Aflatoxin production inhibiting bacterium, aflatoxin production inhibitor produced by aflatoxin production inhibiting bacterium, preparation method of aflatoxin production inhibitor, and aflatoxin pollution control method using aflatoxin production inhibiting bacterium and aflatoxin production inhibitor

Abstract

Provided are: a novel strain of an aflatoxin production-inhibiting bacterium, which is a novel microorganism having a high activity for inhibiting the production of aflatoxin; an aflatoxin production inhibitor produced by an aflatoxin production-inhibiting bacterium comprising the novel strain; and an effective method for producing an inhibitor comprising an agent for improving the effect of the aflatoxin production-inhibiting bacterium. And a method for controlling aflatoxin contamination using the aflatoxin production inhibiting bacteria or the aflatoxin production inhibitor. The present invention relates to an aflatoxin production inhibitor comprising a culture of the genus Klebsiella sp. Or Raoultella sp., or a compound represented by general formula (1), and more specifically, to an aflatoxin production inhibitor comprising a culture of the genus Klebsiella sp. Or Raoultella sp., or a compound represented by general formula (1). In the formula (1), R is a straight-chain or branched alkyl group with 1-4 carbon atoms.

Description

Technical Field

[0001] This invention relates to novel strains of aflatoxin-producing inhibitory bacteria as novel microorganisms, aflatoxin-producing inhibitors produced by aflatoxin-producing inhibitory bacteria containing said novel strains, methods for preparing the same, and methods for preventing aflatoxin contamination using the same. Background Technology

[0002] Among the toxic compounds known as mycotoxins, which are secondary metabolites of molds, aflatoxin is the most problematic.

[0003] As microorganisms that inhibit aflatoxin production, several methods have been discovered, including microorganisms that inhibit aflatoxin production by suppressing the growth of aflatoxin-producing bacteria, microorganisms whose growth is not inhibited but can inhibit aflatoxin production, microorganisms with aflatoxin-degrading activity, and microorganisms that adsorb aflatoxin onto their cell bodies. For Bacillus subtilis (… Bacillus subtilis ), Invading Dwarf Cynodon dactylus ( Nannocystis exedens ), Bacillus pumilus ( Bacillus pumilus ), Pseudomonas syringae ( Pseudomonas syringae Rare Rolstonia ( Ralstonia paucula ) or Burkholderia ( Burkholderia cepacia Bacteria such as *Streptococcus lactis* were found to inhibit the growth of aflatoxin-producing bacteria and the production of aflatoxin. Furthermore, it was found that *Streptococcus lactis* (…) Streptococcus lactis Inhibits aflatoxin production without affecting the growth of aflatoxin-producing bacteria, xylose oxidation alkali-producing bacteria ( Achromobacter xylosoxidans Inhibits the synthesis of aflatoxin by indomethacin ( ) norsolorinic acid The production of chloropicrin by strains of *Kluyveromyces* was also reported. Additionally, the production of chloropicrin by strains of *Kluyveromyces* was also reported. Kluyveromyces genus Candida, genus Candida Candida genus) or Pichia pastoris genus ( Pichia Yeasts of the genus *Flavobacterium* inhibit aflatoxin production. Further investigation into bacteria that decompose aflatoxin revealed *Flavobacterium orangeense* as a possible candidate. Flavobacterium aurantiacum ), Mycobacterium fluorescens ( Mycobacterium fluoranthenivorans ), Rhodococcus redissus ( Rhodococcus erythropolis ) and Orange Myxococcus ( Myxococcus fulvus Lactobacillus rhamnosus, among others, decomposes aflatoxin. Furthermore, Lactobacillus rhamnosus has been found to be a bacterium that adsorbs aflatoxin onto its cells. Lactobacillus rhamnosus Lactic acid bacteria (see Non-Patent Literature 1).

