JP6672441B2 - Novel Bacillus beresensis CJBV and antibacterial composition containing the same - Google Patents

Description

  The present invention provides a novel Bacillus velezensis CJBV strain having a specific antibacterial activity against fungi and food poisoning-inducing bacteria, and an antibacterial composition using a bacillomycin D analog isolated therefrom, and the same. The present invention relates to a pharmaceutical composition for preventing or treating a microbial infectious disease.

  When a fermented soybean food is produced by a traditional method, the food is a food using a microorganism, and therefore, is exposed to the risk of food poisoning due to harmful bacteria contained in the microorganism. However, harmful bacteria such as pathogenic fungi and food poisoning-inducing bacteria cannot be completely killed by general sterilization methods, but produce toxic substances and induce food poisoning.

  Therefore, chemical preservatives or preservatives are used to prevent deterioration and decay and maintain the quality of the food or the like for a long period of time, but the chemical itself acts as an environmental hormone or causes skin allergies. Consumer resistance to chemical preservatives or preservatives tends to be greater because of the potential for this to occur.

  Therefore, interest in natural preservatives, which are microorganisms or metabolites thereof and can inhibit the growth of spoilage microorganisms, is rapidly increasing. As an example of a natural antibacterial substance that can be used as a preservative, interest in bacteriocin or bacteriocin-like substances is increasing. Bacteriocins are natural non-toxic preservatives, antibacterial proteins composed of proteins or carbohydrate complexes with proteins produced by many Gram-positive and Gram-negative bacteria. In particular, since the U.S. FDA approved the use of nisin, a bacteriocin, in processed cheese spread foods in 1998, bacteriocin has been recognized as an antibacterial formulation that can be used in the food industry.

  Studies on the antibacterial substances produced by Bacillus and lactic acid bacteria have been actively conducted, but unlike lactic acid bacteriocins, which are mainly capable of inhibiting the growth of Gram-positive bacteria, bacteriocins produced by Bacillus strains have antibacterial properties. It has a very wide active range and various properties.

  Specifically, the genus Bacillus is known to produce various antibacterial substances (Non-Patent Document 1), but as reported antifungal substances, mycosubtilin (mycosubtilin), eumycin (eumycin) Bacillomycin, surfactin, iturin, aminopolyol, aminopolyol, lipoprotein, etc., and those having both antibacterial and antifungal activities are Bacillus subtilis. Bacilysin produced by Bacillus subtilis has been reported.

  As a prior art relating thereto, Patent Document 1 describes that basilomycin D1, which is an antibacterial substance produced by Bacillus amyloliquefaciens GYL4, has a wide range of activity against phytopathogenic fungi. I have.

  In addition, Patent Document 2 discloses that as antibacterial substances produced by Bacillus subtilis, UV254-B, a novel antibacterial substance completely different from the iturin substance containing basilomycin, is a food poisoning-inducing bacterium Bacillus cereus and Listeria monolith. It is described that it exhibits antibacterial activity specifically to Cytogenes.

  However, the reported bacteriocins have only been reported to have antibacterial activity against specific microorganisms, and are effective against various pathogenic bacteria that induce microbial infectious diseases including various crop diseases and food poisoning. No bacteriocin has been reported. Furthermore, Bacillus amyloliquefaciens described in the above-mentioned literature is a useful fermented bacterium in soybean fermented foods. Therefore, even if the bacteriocin is applied to fermented soybean food, food poisoning-inducing fungi can be obtained. In addition, it is difficult to use it directly in fermented foods because it is not considered to exhibit a specific activity only for bacteria.

  Therefore, the present inventors have conducted intensive studies to develop a method which is harmless to the human body and suppresses various fungi and bacteria, and as a result, Bacillus beresensis CJBV newly isolated from conventional miso tamakogi. Not only has antibacterial activity against at least one kind of various fungi such as pathogenic molds and yeasts, but also has no antibacterial activity against fermentative bacteria of jan (soy), and only major harmful microorganisms of foods such as food poisoning bacteria The present invention has been completed by confirming that it has an antibacterial activity to suppress specifically.

Korean Laid-Open Patent No. 10-2013-0008673 Korean Laid-Open 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 strain of Bacillus belerensis CJBV having specific antibacterial activity against fungi and food poisoning-inducing bacteria.

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

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

  Further, another object of the present invention is to provide a food composition containing the antibacterial composition.

