NOVEL RHIZOPUS OLIGOSPORUS STRAIN AND ANTIBIOTIC COMPOSITION CONTAINING THE SAME

Abstract

The present application relates to the Rhizopus oligosporus CJCC02-20 strain deposited under accession number KCCM12893P and uses thereof.

Description

[Technical field]

[0001] The present application relates to the Rhizopus oligosporus CJCC02-20 strain deposited under accession number KCCM12893P, an antibacterial composition comprising as an active ingredient at least one selected from the group consisting of said strain, spores of said strain, a culture of said strain, a concentrate thereof, and a dried product thereof, a food composition, a feed composition, a quasi-drug composition, or a functional food composition comprising said antibacterial composition, or a pharmaceutical composition for preventing or treating a microbial infection disease caused by a fungus, a bacterium, or a combination thereof, comprising said antibacterial composition. [Background technology]

[0002] Traditionally fermented foods are always vulnerable to fungi and food poisoning bacteria during the storage, manufacturing and distribution of ingredients. Fungi and food poisoning bacteria are known to survive heat treatment and cause food spoilage, putrefaction and food poisoning.

[0003] In order to prevent spoilage and the proliferation of food poisoning bacteria and to maintain the quality of the above-mentioned foods for a long period of time, research on antibacterial substances has been actively conducted. Among antibacterial substances, there is a particularly high need for research on natural preservatives that are excellent in safety and cost-effectiveness.

[0004] Bacteriocins, a representative natural preservative, are a general term for natural antibacterial proteins or protein-based substances produced by microorganisms. Bacteriocins are proteins and are decomposed in the human body, so their safety has been proven. This is in contrast to conventional antibiotics, which are secondary metabolites and cause side effects in the human body, and the need for research on bacteriocins is currently increasing. In relation to the bacteriocins, research on bacteriocins derived from lactic acid bacteria has been actively conducted recently. Bacteriocins derived from lactic acid bacteria have the advantage of being at the GRAS level and their safety has been proven, but their antibacterial activity is specific to gram-positive bacteria, so they cannot be widely used.

[0005] Regarding the antibacterial properties of Rhizopus oligosporus, antibacterial substances such as antibiotic peptide (ABP) and linoleic acid produced from Rhizopus oligosporus culture medium are known to inhibit the gram-positive Straphylococcus and Bascillus genera (Roubos-van den Hil et al., 2010). However, only antibacterial activity against specific gram-positive microorganisms has been reported, and no reports have been published on their effectiveness against various strains of bacteria that cause spoilage or food poisoning. In addition, since Rhizopus oligosporus is a bacterium useful for fermented foods, even if the bacteriocin is applied to fermented foods, it cannot be said that it shows specific activity against spoilage-inducing fungi and food poisoning bacteria, and it is difficult to industrialize the production and distribution of fermented foods. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, the inventors conducted extensive research to develop a method that is harmless to the human body and inhibits various fungi and bacteria, and as a result, they confirmed that Rhizopus oligosporus CJCC02-20, newly isolated from tempe, not only has antibacterial activity against fungi, but also has antibacterial activity that specifically inhibits only major harmful microorganisms in food, such as food poisoning bacteria, and thus completed the present application. [Means for solving the problem]

[0007] The present application relates to the Rhizopus oligosporus CJCC02-20 strain deposited under accession number KCCM12893P, an antibacterial composition comprising as an active ingredient at least one selected from the group consisting of said strain, spores of said strain, a culture of said strain, a concentrate thereof, and a dried product thereof, a food composition, a feed composition, a quasi-drug composition, or a functional food composition comprising said antibacterial composition, or a pharmaceutical composition for preventing or treating a microbial infection disease caused by a fungus, a bacterium, or a combination thereof, comprising said antibacterial composition.

[0008] The present application aims to provide a strain of Rhizopus oligosporus CJCC02-20 deposited under accession number KCCM12893P.

[0009] In addition, the present application aims to provide an antibacterial composition containing at least one selected from the group consisting of the Rhizopus oligosporus CJCC02-20 strain, spores of the strain, a culture of the strain, a concentrate thereof, and a dried product thereof as an active ingredient.

[0010] A further object of the present application is to provide a food composition comprising said antibacterial composition.

[0011] Furthermore, the present application aims to provide a feed composition comprising the antibacterial composition.

[0012] A further object of the present application is to provide a quasi-drug composition containing the antibacterial composition.

[0013] A further object of the present application is to provide a functional food composition comprising said antibacterial composition.

[0014] A further object of the present application is to provide a pharmaceutical composition for preventing or treating a microbial infectious disease caused by a fungus, a bacterium, or a combination thereof, comprising the antibacterial composition. Effect of the Invention

[0015] The antibacterial composition containing the Rhizopus oligosporus CJCC02-20 strain of the present application has properties that exhibit antibacterial activity against fungi and bacteria, and is therefore useful in foods, medicines, quasi-drugs, functional foods, etc. [Brief description of the drawings]

[0016] [Figure 1] The antibacterial activity of Rhizopus oligosporus CJCC02-20 against Aspergillus oryzae, Aspergillus niger, and Escherichia coli was measured using a paper disk assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present application will now be described in more detail.

