KR20160059474A - Novel Bacillus velezensis CJBV and antibacterial and antifungal composition comprising the same - Google Patents

Abstract

The present invention relates to a Bacillus velezensis CJBV strain which is newly isolated from Deposit No. KCCM11528P. The present invention further relates to a bacillomycin D derivative isolated therefrom, and to an antibacterial composition using the strain and the derivative. According to the present invention, the newly isolated Bacillus velezensis CJBV exhibits specific antibacterial effects against both Vibrio ordalii and at least one different type of eumycetes. Thus, the strain of the present invention can be used as antimicrobial agents for plate cultivation, foods containing antibacterial fermented-foods, and products including feeds, functional health foods, quasi-drugs, and drugs for preventing, ameliorating, and treating microbial infection.

Description

Novel Bacillus velezensis CJBV and antibacterial and antifungal composition comprising the same}

The present invention is a novel Bacillus velezensis CJBV (Bacillus velezensis CJBV) strain having specific antimicrobial activity against fungi and food poisoning-causing bacteria, and an antibacterial composition using a bacilomycin D analog isolated therefrom, and a microorganism comprising the same It relates to a pharmaceutical composition for preventing or treating infectious diseases.

In the case of manufacturing fermented soybean food in the traditional way, since it is a food using microorganisms, it is exposed to the risk of food poisoning by harmful bacteria contained in the microorganism. However, harmful bacteria such as pathogenic fungi and food poisoning-causing bacteria are not completely killed by a general sterilization method, and produce toxic substances to cause food poisoning.

Therefore, chemical preservatives or preservatives are used to prevent deterioration or spoilage and maintain the quality of the food for a long time, but the chemical itself may act as an environmental hormone or cause skin allergies. There is a trend of increasing consumer rejection.

Accordingly, interest in natural preservatives that can inhibit the growth of deteriorated microorganisms as microorganisms or metabolites thereof is increasing rapidly. As an example of natural antibacterial substances that can be used as preservatives, interest in bacteriocin or bacteriocin-like substances is increasing. Bacteriocin is a natural non-toxic preservative and is an antimicrobial protein composed of a protein produced by many Gram-positive and Gram-negative bacteria, or a complex of protein and carbohydrate. In particular, since nisin among bacteriocins was approved for use in processed cheese spread foods by the US FDA in 1998, bacteriocins have been recognized as antibacterial agents that can be used in the food industry.

Numerous studies are being conducted on the antibacterial substances produced by the genus Bacillus or lactic acid bacteria, and unlike lactic acid bacteria bacteriocin, which can mainly inhibit the growth of Gram-positive bacteria, bacteriocin produced by Bacillus strains has a very wide range of antibacterial activity and various properties. Has.

Specifically, the genus Bacillus is known to produce a variety of antibacterial substances (Sholberg et al., 1995 Can. J. Microbio. 41, 247-252), and reported antifungal substances include mycosubtilin and yumycin (Sholberg et al., 1995 Can. J. Microbio. 41, 247-252). eumycin), bacillomycin, surfactin, iturin, aminopolyol, lipoprotein, etc. Reports include bacilysin produced by Bacillus subtilis.

As a prior document related to this, Korean Patent Publication 10-2013-0008673 describes that Bacillus amyloliquefaciens strain Bacillus amyloliquefaciens (Bacillus amyloliquefaciens GYL4) has a wide range of activity against phytopathogenic fungi. .

In addition, Korean Patent Laid-Open Publication No. 10-2013-0025453 is an antibacterial substance produced by Bacillus subtilis, and UV254-B, a novel antibacterial substance completely different from the iturine substance containing bacilomycin, is Bacillus cereus, a bacteria that causes food poisoning. And only Listeria monocytogenes, showing antibacterial activity specifically.

However, only the antimicrobial activity of the reported bacteriocin against specific microorganisms has been reported, and there is no report of bacteriocin that exhibits an effect against various pathogenic bacteria that cause microbial infectious diseases, including various crop diseases or food poisoning. Furthermore, Bacillus amyloliquefaciens described in the above document is a useful enteric fermentation bacterium in fermented foods, so when the bacteriocin is applied to fermented soybean food, it is difficult to see that it exhibits specific activity only on fungi and bacteria that cause food poisoning, so it is used directly in fermented foods. There was a problem that was difficult to do.

Accordingly, the inventors of the present invention have made intensive research efforts to develop a method of inhibiting various fungi and bacteria while being harmless to the human body. As a result, Bacillus bellegensis CJBV newly isolated from conventional meju is antibacterial against at least one variety of fungi such as pathogenic fungi and yeast. In addition to having activity, the present invention was completed by confirming that there is no antibacterial activity against enteric fermentation bacteria, and that it has antimicrobial activity that specifically inhibits only major harmful microorganisms of food such as food poisoning bacteria.

The main object of the present invention is to provide a novel isolated Bacillus bellesensis CJBV strain having specific antibacterial activity against fungi and food poisoning-causing bacteria.

Another object of the present invention is to provide an analogue of Bacilomycin D, produced in the Bacillus belegensis CJBV strain.

Another object of the present invention is to provide an antimicrobial composition using the Bacillus bellegensis CJBV strain and the Bacilomycin D analog.

Another object of the present invention is to provide a food composition comprising the antibacterial composition.