  Still another object of the present invention is to provide a feed composition containing the antibacterial composition.

  Still another object of the present invention is to provide an antibacterial agent for plant crops containing the antibacterial composition.

  It is a further object of the present invention to provide a method for controlling phytopathogenic fungi, comprising a step of treating the plant or soil with the antibacterial agent.

  Further, another object of the present invention is to provide a pharmaceutical composition for preventing or treating a microbial infectious disease, comprising the antibacterial composition.

  Still another object of the present invention is to provide a health functional food for preventing or ameliorating a microbial infectious disease containing the antibacterial composition.

  Still another object of the present invention is to provide a quasi-drug containing the antibacterial composition.

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

  The term "Bacillus" in the present invention means an aerobic or facultatively anaerobic Gram-positive Bacillus genus widely distributed in nature, and microorganisms belonging to the genus Bacillus include Bacillus beresensis and the like. The present inventors have elucidated a novel Bacillus bacterium having a specific antibacterial activity against fungi and food poisoning-inducing bacteria and belonging to Bacillus belerensis as follows.

  First, to isolate a Bacillus strain from a conventional type of miso tamako-koji, to screen for pathogenic molds, 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 nucleotide sequence analyzes were performed. The nucleotide sequence of the 16S rDNA gene of the selected strain is shown as SEQ ID NO: 1, which shows 99% homology with Bacillus belerensis. In addition, the gyrA gene base sequence of the selected strain is shown as SEQ ID NO: 2, which shows 100% homology with Bacillus belerensis (Example 1). Accordingly, the strain was identified as Bacillus velezensis, named Bacillus velesensis CJBV, and deposited on April 17, 2014 with the Korean Microbial Conservation Center (KCCM), which is an international depository organization under the Budapest Treaty. Then, accession number KCCM11528P was assigned (Example 1).

  Therefore, the Bacillus belerensis 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 belerensis CJBV is not only excellent in antibacterial activity against fungi such as phytopathogenic fungi and yeast, but also inhibits food poisoning-inducing bacteria, and has various fungi. And antibacterial activity capable of simultaneously acting on food poisoning-inducing bacteria (Example 3, FIGS. 3 and 4).

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

  As used herein, the term "Colletotricum orviculale" refers to a fungus that causes Anthracnose in cucumber, makuwauri, watermelon and other cucumber vegetable crops.

  The term "Fusarium oxysporum" in the present invention means a pathogenic fungus that causes Fusarium wilt on cucumber, makuwauri, watermelon and other cucumber vegetable crops.

  The present inventors have confirmed that the newly isolated Bacillus belerensis CJBV has an antibacterial effect against the coletotricum orbiculae and Fusarium oxysporum. Thus, the novel strain can suppress plant infectious diseases such as anthrax and vine rot in cucumber, cucumber, watermelon and other cucumber plants, and can be used as a microbial preparation for controlling plant diseases. .

  Therefore, the use of newly isolated Bacillus belerensis CJBV as an environmentally friendly non-polluting pesticide is useful for solving ecological destruction and toxicity problems caused by organic synthetic pesticides, and can reduce the financial burden on each farmer. it can. In addition, 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 in order to control phytopathogenic bacteria in agricultural products.

  The term "Candida albicans" in the present invention is a genus name of a fungus belonging to a defective fungus, 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 have vaginal or vulvar Candida vaginosis more than once in their lifetime, and 45% of women have recurrence more than twice a year. Is caused by Candida albicans, but is transmitted when Candida albicans, which is normally present in the oral cavity, intestines, and vagina of healthy humans, when the status of female hormones is abnormal or immunity is severely reduced. .

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

  The term “Digosaccharomyces vili” in the present invention is a yeast belonging to the genus Saccharomyces. In addition, it is a perishable yeast, and produces a large amount of carbon dioxide gas together with alcohol fermentation in beverage products and processed starch products, thereby deteriorating the quality of food.

  The present inventors have confirmed that the newly isolated Bacillus belerensis CJBV has an antibacterial effect on the above-mentioned Digosaccharomyces vali. Thus, the novel strain can improve the preservability of foods such as beverage products and processed starch products, and prevent or treat microbial infectious 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.