[0018] In addition, each description and embodiment disclosed in this application is also applicable to other descriptions and embodiments. In other words, all combinations of various elements disclosed in this application are included in this application. In addition, this application is not limited to the following specific description.

[0019] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present application described herein which equivalents are intended to be encompassed by this application.

[0020] To achieve the above object, one aspect of the present application provides a Rhizopus oligosporus CJCC02-20 strain deposited under accession number KCCM12893P.

[0021] The present inventors have elucidated a novel strain belonging to Rhizopus oligosporus that has excellent antibacterial activity against fungi and food poisoning bacteria that cause deterioration and contamination of fermented foods as follows.

[0022] Various species of Rhizopus oligosporus were isolated from existing tempeh and tested for fungi such as mold and yeast, and food poisoning bacteria to select a strain of Rhizopus oligosporus with excellent antibacterial activity, and gene sequence analysis was performed to identify the selected strain. The 18S rRNA sequence of the selected strain is represented by SEQ ID NO: 1, the ABP (antibiotic peptide) gene sequence is represented by SEQ ID NO: 2, and the Chitinase (Chi2) gene sequence is represented by SEQ ID NO: 3. Based on the gene analysis, the strain was identified as a Rhizopus oligosporus strain, named Rhizopus oligosporus CJCC02-20, and deposited with the Korea Center for Microorganisms (KCCM), an international depository institution under the Budapest Treaty, under the accession number KCCM12893P on December 9, 2020.

[0023] According to one embodiment of the present application, the newly isolated Rhizopus oligosporus CJCC02-20 has excellent antibacterial activity against fungi and food poisoning bacteria, and exhibits antibacterial activity that acts simultaneously against various fungi and food poisoning bacteria (Example 3).

[0024] The fungi and bacteria include, but are not limited to, those of the genera Aspergillus, Bacillus, and Escherichia.

[0025] In one embodiment of the present application, the Rhizopus oligosporus CJCC02-20 has antibacterial activity against fungi Aspergillus oryzae, Aspergillus niger, or a combination thereof, but is not limited to the type of fungi.

[0026] In one embodiment of the present application, the Rhizopus oligosporus CJCC02-20 has antibacterial activity against Bacillus cereus, Escherichia coli, or a combination thereof, but is not limited to the types of bacteria.

[0027] In one embodiment of the present application, the Rhizopus oligosporus CJCC02-20 has specific antibacterial activity against, but is not limited to, Aspergillus oryzae, Aspergillus niger, or Escherichia coli.

[0028] Aspergillus oryzae and Aspergillus niger are known as bacteria that cause deterioration and contamination of fermented foods, and Bacillus cereus and Escherichia coli are known as bacteria that cause food poisoning, so Rhizopus oligosporus CJCC02-20, which has excellent antibacterial activity against them, can be used for various purposes.

[0029] To achieve the above object, another aspect of the present application provides a composition comprising at least one selected from the group consisting of the newly isolated Rhizopus oligosporus CJCC02-20 strain, spores of the strain, a culture of the strain, a concentrate thereof, an extract thereof, and a dried product thereof, and a carrier.

[0030] Here, the carrier may be a naturally occurring or non-naturally occurring carrier.

[0031] In one embodiment of the present application, the dry product may be a freeze-dried product.

[0032] The composition may further include a cryoprotectant. For example, the composition may further include at least one cryoprotectant selected from the group consisting of glycerin, trehalose, maltodextrin, skim milk powder, and starch. The cryoprotectant of the present application may be included in the composition of the present application at 0.01% by weight to 20% by weight, or 0.01% by weight to 10% by weight, based on the total weight of the composition of the present application. Specifically, the glycerin of the present application may be included in the composition of the present application at 5% by weight to 20% by weight, the trehalose of the present application at 2% by weight to 10% by weight, the maltodextrin of the present application at 2% by weight to 10% by weight, the skim milk powder of the present application at 0.5% by weight to 2% by weight, and the starch of the present application at 0.1% by weight to 1% by weight.

[0033] To achieve the above object, yet another aspect of the present application provides an antibacterial composition comprising, as an active ingredient, at least one selected from the group consisting of the newly isolated Rhizopus oligosporus CJCC02-20 strain, spores of the strain, a culture of the strain, a concentrate thereof, an extract thereof, and a dried product thereof.

[0034] Here, the state of the strain may be, but is not limited to, a liquid phase state or a dry state.

[0035] The antibacterial composition has antibacterial activity against fungi, bacteria or a combination thereof, specifically, has specific antibacterial activity against Aspergillus oryzae, Aspergillus niger or Escherichia coli, and has enhanced antibacterial activity against Bacillus cereus, but is not limited thereto.

[0036] In this application, "spore" refers to a reproductive cell of a bacterium or the like, and for the purposes of this application, said spore refers to a reproductive cell of Rhizopus oligosporus CJCC02-20.

[0037] In the present application, the spores of Rhizopus oligosporus CJCC02-20 exhibit antibacterial activity against fungi and food poisoning bacteria, and therefore can be contained in foods, feeds, medicines, quasi-drugs, and functional foods having antibacterial activity.

