KR101629551B1 - Novel Bacillus velezensis CJBV and antibacterial and antifungal composition comprising the same - Google Patents

Abstract

The present invention relates to a novel isolated Bacillus velezensis CJBV strain of Accession No. KCCM11528P, and to an antimicrobial composition using the same, and a novel isolated Bacillus velezensis CJBV Has antimicrobial effect on at least one kind of fungi and food poisoning bacterium. Therefore, the antimicrobial agent for plant crops, the food including fermented food having antimicrobial activity, and the food for prevention, improvement and treatment of microbial infectious diseases Functional foods, quasi-drugs, and medicines.

Description

[0001] The present invention relates to a novel Bacillus velencys CJBV and an antimicrobial composition comprising the same, which comprises the novel antibacterial and antifungal composition,

The present invention relates to novel Bacillus cereus CJBV ( Bacillus spp.) Having a specific antimicrobial activity against fungi and food poisoning- velezensis CJBV), and an antimicrobial composition using the basilomycin D analog separated therefrom, and a pharmaceutical composition for preventing or treating a microbial infection disease comprising the same.

When soybean fermented food is produced in a conventional manner, it is exposed to the risk of food poisoning caused by harmful bacteria contained in microorganisms because it is a food using microorganisms. However, harmful bacteria such as pathogenic fungi and food poisoning-causing bacteria are not completely killed by the general sterilization method, and toxic substances are generated to cause food poisoning.

Therefore, although a chemical preservative or preservative is used to prevent deterioration or corruption and to maintain the quality of the food for a long time, there is a risk that the chemical itself may act as an environmental hormone or cause skin allergy, so that a chemical preservative or preservative Consumer rejection is increasing.

As a result, there is a growing interest in natural preservatives that can inhibit the growth of microorganisms or metabolites thereof. Interest in bacteriocin or bacteriocin-like substances has been growing as one example of a natural antimicrobial substance that can be used as a preservative. Bacteriocin is a natural, non-toxic preservative, an antimicrobial protein composed of a complex of proteins or carbohydrates produced by many Gram-positive bacteria and gram-negative bacteria. In particular, bacteriocin has been recognized as an antibacterial agent that can be used in the food industry since nisin in bacteriocins was approved for use in processing cheese spreads from the US FDA in 1998.

A number of studies have been conducted on the above-mentioned antimicrobial substances produced by the genus Bacillus or lactic acid bacteria. Unlike lactic acid bacteriocins which can inhibit the growth of gram-positive bacteria, the bacteriocins produced by the Bacillus strains have a wide range of antibacterial activity, .

Specifically, the genus Bacillus is known to produce a variety of antimicrobial substances (Sholberg et al., 1995 Can. J. Microbio. 41, 247-252), reported antifungal agents include mycosubtilin, eumycin, bacillomycin, surfactin, iturin, aminopolyol, lipoprotein, and the like, which have antibacterial and antifungal activity at the same time The report includes bacilysin produced by Bacillus subtilis .

In a related prior literature Korea Patent Publication 10-2013-0008673 is Bacillus amyl Lowry query Enschede Pacific strain (Bacillus amyloliquefaciens GYL4), have been shown to have broad activity against phytopathogenic fungi.

Korean Patent Laid-Open No. 10-2013-0025453 discloses an antimicrobial substance produced by Bacillus subtilis. UV254-B, a new antimicrobial substance completely different from an authentic substance containing a basilomycin, is used as a food poisoning- And exhibit anti-bacterial activity specifically against Listeria monocytogenes alone.

However, reported bacteriocin has been reported only for antimicrobial activity against specific microorganisms, and bacteriocin has not been reported to be effective against various pathogenic bacteria causing microbial infectious diseases including various crop diseases or food poisoning. Further, since Bacillus amyloliquefaciens described in the above document is a fermented soybean fermenting bacterium useful in fermentation food, when the bacteriocin is applied to fermented soybean food, it is difficult to show specific activity only for food poisoning-causing fungi and bacteria, There was a difficult problem.

Therefore, the present inventors have made extensive efforts to develop a method of inhibiting various fungi and bacteria by harmless to the human body. As a result, the inventors of the present invention found that Bacillus cereus CJBV newly isolated from conventional meju is effective against one or more kinds of fungi such as pathogenic fungi and yeast The present inventors have completed the present invention by confirming that there is no antimicrobial activity against fermented soybean bacteria and that there is an antimicrobial activity specifically inhibiting only major microorganisms of food such as food poisoning bacteria.

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

Another object of the present invention is to provide a basilomycin D analogue produced in said strain of Bacillus cereus CJBV.

Another object of the present invention is to provide an antimicrobial composition using the above-mentioned Bacillus cereus CJBV strain and the above-mentioned basilomycin D analog.