  As used herein, the term "Bacillus cereus" is a gram-positive bacterium that forms thermostable spores, but causes diarrhea and vomiting when infected. It is an aerobic bacterium and is widely distributed in nature such as soil and cereals. Since the spores of this strain are superior to the spores of other strains in surface adhesion, it is difficult to wash and disinfect. In addition, the Bacillus cereus may grow by germination of spores in meat, vegetables, rice, noodles, etc. left at room temperature for several hours after cooking, and may also germinate in pasteurized milk, cheese, and the like. , Producing heat-resistant toxins and exhibiting resistance to heat, making it difficult to suppress them.

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

  The term "Listeria monocytogenes" in the present invention is a zoonotic infectious disease pathogen, which may be transmitted to humans through livestock foods such as milk and cheese, and has recently been receiving worldwide attention. Is one of the pathogenic food poisoning bacteria. The mortality rate is very high, about 30%, when the infection causes the listeriosis. In particular, in the case of human infection, people with weak immunity or immunodeficiency, such as infants, organ transplant patients, lymphomas and AIDS patients, are susceptible to infection and may induce sepsis, encephalitis and meningitis. The pathogenic bacterium, unlike other pathogenic bacteria, has the property of being able to grow even at low temperatures, and it is known that bacteria grown at 4 ° C are stronger than bacteria grown at 37 ° C. . Due to the characteristics of these fungi, food poisoning due to Listeria monocytogenes can occur in refrigerated foods, refrigerated foods, and preserved foods that are considered safe at home, which is a major problem.

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

  In particular, the present inventors have found that the newly isolated Bacillus beresensis CJBV of the present invention not only has the effect of simultaneously suppressing the Bacillus cereus (Bacillus cereus) and Listeria monocytogenes (Listeria monocytogenes), No antimicrobial activity was confirmed against Bacillus subtilis and Bacillus amyloliquefaciens, which are fermentative bacteria useful in fermented foods (Examples 3 and 4). (Fig. 4).

  As still another specific example, the Bacillus belerensis CJBV comprises an antibiotic Bacillomycin D and a Bacillomycin D analog (analog) represented by the chemical formula 1, that is, C14-bacillomycin D, C15-bacillomycin D, And at least one analog of basilomycin D selected from the group consisting of C16-basilomycin D.

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

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

  The present inventors have separated the antibacterial substance produced and secreted by the newly isolated Bacillus belerensis CJBV of the present invention and measured the UV spectrum and molecular weight. As a result, the separated substances are the three bacillomycin D analogs. This was confirmed (Example 2, FIGS. 1 and 2).

  According to another aspect of the present invention, there is provided a basilomycin D analog produced from the novel Bacillus beresensis CJBV strain.

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

  In the above chemical formula, R is CH3, CH2-CH3, CH2-CH2-CH3, the case of CH3 is named C14-bacillomycin D, and the case of CH2-CH3 is named C15-bacillomycin D; The case of CH2-CH3 was named C16-bacillomycin D.

  The present inventors have separated the antibacterial substance produced and secreted by the newly isolated Bacillus belerensis CJBV of the present invention and measured the UV spectrum and molecular weight. As a result, the separated substances are the three bacillomycin D analogs. This was confirmed (Example 2, FIGS. 1 and 2).

  A basilomycin D analog produced from the novel strain of Bacillus belerensis CJBV was used for plant pathogens including coletotricum orviculae causing anthrax, fusarium oxysporum causing vine disease, dermatomycosis when infected, and Candida. Vaginitis, enteritis, oral inflammation and the like can cause Candida albicans, degrading yeast, Digosaccharomyces baili, food poisoning bacteria Bacillus cereus, Listeria monocytogenes, as a result of being treated to the fungi and bacteria, The antibacterial effect was confirmed (FIGS. 3 and 4). Therefore, a bacillomycin D analog produced from the novel Bacillus beresensis CJBV strain can be used as an antibacterial composition, which is used as a feed, a pharmaceutical composition, and a health function for preventing, ameliorating or treating a microbial infectious disease. It can be effectively used for foods, quasi-drug compositions, foods having antibacterial activity, antibacterial agents for plant crops, and control methods using the same.

  In still another aspect to achieve the above-mentioned object, the present invention provides a method for producing the newly isolated Bacillus belerensis CJBV strain, spores of the strain, a culture of the strain, a concentrate thereof, an extract thereof, and drying thereof. The present invention provides an antimicrobial composition comprising as an active ingredient at least one selected from the group consisting of: The state of the strain is in a liquid state or a dry state, but is not limited thereto.