[0038] The term "culture" in this application means a product obtained by culturing the strain, and may be a culture solution containing the cells, or may be a cultured strain, cells from which the culture supernatant has been removed, or concentrated cells. The composition of the culture may contain not only components necessary for normal cultivation of Rhizopus oligosporus, but also components that act synergistically on the growth of Rhizopus oligosporus, and such compositions can be easily selected by a person having ordinary skill in the art.

[0039] In the present application, the term "concentrate" refers to a concentrated form of the culture.

[0040] The term "extract" in the present application means an extract extracted from the culture or a concentrate thereof, and is not limited to the above, so long as it is an extract that exhibits a specific antibacterial effect against the fungi and bacteria of the present application, and includes an extract, a diluted or concentrated extract, a dried product obtained by drying the extract, or a crude or purified product thereof, and all fractions obtained by fractionating these.

[0041] In the present application, the term "dried product" refers to a product obtained by drying the culture, its concentrate, its extract, or its fraction. Drying methods that can be used include, but are not limited to, forced air drying, natural drying, spray drying, and freeze drying.

[0042] The newly isolated Rhizopus oligosporus CJCC02-20 of the present application can be cultured by a conventional method for culturing Rhizopus oligosporus strains, but is not limited thereto. A natural medium or a synthetic medium can be used as the medium. The carbon source of the medium can be, for example, glucose, sucrose, dextrin, glycerin, starch, etc., and the nitrogen source can be, for example, peptone, meat extract, yeast extract, dry yeast, soybean, ammonium salt, nitrate, and other organic or inorganic nitrogen-containing compounds, but is not limited thereto. The inorganic salt contained in the medium can be, for example, magnesium, manganese, calcium, iron, phosphorus, etc., but is not limited thereto. In addition to the carbon source, nitrogen source, and inorganic salt components, amino acids, vitamins, nucleic acids, and compounds related thereto may be added to the medium.

[0043] The antibacterial composition may also contain other by-products produced during the cultivation of Rhizopus oligosporus CJCC02-20.

[0044] In order to achieve the above object, yet another aspect of the present application provides a food composition comprising the above antibacterial composition.

[0045] The type of the food is not particularly limited, and includes all foods in the usual sense. Examples of foods to which the substance can be added include, but are not limited to, fermented foods such as soy sauces and salted fish, sauces, meats, sausages, breads, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products such as ice cream, various soups, soft drinks, tea, energy drinks, alcoholic beverages, vitamin complexes, etc. When the composition is used as a food additive, the composition may be added as it is or may be used together with other foods or food ingredients, and may be used appropriately in a usual manner.

[0046] The food composition may be specifically a fermented food, more specifically a soy sauce or salted food, and even more specifically tempeh, but is not limited thereto.

[0047] The food composition may be a sauce containing tempeh, but is not limited thereto.

[0048] The food composition may comprise a food-based acceptable carrier.

[0049] The type of the carrier is not particularly limited, and any carrier commonly used in the art can be used.

[0050] The food composition may also contain additional ingredients commonly used in food compositions to improve aroma, taste, visual appearance, etc. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, etc. Minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), etc., and amino acids such as lysine, tryptophan, cysteine, and valine may also be included.

[0051] In addition, the food composition may contain food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (bleaching powder, highly bleached powder, sodium hypochlorite, etc.), antioxidants (butyl hydroxyanisole (BHA), butyl hydroxytoluene (BHT), etc.), colorants (tar dyes, etc.), color formers (sodium nitrite, sodium acetate, etc.), bleaching agents (sodium sulfite), seasonings (monosodium glutamate (MSG), etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavors (vanillin, lactones, etc.), leavening agents (alum, potassium D-hydrogen tartrate, etc.), strengthening agents, emulsifiers, thickeners (thickening agents), coating agents, gum bases, foam inhibitors, solvents, improvers, dispersants, etc. The additives are selected according to the type of food and used in appropriate amounts.

[0052] In particular, the present inventors confirmed that the newly isolated Rhizopus oligosporus CJCC02-20 of the present application has the effect of simultaneously inhibiting Aspergillus oryzae, a fermentation mold for soy sauce, Aspergillus niger, a food contaminant, Bacillus cereus, a food poisoning bacterium, and Escherichia coli, an enteropathogenic bacterium (Example 3, Figure 1). In addition, when the antibacterial composition was applied to tempeh, a fermented food, it was confirmed that the antibacterial effect of controlling food poisoning bacteria could be maintained while realizing the traditional flavor (Example 4). Therefore, these can be directly used in all foods, including fermented foods.

[0053] In order to achieve the above object, yet another aspect of the present application provides a pharmaceutical composition for preventing or treating a microbial infectious disease, comprising the above antibacterial composition.

[0054] In the present application, "prevention" refers to any action of suppressing or delaying the onset of a microbial infectious disease by administering to an individual a composition for preventing or treating a microbial infectious disease according to the present application.

[0055] In the present application, "treatment" refers to any act of ameliorating or beneficially altering the symptoms of a microbial infectious disease by administering the composition of the present application to an individual suspected of developing the microbial infectious disease.

[0056] By "amelioration" in this application is meant any action that at least reduces a parameter related to the condition being treated, such as the severity of a symptom.