It is a further object of the present invention to provide a food composition comprising the antimicrobial composition.

It is still another object of the present invention to provide a feed composition comprising the antimicrobial composition.

It is still another object of the present invention to provide an antimicrobial agent for plant crops containing the antimicrobial composition.

It is still another object of the present invention to provide a method for controlling phytopathogenic fungi, which comprises treating the antimicrobial agent to a plant or soil.

It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating a microbial infection disease comprising the above-mentioned antimicrobial composition.

It is still another object of the present invention to provide a health functional food for preventing or ameliorating a microbial infectious disease comprising the antimicrobial composition.

It is still another object of the present invention to provide a quasi-drug comprising the antimicrobial composition.

In order to achieve the above object, the present invention provides a novel isolated Bacillus cereus CJBV ( Bacillus spp.) Having a specific antimicrobial activity against fungi and food poisoning- velezensis CJBV).

In the present invention, the term Bacillus is an aerobic or thermophilic anaerobic gram-positive bacterium widely distributed in nature, and Bacillus bacteria such as microorganisms belonging to the genus Bacillus. The present inventors have identified a novel strain belonging to Bacillus blazensis as Bacillus sp. Having a specific antimicrobial activity against fungi and food poisoning-causing bacteria as follows.

First, Bacillus strains were isolated from conventional meju, tested for fungi such as pathogenic fungi and yeast, and fungi of food poisoning, and strains having excellent antimicrobial activity were selected, and 16S rDNA and gyrA gene sequence analysis was performed in order to identify selected strains . The 16S rDNA gene sequence of the selected strain is shown in SEQ ID NO: 1, which shows 99% homology with Bacillus cereus. In addition, the gyrA gene sequence of the selected strain is shown in SEQ ID NO: 2, which is 100% homologous to Bacillus cereus (Example 1). Thus, the strain was identified as Bacillus velezensis and designated as Bacillus breezensis CJBV, deposited on April 17, 2014 under the Budapest Treaty with the Korean Society for Microbiological Protection (KCCM) KCCM11528P (Example 1).

Therefore, the Bacillus cereus CJBV has the 16s rDNA nucleotide sequence shown in SEQ ID NO: 1.

According to one aspect of the present invention, the newly isolated Bacillus brecesens CJBV has excellent antimicrobial activity against fungi such as plant pathogenic fungi and yeast, inhibits food poisoning-causing bacteria, and is effective against various fungi and food poisoning- (Example 3, Fig. 3 and Fig. 4).

In one embodiment, the fungus is Collet sat tree glutamicum Ob particulate rare (Colletotrichum orbiculare , Fusarium < RTI ID = 0.0 > oxysporum), Candida albicans (Candida albicans , and Zygosaccharomyces < RTI ID = 0.0 > bailii ), but the present invention is not limited thereto.

In the present invention, the term choletotrichum orbicule refers to a fungus causing anthracnose to a plant or vegetable crop such as cucumber, melon, watermelon and the like.

In the present invention, the term fusarium oxysporum means a pathogenic fungus that causes fusarium wilt against foliage and vegetable crops such as cucumber, melon, watermelon and the like.

The inventors of the present invention confirmed that the newly isolated Bacillus bellegensis CJBV has antimicrobial effect against the choletotrichum oryziculare and fuarium oxysporum, It can be used as a microorganism preparation for controlling plant diseases by inhibiting plant infectious diseases such as anthrax and vine fungus on poultry and vegetable crops.

Therefore, the use of the newly isolated Bacillus brecesens CJBV as an environmentally friendly non - polluting biological pesticide can be useful for eliminating ecosystem destruction and toxicity problems caused by organic synthetic pesticides and reducing the financial burden on each farm. In addition, due to the nature of the microorganism, it can be sustained and its long-term benefits can be seen in the future.

On the other hand, the biological pesticide is defined as a product obtained by isolating microorganisms isolated in a natural environment in order to control plant pathogens of crops.

In the present invention, the term Candida albicans is a genus of fungi belonging to the incomplete fungus, causing skin fungus, Candida oral cavity, enteric diseases, vaginitis and the like. Candida albicans is a common condition in which about 75% of women experience candida vaginitis in vagina and vulva during their lifetime, and 45% of women have two or more recurrences a year, most of which are caused by Candida albicans. Candida albicans, which is normally found in the oral cavity, bowel, vagina, etc. of a healthy person, can be infected when the condition of the female hormone is abnormal or the immune system is extremely weak.

The inventors of the present invention confirmed that the newly isolated Bacillus bellegensis CJBV has an antibacterial effect against the Candida albicans. As a result, the novel strain can effectively prevent or prevent skin fungus, candida vaginitis, microbial infectious oral cavity, Can be treated.