Another object of the present invention is to provide a feed composition comprising the antibacterial composition.

Another object of the present invention is to provide an antibacterial agent for plant crops comprising the antibacterial composition.

Another object of the present invention is to provide a method for controlling phytopathogenic fungi comprising the step of treating plants or soil with the antimicrobial agent.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating microbial infectious diseases comprising the antimicrobial composition.

Another object of the present invention is to provide a health functional food for preventing or improving microbial infectious diseases comprising the antibacterial composition.

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

As an aspect for achieving the above object, the present invention provides a novel isolated Bacillus velezensis CJBV (Bacillus velezensis CJBV) strain of accession number KCCM11528P, having specific antibacterial activity against fungi and food poisoning-causing bacteria.

In the present invention, the term Bacillus refers to a genus of aerobic or anaerobic Gram-positive bacillus widely distributed in nature, and microorganisms belonging to the genus Bacillus include Bacillus beregensis. The present inventors have identified a novel strain belonging to Bacillus belegensis as a Bacillus bacterium having specific antibacterial activity against fungi and food poisoning-causing bacteria as follows.

First, Bacillus strains were isolated from conventional meju and tested for fungi and food poisoning bacteria such as pathogenic fungi and yeast to select strains with excellent antibacterial activity, and to identify the selected strains, 16S rDNA and gyrA gene sequence analysis was performed. . The 16S rDNA gene nucleotide sequence of the selected strain was described in SEQ ID NO: 1, which showed 99% homology with Bacillus belegensis. In addition, the nucleotide sequence of the gyrA gene of the selected strain was described in SEQ ID NO: 2, which showed 100% homology with Bacillus belegensis (Example 1). Therefore, the strain was identified as Bacillus velezensis and named as Bacillus velezensis CJBV, deposited on April 17, 2014 to the Korea Microbiological Conservation Center (KCCM), an international depository under the Budapest Treaty, and accession number It was given KCCM11528P (Example 1).

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

According to an aspect of the present invention, the newly isolated Bacillus belegensys CJBV not only has excellent antibacterial activity against fungi such as phytopathogenic fungi and yeast, but also inhibits food poisoning-causing bacteria, and simultaneously protects against various fungi and food poisoning-causing bacteria. It shows an antimicrobial activity that can act (Example 3, Figs. 3 and 4).

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

In the present invention, the term Choletotricum orbiculae refers to a fungus that causes anthracnose on gourd and vegetable crops such as cucumber, melon, and watermelon.

In the present invention, the term Fusarium oxisporum refers to pathogenic fungi that cause Fusarium wilt on gourd and vegetable crops such as cucumber, melon, and watermelon.

The inventors of the present invention confirmed that the newly isolated Bacillus belegensys CJBV has an antibacterial effect against the Coletotricum obiculae and Fusarium oxysporum. Using the new strain, such as cucumber, melon, watermelon, etc. It can be used as a microbial agent for controlling plant diseases by inhibiting plant infectious diseases such as anthrax and creeping vines on gourd and vegetable crops.

Therefore, when using the newly isolated Bacillus bellegensys CJBV as an eco-friendly, non-polluting biological pesticide, it can be useful in solving the problem of ecosystem destruction and toxicity caused by organic synthetic pesticides, and it can reduce the financial burden of each farmer. In addition, due to the nature of microorganisms, it is sustainable and can provide long-term benefits in the future.

Meanwhile, the biological pesticide may be defined as a commercialization of microorganisms separately collected in a natural environment in order to control plant pathogens of agricultural crops.

In the present invention, the term Candida albicans is a generic name of a fungus belonging to an incomplete fungus, and causes dermatosis, Candida oral, intestinal diseases, and Candida vaginitis. In particular, Candida vaginitis is a common disease in which about 75% of women suffer from candidal vaginitis of the vagina and vulva more than once during their lives, and 45% of women experience recurrence more than twice a year. Most of them are caused by Candida albicans. And, Candida albicans, which is normally held in the oral, intestine, and vagina of a healthy person, can cause infection when the female hormone status is abnormal or the immunity is extremely weak.

The present inventors confirmed that the newly isolated Bacillus bellegensis CJBV has an antibacterial effect against the Candida albicans, and the novel strain effectively prevents skin mycosis, candidal vaginitis, microbial infectious oral and intestinal diseases, or It can be cured.

In the present invention, the term Zygo Saccharomyces biley is a yeast of the genus Saccharomyces. As a deteriorating yeast, it produces a large amount of carbon dioxide gas along with alcohol fermentation in beverage products and processed starch products, thereby reducing the quality of food.

The present inventors confirmed that the newly isolated Bacillus bellegensys CJBV has an antimicrobial effect against the Xygo Saccharomyces viley. By using the new strain, the storage properties of food such as beverage products and starch processed products are improved. It is possible to prevent or treat microbial infectious diseases such as food poisoning that may arise from this.

In another embodiment, the food poisoning-causing bacteria is Bacillus cereus or Listeria monocytogenes ( Listeria monocytogenes ), but is not limited thereto.