[0057] In this application, "individual" refers to any animal, including a human, that is affected or at risk of developing a microbial infectious disease.

[0058] Here, the microbial infection disease is a disease caused by infection with a microorganism, and is preferably a disease caused by infection with a fungus or a bacterium.

[0059] In one embodiment of the present application, the fungus may be, but is not limited to, Aspergillus oryzae, Aspergillus niger, or a combination thereof.

[0060] In one embodiment of the present application, the bacterium may be, but is not limited to, Bacillus cereus, Escherichia coli, or a combination thereof.

[0061] In addition, as a specific example of the present application, the microbial infection disease is at least one selected from the group consisting of food poisoning, diarrhea, vomiting, enteritis, gastroenteritis, constipation, abdominal pain and abdominal distension, but is not limited to these.

[0062] In one embodiment of the present application, enhanced antibacterial activity against Bacillus cereus, a bacterium that induces food poisoning, and specific antibacterial activity against enteropathogenic Escherichia coli were confirmed, and specific antifungal activity against Aspergillus oryzae and Aspergillus niger, which are mold fungi, was confirmed, so that the antibacterial composition of the present application is useful as a pharmaceutical composition for preventing or treating microbial infectious diseases.

[0063] The composition for preventing or treating a microbial infectious disease according to the present application may further contain a pharma- ceutically acceptable carrier, and may be formulated together with the carrier and provided as a food, functional food, medicine, quasi-drug, feed additive, drinking water additive, etc.

[0064] In this application, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate an organism and does not destroy the biological activity and properties of the compound being administered.

[0065] The type of the carrier used in the present application is not particularly limited, and any carrier that is commonly used in the art and is pharma- ceutically acceptable can be used. Examples of the carrier include, but are not limited to, saline, sterile water, Ringer's solution, buffered saline, albumin injection, glucose solution, maltodextrin solution, glycerin, ethanol, etc. These may be used alone or in combination of two or more.

[0066] If necessary, other conventional additives such as antioxidants, buffer solutions, bacteriostatic agents, etc. may be added, and diluents, dispersants, surfactants, binders, lubricants, etc. may be further added to the composition to be formulated into injectable dosage forms such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, tablets, etc.

[0067] The method of administration of the composition for preventing or treating microbial infection diseases according to the present application is not particularly limited, and may be a method commonly used in the art. As the administration method, for example, the composition may be administered orally or parenterally, but is not limited thereto. The composition for preventing, improving or treating microbial infection diseases according to the present application may be prepared in various dosage forms depending on the intended administration method.

[0068] The pharmaceutical dosage forms of the antibacterial agents of the present application may be used in the form of their pharma- ceutically acceptable salts, either alone or in combination or suitable association with other pharma- ceutical active compounds.

[0069] Yet another aspect of the present application provides a method for preventing or treating a microbial infectious disease, comprising administering to an individual the composition for preventing or treating the microbial infectious disease, wherein "individual" in the present application means any animal, including a human, suffering from or at risk of suffering from a microbial infectious disease.

[0070] The prophylactic or therapeutic method of the present application may specifically comprise the step of administering a pharma- tically effective amount of the composition to an individual suffering from or at risk of suffering from a microbial infectious disease.

[0071] The total suitable daily dose of the composition containing said extract may be determined by the attending physician within the scope of sound medical judgment and may be administered in one or several doses. However, for the purposes of this application, it is preferred that the specific therapeutically effective amount for a particular patient will vary depending on a variety of factors, including the type and extent of the response to be achieved, whether other preparations are used, the specific composition, the patient's age, weight, general health, sex, diet, time of administration, route of administration, excretion rate of the composition, duration of treatment, drugs administered with or simultaneously with the specific composition, and similar factors well known in the medical art.

[0072] The antibacterial agent of the present application can be administered to mammals such as rats, mice, livestock, humans, etc., by various routes, including oral, rectal, or intravenous, intramuscular, subcutaneous, intrauterine, dural, or intracerebroventricular injection, although any method of administration is possible.

[0073] Yet another aspect of the present application provides a functional food for preventing or ameliorating a microbial infectious disease, comprising the antibacterial composition.

[0074] Here, the prevention and amelioration of microbial infectious diseases are as described above.

[0075] In this application, the term "functional food (health functional food or nutraceutical)" is synonymous with specific health foods, functional health foods, and health foods, and refers to foods with high medical and therapeutic effects that are processed to efficiently exhibit bioregulatory functions in addition to providing nutrients. The foods can be manufactured in various forms such as tablets, capsules, powders, granules, liquids, and pills to obtain useful effects in preventing or improving microbial infectious diseases.

[0076] The functional food may include a nutrient-acceptable carrier, as described above.

[0077] The functional food of the present application can be produced by a method commonly used in the art, and can be produced by adding raw materials and ingredients commonly added in the art. The dosage form of the functional food is not limited as long as it is a dosage form recognized as a functional food. The functional food of the present application can be produced in various dosage forms, and unlike general medicines, it uses food as a raw material, so it has the advantage of being free of side effects that may occur when taking medicines for a long period of time, and is highly portable, so the functional food of the present application can be taken as an adjuvant to enhance the preventive or ameliorative effects of microbial infectious diseases.