[0004] In addition, non-patent literature 2 reports Klebsiella spp. ( Klebsiella sp. ) and Klebsiella pneumoniae ( Klebsiella pneumoniae (and its culture supernatant decomposes aflatoxin)

[0005] In addition, some diketopiperazines are known as compounds that inhibit aflatoxin production, such as xylose-oxidizing alkali-producing bacteria described in Non-Patent Literature 3. Achromobacter xylosoxidans The cyclo(L-Leu-L-Pro) produced by ) is described in Non-Patent Literature 4 as belonging to the genus Oligoatomum ( Stenotrophomonas cyclo(L-Ala-L-Pro) and cyclo(L-Val-L-Pro) produced by sp.)

[0006] Existing technical documents Non-patent literature Non-patent document 1: Shohei Sakuda: Pollution Control Mycotoxins, 63(2), 217-224 2013) Non-patent literature 2: Ning, M., Zhang, S., Xie, Y., Wang, W., Gao, Y.: Aflatoxin B1removal by three bacterial strains and optimization offermentation process parameters. Biotechnology and Applied Biochemistry, 66(6), 930-938(2019) Non-patent literature 3: Yan, PS, Song, Y., Sakuno, E., Nakajima, H., Nakagawa, H., Yabe, K.: Cyclo (L-Leucyl-L-Prolyl) produced by Achromobacterxylosoxidans inhibits aflatoxin production by Aspergillus parasiticus. ApplEnviron Microbiol, 70, 7466-7473(2004) Non-patent document 4: Jermnak, U., Chinaphuti, A., Poapolathep, A., Kawai, R., Nagasawa, H., Sakuda, S.: Prevention of aflatoxin contamination by a soilbacterium of Stenotrophomonas sp. that produces aflatoxin productioninhibitors. Microbiology, 159,902-912(2013) Summary of the Invention

[0007] (a) Technical problems to be solved The technical problem of the present invention is to provide a novel strain of aflatoxin-producing inhibitory bacteria, which is a novel microorganism with high activity in inhibiting aflatoxin production; an effective preparation method of an aflatoxin-producing inhibitor produced by the aflatoxin-producing inhibitory bacteria containing the novel strain and an inhibitor containing an agent that enhances its effect; and a method for preventing aflatoxin contamination using these aflatoxin-producing inhibitory bacteria or aflatoxin-producing inhibitors.

[0008] (II) Technical Solution The above-mentioned technical problems can be solved by the following invention.

[0009] [1] An aflatoxin production inhibitor comprising Klebsiella spp. ( Klebsiella sp. ) or Raoulbacterium genus ( Raoultella sp. ) culture.

[0010] [2] An aflatoxin production inhibitor comprising a compound represented by the following general formula (1), [Chemical Formula 1] In formula (1), R is a straight-chain or branched alkyl group with 1 to 4 carbon atoms.

[0011] [3] A method for preparing an aflatoxin production inhibitor, comprising inhibiting Klebsiella pneumoniae (Klebsiella spp.) Klebsiella sp. ) or Raoulbacterium genus ( Raoultella sp. The process of cultivation.

[0012] [4] The preparation method according to [3] further includes: a step of purifying the compound represented by the following general formula (1) from the culture obtained in the culture step. [Chemical Formula 2] In formula (1), R is a straight-chain or branched alkyl group with 1 to 4 carbon atoms.

[0013] [5] A method for inhibiting aflatoxin production, characterized in that an aflatoxin production inhibitor of [1] or [2] is used.

[0014] [6] A method for preventing aflatoxin contamination, characterized in that an aflatoxin production inhibitor of [1] or [2] is used to inhibit the production of aflatoxin caused by aflatoxin-producing bacteria.

[0015] [7] A compound represented by the following general formula (1), [Chemical Formula 3] In formula (1), R is a methyl group.

[0016] [8] A type of Klebsiella pneumoniae ( Klebsiella aerogenes (), its accession number is NITE BP-03899.

