection disease according to the present invention is not particularly limited, and a method commonly used in the art can be employed. As an example of the administration method, the composition can be administered by oral administration or parenteral administration. The composition for preventing, improving or treating the microbial infection disease of the present invention can be prepared in various dosage forms according to the intended administration method.

  The pharmaceutical dosage form of the antimicrobial substance of the present invention may be used in the form of these pharmaceutically acceptable salts. Moreover, it may be used alone, and may be used in an appropriate combination as well as binding to other pharmaceutically active compounds.

  As yet another aspect, the present invention provides a method for preventing or treating a microbial infection disease, comprising the step of administering to the individual the composition for the prevention or treatment of a microbial infection disease. The term “individual” in the present invention means any animal including a human who has or is likely to develop a microbial infection disease.

  Specifically, the prevention or treatment method of the present invention may include a step of administering the composition in a pharmaceutically effective amount to an individual who has developed a microbial infection disease or an individual who is likely to develop the disease.

  A suitable total daily usage of the composition comprising the extract is determined by the attending physician within the scope of sound medical judgment and can be administered in one or several divided doses. However, for the purposes of the invention, the specific therapeutically effective amount for a particular patient is the specific composition, including the type and extent of the response to be achieved and, in some cases, whether other formulations are used. Product, patient age, weight, general health, sex, diet, administration time, route of administration, secretion rate of the composition, duration of treatment, drugs used in combination with or co-administered with a specific composition, etc. It is preferable to apply different amounts depending on the various factors and similar factors well known in the pharmaceutical art.

  The antibacterial substance of the present invention can be administered to mammals such as rats, mice, domestic animals and humans by various routes. Any method of administration is possible, but can be administered, for example, by oral, rectal, or intravenous, intramuscular, subcutaneous, intrauterine dura or intracerebroventricular injection.

  As yet another aspect, the present invention provides a health functional food for preventing or ameliorating a microbial infection disease comprising the antibacterial composition.

  Here, the meanings of microbial infection disease, prevention and improvement are as described above.

  As yet another aspect, the present invention provides a quasi-drug composition comprising the antibacterial composition.

  In the present invention, the term “quasi-drug” means a fiber, rubber product or the like used for the purpose of treating, reducing, treating or preventing human or animal diseases, weak action on the human body, Articles that fall under any of the following categories: non-instrumental or non-instrumental or similar products, or products used for sterilization, insecticidal and similar uses for infectious prophylaxis. Articles used for the purpose of diagnosing, treating, mitigating, treating, or preventing diseases that are not instruments, machines or devices, and articles used for the purpose of pharmacologically affecting the structure and function of humans or animals Means an article that is not a device. The quasi-drug includes skin external preparations and personal hygiene products. Hand disinfectants, shower foams, mouth washes, wet tissues, detergents, soaps, hand wash or ointments are preferred but not limited.

  The present inventors have confirmed that the newly isolated Bacillus berescensis CJBV has an antibacterial effect on the Candida albicans. Therefore, the antibacterial composition using the novel strain can be effectively used as a quasi-drug for the treatment, reduction, treatment or prevention of microbial infection diseases including dermatomycosis and candida vaginitis.

  The newly isolated Bacillus beresensis CJBV of the present invention has an antibacterial effect against one or more different types of fungi and bacteria such as pathogenic fungi and yeast, and therefore has an antibacterial agent for plant crops and an antibacterial activity It can be used as a food, feed for prevention, improvement and treatment of microbial infection disease, health functional food, quasi-drug, and pharmaceutical.

  In particular, the newly isolated Bacillus bereshensis CJBV of the present invention has an antibacterial activity that specifically inhibits only the main harmful microorganisms of foods such as food poisoning bacteria, and therefore can be used directly in fermented foods.

It is a figure which shows the process of isolate | separating the basilomycin D analog which is an antibacterial active ingredient from the Bacillus bereshensis CJBV of this invention. It is a figure which shows the ESI-MS and UV spectrum of the antimicrobial substance produced from the strain by this invention. It is a figure which shows the antibacterial activity with respect to four types of fungi, Colletotricum orbiclare, Fusarium oxysporum, Candida albicans, Digosaccharomyces baili. It is a figure which shows the antibacterial activity with respect to Bacillus cereus and Listeria monocytogenes which are food poisoning induction bacteria, and Bacillus subtilis and Bacillus amyloliquefaciens which are jean (soy) fermenting bacteria.

  Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by the following examples.

Example 1: Isolation and identification of strains producing antibacterial substances (1) Separation and selection of strains In order to select strains having antibacterial activity, Bacillus strains isolated from conventional miso onions were used. The strain was precultured in a soybean fermentation liquid medium at 37 ° C. for 36 hours. A paper disk having a diameter of 8 mm was placed on an agar solid medium in which pathogenic fungi and food poisoning-inducing bacteria are lawn cells, and inoculated with a pre-cultured strain. Strains that were cultured for 12 hours and then formed transparent circles simultaneously against fungi and bacteria around the strains inoculated into the medium were selected.