[0078] Specifically, the food is food in which the antibacterial composition is added to food ingredients such as beverages, teas, spices, gum, and confectionery, or made into capsules, powders, suspensions, and the like, and examples thereof include various foods, beverages, gums, teas, vitamin complexes, and dietary supplements.

[0079] The food can be manufactured into dosage forms such as tablets, granules, powders, capsules, liquid solutions, pills, etc., by known manufacturing methods, and the content of the composition according to the present application can be adjusted according to the dosage form. Other than containing the antibacterial composition according to the present application as an active ingredient, there are no particular limitations on other ingredients, and various common flavoring agents, natural carbohydrates, etc. may be contained as additional ingredients.

[0080] Yet another aspect of the present application provides a quasi-drug composition comprising the antibacterial composition.

[0081] In the present application, the term "quasi-drug" refers to any of the following: fibers, rubber products, or similar products used for the purpose of treating, mitigating, treating, or preventing diseases in humans or animals; products that have a weak effect on the human body or do not directly act on the human body and are not instruments or machines or similar products; and preparations used for sterilization, insecticide, and similar purposes for infection prevention, excluding those that are not instruments, machines, or devices among those used for the purpose of diagnosing, treating, mitigating, treating, or preventing diseases in humans or animals, and those that are not instruments, machines, or devices among those used for the purpose of pharmacologically affecting the structure and function of humans or animals. The quasi-drug also includes skin topical agents and personal hygiene products. Disinfectants, shower foams, mouthwashes, wet tissues, detergents, soaps, hand washes, and ointments are preferred, but are not limited thereto.

[0082] The quasi-drug composition may contain a pharma- ceutically acceptable carrier, as described above.

[0083] The inventors have confirmed that the newly isolated Rhizopus oligosporus CJCC02-20 has antibacterial effects against the above fungi and bacteria, and therefore an antibacterial composition using the novel strain is useful as a quasi-drug.

[0084] In order to achieve the above object, yet another aspect of the present application provides a feed composition comprising the above antibacterial composition.

[0085] By "feed" in this application is meant any natural or artificial diet, meal, or component of such a meal, intended or suitable for consumption, ingestion and digestion by an animal.

[0086] The type of the feed is not particularly limited, and feeds commonly used in the technical field can be used. The feed composition may contain a feed additive. The feed additive of the present application corresponds to a supplementary feed under the Feed Management Law, and may contain a live bacteria agent. The feed includes, for example, vegetable feeds such as grains, roots and fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, meals, and grain by-products, and animal feeds such as proteins, inorganic substances, oils and fats, minerals, single-cell proteins, zooplankton, and food and drink, but is not limited thereto. These may be used alone, or two or more types may be mixed together.

[0087] Here, the feed composition containing the antibacterial composition of the present application may further contain an excipient, a diluent, or an additive.

[0088] The newly isolated Rhizopus oligosporus CJCC02-20 of the present application has been confirmed to have the effect of simultaneously inhibiting the mold fungi Aspergillus oryzae and Aspergillus niger, as well as the food poisoning bacteria Bacillus cereus and Escherichia coli. Therefore, the present application provides a feed composition having antibacterial and antifungal activities, thereby making it possible to prevent microbial infectious diseases, including food poisoning in livestock.

[0089] To achieve the above object, yet another aspect of the present application provides a method for producing an antibacterial composition, comprising a step of culturing the newly isolated Rhizopus oligosporus CJCC02-20 in a medium.

[0090] "Culturing" in the present application means growing Rhizopus oligosporus CJCC02-20 of the present application under appropriately adjusted environmental conditions. The culturing process of the present application can be carried out in suitable media and culture conditions known in the art. Such a culturing process can be easily adjusted and used by those skilled in the art depending on the strain selected. Specifically, the culturing may be batch, continuous and / or fed-batch culture, but is not limited thereto.

[0091] The term "culture medium" in the present application means a mixture of nutrients necessary for culturing the present Rhizopus oligosporus CJCC02-20 as the main components, and supplies nutrients such as water essential for survival and growth, growth factors, etc. Specifically, the medium and other culture conditions used for culturing the present Rhizopus oligosporus CJCC02-20 may be any medium used for culturing ordinary microorganisms, and the present Rhizopus oligosporus CJCC02-20 can be cultured in an ordinary medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids and / or vitamins under aerobic conditions by adjusting temperature, pH, etc.

[0092] The carbon source in the present application may be carbohydrates such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc., sugar alcohols such as mannitol, sorbitol, etc., organic acids such as pyruvic acid, lactic acid, citric acid, etc., amino acids such as glutamic acid, methionine, lysine, etc. Also, natural organic nutrient sources such as starch hydrolysates, molasses, blackstrap molasses, rice bran, cassava, bagasse, corn steeping liquid, etc. may be used, specifically, carbohydrates such as glucose and sterilized pretreated molasses (i.e., molasses converted into reducing sugars) may be used, and any other suitable carbon source may be used. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0093] The nitrogen source may be an inorganic nitrogen source such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, or ammonium nitrate, or an organic nitrogen source such as an amino acid such as glutamic acid, methionine, or glutamine, peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its decomposition product, or defatted soybean cake or its decomposition product. These nitrogen sources may be used alone or in combination of two or more kinds, but are not limited thereto.