In the present invention, the term Bacillus cereus is a Gram-positive bacterium that forms heat-resistant spores, causing diarrhea and vomiting during infection. As aerobic bacteria, they are widely distributed in the natural world such as soil and grains. The spores of this strain have superior surface adhesion properties compared to spores of other strains, making it difficult to clean and disinfect. In addition, the Bacillus cereus can be multiplied by spore germination in meat, vegetables, rice, noodles, etc., left at room temperature for several hours after cooking, and can germinate in milk, cheese, etc. after pasteurization, and produce heat-resistant toxins. Therefore, it is not easy to suppress it because it shows resistance to heat.

The present inventors have confirmed that the newly isolated Bacillus bellegensis CJBV has an antimicrobial effect against the Bacillus cereus, and the new strain can be used to prevent or treat food poisoning, diarrhea or vomiting caused by infection.

In the present invention, the term Listeria monocytogenes is a common infectious pathogen and can be transmitted to humans through livestock foods such as milk or cheese, and thus is one of the pathogenic food poisoning bacteria that are receiving worldwide attention in recent years. When listeriosis occurs due to infection with the above bacteria, the mortality rate is very high, about 30%. In particular, human infections are susceptible to infection by people with weak immunity or immunodeficiency, such as infants, organ transplant patients, lymphoma, and AIDS patients, and can cause sepsis, encephalitis and meningitis. The pathogens are known to have characteristics capable of growing at low temperatures, unlike other pathogenic bacteria, and are known to have stronger pathogenicity of bacteria grown at 4°C than bacteria grown at 37°C. Due to the characteristics of these bacteria, food poisoning by Listeria monocytogenes may occur in foods in refrigerated storage, refrigerated foods and stored foods that are considered safe at home, which is a big problem.

The present inventors confirmed that the newly isolated Bacillus bellegensis CJBV has an antibacterial effect against the Listeria monocytogenes, and the novel strain can be used to prevent or treat microbial infections such as food poisoning and Listeriosis.

In particular, the present inventors believe that the novel isolated Bacillus belegensys CJBV of the present invention has the effect of simultaneously inhibiting the Bacillus cereus and Listeria monocytogenes , as well as useful in fermented foods. It was confirmed that there was no antibacterial activity against the enteric fermentation bacteria Bacillus subtilis and Bacillus amyloliquefaciens (Example 3, FIG. 4).

In addition, as an embodiment, the Bacillus belegensys CJBV is an antibiotic, Bacilomycin D, and a bacilomycin D analog represented by Chemical Formula 1, that is, C14-bacilomycin D, C15- It is possible to produce and secrete any one or more analogs of bacilomycin D selected from the group consisting of bacilomycin D and C16-bacilomycin D.

[Formula 1]

In the above formula, wherein R is, in the case of erythromycin D by Vasil C14- CH _3, in the case of erythromycin D as C15- Basil CH ₂ -CH _3, in the case of erythromycin D C16- Basil CH ₂ -CH ₂ -CH ₃ .

In the present invention, the term bacilomycin D is a substance belonging to the iturine family, which is one of the cyclic peptide group, and includes 7 α-amino acids (2 Asn, Glu, Tyr, Pro, Ser, Thr) and one It is a ring structure composed of β-amino acids and has antibiotic activity.

The present inventors measured the UV spectrum and molecular weight by separating the antimicrobial substances produced and secreted by the newly isolated Bacillus bellegensis CJBV of the present invention, and as a result, it was confirmed that the separated substances were three analogs of the bacilomycin D ( Example 2, Figs. 1 and 2).

As another aspect for achieving the above object, the present invention provides a bacilomycin D analog, produced in the novel Bacillus bellegensis CJBV strain.

The bacilomycin D analog is represented by the following formula (1).

[Formula 1]

In the above formula, R is CH ₃ , CH ₂ -CH ₃ or CH ₂ -CH ₂ -CH ₃ , and _{in the case of CH 3} , C14-bacilomycin D, and in the case of _{CH 2} -CH _{3, C15-basilo} In the case of mycin D, CH ₂ -CH ₂ -CH ₃ , it was named C16-bacilomycin D.

The present inventors measured the UV spectrum and molecular weight by separating the antimicrobial substances produced and secreted by the newly isolated Bacillus bellegensis CJBV of the present invention, and as a result, it was confirmed that the separated substances were three analogs of the bacilomycin D ( Example 2, Figs. 1 and 2).

Plant pathogens, including coletotricum obiculae, which are the cause of anthrax, and Fusarium oxisporum, which are the cause of vines, are infected with the bacilomycin D analog, which is produced from the new Bacillus belegensis CJBV strain. Candida albicans, which can cause cutaneous mycosis, candidal vaginosis, enteritis, oral inflammation, etc., Xygosaccharomyces bailey, a deteriorating yeast, Bacillus cereus, and Listeria monocytogenes, which are food poisoning bacteria. And it was confirmed that there is an excellent antibacterial effect against bacteria (Figs. 3 and 4). Therefore, it is possible to use a bacilomycin D analog, produced in the novel Bacillus belegensis CJBV strain, as an antibacterial composition, which is feed for the prevention, improvement or treatment of microbial infectious diseases, pharmaceutical compositions, health functional foods, quasi-drug compositions, It can be effectively used in foods having antibacterial activity, antibacterial agents for plant crops, and control methods using the same.