  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 refers to a germ cell of Bacillus belerensis CJBV. In the present invention, since the spores of Bacillus belerensis CJBV exhibit antibacterial activity against fungi and food poisoning bacteria, they can be used in microbial preparations for controlling plant diseases, foods having antibacterial activity, feed, pharmaceuticals, quasi-drugs, and health functional foods. It can be used by incorporating it.

  The term "culture" in the present invention means a product obtained by culturing the strain, and may be a stock solution containing the cells, or may be a cultured strain or a cell from which the culture supernatant has been removed. Or concentrated cells. The composition of the culture may further include components necessary for normal Bacillus culture, as well as components that act synergistically on the growth of Bacillus, and their composition may be easily determined by those having ordinary knowledge in the art. Can be selected.

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

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

  The term “dried product” in the present invention means a dried product of the above-mentioned culture, its concentrate, its extract, and its fraction. As the drying method, ventilation drying, natural drying, spray drying, and freeze drying can be used, but the drying method is not limited thereto.

  The newly isolated Bacillus belerensis CJBV of the present invention can be cultured by an ordinary method for culturing a Bacillus strain, but is not limited thereto. As a 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.As the nitrogen source, peptone, meat extract, yeast extract, dried yeast, soybean, ammonium salt, nitrate , And other organic or inorganic nitrogen-containing compounds can be used, but are not limited to these components. Examples of the inorganic salt contained in the medium include, but are not limited to, magnesium, manganese, calcium, iron, phosphorus and the like. In addition to the components of the carbon source, nitrogen source and inorganic salt, amino acids, vitamins, nucleic acids and compounds related thereto may be added to the medium.

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

  According to yet another aspect of the present invention to achieve the above object, the present invention provides a food composition comprising the antimicrobial 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, candies, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice creams, various soups, Examples include soft drinks, tea, drinks, alcoholic beverages, and vitamin composites. When the composition is used as a food additive, the composition may be added as it is, may be used together with other foods or food components, or may be appropriately used in a usual manner.

  The food composition may include a pharmaceutically acceptable carrier.

  In particular, the present inventors have the effect that the newly isolated Bacillus belerensis CJBV of the present invention can simultaneously suppress the perishable 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 bacteria for fermented foods. (Example 3 and FIG. 4). Therefore, the antimicrobial composition can be directly used for all foods including fermented foods.

  According to yet another aspect of the present invention to achieve the above object, the present invention provides a feed composition comprising the antibacterial composition.

  The term "feed" in the present invention means any natural or artificial diet, meal, etc., or component of said meal, for eating, ingestion and digestion by or suitable for an animal.

  The type of the feed is not particularly limited, and a feed generally used in the art 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 probiotic agent. Examples of the feed, cereals, nuts, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, plant feeds such as cucurbitaceous plants or cereal by-products, Examples include proteins, minerals, fats and oils, minerals, single-cell proteins, zooplankton, and animal feed such as food and drink. These may be used alone or in combination of two or more.

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

  It has been confirmed that the newly isolated Bacillus belerensis CJBV of the present invention has the effect of simultaneously suppressing the perishable yeast Digosaccharomyces bairi, the food poisoning bacteria Bacillus cereus, and Listeria monocytogenes. . Therefore, the present invention can prevent a microbial infectious disease including food poisoning of livestock by providing a feed composition having antibacterial and antifungal activities.

  As yet another aspect of the present invention to achieve 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 belerensis CJBV of the present invention has an inhibitory effect on plant pathogenic bacteria including coletotricum orbiculae causing anthrax, fusarium oxysporum causing vine disease and the like. It was confirmed (FIG. 3). Therefore, the antibacterial composition of the present invention is used as an environmentally friendly non-polluting pesticide, is useful for solving ecological destruction and toxicity problems caused by organic synthetic pesticides, and can reduce the financial burden of each farmer. . In addition, long-term benefits are expected in the future because they are sustainable due to the characteristics of microorganisms.

  As yet another aspect of the present invention to achieve the above object, the present invention provides a method for controlling phytopathogenic fungi, comprising a step of applying the plant crop antibacterial agent to a plant or soil.

  The phytopathogenic fungi are preferably, but are not limited to, coletotricum orbiculare, which causes anthrax, and Fusarium oxysporum, which causes vine disease.