(2) Identification of strains The 16S rDNA and gyrA nucleotide sequences of the selected strains were analyzed by a conventional method known in the art. NCBI BLAST was used to compare the GenBank database with the 16S rDNA base sequence (SEQ ID NO: 1) and gyrA base sequence (SEQ ID NO: 2) of the selected strains. As a result, the 16S rDNA base sequence of this strain showed 99% homology with Bacillus berescensis, and the gyrA base sequence showed 100% homology with Bacillus berescensis.

配列番号２：

From these results, the strain obtained according to the present invention was identified as the Bacillus beresensis strain and named as Bacillus beresensis CJBV. The accession number KCCM11528P was given.

SEQ ID NO: 1:

SEQ ID NO: 2:

Example 2: Separation and Purification of Antibacterial Substance An experiment was conducted to separate the antibacterial substance produced by newly separated Bacillus beresensis CJBV. The process is shown in FIG.

  The strain was inoculated into 400 μL of soybean fermentation liquid medium, and then cultured with shaking at 200 rpm at 37 ° C. for 48 hours. The culture solution was centrifuged at 8000 × g for 20 minutes to obtain a supernatant, and then extracted using butanol.

  The separated extract was concentrated under reduced pressure, and then chromatographed using a YMC HPLC column and a C18 sep-pak cartridge. Each fraction was developed by mixing 80% v / v methanol at a predetermined ratio, and fractions having antibacterial activity were separated and purified.

  In order to examine the UV spectrum and molecular weight (Mass) of the separated active substance, the purified sample was dissolved in a methanol solvent and measured by the ESI-MS method.

  FIG. 2 is a diagram showing the ESI-MS and UV spectrum of the separated antibacterial substance.

  From the above analysis, it was confirmed that the substances separated according to the present invention were three kinds of analogs of basilomycin D represented by the following chemical formula 1.

  The R is CH3 in the case of C14-bacilomycin D, CH2-CH3 in the case of C15-bacilomycin D, and CH2-CH2-CH3 in the case of C16-bacilomycin D.

Example 3: Antibacterial activity test against fungi and food poisoning-inducing bacteria To confirm whether the newly isolated Bacillus beresensis CJBV has an inhibitory effect on fungi and food poisoning-inducing bacteria, the following antibacterial activity The experiment was conducted.

(1) Antibacterial activity test against yeasts and molds (fungi) First, the molds Choletotricum orbiclare, Fusarium oxysporum f. sp. Experiments were conducted to determine whether Bacillus berescensis CJBV newly isolated against Lycopersici JCM12575 (Fusarium oxysporum f.sp. lycopersici JCM12575), Candida albicans, and the pathogenic yeast Digosaccharomyces baili exhibited antibacterial activity.

  Each of the four fungi was cultured on a potato agar (Potato dextrose agar, PDA) medium. Thereafter, a butanol extract of Bacillus bereshensis CJBV culture solution, which is an antibacterial composition concentrated under reduced pressure according to Example 2, was dissolved in ethanol, and then a paper disk having a diameter of 8 mm on each plate medium smeared with the above four strains. Then, a predetermined amount of 25 μL of the antibacterial substance was added and cultured at 30 ° C. for 24 to 48 hours to observe whether or not a growth inhibition circle was formed by the antibacterial substance produced by the isolate.

(2) Antibacterial activity test against food poisoning-inducing bacteria In order to examine whether the newly isolated Bacillus beresensis CJBV has antibacterial activity specifically only against food poisoning-inducing bacteria in fermented foods, the following experiment was conducted.

  TSA (Tryptic soy agar) medium containing Bacillus cereus and Listeria monocytogenes, which are food poisoning-inducing bacteria, and Bacillus subtilis and Bacillus amyloliquefaciens, which are fermented jean bacteria. In culture.

  Thereafter, the butanol extract (Extraction with BuOH) of the culture solution of Bacillus berescensis CJBV, which is an antibacterial composition concentrated in vacuo according to Example 2, was dissolved in ethanol, and then placed on each plate medium smeared with the above four strains. A predetermined amount of 25 μL of the antibacterial substance was added by placing a paper disk having a diameter of 8 mm, and cultured at 37 ° C. for 24 hours to observe whether or not a growth inhibition circle was formed by the antibacterial substance produced by the isolate.

  In the two kinds of jean fermenting bacteria, butanol (BuOH) was used in place of the butanol extract of the Bacillus bereshensis CJBV culture solution as a control group in addition to the experimental group.

  The results of Example 3 are shown in FIG. 3 and FIG. 4, and the degree of antibacterial activity depending on the degree of formation of the growth inhibition circle is shown in Table 1 below.

  “-” In Table 1 is a case where growth cannot be inhibited, “+” is a case where the diameter of the growth inhibition circle is 3 mm to 5 mm, and “++” is a case where the diameter of the growth inhibition circle is 6 mm to 10 mm. “++++” is the case where the diameter of the growth inhibition circle is 10 mm or more.