As another aspect for achieving the above object, the present invention is a group consisting of the newly isolated Bacillus bellesensis CJBV strain, spores of the strain, cultures of the strain, concentrates thereof, extracts thereof, and dried products thereof. It provides an antibacterial composition comprising any one or more selected from as an active ingredient. The state of the strain may be in a liquid state or a dry state, but is not limited thereto.

In the present invention, the term "spore" refers to propagating cells such as bacteria, and for the purposes of the present invention, the spores refer to propagating cells of Bacillus bellegensis CJBV. In the present invention, the spores of Bacillus bellegensis CJBV exhibit antibacterial activity against fungi and food poisoning bacteria, and thus can be used in microbial preparations for controlling plant diseases, foods with antibacterial activity, feeds, pharmaceuticals, quasi-drugs, and health functional foods. .

In the present invention, the term "culture" refers to a product obtained after culturing the strain, and may be a culture stock solution including cells, and may also be a culture strain, a culture supernatant removed or concentrated. The composition of the culture may additionally include components necessary for normal Bacillus culture, as well as components synergistically acting on the growth of Bacillus, and the composition accordingly can be easily selected by a person having ordinary skill in the art. I can.

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

In the present invention, the term "extract" means extracted from the culture or its concentrate, and as long as it is an extract capable of exhibiting a specific antibacterial effect on the fungi and food poisoning-causing bacteria of the present invention, the extract is not limited thereto. , Diluted liquid or concentrated liquid of the extract, dried product obtained by drying the extract, or these crude or purified products, and fractions obtained by fractionating them may all be included.

In the present invention, the term "dried product" means drying the culture, its concentrate, its extract, and its fraction. The drying method may be air drying, natural drying, spray drying, and freeze drying, but is not limited thereto.

The newly isolated Bacillus bellegensis CJBV of the present invention can be cultured through a conventional method of culturing a Bacillus strain, but is not limited thereto. As a medium, natural or synthetic medium may be used. As the carbon source of the medium, for example, glucose, sucrose, dextrin, glycerol, starch, etc. may be used, and as the nitrogen source, peptone, meat extract, yeast extract, dried yeast, soybean, ammonium salt, nitrate and other organic or inorganic Nitrogen-containing compounds may be used, but are not limited to these components. Inorganic salts included in the medium may include magnesium, manganese, calcium, iron, phosphorus, etc., 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 compounds related thereto may be added to the medium.

The antimicrobial composition may also contain other by-products produced in the process of culturing Bacillus bellegensis CJBV.

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

The type of food is not particularly limited, and may include all foods in a conventional sense. Non-limiting examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea. , Drinks, alcoholic beverages, and vitamin complexes. When the composition is used as a food additive, the composition may be added as it is or may be used with other foods or food ingredients, and may be appropriately used according to a conventional method.

The food composition may include a food pharmaceutically acceptable carrier.

In particular, the inventors of the present invention have the effect of simultaneously inhibiting the newly isolated Bacillus belegensys CJBV of the present invention, a degenerative yeast, Zygo Saccharomyces bily, a food poisoning bacterium Bacillus cereus, and Listeria monocytogenes. In addition, it was confirmed that there was no antibacterial activity against Bacillus subtilis , Bacillus amyloliquefaciens , which are useful enteric fermentation bacteria in fermented food (Example 3, FIG. 4), the antibacterial The composition can be used directly in any food product, including fermented foods.

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

In the present invention, the term "feed" may mean any natural or artificial diet, one meal meal, etc., or a component of the one meal meal, for animals to eat, ingest, and digest.

The kind of feed is not particularly limited, and feed commonly used in the art may be used. The feed composition may include a feed additive. The feed additive of the present invention corresponds to an auxiliary feed according to the feed management method, and may contain a probiotic. Non-limiting examples of the feed include vegetable feeds such as grains, root fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, meals or grain by-products; And animal feeds such as proteins, inorganic logistics, oils, minerals, oils and fats, single cell proteins, zooplanktons or food. These may be used alone or in combination of two or more.

At this time, the feed composition comprising the antibacterial composition of the present invention may further include excipients, diluents, and additives.

It was confirmed that the novel isolated Bacillus bellegensis CJBV of the present invention has the effect of simultaneously inhibiting the degenerative yeast Zygo Saccharomyces biley, the food poisoning bacteria Bacillus cereus, and Listeria monocytogenes. The present invention provides a feed composition having antibacterial and antifungal activity, and can prevent microbial infectious diseases including food poisoning of livestock.

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

The inventors of the present invention have the inhibitory effect on plant pathogens, including the newly isolated Bacillus bellegensis CJBV of the present invention, Choletotricum orbiculae, which is the cause of anthrax, and Fusarium oxysporum, which is the cause of vines. 3), the antimicrobial composition of the present invention is used as an eco-friendly, non-polluting biological pesticide, and may be useful for destroying the ecosystem and solving toxicity problems by organic synthetic pesticides, and it is possible to reduce the financial burden of each farmer. . In addition, due to the nature of microorganisms, it is sustainable and can provide long-term benefits in the future.

As another aspect for achieving the above object, the present invention provides a method for controlling phytopathogenic fungi comprising the step of treating the plant or soil with the antimicrobial agent for plant crops.

Preferably, the phytopathogenic fungi may be Coletotricum orbiculae, which causes anthrax, or Fusarium oxysporum, which causes vines, but is not limited thereto.