In the present invention, the term Zygosaccharomyces cerevisiae is a yeast of the genus Saccharomyces. Decomposable yeast produces a large amount of carbon dioxide gas as well as alcohol fermentation in beverage products and starch processed products, thereby deteriorating the quality of food.

The present inventors confirmed that the newly isolated Bacillus brecesensis CJBV has an antibacterial effect against the above-mentioned Zygosaccharomyces cerevisiae. By using the novel strain, the storage stability of foods such as beverage products and starch processed products can be improved It is possible to prevent or treat microbial infectious diseases such as food poisoning which may occur therefrom.

In yet another embodiment, the cause of food poisoning bacteria are Bacillus cereus (Bacillus cereus) or L. monocytogenes jeneseu (Listeria monocytogenes , but are not limited thereto.

In the present invention, the term Bacillus cereus is a Gram-positive bacterium that forms heat-resistant spores, which cause diarrhea and vomiting when infected. It is an aerobic bacterium widely distributed in the natural world such as soil and cereal. The spores of this strain are more resistant to washing and disinfection than the other spores because of their excellent surface adhesion ability. The above-mentioned Bacillus cereus can be propagated by germination of spores in meat, vegetables, rice, cotton, etc. left at room temperature for several hours after cooking, germinated in milk and cheese after pasteurization, and produced heat resistant toxin So that it is not easy to inhibit it.

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

In the present invention, the term Listeria monocytogenes is a common pathogen of acquired infectious diseases and can be transmitted to humans through livestock foods such as milk and cheese, and is one of pathogenic food poisoning bacteria which has recently attracted worldwide attention. If the bacteria are infected and listeriosis occurs, the mortality rate is as high as about 30%. In particular, human infections are susceptible to infectious diseases such as infants, organ transplants, lymphomas, and AIDS patients who are immunocompromised or immunodeficient, and may cause sepsis, encephalitis and meningitis. Unlike other pathogenic fungi, the pathogenic fungi are capable of growth even at low temperatures. It is known that the pathogenic fungi grown at 4 ° C are more virulent than those grown at 37 ° C. Due to the characteristics of these bacteria, food poisoning caused by Listeria monocytogenes can occur in refrigerated foodstuffs and refrigerated foodstuffs and stored foods which are considered to be safe at home, which is a serious problem.

The present inventors have confirmed that the newly isolated Bacillus brecesensis CJBV has an antimicrobial effect against the Listeria monocytogenes. As a result, the novel strain can prevent or treat microbial infectious diseases such as food poisoning and listeriosis.

In particular, the present inventors newly separated Bacillus Belle Zensys CJBV said Bacillus cereus (Bacillus of the present invention cereus), Listeria monocytogenes jeneseu (Listeria monocytogenes) to be only effective as that to suppress the same time, the useful soy sauce fermentation bacteria in the fermented food Bacillus subtilis (Bacillus subtilis), Bacillus amyl Lowry query Pacific Enschede (Bacillus amyloliquefaciens ) was found to have no antimicrobial activity (Example 3, Fig. 4).

In one embodiment, the Bacillus cereus CJBV is selected from the group consisting of antibiotic Bacillomycin D, and a benzylmyrimine D analogue represented by formula 1, namely C14-barylomycin D, C15- It is possible to generate and secrete any one or more of the basilomycin D analogues selected from the group consisting of VILIMIN D,

[Chemical Formula 1]

In the above formula, R is CH ₃ for C ₁₄ -varymylin D, CH ₂ -CH _{3 for} C ₁₅ -varymylin D, and CH ₂ -CH ₂ -CH ₃ for C ₁₆ -varymylin D .

In the present invention, the term " basilomycin D " refers to a substance belonging to the iturin family, which is one of the cyclic peptide groups, and includes seven? -Amino acids (2 Asn, Glu, Tyr, Pro, Ser, Thr) It has a ring structure composed of? -amino acid and has antibiotic activity.

The present inventors isolated the antimicrobial substance produced and secreted by the newly isolated Bacillus blazensis CJBV of the present invention and measured the UV spectrum and the molecular weight. As a result, it was confirmed that the isolated substance was the analog of the above-mentioned basilomyces D Example 2, Figures 1 and 2).

In another aspect to achieve the above object, the present invention provides a basilomycin D analog produced from the novel Bacillus buretzensis CJBV strain.

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

[Chemical Formula 1]

In the above formula, wherein R is CH _3, and CH ₂ -CH ₃ or CH ₂ -CH ₂ -CH _3, CH ₃ when the case is in the C14- Basil mitomycin D, CH ₂ -CH ₃ is a C15- Basil MICIN D, and CH ₂ -CH ₂ -CH ₃ .