  The above-mentioned microbial preparation is obtained by foliar treatment on the aerial part of the plant body and by irrigation on the root part of the plant body, 15 days after planting, or during the rainy season and immediately before the onset of disease, when clearing the soil before planting the crop. Spraying is preferred, but not limited thereto.

  In yet another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating a microbial infectious disease, comprising the antimicrobial composition.

  The term "prevention" in the present invention means any act of suppressing or delaying the onset of a microbial infectious disease by administering a composition for preventing or treating a microbial infectious disease according to the present invention to an individual.

  The term “treatment” in the present invention means any act of improving or suitably changing the symptoms of a microbial infectious disease by administering the composition of the present invention to an individual suspected of having a microbial infectious disease.

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

  As used herein, the term "individual" means any animal, including humans, that has, or is likely to develop, a microbial infectious disease.

  Here, the microbial infectious disease is a disease caused by infection by a microorganism, and is preferably a disease caused by infection by a fungus or a food poisoning-inducing bacterium.

  By way of example, the fungi may be Colletotricum orbiculae, Fusarium oxysporum f. sp. It may be selected from the group consisting of Laico Persisi JCM 12575, Candida albicans, and Digosaccharomyces vaili.

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

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

  Cellulitis refers to acute purulent inflammation that appears in the dermis and subcutaneous tissue, when purulent inflammation occurs in the tissue and intense neutrophil infiltration spreads chronically and extensively into the interstitial space. Yes, with redness and edema.

  Felon refers to acute purulent inflammation of the fingers or toes.

  The present inventors have found that Candida albicans, a perishable yeast which is present in the oral cavity, intestines, and vagina of animals including humans and which can cause dermatomycosis, Candida vaginitis, enteritis, oral inflammation, etc. Digosaccharomyces baili, which can cause food poisoning, vomiting, diarrhea, fever, etc., Bacillus cereus, which can cause food poisoning, diarrhea, vomiting, etc., when infected, can cause food poisoning, listeriosis, sepsis, encephalitis, meningitis, etc. The excellent inhibitory effect of the strain of the present invention on Listeria monocytogenes was confirmed (FIGS. 3 and 4). Therefore, the antibacterial composition of the present invention can be effectively used for a pharmaceutical composition for preventing or treating a microbial infectious disease.

  The composition for preventing or treating a microbial infectious disease according to the present invention may further include a pharmaceutically acceptable carrier, and may be formulated together with the carrier to prepare a food, a health food, a pharmaceutical, a quasi-drug, or a feed additive. And as a drinking water additive. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate the organism and does not interfere with 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 commonly used in the art. Examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerin, ethanol and the like. These may be used alone or in combination of two or more.

  If necessary, other conventional additives such as antioxidants, buffers and / or bacteriostats may be added and used, and diluents, dispersants, surfactants, binders, lubricants The drug may be further added and formulated into an injection form such as an aqueous solution, suspension, or emulsion, a pill, a capsule, a granule, a tablet, or the like.

  The method for administering the composition for preventing or treating a microbial infectious disease according to the present invention is not particularly limited, and a method generally used in the art can be employed. As an example of the administration method, the composition can be administered orally or parenterally. The composition for preventing, ameliorating or treating a microbial infectious disease of the present invention can be prepared in various dosage forms depending on the intended administration method.

  The pharmaceutical dosage forms of the antimicrobial substances of the present invention may be used in the form of their pharmaceutically acceptable salts. In addition, they may be used alone, and may be used not only in combination with other pharmaceutically active compounds but also in an appropriate combination.

  In still another aspect, the present invention provides a method for preventing or treating a microbial infectious disease, comprising a step of administering the composition for preventing or treating a microbial infectious disease to an individual. As used herein, the term "individual" means any animal, including humans, that has developed or is at risk of developing a microbial infectious disease.

  Specifically, the method for preventing or treating according to the present invention may include a step of administering the composition in a pharmaceutically effective amount to a solid or a person at risk of developing a microbial infectious disease.

  A suitable total daily usage of the composition containing the extract will be determined by the attending physician within the scope of sound medical judgment and may be administered in one or several divided doses. However, for purposes of the invention, the specific therapeutically effective amount for a particular patient will depend on 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's age, body weight, general health, gender, diet, time of administration, route of administration, secretion rate of the composition, treatment period, including drugs used in combination with or co-administered with the specific composition It is preferred to apply different amounts depending on the various factors involved 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, livestock, and humans by various routes. Any method of administration is possible and can be administered, for example, orally, rectally, or by intravenous, intramuscular, subcutaneous, intrauterine or intracerebroventricular injection.