[0017] (III) Beneficial Effects According to the present invention, it is possible to provide a novel strain of aflatoxin-producing inhibitory bacteria that is a novel microorganism with higher efficacy, an aflatoxin-producing inhibitor with higher activity produced by the aflatoxin-producing inhibitory bacteria containing the novel strain, an effective preparation method thereof, and a method for preventing aflatoxin contamination using these aflatoxin-producing inhibitory bacteria or aflatoxin-producing inhibitors. Attached Figure Description

[0018] Figure 1 This demonstrates the inoculation and culture of aflatoxin-producing bacteria into Klebsiella pneumoniae (Klebsiella pneumoniae) that has been impregnated with aflatoxin-producing bacteria. Klebsiella aerogenes The graph shows the results of analyzing the aflatoxin concentration in peanuts after diluting the KTTM strain culture medium into liquids at 10-fold gradients.

[0019] Figure 2 A graph showing the results of analyzing aflatoxin concentrations in peanuts after inoculating and culturing aflatoxin-producing bacteria in a liquid that had been diluted 10-fold in a culture medium containing various microorganisms belonging to the genus Klebsiella or Raoultella.

[0020] Figure 3The structural formulas of various diketopiperazines for evaluating their activity in inhibiting aflatoxin production are shown, along with the concentrations (IC50) of each evaluation sample that inhibited aflatoxin B1 production to 1 / 2. 50 (The image is missing.) Detailed Implementation

[0021] (A novel strain of bacteria that inhibits aflatoxin production) The novel aflatoxin-producing inhibitor of this invention is Klebsiella pneumoniae (Klebsiella pneumoniae). Klebsiella aerogenes The KTTM strain (accession number NITE BP-03899) is an aflatoxin-producing inhibitory bacterium. Cultures of this bacterium (e.g., culture medium, culture supernatant, bacterial cells, or fragments of bacterial cells, or extracts of these cultures) exhibit aflatoxin-inhibiting activity. Therefore, it can be used as a bacterium producing an aflatoxin-producing inhibitor.

[0022] The mycological characteristics of the KTTM strain are as follows: The colony morphology on nutrient agar was: creamy, round, lenticular, entire, smooth, opaque, and buttery. It was identified as [specific species / organism] by 16S rDNA sequence analysis. Klebsiella aerogenes (Klebsiella pneumoniae). It is a motile, Gram-negative bacillus, positive for catalase but negative for oxidase, capable of fermenting glucose and producing gas. It possesses β-galactosidase, lysine decarboxylase, and ornithine decarboxylase activities, but lacks arginine dihydrolase, urease, and gelatinase activities. It can oxidize various sugars. It can grow at 37°C and 45°C.

[0023] (Aflatoxin-inhibiting bacteria) In this invention, for example, to inhibit aflatoxin production, prevent aflatoxin contamination, and prepare aflatoxin production inhibitors, in addition to the KTTM strain, microorganisms belonging to the Klebsiella genus (Klebsiella spp.) and the Raoultella genus (Rauvolfia spp.) can be used. Raoultella Microorganisms belonging to the genus *Rauvolfia* (Raoultella). Examples of microorganisms belonging to the genus *Klebsiella* include... Klebsiella aerogenes (Klebsiella pneumoniae) Klebsiella pneumoniae (Klebsiella pneumoniae) Klebsiella oxytoca (Klebsiella acidogenic bacteria). As microorganisms belonging to the genus *Laurella*, examples include... Raoultella planticola (Phytophyte Raoultella), Raoultella terrigena (Native Raoulbacterium) Raoultella ornithinolytica (Ornithine-derived Raoulbacterium).

[0024] (Aflatoxin production inhibitor) The aflatoxin production inhibitor of the present invention comprises at least a culture of Klebsiella or Raoultella, and / or a compound represented by the following general formula (1), and further comprises other ingredients as needed.

[0025] [Chemical Formula 4] (In formula (1), R is a straight-chain or branched alkyl group with 1 to 4 carbon atoms) The aflatoxin production inhibitor of the present invention may (i) consist solely of a culture of Klebsiella or Raoultella, or (ii) consist solely of one or more compounds represented by general formula (1), or (iii) combine a culture of Klebsiella or Raoultella with one or more compounds represented by general formula (1).