The present inventors isolated the antimicrobial substance produced and secreted by the newly isolated Bacillus blazensis CJBV of the present invention and measured the UV spectrum and the molecular weight. As a result, it was confirmed that the isolated substance was the analog of the above-mentioned basilomyces D Example 2, Figures 1 and 2).

A plant pathogenic bacterium including a choletotrichum oviculare causative of anthrax, a fructitus oxysporum causing a fission of the fungus, and the like, a plant pathogenic bacterium which is produced by the novel Bacillus buretzensis CJBV strain, Candida albicans, which can cause skin fungus, candida vaginitis, enteritis and oral inflammation, Zygosaccharomyces cerevisiae, a transforming yeast, Bacillus cereus, and Listeria monocytogenes, food poisoning bacteria, And antibacterial effect against bacteria (FIGS. 3 and 4). Therefore, it is possible to use the basilomycin D analogue produced from the novel Bacillus brecesens CJBV strain as an antimicrobial composition, and to provide a feed, a pharmaceutical composition, a health functional food, a quasi-drug composition for preventing, Can be effectively used for foods having antimicrobial activity, antimicrobial agents for plant crops, and methods of controlling them using them.

According to another aspect of the present invention, there is provided a method for producing a Bacillus sp. Strain CJBV strain, which comprises the newly isolated Bacillus brecesens CJBV strain, a spore of the strain, a culture of the strain, a concentrate thereof, an extract thereof, Or a pharmaceutically acceptable salt thereof, as an active ingredient. The state of the strain may be a liquid state or a dry state, but is not limited thereto.

The term "spore" in the present invention refers to a reproductive cell such as a bacterium. For the purpose of the present invention, the spore refers to a reproductive cell of Bacillus cereus CJBV. In the present invention, the spores of Bacillus brecesens CJBV exhibit antimicrobial activity against fungi and food poisoning bacteria and can be used as a microorganism preparation for controlling plant diseases, foods having antimicrobial activity, feeds, medicines, quasi-drugs, and health functional foods .

In the present invention, the term "culture product" means a product obtained by culturing the strain, and may be a culture solution containing cells, and may be a culture or a culture obtained by removing or concentrating the culture supernatant. The composition of the culture may additionally contain not only the components necessary for culturing normal Bacillus but also a component acting synergistically with the growth of bacillus, and the composition thereof may be easily selected by those skilled in the art .

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

As used herein, the term "extract" means an extract from the culture or a concentrate thereof, and is not limited to the extracts capable of exhibiting a specific antimicrobial effect on the fungi and food poisoning-causing bacteria of the present invention, , A diluted solution or concentrate of the extract, a dried product obtained by drying the extract, or a fraction prepared by fractionating these preparations or purified products.

In the present invention, the term "dried material" means that the culture, its concentrate, its extract, and its fractions are dried. Drying methods include, but are not limited to, air drying, natural drying, spray drying and freeze drying.

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

The antimicrobial composition may include other by-products generated during the cultivation of Bacillus cereus CJBV.

In another aspect of the present invention, there is provided a food composition comprising the above-mentioned antimicrobial composition.

The kind of the food is not particularly limited, and may include foods in a conventional sense. Non-limiting examples of the food to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, , A drink, an alcoholic beverage, and a vitamin complex. When the composition is used as a food additive, the composition may be added as it is or may be used together with other food or food ingredients, and may be suitably used according to a conventional method.

The food composition may comprise a pharmaceutically acceptable carrier.

Particularly, the inventors of the present invention have found that the newly isolated Bacillus breculens CJBV of the present invention can simultaneously inhibit Zygosaccharomyces cerevisiae as a defective yeast, Bacillus cereus as a food poisoning bacterium, and Listeria monocytogenes as well rather, Bacillus subtilis (Bacillus subtilis) useful bacteria in fermented foods fermented soy sauce, Bacillus amyl Lori query Pacifico Enschede (Bacillus amyloliquefaciens ) (Example 3, Fig. 4), the antimicrobial composition can be directly used for all foods including fermented foods.

In another aspect of the present invention, the present invention provides a feed composition comprising the above-described antimicrobial composition.

The term "feed" as used herein in the context of the present invention may mean any natural or artificial diet, single meal, or the like ingredients for feeding, ingesting, digesting or suitable for the animal.

The kind of the feed is not particularly limited, and feeds conventionally 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 control method, and may include a probiotic agent. Non-limiting examples of such feeds include vegetable feeds such as cereals, muscle roots, food processing busines logistics, algae, fibers, pharmaceutical buses, oils, fats, pastes or grain by-products; Animal feeds such as proteins, inorganic substances, fats, oils, fats, oils, monocellular proteins, animal plankton or foods. These may be used alone or in combination of two or more.