  In still another aspect, the present invention provides a health food for preventing or ameliorating a microbial infectious disease, comprising the antimicrobial composition.

  Here, the meaning of microbial infectious disease, prevention and improvement is as described above.

  In yet another aspect, the present invention provides a quasi-drug composition comprising the antimicrobial composition.

  In the present invention, the term "quasi-drug" refers to fibers, rubber products or the like used for the purpose of treating, reducing, treating or preventing diseases of humans and animals, having a weak effect on the human body, A product that does not act directly and is not a device or a machine, or a product similar to a product used for disinfection, insecticide, and similar uses to prevent infectious disease, and that is a human or animal disease Of instruments, machines or devices used for the purpose of diagnosing, treating, alleviating, treating or preventing, and instruments, machines or devices used for the purpose of pharmacologically affecting the structure and function of humans or animals Articles other than those that are not devices. The quasi-drugs also include 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 thereto.

  The present inventors have confirmed that the newly isolated Bacillus belerensis 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 treating, reducing, treating or preventing microbial infectious diseases including dermatomycosis and Candida vaginitis.

  The newly isolated Bacillus belerensis CJBV of the present invention has an antibacterial effect against one or more different types of fungi and bacteria such as pathogenic molds and yeasts, and thus has an antibacterial agent for plant crops and an antibacterial activity. It can be used as food, feed for prevention, improvement, and treatment of microbial infectious diseases, health functional foods, quasi-drugs, and pharmaceuticals.

  In particular, the newly isolated Bacillus belerensis CJBV of the present invention has an antibacterial activity that specifically suppresses only major harmful microorganisms of food such as food poisoning bacteria, and thus can be directly used for fermented foods.

FIG. 2 is a view showing a process of separating a basilomycin D analog, which is an antibacterial active ingredient, from Bacillus beresensis CJBV of the present invention. FIG. 2 shows ESI-MS and UV spectrum of an antibacterial substance produced from a strain according to the present invention. It is a figure which shows the antimicrobial activity with respect to four types of fungi, Colletotricum orbiculae, Fusarium oxysporum, Candida albicans, and Digosaccharomyces vali. 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) fermentation 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 a strain producing an antibacterial substance (1) Isolation and selection of a strain A Bacillus strain isolated from a conventional miso tamako-koji was used to select a strain having antibacterial activity. The strain was pre-cultured in a fermented soybean liquid medium at 37 ° C. for 36 hours. An 8 mm diameter paper disk was placed on a solid agar medium containing lawn cells containing pathogenic mold and food poisoning-inducing bacteria, and the precultured strain was inoculated. After culturing for 12 hours, strains which simultaneously formed a transparent circle against fungi and bacteria around the strain inoculated in the medium were selected.

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

配列番号２：

From these results, the strain obtained according to the present invention was identified as a Bacillus belerensis strain and named Bacillus belerensis CJBV, and the strain was deposited with the Korea Microbial Conservation Center (KCCM) on April 17, 2014. , Accession number KCCM11528P.

SEQ ID NO: 1:

SEQ ID NO: 2:

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

  The strain was inoculated into 400 μL of a soybean fermentation liquid medium, and then cultured at 37 ° C. for 48 hours with shaking at 200 rpm. The culture 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 the fraction having antibacterial activity was separated and purified.

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

  FIG. 2 shows ESI-MS and UV spectrum of the separated antibacterial substance.

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

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

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

(1) Antibacterial activity test against yeasts and molds (fungi) First, fungi such as Colletotricum orbiculae, Fusarium oxysporum f. sp. It was tested whether Bacillus beresensis CJBV, newly isolated against Fusarium oxysporum f.sp. lycopersici JCM12575, Candida albicans, and the pathogenic yeast Digosaccharomyces vail, shows antibacterial activity.

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

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

  Bacillus cereus and Listeria monocytogenes, which are food poisoning-inducing bacteria, and Bacillus subtilis and Bacillus amyloliquefaciens, which are fermentative germs of Jean (soy), are each in TSA (Tryptic soy agar) medium. Was cultured.