[0026] There are no particular limitations on the preparation method of the aflatoxin production inhibitor, and it can be appropriately selected according to the purpose. It can be prepared appropriately using the preparation method of the aflatoxin production inhibitor of the present invention described later.

[0027] The culture used as the aflatoxin production inhibitor of the present invention is not particularly limited as long as it is a culture of Klebsiella or Raoultella, and can be appropriately selected according to the purpose. For example, culture medium, culture supernatant, cultured cells, or fragments of cultured cells can be listed.

[0028] In addition, it can be an extract of the culture. An extract of the culture may include, for example, the supernatant obtained by adding a suitable organic solvent (e.g., ethanol, methanol, acetone, etc.) to the culture, suspending it, and then separating it from the bacterial cells by centrifugation or membrane filtration, or a substance obtained by performing conventional separation and purification treatments on the supernatant.

[0029] They can be used alone or in combination with two or more. Among them, culture medium, culture supernatant or cultured cells are preferred.

[0030] In addition, the Klebsiella or Raoult bacteria can also be treated to enhance their activity in inhibiting aflatoxin production by subjecting them to radiation irradiation or other mutagenesis treatments.

[0031] The content of the culture used as an inhibitor of aflatoxin production is not particularly limited and can be appropriately selected according to the purpose. Furthermore, the inhibitor of aflatoxin production can be the culture itself.

[0032] As the aflatoxin production inhibitor of the present invention, the compound represented by the general formula (1) is not particularly limited as long as R is a straight-chain or branched alkyl group with 1 to 4 carbon atoms. Examples include cyclo(L-Ala-Gly) where R is methyl, cyclo(L-Abu(2)-Gly) where R is ethyl, cyclo(L-Val-Gly) where R is isopropyl, cyclo(L-Nva-Gly) where R is n-propyl, cyclo(L-NorLeu-Gly) where R is n-butyl, cyclo(L-Leu-Gly) where R is isobutyl, cyclo(L-Ile-Gly) where R is sec-butyl, and cyclo(L-tert-Leu-Gly) where R is tert-butyl.

[0033] As R, methyl, ethyl, isopropyl, n-propyl, and isobutyl are more preferred, methyl, ethyl, isopropyl, and n-propyl are even more preferred, ethyl, isopropyl, and n-propyl are even more preferred, ethyl and isopropyl are even more preferred, and ethyl is particularly preferred.

[0034] The amount of the compound represented by general formula (1) in the aflatoxin production inhibitor is not particularly limited and can be appropriately selected according to the purpose. Furthermore, the aflatoxin production inhibitor can be the compound represented by general formula (1) itself.

[0035] The compound represented by the general formula (1) can be appropriately selected according to the purpose, and can be prepared by the preparation method of the aflatoxin production inhibitor of the present invention, which includes the culturing and purification steps, as described later, or by chemical synthesis.

[0036] There are no particular limitations on the method of preparation using the aforementioned chemical synthesis, and a suitable method can be selected from known methods depending on the purpose. For example, the method described in Thajudeen, H.; Park, K.; Moon, SS; Hong, IS An efficient green synthesis of proline-based cyclic dipeptides under water-mediated catalyst-free conditions. Tetrahedron Lett. 2010, 51, 1303-1305. can be cited.

[0037] Specifically, after coupling a methyl ester (e.g., L-Ala-OCH3) of an amino acid constituting the compound represented by general formula (1) with Boc-Gly using a dehydrating agent to obtain a dipeptide protected by a Boc group (tert-butyloxycarbonyl) and a methyl ester group, these protecting groups are removed and cyclized, thereby enabling the synthesis of the compound represented by general formula (1) (e.g., cyclo(L-Ala-Gly)).

[0038] Other components in the aflatoxin production inhibitor are not particularly limited as long as they do not impair the effect of the present invention; for example, any pesticide components, pesticide adjuvants, etc. can be listed.

[0039] The content of the other components that are the aflatoxin production inhibitors is not particularly limited and can be appropriately selected according to the purpose.