Here, the feed composition comprising the antimicrobial composition of the present invention may further contain excipients, diluents, and additives.

It was confirmed that the newly isolated Bacillus blazezens CJBV of the present invention has the effect of simultaneously inhibiting the defective yeasts, Zygosaccharomyces bailei, Bacillus cereus, and Listeria monocytogenes, which are food poisoning bacteria, The invention provides a feed composition with antimicrobial and antifungal activity to prevent microbial infectious diseases including food poisoning of livestock.

According to another aspect of the present invention, there is provided an antimicrobial agent for plant crops comprising the antimicrobial composition.

The inventors of the present invention found that the newly isolated Bacillus buretzensis CJBV of the present invention has an inhibitory effect on plant pathogens including choletotrichum oviculare causing anthrax and fuarium oxysporum causing vine fission. (FIG. 3), the antimicrobial composition of the present invention can be used as an environmentally friendly non-pollution biological pesticide, and can be useful for eliminating ecosystem destruction and toxicity problems caused by organic synthetic pesticides, and can reduce the financial burden on each farm . In addition, due to the nature of the microorganism, it can be sustained and its long-term benefits can be seen in the future.

In another aspect of the present invention, the present invention provides a method for controlling phytopathogenic fungi, which comprises treating the plant or soil with the antimicrobial agent for plant crops.

Preferably, the phytopathogenic fungus may be, but is not limited to, choletotrichum oviculare, an cause of anthrax, and fusarium oxysporum, which causes vine fission.

It is preferable that the above-mentioned microorganism preparation is sprayed by using the foliar treatment method and the root of the plant roots at the ground surface of the plant from the 15th day after the preparation, or just before the rainy season and the onset of the disease, It is not limited.

In another aspect for achieving the above object, the present invention provides a pharmaceutical composition for preventing or treating a microbial infection disease, which comprises the above-mentioned antimicrobial composition.

As used herein, the term "prevention" may refer to any action that inhibits or delays the onset of a microbial infectious disease by administering to a subject a composition for the prevention or treatment of a microbial infectious disease according to the present invention.

The term "treatment" as used in the present invention may mean all actions that cause the symptom of a microbial infectious disease to be improved or benefited by administering the composition of the present invention to a suspected microbial infectious disease subject.

As used herein, the term "improvement" may mean any action that at least reduces the degree of symptom associated with the condition being treated.

As used herein, the term "individual" may refer to all animals, including humans, who have developed or are likely to develop a microbial infectious disease.

In this case, the microbial infectious disease is a disease caused by infection with microorganisms, preferably a disease caused by infection with fungi or food poisoning-causing bacteria.

As an example, the fungi may be selected from the group consisting of choletotrichum oviculare, fusarium oxysporum f. Lycopersicus JCM12575, Candida albicans, and Zygosaccharomyces < RTI ID = 0.0 > Vylli. ≪ / RTI >

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

In another embodiment, the microbial infectious disease is selected from the group consisting of food poisoning, skin fungus, vaginitis, diarrhea, vomiting, enteritis, gastroenteritis, constipation, abdominal pain, abdominal distension, cholera, listeriosis, Inflammatory bowel disease, inflammatory bowel disease, pneumonia, pyogenic inflammation, or sepsis.

Cellulitis refers to acute, purulent inflammation of the dermis and subcutaneous tissues, with the presence of pyritic salts in the tissues and chronic neutrophil infiltration extending chronicly and extensively into the interstitial space, accompanied by congestion or species.

Fleon refers to acute fungal infestation of the fingers or toes.

The present inventors have found that Candida albicans, which is present in the oral cavity, intestines and vagina of an animal including a human being and is capable of causing skin fungus infection, candidiasis vaginitis, enteritis, oral inflammation and the like during infestation and food poisoning, vomiting, diarrhea, Which may cause food poisoning, diarrhea, vomiting and the like, which may cause fever and the like, can cause food poisoning, listeriosis, sepsis, encephalitis, meningitis and the like, which may cause food poisoning, diarrhea, (FIG. 3 and FIG. 4), the antimicrobial composition of the present invention can be effectively used in a pharmaceutical composition for preventing or treating a microbial infection disease.

The composition for preventing or treating a microbial infectious disease according to the present invention may further comprise a pharmaceutically acceptable carrier and may be formulated together with the carrier to provide a food, a health functional food, a medicine, a quasi-drug, a feed additive, . ≪ / RTI > As used herein, the term "pharmaceutically acceptable carrier" may refer to a carrier or diluent that does not interfere with the biological activity and properties of the compound being administered, without irritating the organism.