The above microbial agents are preferably sprayed on the above-ground part of the plant from the 15th day after planting or in the rainy season and immediately before the onset when the soil is cultivated before planting the crop, using a foliar treatment method and a drench on the root of the plant. Is not limited.

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

The term "prevention" used in the present invention may mean any action of inhibiting or delaying the onset of a microbial infectious disease by administering the composition for preventing or treating a microbial infectious disease according to the present invention to an individual.

The term "treatment" used in the present invention may refer to any act of administering the composition of the present invention to an individual suspected of developing a microbial infection to improve or benefit the symptoms of a microbial infection.

As used herein, the term "improvement" may mean any action that at least reduces the severity of a parameter related to a condition to be treated, for example, a symptom.

The term "individual" used in the present invention may mean all animals including humans who have or are likely to develop a microbial infection.

In this case, the microbial infectious disease is a disease caused by infection by a microorganism, and preferably may be a disease caused by infection by a fungus or food poisoning-causing bacteria.

As an example, the fungus is Coletotricum orbiculae, Fusarium oxysporum f.sp. Lycopulcisyl JCM12575, Candida albicans, and Zygosaccharomyces biley.

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

As another example, the microbial infectious disease is food poisoning, skin mycosis, vaginitis, diarrhea, vomiting, enteritis, gastroenteritis, constipation, abdominal pain, abdominal distention, cholera, listeriosis, cellulitis, urinary tract infection, meningitis, peritonitis, cystitis, lymphatic vessels. Salt, superficial, otitis media, respiratory disease, pneumonia, purulent inflammation, or sepsis.

Cellulitis refers to an acute purulent inflammation that appears in the dermis and subcutaneous tissue, and purulent inflammation occurs in the tissues, and strong neutrophil infiltration is a chronic and widespread condition in the tissue gap, accompanied by congestion or edema.

Felon refers to acute purulent inflammation of the finger or toe.

The inventors of the present invention are Candida albicans, which is present in the oral cavity, intestine, and vagina of animals including humans and can cause skin mycosis, candidal vaginitis, enteritis, oral inflammation, etc. upon infection. Xygosaccharomyces biley, which can cause fever, Bacillus cereus, which can cause food poisoning, diarrhea, and vomiting when infected, Listeria monocyto, which can cause food poisoning, listeriosis, sepsis, encephalitis, and meningitis when infected. It was confirmed that the excellent inhibitory effect of the strain of the present invention on Genes was confirmed (FIGS. 3 and 4), and the antimicrobial composition of the present invention can be effectively used in a pharmaceutical composition for preventing or treating microbial infectious diseases.

The composition for preventing or treating microbial infectious diseases according to the present invention may additionally include a pharmaceutically acceptable carrier, and is formulated with the carrier to be formulated with foods, health functional foods, pharmaceuticals, quasi-drugs, feed additives, drinking water additives, etc. Can be provided. As used herein, the term "pharmaceutically acceptable carrier" may mean a carrier or diluent that does not stimulate an organism and does not inhibit the biological activity and properties of the administered compound.

The kind of the carrier that can be used in the present invention is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable can be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These may be used alone or in combination of two or more.

In addition, if necessary, other conventional additives such as antioxidants, buffers and/or bacteriostatic agents can be added and used, and diluents, dispersants, surfactants, binders, lubricants, etc. can be additionally added to Formulations, pills, capsules, granules, or tablets can be formulated and used.

The administration method of the composition for preventing or treating microbial infectious diseases according to the present invention is not particularly limited, and may be according to a method commonly used in the art. As a non-limiting example of the mode of administration, the composition may be administered orally or parenterally. The composition for preventing, improving or treating the microbial infectious disease of the present invention may be prepared in various formulations 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 a pharmaceutically acceptable salt thereof, and may be used alone or in combination with other pharmaceutically active compounds, as well as in an appropriate set.

As another aspect, the present invention provides a method for preventing or treating a microbial infectious disease comprising administering the composition for preventing or treating the microbial infectious disease to an individual. The term "individual" used in the present invention may mean all animals including humans who have or are likely to develop a microbial infection.

The prevention or treatment method of the present invention may specifically include administering the composition in a pharmaceutically effective amount to an individual who has developed or is at risk of developing a microbial infection.

The appropriate total daily use amount of the composition containing the extract may be determined by the treating physician within the range of correct medical judgment, and may be administered once or divided into several times. However, for the purposes of the present invention, a specific therapeutically effective amount for a specific patient is a specific composition, including the type and degree of reaction to be achieved, whether other agents are used in some cases, the patient's age, weight, general health condition, It is preferable to apply differently according to various factors including sex and diet, administration time, administration route and secretion rate of the composition, treatment period, drugs used with or concurrently with the specific composition, and similar factors well known in the medical field.

The antimicrobial substance of the present invention can be administered to mammals such as rats, mice, livestock, and humans by various routes. All modes of administration can be expected and can be administered by, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dura mater or intracerebroventricular injection.

As another aspect, the present invention provides a health functional food for preventing or improving microbial infectious diseases comprising the antibacterial composition.

At this time, the meaning of microbial infectious diseases, prevention, and improvement is as described above.