[0040] There are no particular restrictions on the pesticide ingredients, and they can be appropriately selected according to the purpose. For example, the following active ingredients of pesticides can be listed.

[0041] Examples of pesticides include, for example, fungicides, bactericides, antiviral agents, plant resistance inducers, insecticides, acaricides, nematicides, insect growth regulators, insect attractants, herbicides, plant growth regulators, synergists, pesticide damage reducers, bird repellents, fertilizers, and soil conditioners. They can be used individually or in combination.

[0042] The pesticide adjuvant contains a carrier, surfactants and other adjuvants, and may further contain other ingredients as needed.

[0043] As for the carrier, any carrier suitable for agricultural or horticultural use is acceptable, with no particular restrictions. It can be selected appropriately according to the purpose, such as liquid carriers and solid carriers. One type can be used alone, or two or more types can be used simultaneously.

[0044] Examples of liquid carriers include water; alcohols such as isopropanol and ethylene glycol; ketones such as cyclohexanone and methyl ethyl ketone; ethers such as propylene glycol monomethyl ether and diethylene glycol monobutyl ether; aliphatic hydrocarbons such as kerosene and light oil; aromatic hydrocarbons such as xylene, trimethylbenzene, tetramethylbenzene, methylnaphthalene, and solvent naphtha; amides such as N-methyl-2-pyrrolidone; esters such as fatty acid glycerides; and vegetable oils such as soybean oil and rapeseed oil.

[0045] Examples of solid carriers include, for instance, animal and plant powders such as starch, activated carbon, soybean flour, wheat flour, wood flour, fish meal, and milk powder; and mineral powders such as talc, kaolin, bentonite, zeolite, diatomaceous earth, white carbon, clay, alumina, calcium carbonate, potassium chloride, and ammonium sulfate.

[0046] There are no particular limitations on the surfactants used, and they can be appropriately selected according to the purpose. Examples include nonionic surfactants, anionic surfactants, cationic surfactants, and amphoteric surfactants. These surfactants can be used alone or in combination with two or more.

[0047] Examples of nonionic surfactants include polyoxyethylene alkyl ethers, polyoxyethylene alkylaryl ethers, polyoxyethylene styrene phenyl ethers, polyoxyethylene alkyl esters, polyoxyethylene sorbitan alkylates, polyoxyethylene phenyl ether polymers, polyoxyethylene alkylene aryl phenyl ethers, polyoxyethylene alkyl glycols, and polyoxyethylene polyoxypropylene block polymers.

[0048] Examples of anionic surfactants include lignin sulfonates, alkyl aryl sulfonates, dialkyl sulfosuccinates, polyoxyethylene alkyl aryl ether sulfates, alkyl naphthalene sulfonates, and polyoxyethylene styrene phenyl ether sulfates.

[0049] Examples of cationic surfactants include, for example, alkylamine salts.

[0050] Examples of such amphoteric surfactants include quaternary ammonium salts of alkyl betaine and amine oxides.

[0051] Other adjuvants are not particularly limited and can be selected appropriately according to the purpose. Examples include binders, thickeners, fixatives, preservatives and mildew inhibitors, solvents, pesticide active ingredient stabilizers, antioxidants, UV stabilizers, anti-crystallization agents, defoamers, property modifiers, and colorants.

[0052] There are no particular limitations on the binders, tackifiers, and fixatives used, and they can be appropriately selected according to the purpose. Examples include dextrin, cellulose, methylcellulose, ethylcellulose, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethyl starch, pullulan, sodium alginate, ammonium alginate, propylene glycol alginate, guar gum, locust bean gum, gum arabic, xanthan gum, gelatin, casein, polyvinyl alcohol, polyethylene oxide, polyethylene glycol, ethylene-propylene block polymer, sodium polyacrylate, and polyvinylpyrrolidone.

[0053] There are no particular limitations on the dosage form of the aflatoxin production inhibitor, and it can be appropriately selected according to the purpose. Examples include emulsions, suspensions, wettable powders, water-soluble solutions, liquids, colloidal suspensions (flowing agents), wettable granules, powders, microparticles, granules, tablets, oils, sprays, aerosols, and ointments. Among these, liquids are preferred.