The type of the carrier that can be used in the present invention is not particularly limited, and any carrier conventionally used in the art and pharmaceutically acceptable may be used. Non-limiting examples of the carrier include saline, sterilized 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 an antioxidant, a buffer and / or a bacteriostatic agent can be added and used. A diluent, a dispersant, a surfactant, a binder, a lubricant, Pills, capsules, granules or tablets, and the like.

The method of administering the composition for preventing or treating a microbial infectious disease according to the present invention is not particularly limited and may be conventionally used in the art. As a non-limiting example of such a mode of administration, the compositions may be administered orally or parenterally. The composition for preventing, ameliorating or treating the microbial infectious disease of the present invention may be manufactured into various formulations according to the intended administration mode.

The pharmaceutical dosage forms of the antimicrobial agents of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable set.

In another aspect, the present invention provides a method for preventing or treating a microbial infectious disease, comprising the step of administering to a subject a composition for preventing or treating the microbial infectious disease. As used herein, the term "individual" may refer to all animals, including humans, who have developed or are likely to develop a microbial infectious disease.

The preventive or therapeutic method of the present invention may specifically include administering the composition in a pharmaceutically effective amount to a subject suffering from or at risk of developing a microbial infectious disease.

The appropriate total daily usage of the composition containing the extract may be determined by the treatment within the scope of appropriate medical judgment, and may be administered once or several times. For purposes of the present invention, however, the specific therapeutically effective amount for a particular patient will depend upon the nature and extent of the reaction to be achieved, the specific composition, including whether or not other agents are used, the age, weight, Sex and diet of the patient, the time of administration, the route of administration and the rate of administration of the composition, the duration of the treatment, the drugs used or concurrently used with the specific composition, and similar factors well known in the medical arts.

The antimicrobial substance of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

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

In this case, the meaning of microbial infection disease, prevention and improvement is as described above.

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

The term "quasi-drug" in the present invention means a fiber, a rubber product or the like used for treating, alleviating, treating or preventing a disease of a human or an animal, Or products similar to those that are not machinery, preparations used for sterilization, insecticides and similar uses for the prevention of infectious diseases, for the purpose of diagnosing, treating, alleviating, treating, or preventing diseases of human beings or animals Means an article, except machinery, machinery or equipment, of the commodity used, and not used for the purpose of giving pharmacological effects to the structure and function of humans or animals; The quasi-drug may also include external preparations for skin and personal hygiene products. But is not limited to, a disinfectant cleaner, a shower foam, a guarine, a wet tissue, a detergent soap, a hand wash, or an ointment.

The present inventors confirmed that the newly isolated Bacillus bellegens CJBV has an antibacterial effect against the Candida albicans. As a result, the antimicrobial composition using the novel strain is useful for the treatment and alleviation of microbial infectious diseases including skin fungus, Candida albicans, , Can be effectively used for quasi-drugs for treatment or prevention.

The newly isolated Bacillus breeselensis CJBV of the present invention has antimicrobial effect against at least one kind of fungi and bacteria such as pathogenic fungi and yeast, so that the antimicrobial agent for plant crops, the food having antimicrobial activity, , Feed for improvement, treatment, health functional foods, quasi-drugs, and medicines.

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

Brief Description of the Drawings Fig. 1 shows a process of isolating an antimicrobial active ingredient, a basilomyces D analogue, from Bacillus cereus CJBV of the present invention.
2 shows ESI-MS and UV spectrum of the antimicrobial substance produced in the strain according to the present invention.
Fig. 3 shows the antimicrobial activity against four fungi choletotrichum orycticum, fusarium oxysporum, Candida albicans, and Zygosaccharomyces var.
Fig. 4 shows antibacterial activities against food poisoning-causing bacteria such as Bacillus cereus, Listeria monocytogenes, and Bacillus subtilis, which is a fermented bacterium, and Bacillus amyloliquefaciens.

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

Example 1: Isolation and identification of strains producing antimicrobial substances

(1) Isolation and selection of strains

Bacillus strains isolated from conventional meju were used to select strains with antibacterial activity. The strains were preincubated in soybean fermentation broth for 36 hours at 37 ° C. A 8 mm diameter disc was placed on agar solid medium containing lawn cells for pathogenic fungi and food poisoning bacteria, and the pre-cultured strains were inoculated. After culturing for 12 hours, strains were selected which produced a ring that was transparent to both fungi and bacteria around the strain inoculated on the medium.

(2) Identification of the strain

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

From the above results, the strain obtained in the present invention was identified as Bacillus breezensis strain and named Bacillus breezensis CJBV. The strain was deposited with the Korean Society for Microbiological Research (KCCM) on Apr. 17, 2014 and deposited with the accession number KCCM11528P .