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

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

The present inventors confirmed that the newly isolated Bacillus bellegensys CJBV has an antibacterial effect against the Candida albicans, and the antimicrobial composition using the new strain is used to treat and alleviate microbial infectious diseases including cutaneous mycosis and Candida vaginitis. , It can be effectively used in quasi-drugs for treatment or prevention.

The newly isolated Bacillus bellegensis 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, so it is an antibacterial agent for plant crops, food with antibacterial activity, and prevention of microbial infectious diseases. It can be used as feed for improvement, treatment, health functional food, quasi-drugs, and medicines.

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

FIG. 1 shows a process of separating an antibacterial active ingredient, Bacilomycin D analog, from Bacillus belegensys CJBV of the present invention.
Figure 2 shows the ESI-MS and UV spectrum of the antimicrobial material produced in the strain according to the present invention.
Figure 3 shows the antimicrobial activity against four types of fungi Choletotricum obiculae, Fusarium oxysporum, Candida albicans, and Xygosaccharomyces viley.
Figure 4 shows the antibacterial activity against food poisoning-causing bacteria Bacillus cereus, Listeria monocytogenes, and enteric fermentation bacteria Bacillus subtilis and Bacillus amyloliquefaciens.

Hereinafter, the present invention is described in detail by 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) Isolation and selection of strains

In order to select a strain having antibacterial activity, Bacillus strains isolated from conventional meju were used. The strains were pre-cultured in a soybean fermentation liquid medium at 37°C for 36 hours. Pathogenic fungi and food poisoning-causing bacteria were placed on an agar solid medium consisting of lawn cells, and then a paper disk having a diameter of 8 mm was placed on it, and the pre-cultured strain was inoculated. After culturing for 12 hours, strains that simultaneously produced transparent rings against fungi and bacteria were selected around strains inoculated on the medium.

(2) Identification of strain

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

From the above results, the strain obtained in the present invention was identified as a Bacillus belegensis strain and named as Bacillus belegensis CJBV, and the strain was deposited with the Korea Microbial Conservation Center (KCCM) on April 17, 2014, and accession number KCCM11528P. Was given.

Example 2: Isolation and purification of antibacterial substances

An experiment was conducted to isolate an antimicrobial substance produced by the newly isolated Bacillus bellegensis CJBV. The process is shown in FIG. 1.

The strain was inoculated into 400 µl of soybean fermentation liquid medium and then cultured with shaking at 200 rpm for 48 hours at 37°C. The culture solution was centrifuged at 8000 x 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 in a certain ratio, and fractions having antibacterial activity were separated and purified.

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

Figure 2 shows the ESI-MS and UV spectrum of the separated antimicrobial material.

Through the above analysis, it was confirmed that the substances isolated in the present invention were three types of bacilomycin D analogs represented by the following formula (1).

[Formula 1]

Wherein R is, in the case of erythromycin D by Vasil C14- CH _3, in the case of erythromycin D as C15- Basil CH ₂ -CH _3, in the case of erythromycin D C16- Basil is CH ₂ -CH ₂ -CH _3.

Example 3: Antibacterial activity test against fungi and food poisoning-causing bacteria

In order to confirm whether the newly isolated Bacillus belegensys CJBV has an inhibitory effect on fungi and food poisoning-causing bacteria, the following antimicrobial activity experiments were conducted.

(1) Antibacterial activity test against yeast and mold (fungi)

First, the fungus Colletotrichum orbiculae orbiculare ), Fusarium oxysporum f.sp. Lycopulcisyl JCM12575 ( Fusarium oxysporum f.sp. lycopersici JCM12575), Candida albicans ( Candida albicans ), the pathogenic yeast Zygosaccharomyces bailii ) was tested to see if the newly isolated Bacillus belegensis CJBV had antibacterial activity.

Each of the four fungi was cultured on a potato dextrose agar (PDA) medium. Thereafter, after dissolving the butanol extract of the Bacillus bellegensis CJBV culture solution, which is an antimicrobial composition concentrated under reduced pressure according to Example 2, in ethanol, an 8 mm diameter paper disk was placed on each plate medium plated with the four strains. After adding the antimicrobial substance at a constant rate of 25 µl, it was incubated at 30° C. for 24 to 48 hours to observe whether or not the growth inhibition ring was produced by the antibacterial substance produced by the isolated strain.

(2) Antibacterial activity test against food poisoning-causing bacteria

In addition, the following experiment was conducted to find out whether the newly isolated Bacillus bellegensis CJBV has antibacterial activity specifically only for food poisoning-causing bacteria in fermented foods.

Bacillus cereus , Listeria monocytogenes , and Bacillus subtilis , Bacillus amyloliquefaciens , and Tryptic soy (TSA) respectively agar) medium.

Thereafter, after dissolving the butanol extract (Extraction with BuOH) of Bacillus bellegensis CJBV culture solution, which is an antimicrobial composition concentrated under reduced pressure according to Example 2, in ethanol, 8 mm diameter on each plate medium plated with the four strains After placing a paper disc of the antibacterial substance at a constant rate of 25 µl, it was cultured at 37°C for 24 hours to observe whether or not the growth inhibition ring was produced by the antimicrobial substance produced by the isolated strain.