[0054] There are no particular restrictions on the preparation method of the aforementioned agents; they can be prepared using known methods.

[0055] The aflatoxin production inhibitor can also be used in combination with or simultaneously with other fungicides (fungicides, bactericides, antiviral agents, plant resistance inducers, etc.), insecticides, acaricides, nematicides, insect growth regulators, insect attractants, herbicides, plant growth regulators, synergists, pesticide damage reducers, bird repellents, fertilizers, soil conditioners, etc.

[0056] (Preparation method of aflatoxin production inhibitor) In the preparation method of the aflatoxin production inhibitor of the present invention, the preparation method of the aflatoxin production inhibitor containing a culture of Klebsiella spp. (preferably the KTTM strain) includes at least a step of culturing Klebsiella spp. or Raoultella spp. (culturing step), and may further include other steps as needed.

[0057] Furthermore, in the preparation method of the aflatoxin production inhibitor of the present invention, the preparation method of the aflatoxin production inhibitor containing the compound represented by the general formula (1) (especially cyclo(L-Ala-Gly)) includes at least a step of culturing Klebsiella spp. (preferably the KTTM strain) or Raoultella spp. (culturing step), and a step of purifying the compound represented by the general formula (1) from the culture obtained in the culturing step (purification step), and may further include other steps as needed.

[0058] The cultivation process is carried out by inoculating Klebsiella spp. (preferably the KTTM strain) or Raoultella spp. into a nutrient medium (hereinafter, sometimes simply referred to as "medium") and culturing it at a temperature that is conducive to the production of the aflatoxin production inhibitor.

[0059] There are no particular limitations on the nutrient medium used, and it can be appropriately selected according to the purpose. For example, known nutrient media that can be used for the culture of Klebsiella (preferably Klebsiella aerogenes) or Raoulbacterium can be used.

[0060] There are no particular restrictions on the nutrient sources added to the nutrient culture medium, and appropriate sources can be selected according to the purpose. For example, commercially available nitrogen sources such as peptone, yeast extract, meat extract, corn steep liquor, cottonseed meal, peanut meal, soybean meal, NZ-amine, casein, sodium nitrate, ammonium nitrate, and ammonium sulfate can be listed; carbon sources such as tomato sauce, glycerol, sucrose, starch, glucose, galactose, mannose, dextrin, molasses, and fat can be listed; and inorganic salts such as salt, phosphate, calcium carbonate, and magnesium sulfate can be listed.

[0061] In addition to the nutrient source, trace amounts of metal salts, animal oils, vegetable oils, mineral oils, etc., as defoaming agents may also be added to the culture medium.

[0062] For these materials, all known culture materials can be used as long as they are useful for the production of aflatoxin inhibitors when utilized by Klebsiella (preferably the KTTM strain) or Raoultella.

[0063] There are no particular restrictions on the seed culture used for the production of aflatoxin inhibitors, and appropriate selection can be made according to the purpose. For example, Klebsiella spp. (preferably the KTTM strain) or Raoult spp. grown on agar medium can be listed.

[0064] There are no particular restrictions on the conditions for cultivation, and they can be appropriately selected according to the purpose, with aerobic conditions being preferred.

[0065] The cultivation method can be any of solid (agar) culture, such as slant culture or plate culture, or liquid culture, but from the perspective of producing a large amount of aflatoxin inhibitor, liquid culture is preferred. The liquid culture can be any of shaking culture, static culture, or stirred culture, with shaking culture being preferred, and rotary shaking culture being more preferred. Furthermore, when conducting large-scale cultivation, fermenters or similar devices can also be used.

[0066] The culture temperature is not particularly limited as long as it is within a range that does not substantially inhibit the development of Klebsiella (preferably the KTTM strain) or Raoultella and can produce aflatoxin production inhibitors. It can be appropriately selected according to the producing strain used, preferably 20°C to 37°C.