[0094] The phosphorus source may be potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or a sodium-containing salt equivalent thereto. The inorganic compound may be sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, or other suitable compounds, such as amino acids, vitamins, and / or suitable precursors. These components or precursors may be added to the medium in a batch or continuous manner. However, they are not limited thereto.

[0095] In addition, the pH of the medium can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid to the medium in a suitable manner during the cultivation of Rhizopus oligosporus CJCC02-20 of the present application. Furthermore, during the cultivation, foam generation can be suppressed using an antifoaming agent such as fatty acid polyglycol ester. Furthermore, in order to maintain the aerobic state of the medium, oxygen or oxygen-containing gas may be injected into the medium, and in order to maintain the anaerobic and microaerobic state, no gas may be injected, and nitrogen, hydrogen, or carbon dioxide gas may be injected, but is not limited thereto.

[0096] In the present invention, the culture temperature may be maintained at 20 to 45° C., specifically 25 to 40° C., and the culture may be performed for about 10 to 160 hours, but is not limited thereto.

[0097] The method for producing the antibacterial composition of the present application may further include a step of preparing the Rhizopus oligosporus CJCC02-20 strain of the present application, a step of preparing a medium for culturing the strain, or a combination thereof (in any order), for example prior to the culturing step.

[0098] The method for producing the antibacterial composition of the present application may further include a step of recovering the antibacterial substance from the culture medium (culture medium) or the Rhizopus oligosporus CJCC02-20 strain of the present application as described above. The recovering step may be further included after the culturing step.

[0099] Here, the antibacterial substance may be any substance that has antibacterial activity exhibited by the Rhizopus oligosporus CJCC02-20 strain, and specific examples thereof include crude extracts and compounds, but are not limited to these.

[0100] The recovery may involve collecting the antibacterial substance of interest using a suitable method known in the art depending on the culture method of the microorganism of the present application, such as batch, continuous, or fed-batch culture. For example, centrifugation, filtration, crystallization, treatment with a protein precipitant (salting out), extraction, ultrasonic disruption, ultrafiltration, dialysis, various chromatographies such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, and affinity chromatography, HPLC, or a combination thereof may be used to recover the antibacterial substance of interest from the medium or the microorganism using a suitable method known in the art.

[0101] The method for producing the antibacterial composition of the present application may further include a purification step. The purification may be performed by any suitable method known in the art. For example, when the method for producing the antibacterial composition of the present application includes both a recovery step and a purification step, the recovery step and the purification step may be performed at different times (or consecutively) regardless of the order, or may be performed simultaneously or integrated into one step, but is not limited thereto.

[0102] Yet another aspect of the present application provides a method for producing a fermented product, the method comprising culturing the Rhizopus oligosporus CJCC02-20 strain in a medium and fermenting it.

[0103] Yet another aspect of the present application provides a fermentate produced by said method.

[0104] The Rhizopus oligosporus CJCC02-20 strain is as described above, and the step of culturing the microorganism in a medium is as described above.

[0105] In the present application, the term "fermented product" refers to a composition obtained by culturing the microorganism of the present application.

[0106] Furthermore, the fermented product includes a liquid or powder composition obtained by culturing the microorganism and then carrying out an appropriate post-treatment process. Here, the appropriate post-treatment process may include, for example, a process of culturing the microorganism, a process of removing bacteria, a process of concentrating, a process of filtering, and a process of mixing with a carrier, and may further include a drying process. In some cases, the post-treatment process may not include a purification process.

[0107] Furthermore, "the fermented product" does not exclude a composition obtained by culturing the microorganism of the present application, which further contains a mixture of substances obtained by non-fermentative processes and / or other substances obtained by non-natural processes.

[0108] In order to achieve the above object, yet another aspect of the present application provides a food composition comprising the newly isolated Rhizopus oligosporus CJCC02-20 strain or a fermentation product using the strain.

[0109] The Rhizopus oligosporus CJCC02-20 and the food are as described above.

[0110] The fermented product refers to a medium obtained by inoculating and culturing Rhizopus oligosporus CJCC02-20 strain in a medium containing a main ingredient of food, or a culture containing a microorganism cultured together with the medium.

[0111] The fermented product may be a culture obtained by inoculating a food material with Rhizopus oligosporus CJCC02-20 strain and culturing the same. The food material may be, but is not limited to, grains.

[0112] In one embodiment of the present application, the cereal grains are rice, barley, wheat, corn, etc., specifically wheat, wheat bran, and rice, but are not limited thereto.

[0113] The fermentation product is understood to comprehensively include, but is not limited to, filtrates, dilutions, concentrates thereof, crude products thereof, purified products, dried products, crushed products, etc. of the medium or culture.

[0114] In order to achieve the above object, yet another aspect of the present application provides a method for producing a food product, comprising a step of inoculating the newly isolated strain Rhizopus oligosporus CJCC02-20.

[0115] The Rhizopus oligosporus CJCC02-20 and the food are as described above.