Example  2: Separation and purification of antimicrobial substances

Experiments were conducted to isolate the antimicrobial substances produced by the newly isolated Bacillus brecesensis CJBV. The process is shown in Fig.

The strain was inoculated into 400 mu l of soybean fermentation broth and incubated at 37 DEG C for 48 hours with shaking at 200 rpm. The culture broth was centrifuged at 8000 x g for 20 minutes to obtain supernatant, which was then extracted with butanol.

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

To determine the UV spectrum and molecular mass (mass) of the separated active material, the purified sample was dissolved in methanol and measured by ESI-MS.

Figure 2 shows the ESI-MS and UV spectra of the isolated antimicrobial material.

Through the above analysis, it was confirmed that the substance isolated in the present invention is three kinds of basilomycin D analog represented by the following formula (1).

[Chemical Formula 1]

The R is CH ₃ for C ₁₄ -varylmycin D, CH ₂ -CH _{3 for} C ₁₅ -varymylin D, and CH ₂ -CH ₂ -CH ₃ for C ₁₆ -varymylin D.

Example  3: Antimicrobial activity test against fungi and food poisoning bacteria

The following antibacterial activity experiments were carried out to confirm whether the newly isolated Bacillus bellheensis CJBV has an inhibitory effect against fungi and food poisoning-causing bacteria.

(1) Antibacterial activity test for yeast and fungi (fungi)

First, the mold, Colletotrichum < RTI ID = 0.0 > orbiculare ), fusarium oxysporum f. sp. Lycopersicus JCM12575 ( Fusarium oxysporum f.sp. lycopersici JCM12575), Candida albicans , and Zygosaccharomyces bailii , a pathogenic yeast, were tested for their antimicrobial activity against newly isolated Bacillus brecesensis CJBV.

The four fungi were each cultured in a potato dextrose agar (PDA) medium. Thereafter, the butanol extract of Bacillus cereus CJBV culture solution, which is the reduced-pressure concentrated antimicrobial composition according to Example 2, was dissolved in ethanol, and a paper disk having a diameter of 8 mm was placed on each flat plate medium on which the above- The antimicrobial material was added at a constant rate of 25 and then cultured at 30 캜 for 24 to 48 hours to observe whether or not the antimicrobial substance produced by the isolate produced the inhibition of growth.

(2) Antibacterial activity test for food poisoning bacteria

In order to investigate whether the newly isolated Bacillus brecesens CJBV has antimicrobial activity specifically for foodborne pathogens in fermented foods, the following experiment was conducted.

Cause food poisoning bacterium Bacillus cereus (Bacillus cereus), Listeria monocytogenes jeneseu (Listeria monocytogenes) and soy sauce fermentation bacteria, Bacillus subtilis (Bacillus subtilis and Bacillus amyloliquefaciens were cultured in TSA (Tryptic soy agar) medium, respectively.

 Then, the butanol extract (Extraction with BuOH) of the Bacillus cereus CJBV culture solution, which is the concentrated and concentrated antimicrobial composition according to Example 2, was dissolved in ethanol, and then the 4 kinds of strains were plated on the flat plate medium with 8 mm diameter , And then incubated at 37 占 폚 for 24 hours to observe whether or not the antimicrobial substance produced by the isolate produced the inhibition of growth.

As the control group, butanol (BuOH) was used instead of the butanol extract of the Bacillus cereus CJBV culture in addition to the test group for the two fermented soybean cultivars.

The results according to Example 3 are shown in FIG. 3 and FIG. 4, and the degree of antimicrobial activity according to the degree of generation of the growth inhibition ring is shown in Table 1 below.

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

In the case of '+', the diameter of the growth inhibition ring is 3 mm to 5 mm or less, '++' is the growth inhibition diameter is 6 mm to 10 mm And in the case of '+++', the growth inhibition diameter is 10 mm or more.

As shown in the upper part of FIG. 3, the present inventors have found that a novel isolated Bacillus breezensis CJBV strain of the present invention, a spore of the strain, a culture of the strain, a concentrate thereof, an extract thereof, When the antimicrobial composition comprising any one or more as an active ingredient is treated with fungal growth inhibitory activity of fungal growth inhibitory activity, the antifungal activity of inhibiting the growth of fungi is excellent Respectively.

Therefore, the newly isolated Bacillus blazezens CJBV and the antimicrobial composition of the present invention inhibit plant infectious diseases such as anthrax and vine fungus against foliage and vegetable crops such as cucumber, melon, watermelon and the like, It can be effectively used for a method for controlling pathogenic fungi.