[0067] There is no particular limitation on the incubation time; it can be appropriately selected based on the accumulation of aflatoxin production inhibitors. Typically, the accumulation of aflatoxin production inhibitors reaches its peak between day 3 and day 7 of incubation.

[0068] The purification step is a process of purifying the compound represented by the general formula (1) from the culture obtained in the culturing step. For the culture, the description relating to the culture in the aflatoxin production inhibitor of the present invention can be directly applied.

[0069] There are no particular limitations on the method for purifying the compound represented by the general formula (1) from the culture, and appropriate methods can be selected according to the purpose. Examples include solvent extraction, partition or adsorption chromatography, high performance liquid chromatography, reversed phase chromatography, etc. These methods can also be combined.

[0070] The purification process can be performed once, but from the perspective of improving the yield of the compound represented by the general formula (1), it is preferable to perform it multiple times.

[0071] There are no particular limitations on the method for confirming that the substance obtained in the purification process is a compound represented by the general formula (1), and it can be appropriately selected according to the purpose. For example, methods such as using ESI-time-of-flight mass spectrometry (ESI-TOFMS), measuring optical rotation using a polarimeter, and proton nuclear magnetic resonance spectroscopy (PTN) can be listed. 1 H-NMR), carbon nuclear magnetic resonance spectroscopy ( 13 Methods such as C-NMR (for analysis) are used.

[0072] (Methods to prevent aflatoxin contamination) The method for preventing aflatoxin contamination of the present invention uses the aflatoxin production inhibitor described in this invention to inhibit the production of aflatoxin caused by aflatoxin-producing bacteria. There are no particular limitations on the method of applying the aflatoxin production inhibitor to objects that are attached to and / or infected with the aflatoxin-producing bacteria, or to objects on which it is desired to prevent attachment and / or infection by the aflatoxin-producing bacteria. The appropriate method can be selected according to the purpose. The method for preventing aflatoxin contamination of the present invention is not limited to this; by inhibiting the production of aflatoxin caused by aflatoxin-producing bacteria, aflatoxin contamination can be prevented.

[0073] There are no particular restrictions on the objects mentioned, and they can be selected appropriately according to the purpose. For example, plants, crops, etc. can be listed.

[0074] Examples of crops mentioned include grains such as corn, rice, buckwheat, and Job's tears; nuts such as peanuts, pistachios, and Brazil nuts; spices such as nutmeg, chili peppers, and bell peppers; beans such as coffee beans; sesame seeds; cottonseed; and so on.

[0075] There are no particular limitations on the method of applying the aflatoxin production inhibitor, and it can be appropriately selected according to the purpose. For example, methods such as dispersing the original aflatoxin production inhibitor or the aflatoxin production inhibitor diluted with water (e.g., spraying, misting, atomizing, dusting, scattering, water surface application, box application, etc.), applying it to the soil (e.g., mixing, irrigating, etc.), applying it to the surface (e.g., coating, covering, etc.), and impregnation are all possible methods.

[0076] There are no particular restrictions on the amount of aflatoxin production inhibitor to be applied. It can be appropriately selected based on various conditions such as the concentration of the active ingredient in the aflatoxin production inhibitor, the form of the formulation, the type of disease or crop targeted, the degree of damage caused by the disease, the application location, application method, application period, the type or amount of pesticides or fertilizers used in combination or simultaneously, and weather conditions.

[0077] There are no particular restrictions on the concentration at which the aflatoxin production inhibitor is applied; it can be selected appropriately according to the purpose.

[0078] Example The present invention will be specifically described below using examples, but these examples do not limit the scope of the present invention.

[0079] Example 1: Culture of Aflatoxin-Inhibiting Bacteria In this embodiment, Klebsiella pneumoniae (Klebsiella pneumoniae) was prepared. Klebsiella aerogenes Culture medium of KTTM strain.