In addition to the above experimental group, butanol (BuOH) was used instead of the butanol extract of Bacillus bellegensis CJBV culture medium as a control group for the two types of enteric fermentation bacteria.

The results according to Example 3 are shown in Figs. 3 and 4, and the degree of antimicrobial activity is shown in Table 1 according to the degree of production of the growth-stopping ring.

microbe Antibacterial activity Colletotrichum orbiculare +++ Fusarium oxysporum f.sp. lycopersici +++ Candida albicans ++ Zygosaccharmyces bailii ++ Bacillus cereus + Listeria monocytogenes + Bacillus subtilis - Bacillus amyloliquefaciens -

In the case of'-' in Table 1, when growth is not inhibited, in the case of'+', the diameter of the growth-stop ring is 3mm to 5 mm or less, and in the case of'++', when the diameter of the growth-stop ring is 6mm to 10mm In the case of'+++', the diameter of the growth stop ring is 10 mm or more.

As shown at the top of FIG. 3, selected from the group consisting of the newly isolated Bacillus bellegensis CJBV strain of the present invention, spores of the strain, cultures of the strain, concentrates thereof, extracts thereof, and dried products thereof. As a result of treatment of an antimicrobial composition containing one or more as an active ingredient on pathogenic fungi Choletotricum obiculae and Fusarium oxysporum, a growth-stopping ring was formed over 10 mm, which has excellent antibacterial activity to inhibit the growth of fungi. Was confirmed.

Therefore, the novel isolated Bacillus bellegensis CJBV and antimicrobial composition of the present invention suppress plant infectious diseases such as anthrax and vines on gourd and vegetable crops such as cucumber, melon, watermelon, etc., and microbial preparations for controlling plant diseases and plants using the same It can be effectively used in the control method of pathogenic fungi.

In addition, as shown in the lower part of Figure 3, the newly isolated Bacillus belegensys CJBV of the present invention, the bacilomycin D analog produced in the strain, or the antimicrobial composition of the pathogenic fungi Candida albicans, Zygosaccharomyces As a result of treatment with Seth Bailey, it was confirmed that the growth-stopping ring was formed from 6mm to 10mm, and the antibacterial activity was excellent for inhibiting the growth of fungi.

Accordingly, the novel isolated Bacillus bellegensis CJBV of the present invention, the bacilomycin D analog produced from the strain, and the antibacterial composition are prevented, improved, and prevented against microbial infectious diseases such as skin mycosis, candidal vaginitis, oral cavity, intestine, etc. It exhibits a therapeutic effect and can be effectively used in medicines, quasi-drugs, health functional foods, and feeds for this purpose. In addition, it can be effectively used as a food with a low risk of food poisoning.

In addition, as shown in FIG. 4, as a result of simultaneously treating the novel isolated Bacillus belegensys CJBV or antimicrobial composition of the present invention with food poisoning-causing bacteria and useful bacteria used during enteric fermentation, food poisoning-causing bacteria Bacillus cereus and Listeria It was confirmed that the monocytogenes exhibited excellent antibacterial activity, but did not inhibit the growth of enteric fermentation bacteria.

Bacteriocins reported to be produced by conventional microorganisms do not exhibit antimicrobial activity specifically only for food poisoning-causing bacteria, and have an effect on the growth of enteric fermentation bacteria, making it difficult to use directly in fermented foods.

On the other hand, the newly isolated Bacillus belegensys CJBV of the present invention, the bacilomycin D analog produced from the strain, and the antimicrobial composition using the same have no antibacterial activity against enteric fermentation bacteria, and are specific only to major harmful microorganisms of food such as food poisoning bacteria Since it has antibacterial activity that inhibits it, it can be used directly in fermented foods.

Furthermore, it was confirmed that it has excellent antimicrobial activity against various food contaminants, the newly isolated Bacillus belegensis CJBV of the present invention, the bacilomycin D analog produced from the strain, and the antimicrobial composition using the same, food poisoning, diarrhea, It has the effect of preventing, improving, and treating microbial infectious diseases including vomiting, so it can be effectively used in foods, medicines, quasi-drugs, health functional foods, and feeds for this purpose.

As described above, the newly isolated Bacillus belegensys CJBV of the present invention, and the bacilomycin D analog produced from the strain, have antibacterial effects against one or more different types of fungi and bacteria, so that the antibacterial agent for plant crops, antibacterial It can be used as an active food and feed for the prevention, improvement, and treatment of microbial infectious diseases, health functional foods, quasi-drugs, and pharmaceuticals.

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 changing the technical spirit or essential features thereof. In this regard, the embodiments described above are illustrative in all respects and should be understood as non-limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description and equivalent concepts are included in the scope of the present invention.