[0116] In one embodiment of the present application, the method for producing the food may be a method for producing a fermented food, comprising the step of inoculating and culturing the Rhizopus oligosporus CJCC02-20 in grains. The fermented food obtained by inoculating and culturing the strain is specifically, but not limited to, a soy sauce or a salted fish, more specifically, tempeh or a sauce containing tempeh.

[0117] To achieve the above object, yet another aspect of the present application provides a feed composition comprising the newly isolated strain Rhizopus oligosporus CJCC02-20.

[0118] The Rhizopus oligosporus CJCC02-20 and the feed were as described above.

[0119] To achieve the above object, yet another aspect of the present application provides an antibacterial use of the newly isolated strain Rhizopus oligosporus CJCC02-20, a composition comprising at least one selected from the group consisting of spores of the strain, a culture of the strain, a fermentation product thereof, a concentrate thereof, an extract thereof and a dried product thereof. EXAMPLES

[0120] The configuration and effects of the present application will be described in more detail below with reference to examples. However, these examples are merely illustrative of the present application, and the present application is not limited to these examples. EXAMPLES

[0121] Isolation and identification of strains producing antibacterial substances To select strains with antibacterial activity, existing tempeh was cultured in PDA (Potato Dextrose Agar) medium at 30°C for 5 days, and then primary selection was performed using the morphological characteristics of the Rhizopus genus. The selected strains were then cultured in PDA medium containing 5% Kanamycin and 1,000 ppm at 30°C for 5 days to isolate them.

[0122] The nucleotide sequences of the 18S rRNA, ABP (ABP1), and Chitinase (Chi2) genes of the strains selected in the above steps were analyzed. The nucleotide sequences were compared with the GenBank database using NCBI BLAST (18S rRNA: SEQ ID NO: 1, ABP1: SEQ ID NO: 2, Chi2: SEQ ID NO: 3).

[0123] Specifically, primers SEQ ID NO: 4 and 5 were synthesized based on the nucleotide sequence of the ABP1 gene, and primers SEQ ID NO: 6 and 7 were synthesized based on the nucleotide sequence of the Chi2 gene, and their respective nucleotide sequences were confirmed by PCR. As a result, the nucleotide sequence of this strain showed 100% homology to Rhizopus oligosporus 18S rRNA, 99.9% homology to the ABP Gene, and 100% homology to the Chitinase Gene.

[0124] Based on these results, the strain obtained by this application was identified as a Rhizopus oligosporus strain, named Rhizopus oligosporus CJCC02-20, and deposited with the Korea Center for Microorganisms (KCCM), an international depository institution under the Budapest Treaty, on December 9, 2020 under accession number KCCM12893P. EXAMPLES

[0125] Separation and purification of antibacterial substances To isolate the antibacterial substance produced by the newly isolated Rhizopus oligosporus strain CJCC02-20, the strain was inoculated into 10-15g of wheat bran and then cultured at 30℃ for 5 days and 35℃ for 1 day. The culture was diluted 50 times by weight with distilled water and then extracted at 30℃ and 150 rpm for 60 minutes. The crude extracts were filtered under reduced pressure through 5.0μm filter paper using diatomaceous earth. The filtrate was freeze-dried at -20℃ for 48 hours and purified as a dry product. EXAMPLES

[0126] Antibacterial activity test against fungi and food poisoning bacteria To confirm whether the newly isolated Rhizopus oligosporus strain CJCC02-20 has the effect of inhibiting fungi and food poisoning-causing bacteria, the following antibacterial activity experiment was carried out.

[0127] Aspergillus oryzae, a fermentation mold used in soy sauce, and Aspergillus niger, a food contaminant, were cultured on PDA (Potato dextrose agar) medium.

[0128] In addition, the pathogenic yeast Candida albicans and the food contaminant Zygosaccharomyces bailii were cultured on YPD medium (Yeast extract peptone dextrose agar).

[0129] In addition, food poisoning bacteria Bacillus cereus, enteropathogenic Escherichia coli, and soybean fermentation bacteria Bacillus subtilis and Bacillus velezensis were cultured in TSA (tryptic soy agar) medium.

[0130] The antibacterial substance dried in Example 2 was dissolved in distilled water, and then a paper disk with a diameter of 8 mm was placed on the plate medium on which the aforementioned strain had been smeared, and 30 μl of the antibacterial substance was added at regular intervals. The medium was then cultured at 30°C for 24 to 48 hours, and it was observed whether a growth inhibition circle was formed by the antibacterial substance produced by the isolated strain. The results are shown in Table 1 and Figure 1. In Table 1, "-" indicates that growth could not be inhibited, "+" indicates that the diameter of the growth inhibition circle was 3 mm to 5 mm or less, "++" indicates that the diameter of the growth inhibition circle was 6 mm to 10 mm, and "+++" indicates that the diameter of the growth inhibition circle was 10 mm or more.

[0131] In addition to the Rhizopus oligosporus CJCC02-20 strain, four Rhizopus oligosporus strains isolated from existing tempeh (tempeh produced by traditional methods in Indonesia and Vietnam) were used as Comparative Examples 1 to 4 to compare their antibacterial activities.