  Thereafter, a butanol extract (Extraction with BuOH) of a Bacillus belerensis CJBV culture solution, which is an antibacterial composition concentrated under reduced pressure according to Example 2, was dissolved in ethanol, and then the four types of strains were spread on each plate medium. A paper disc having a diameter of 8 mm was placed, a predetermined amount of the antibacterial substance was added in 25 μL portions, and the mixture was cultured at 37 ° C. for 24 hours to observe whether a growth-inhibitory circle was formed by the antibacterial substance produced by the isolated strain.

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

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

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

  As shown in the upper part of FIG. 3, a newly isolated Bacillus belerensis 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 active ingredients as an active ingredient to the pathogenic fungi, coletotrichum orbiculale and fusarium oxysporum, a growth-inhibitory circle is formed at least 10 mm, thereby inhibiting the fungal growth It was confirmed that it was excellent.

  Therefore, the newly isolated Bacillus belerensis CJBV and the antibacterial composition of the present invention can suppress plant infectious diseases such as anthrax and vine rot in cucumber, makuwauri, and watermelon vegetable crops. It 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 belerensis CJBV, the bacillomycin D analog produced from the above strain, or the antibacterial composition was used as a pathogenic fungi such as Candida albicans and Digo. As a result of treating the Saccharomyces bilii, a growth inhibition circle of 6 mm to 10 mm was formed, and it was confirmed that the growth inhibition circle was excellent in inhibiting the growth of fungi.

  Therefore, the newly isolated Bacillus belerensis CJBV of the present invention, a bacillomycin D analog produced from the strain, and an antibacterial composition are useful for prevention of microbial infectious diseases such as dermatomycosis, candida vaginitis, oral cavity, and intestine. It shows improvement and therapeutic effects, and can be effectively used for pharmaceuticals, quasi-drugs, health foods, feeds and the like. Further, it can be effectively used as a food with a low risk of food poisoning.

  Further, as shown in FIG. 4, the newly isolated Bacillus belerensis CJBV or the antibacterial composition of the present invention was simultaneously treated with food poisoning-inducing bacteria and useful bacteria used in fermentation of Jan (soy), resulting in food poisoning-inducing bacteria. Bacillus cereus and Listeria monocytogenes showed excellent antibacterial activity, but did not inhibit the growth of fermented bacteria of Jeans (soy).

  Bacteriocin, which has been reported to be produced by conventional microorganisms, does not specifically exhibit antibacterial activity only against food poisoning-inducing bacteria, so it affects the growth of fermenting bacteria such as jeans (soy). There is a problem that it is difficult to use it directly for food.

  In contrast, the newly isolated Bacillus belerensis CJBV, the bacillomycin D analog produced from the above strain, and the antibacterial composition using the same are antibacterial against the fermentation bacterium of Jean (soy) class. Since it has no activity and has an antibacterial activity that specifically suppresses only major harmful microorganisms of food such as food poisoning bacteria, it can be used directly in fermented foods.

  Furthermore, it was confirmed that it has excellent antibacterial activity against various food contamination sources. Therefore, the newly isolated Bacillus belerensis CJBV of the present invention, a bacillomycin D analog produced from the strain, and an antibacterial composition using the same are useful for prevention, improvement, and improvement of microbial infectious 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 belerensis CJBV of the present invention, and the bacillomycin D analog produced from said strain have antibacterial effects against at least one different kind of fungi and bacteria, and It can be used as an antibacterial agent for crops, a food having an antibacterial activity, a feed for preventing, improving, and treating microbial infectious 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 appreciate that the present invention may be embodied in other specific forms without altering its technical spirit or essential characteristics. It should be understood that the above embodiments are illustrative in all aspects and not restrictive. It is to be understood that the scope of the present invention is not intended to include the above detailed description, but to include any modifications or modified forms derived from the meaning and scope of the claims and equivalents thereof.

Depositary name: Korea Microbial Preservation Center (outside Japan)
Accession number: KCCM11528P
Contract date: 20140417

Claims (2)

(I) Bashiromaishin D analogues of the following Formula (1), Bashiromaishin D analog of the following formula (2), and bar Shiromaishin D analogues of the following Formula (3), or (ii) Bacillus Berezenshisu CJBV accession number KCCM11528P An antibacterial composition against a food poisoning-inducing bacterium, comprising an extract of a culture of the (Bacillus velezensis CJBV) strain or a dried product thereof.

(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 of claim 1 , wherein the antimicrobial composition controls the growth of Bacillus cereus, Listeria monocytogenes, or a combination thereof.

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