Korea Microorganism Conservation Center (overseas) KCCM11528P 20140417

<110> CJ CheilJedang Corporation <120> Novel Bacillus velezensis CJBV and antibacterial and antifungal composition comprising the same <130> KPA140376-KR-D1 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 1493 <212> DNA <213> Bacillus velezensis CJBV <400> 1 ttgctcagga cgaacgctgg cggcgtgcct aatacatgca agtcgagcgg acagatggga 60 gcttgctccc tgatgttagc ggcggacggg tgagtaacac gtgggtaacc tgcctgtaag 120 actgggataa ctccgggaaa ccggggctaa taccggatgg ttgtttgaac cgcatggttc 180 agacataaaa ggtggcttcg gctaccactt acagatggac ccgcggcgca ttagctagtt 240 ggtgaggtaa cggctcacca aggcaacgat gcgtagccga cctgagaggg tgatcggcca 300 cactgggact gagacacggc ccagactcct acgggaggca gcagtaggga atcttccgca 360 atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg aaggttttcg gatcgtaaag 420 ctctgttgtt agggaagaac aagtgccgtt caaatagggc ggcaccttga cggtacctaa 480 ccagaaagcc acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt 540 gtccggaatt attgggcgta aagggctcgc aggcggtttc ttaagtctga tgtgaaagcc 600 cccggctcaa ccggggaggg tcattggaaa ctggggaact tgagtgcaga agaggagagt 660 ggaattccac gtgtagcggt gaaatgcgta gagatgtgga ggaacaccag tggcgaaggc 720 gactctctgg tctgtaactg acgctgagga gcgaaagcgt ggggagcgaa caggattaga 780 taccctggta gtccacgccg taaacgatga gtgctaagtg ttagggggtt tccgcccctt 840 agtgctgcag ctaacgcatt aagcactccg cctggggagt acggtcgcaa gactgaaact 900 caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg 960 cgaagaacct taccaggtct tgacatcctc tgacaatcct agagatagga cgtccccttc 1020 gggggcagag tgacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt 1080 aagtcccgca acgagcgcaa cccttgatct tagttgccag cattcagttg ggcactctaa 1140 ggtgactgcc ggtgacaaac cggaggaagg tggggatgac gtcaaatcat catgcccctt 1200 atgacctggg ctacacacgt gctacaatgg acagaacaaa gggcagcgaa accgcgaggt 1260 taagccaatc ccacaaatct gttctcagtt cggatcgcag tctgcaactc gactgcgtga 1320 agctggaatc gctagtaatc gcggatcagc atgccgcggt gaatacgttc ccgggccttg 1380 tacacaccgc ccgtcacacc acgagagttt gtaacacccg aagtcggtga ggtaaccttt 1440 taggagccag ccgccgaagg tgggacagat gattggggtg aagtcgtaca ggg 1493 <210> 2 <211> 966 <212> DNA <213> Bacillus velezensis CJBV <400> 2 cgtatcccgg gcgcttccgg atgtgcgtga cggtctgaag ccggttcaca gacggatttt 60 gtacgcaatg aatgatttag gcatgaccag tgacaaacca tataaaaaat ctgcccgtat 120 cgtcggtgaa gttatcggta agtaccaccc gcacggtgac tcagcggttt acgaatcaat 180 ggtcagaatg gcgcaggatt ttaactaccg ctacatgctt gttgacggac acggcaactt 240 cggttcggtt gacggcgact cagcggccgc gatgcgttac acagaagcga gaatgtcaaa 300 aatcgcaatg gaaattctgc gtgacattac gaaagacacg attgactatc aagataacta 360 tgacggttca gaaagagagc ctgccgtcat gccttcgaga tttccgaatc tgctcgtaaa 420 cggggctgcc ggtattgcgg tcggaatggc gacaaacatt cctccccatc agcttggaga 480 agtcattgaa ggcgtgcttg ccgtaagtga gaatcctgag attacaaacc aggagctgat 540 ggaatacatc ccgggcccgg attttccgac tgctggtcag attttgggcc ggagcggcat 600 ccgcaaggca tatgaatccg gacggggatc aatcacaatc cgggctaagg ctgaaatcga 660 agagacatca tcaggaaaag aaagaattat tgttacggaa cttccttatc aggtgaacaa 720 agcgagatta attgaaaaaa tcgcagatct tgtccgggac aaaaaaatcg aaggaattac 780 cgacctgcga gacgaatccg accgtaacgg aatgagaatc gtcattgaga tccgccgtga 840 cgccaatgct cacgtcattt tgaataacct gtacaaacaa acggccctgc agacgtcttt 900 cggaatcaac ctgctggcgc tcgttgacgg acagccgaag gtgctgagcc tgaagcaatg 960 cctgga 966

Claims (7)

A compound represented by the following formula (1):
[Chemical Formula 1]

Wherein R is CH ₃ , CH ₂ -CH ₃ or CH ₂ -CH ₂ -CH ₃ .
2. The method of claim 1, wherein R is CH ₂ -CH ₂ -CH ₃ in the compounds.
An antimicrobial composition for fungi, food poisoning-causing bacteria, or combinations thereof, comprising a compound according to claim 1.
An antimicrobial composition for food poisoning-causing bacteria, comprising the compound according to claim 1.
An antimicrobial composition for food poisoning-causing bacteria comprising the compound according to claim 2.
4. The composition of claim 3, wherein the antimicrobial composition is selected from the group consisting of Colletotrichum orbiculare , Fusarium &lt; RTI ID = 0.0 &gt; oxysporum), Candida albicans (Candida albicans , Zygosaccharomyces bailii , Bacillus cereus , Listeria monocytogenes , or a combination thereof.
4 according to any one of claims 5, wherein the antimicrobial composition comprises Bacillus cereus (Bacillus cereus), Listeria monocytogenes jeneseu (Listeria monocytogenes ) or a combination thereof.

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