3, the newly isolated Bacillus brecesens CJBV of the present invention, the basilomycin D analogue produced from the strain, or the antimicrobial composition can be used as pathogenic fungi Candida albicans, As a result of treatment with Sesame seedlings, it was confirmed that the growth inhibition ring was formed to 6 mm to 10 mm, and the antimicrobial activity suppressing the growth of fungi was excellent.

Therefore, the newly isolated Bacillus bellegensis CJBV of the present invention, the basilomycin D analogue produced from the strain, and the antimicrobial composition are useful as preventive, ameliorative, and ameliorative agents against microbial infectious diseases against skin fungus, candidiasis vaginitis, oral cavity, It can be effectively used for medicines, quasi-drugs, health functional foods, feeds and the like. It can also be effectively used as a food with a low risk of food poisoning.

Further, as shown in FIG. 4, when the newly isolated Bacillus cereus CJBV or the antimicrobial composition of the present invention was simultaneously treated with food poisoning-causing bacteria and useful bacteria used in fermentation of soy sauce, the food poisoning-causing bacteria Bacillus cereus and Listeria It was confirmed that the antimicrobial activity was excellent for monocytogenes, but the growth of the fermenting bacteria was not inhibited.

The bacteriocins reported to be produced by conventional microorganisms do not exhibit antibacterial activity specifically for food poisoning-causing bacteria, but also affect the growth of fermenting bacteria. Thus, there is a problem that the bacteriocins are difficult to be directly used in fermented foods.

On the other hand, the newly isolated Bacillus brecesens CJBV of the present invention, the basilomycin D analog produced from the strain, and the antimicrobial composition using the same have no antimicrobial activity against the fermenting bacteria of the soybean flour and only the major microorganisms of the food such as food poisoning bacteria It can be directly used for fermented food.

Further, it was confirmed that the present invention has excellent antibacterial activity against various food pollutants. As a result, the newly isolated Bacillus breezens CJBV of the present invention, the basilomycin D analog produced by the strain, and the antimicrobial composition using the same, It can be effectively used for foods, medicines, quasi-drugs, health functional foods, feeds and the like for the purpose of preventing, improving and treating the microbial infectious diseases including vomiting.

Thus, the newly isolated Bacillus bellegensis CJBV of the present invention and the basilomycin D analog produced from the strain have antimicrobial effects against at least one kind of fungi and bacteria, Food having activity, and food for the prevention, improvement and treatment of microbial infectious diseases, health functional food, quasi-drug, and pharmaceutical product.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

Korea Microorganism Conservation Center (overseas) KCCM11528P 20140417

<110> CJ CheilJedang Corporation <120> Novel Bacillus velezensis and antibacterial and antifungal          composition comprising the same <130> KPA140376-KR <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 (16)

Bacillus velezensis CJBV strain of Accession No. KCCM11528P.
The method according to claim 1,
Wherein the strain does not have antibacterial activity against Bacillus subtilis , Bacillus amyloliquefaciens , or a combination thereof, which is a fermented bacterium of the genus Bacillus strains CJBV.
The method according to claim 1,
Wherein said strain is selected from the group consisting of Colletotrichum orbiculare , Fusarium oxysporum , Candida albicans , and Zygosaccharomyces bailii . Wherein said strain has a specific antimicrobial activity against at least one fungus.
The method according to claim 1,
Wherein said strain has a specific antimicrobial activity on Bacillus cereus , Listeria monocytogenes or a combination thereof.
A method for producing a Bacillus spp. Strain of CJBV according to any one of claims 1 to 4, a spore of said strain, a culture of said strain, a concentrate thereof, an extract thereof, and a dried product thereof As an active ingredient, an antimicrobial composition for fungi, food-borne-causing bacteria or a combination thereof.
A food composition comprising an antimicrobial composition according to claim 5.
A feed composition comprising an antimicrobial composition according to claim 5.
An antimicrobial agent for plant crops comprising the antimicrobial composition according to claim 5, wherein the antimicrobial agent has an antimicrobial activity against fungi.
A pharmaceutical composition for preventing or treating a microbial infectious disease caused by fungi, food poisoning-causing bacteria or a combination thereof, comprising the antimicrobial composition according to claim 5.
delete 10. The method of claim 9,
The microbial infectious disease may be selected from the group consisting of food poisoning, skin fungus, vaginitis, diarrhea, vomiting, enteritis, gastroenteritis, constipation, abdominal pain, abdominal distension, cholera, listeriosis, cellulitis, urinary tract infection, meningitis, peritonitis, Felon, otitis media, respiratory disease, pneumonia, purulent inflammation, and sepsis.
A health functional food for preventing or ameliorating a microbial infectious disease caused by fungi, food poisoning-causing bacteria or a combination thereof, comprising the antimicrobial composition according to claim 5.
A quasi-drug composition comprising an antimicrobial composition according to claim 5.
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