  As shown in the upper part of FIG. 3, the newly selected Bacillus beresensis CJBV strain of the present invention, a spore of the strain, a culture of the strain, a concentrate thereof, an extract thereof, and a dried product thereof are selected. As a result of treating the antibacterial composition containing at least one of the above as an active ingredient with the pathogenic fungi Coretotricum orbiclare and Fusarium oxysporum, a growth inhibition circle of 10 mm or more is formed, and the antibacterial activity that suppresses the growth of fungi It was confirmed to be excellent.

  Therefore, the newly isolated Bacillus berescensis CJBV and antibacterial composition of the present invention can suppress plant infectious diseases such as anthracnose and vine split disease in cucumber, cucumber and watermelon vegetable crops. The present invention can be effectively used in a microbial preparation for controlling plant diseases and a method for controlling phytopathogenic fungi using the same.

  In addition, as shown in the lower part of FIG. 3, the newly isolated Bacillus beresensis CJBV of the present invention, the basilomycin D analog produced from the strain, or the antibacterial composition is converted into a pathogenic fungus Candida albicans, Digo As a result of treatment with Saccharomyces baili, growth inhibition circles of 6 mm to 10 mm were formed, and it was confirmed that they were excellent in antibacterial activity to suppress fungal growth.

  Therefore, the newly isolated Bacillus berescensis CJBV of the present invention, the basilomycin D analog produced from the strain, and the antibacterial composition are dermatomycosis, candida vaginitis, prevention against microbial infection diseases in the oral cavity, intestine, etc. It shows improvement and therapeutic effects, and can be effectively used for pharmaceuticals, quasi drugs, health functional foods, feeds, and the like. Moreover, it can be effectively used as a food with a low risk of food poisoning.

  In addition, as shown in FIG. 4, the newly isolated Bacillus beresensis CJBV or antibacterial composition of the present invention was simultaneously treated with food poisoning-inducing bacteria and useful bacteria used during fermentation of soy sauce, resulting in food poisoning-inducing bacteria. It has been confirmed that it exhibits excellent antibacterial activity against Bacillus cereus and Listeria monocytogenes, but does not inhibit the growth of fermented bacteria.

  Since bacteriocin reported to be produced by conventional microorganisms does not specifically show antibacterial activity only for food poisoning-inducing bacteria, it affects the growth of jean-fermenting bacteria. There was a problem that it was difficult to use directly in food.

  On the other hand, the newly isolated Bacillus berencensis CJBV of the present invention, the basilomycin D analog produced from the above strain, and the antibacterial composition using the same are antibacterial against jean fermenting bacteria. It has no activity and has antibacterial activity that specifically inhibits only the main harmful microorganisms of food such as food poisoning bacteria, so it can be used directly in fermented foods.

  Furthermore, it has been confirmed that it has excellent antibacterial activity against various food contamination sources. Therefore, the newly isolated Bacillus berescensis CJBV of the present invention, the basilomycin D analog produced from the strain, and the antibacterial composition using the same are preventive, improved against microbial infection diseases including food poisoning, diarrhea and vomiting, It shows a therapeutic effect and can be effectively used for foods, pharmaceuticals, quasi drugs, health functional foods, feeds and the like.

  Thus, the newly isolated Bacillus berescensis CJBV of the present invention and the basilomycin D analog produced from the strain have an antibacterial effect on at least one different type of fungi and bacteria. It can be used as an antibacterial agent for crops, a food having antibacterial activity, and a feed for preventing, ameliorating and treating microbial infection diseases, a health functional food, a quasi-drug, and a pharmaceutical.

  From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without altering its technical idea or essential features. It should be understood that the embodiments are illustrative in all aspects and not limiting. The scope of the present invention should be construed to include all modifications and variations derived from the meaning and scope of the claims and their equivalents rather than the detailed description.

Depositary name: Korea Microbial Preservation Center (Overseas)
Accession number: KCCM11528P
Date of entrustment: 20144017

Claims (4)

(I) an antibacterial composition comprising an extract of a basilomycin D analog of the following chemical formula (1), or (ii) an extract of a Bacillus velezensis CJBV strain having the accession number KCCM11528P or a dried product thereof. ,
The said extract or its dried product is an antibacterial composition containing the basilomycin D analog of following Chemical formula (1).

(In the formula, R is CH ₂ —CH ₂ —CH _3. )   The antibacterial composition includes Colletotrichum orbiculare, Fusarium oxysporum, Candida albicans, Zygosaccharomyces bailii, Bacillus cereus, Bacillus cereus, The antibacterial composition according to claim 1, which controls the growth of Listeria monocytogenes. (I) a bacilomycin D analog of any one of the following chemical formulas (1) to (3), or (ii) an extract of a culture of Bacillus velezensis CJBV (Accession No. KCCM11528P) or a dried product thereof. An antibacterial composition against food poisoning-inducing bacteria comprising
The antibacterial composition against food poisoning-inducing bacteria, wherein the extract or a dried product thereof contains a basilomycin D analog of any one of the following chemical formulas (1) to (3).

(In the formula, R is CH ₂ —CH ₂ —CH _3. )

(In the formula, R is CH ₂ —CH _3. )

(In the formula, R is CH _3. )   The antimicrobial composition according to claim 3, wherein the antimicrobial composition controls the growth of Bacillus cereus, Listeria monocytogenes or a combination thereof.

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