[0132] [Table 1]

[0133] As can be seen from Table 1 and Figure 1, the purified substance obtained from Rhizopus oligosporus CJCC02-20 strain does not show antibacterial activity against yeasts, but it does show excellent antifungal activity that can selectively control Aspergillus oryzae and Aspergillus niger mold fungi, which are difficult to inhibit in soy sauces or general foods.

[0134] In addition, it was confirmed that it exhibited selective antibacterial activity against enteropathogenic Escherichia coli and had superior antibacterial activity against the food poisoning bacterium Bacillus cereus compared to other Rhizopus oligosporus strains. EXAMPLES

[0135] Confirmation of the effectiveness of food poisoning bacteria control in the fermented product tempeh In order to confirm the food poisoning bacteria control effect of the fermented product of Rhizopus oligosporus CJCC02-20 identified in Example 1, Rhizopus oligosporus CJCC02-20 strain was inoculated into 15-20g of unpolished barley, and then cultured at 30°C for 6 days and at 40°C for 1 day. The culture was powdered using a grinder.

[0136] In order to develop various fermented grain products in line with the changing consumption trends every year, steamed soybeans, steamed soybeans and rice, steamed soybeans and wheat flour, and steamed wheat flour were inoculated and mixed with the powder of the culture liquid and 4% of a commercially available Rhizopus oligosporus strain (Ragi Tempe Raprima, Bandung, Indonesia), and then cultured at 30℃ for 1-2 days under a humidity control of 60%, to produce the fermented product tempeh.

[0137] The cultured tempeh and existing commercially available tempeh were cultured for 1 day at 30°C in TSA (Tryptic soy agar) medium and Bacillus cereus selective medium supplemented with Polymyxin B and Trimethoprim. It was confirmed that food poisoning-inducing bacteria Bacillus cereus and enteropathogenic Escherichia coli were not detected in the tempeh inoculated with the Rhizopus oligosporus CJCC02-20 strain isolated in Example 1, whereas Bacillus cereus was detected in the tempeh inoculated with the commercially available strain and existing tempeh (see Table 2).

[0138] [Table 2]

[0139] These results confirmed that the Rhizopus oligosporus CJCC02-20 strain is an excellent tempeh fermentation strain that has excellent effects in controlling food poisoning bacteria while still achieving the traditional flavor.

[0140] Therefore, the antibacterial composition containing the novel Rhizopus oligosporus CJCC02-20 strain of the present application, spores of the strain, cultures of the strain, concentrates thereof, dried products thereof, etc., has excellent antibacterial effects against fungi and food poisoning bacteria, and is therefore useful for foods, quasi-drugs, pharmaceuticals, etc. containing the same.

[0141] From the above description, a person skilled in the art to which the present application pertains will understand that the present application can be implemented in other specific forms without changing its technical ideas or essential features. It should be understood that the above examples are merely illustrative and not limiting. The present application should be interpreted as including all modifications and variations derived from the meaning and scope of the claims and their equivalent concepts, rather than the specification.

[0142] Name of depository institution: Korea Microorganism Collection Center Accession number: KCCM12893P Entrustment date: December 9, 2020

[0143] TIFF0007635386000003.tif218170

Claims

1. Rhizopus oligosporus CJCC02-20 strain deposited under accession number KCCM12893P.

2. The strain of claim 1 , wherein the strain has antibacterial activity against fungi, bacteria, or a combination thereof.

3. 3. The strain of claim 2, wherein the fungus is Aspergillus oryzae, Aspergillus niger, or a combination thereof.

4. The strain of claim 2 , wherein the bacterium is Bacillus cereus, Escherichia coli, or a combination thereof.

5. The strain according to claim 1 , wherein the strain has specific antibacterial activity against Aspergillus oryzae, Aspergillus niger or Escherichia coli.

6. An antibacterial composition comprising at least one selected from the group consisting of a culture of the Rhizopus oligosporus CJCC02-20 strain according to any one of claims 1 to 5, a concentrate thereof, and a dried product thereof as an active ingredient.

7. The composition of claim 6, wherein the composition has antimicrobial activity against fungi, bacteria, or a combination thereof.

8. A food composition comprising one or more selected from the group consisting of the Rhizopus oligosporus CJCC02-20 strain described in any one of claims 1 to 5, a culture of said strain, a concentrate thereof, and a dried product thereof.

9. The food composition according to claim 8, wherein the food product is tempeh or a sauce containing tempeh.

10. A feed composition comprising one or more selected from the group consisting of the Rhizopus oligosporus CJCC02-20 strain described in any one of claims 1 to 5, a culture of said strain, a concentrate thereof, and a dried product thereof.

11. A pharmaceutical composition for preventing or treating a microbial infectious disease caused by a fungus, a bacterium or a combination thereof, comprising the antibacterial composition according to claim 6.

12. The pharmaceutical composition according to claim 11, wherein the microbial infection disease is at least one selected from the group consisting of food poisoning, diarrhea, vomiting, enteritis, gastroenteritis, constipation, abdominal pain and abdominal distension.

13. A functional food composition for preventing or ameliorating a microbial infectious disease caused by a fungus, a bacterium or a combination thereof, comprising the antibacterial composition according to claim 6.

14. A quasi-drug composition comprising the antibacterial composition according to claim 6.

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