[0080] Take a platinum ring from a colony of KTTM strain grown on Bennett solid medium (1.5% agar, 1% glucose, 0.2% peptone, 0.1% meat extract, 0.1% yeast extract, pH 7.2) and inoculate it into 7 mL of Bennett liquid medium (1% glucose, 0.2% peptone, 0.1% meat extract, 0.1% yeast extract, pH 7.2) already filled into a test tube. Incubate at 27.5°C with shaking (150 rpm) for 2 days. Add 80% glycerol aqueous solution to the resulting culture medium to adjust the glycerol concentration to 20% and store at -80°C.

[0081] 0.21 mL of the above-mentioned KTTM strain in 20% glycerol culture solution was inoculated into 7 mL of Bennett liquid medium that had been filled into the test tube, and cultured with shaking (150 rpm) at 27.5 °C for 3 days.

[0082] Experimental Example 1: Evaluation of Activity in Inhibiting Aflatoxin Production (1) (1) Preparation of spore suspension Aspergillus flavus, which is a producer of aflatoxin B1 ( Aspergillus flavus IMF47798 (purchased from the Reference Strain of the International Union of Microbiological Societies) was cultured on potato dextrose agar (PDA medium; manufactured by Difco, MD, USA) plates at 27°C for 14 days. Spores were then scraped from the colony using a platinum ring and suspended in distilled water containing 0.1% Tween 20 to a concentration of 1.1 × 10⁻⁶. 5 CFU / μL was used to prepare spore suspensions.

[0083] (2) Evaluation of the activity in inhibiting aflatoxin production Add 10 mL of distilled water to a beaker (100 mL capacity) containing 100 dried peanuts (approximately 0.5 g / peanut, Chiba Hanritachi peanuts, Midorikawa Shoten, Chiba) with the shells and skins removed, and autoclave at 120°C for 20 minutes. Immerse the autoclaved peanuts in a solution serially diluted with sterilized ultrapure water (Millipore water) to the culture medium prepared in Example 1, and then transfer the solution to a 24-well microplate, one well per plate.

[0084] As a control, instead of the culture medium dilution in Example 1, peanuts were immersed in sterilized ultrapure water (Millipore water) and then transferred to a 24-well microplate, one well per plate.

[0085] The spore suspension prepared in Example 1(1) of peanut inoculation experiment was loaded into these wells at 5 μL / well and incubated at 25°C for 14 days.

[0086] Peanuts with mold growth were individually transferred into Falcon tubes (15 mL), and 5.0 mL of 90% (v / v) acetonitrile aqueous solution was added. The mixture was then crushed and extracted using a spatula. 2.0 mL of the resulting crushed extract was added to a column for aflatoxin purification (Autoprep MF-A 1000, Resonac, Tokyo). 1.0 mL of the column pass solution was transferred to a Falcon tube (15 mL) and lyophilized. The lyophilized residue was dissolved in 0.5 mL of 10% (v / v) acetonitrile aqueous solution. The resulting solution was filtered (Minisart RC4, Sartorius, Göttingen, Germany), and the mass of aflatoxin B1 was determined by high-performance liquid chromatography (HPLC) under the following conditions.

[0087] [HPLC determination conditions] -HPLC- • Apparatus: SHIMADZU 20A HPLC system (pump: LC20AD, autosampler: SIL-20AC, column oven: CTO-20AC, fluorescence detector: RF-20A xs) • Chromatographic column: Capcell-pak C 18 Column UG120 (diameter 4.6mm, length 250mm, manufactured by Osaka Soda) • Elution: Acetonitrile:methanol:water (1:3:6, V / V / V) isocratic elution • Flow rate: 1.0 mL / min • Retention time: Aflatoxin B1 (12.5 minutes) • Detection: Fluorescence at 450 nm (excitation wavelength: 365 nm) The results are shown inFigure 1 The aflatoxin-producing bacteria were inoculated into the culture medium pre-impregnated with the KTTM strain (cell count 2.3 × 10⁻⁶). 9 Peanuts were cultured after being diluted 10-fold (per liter / mL) in a series of 10-fold serial dilutions. The concentration of aflatoxin in the peanuts was then analyzed. Even in 100,000-fold dilutions (dilution ratio 10), the concentration of aflatoxin was determined. 5 In the case of immersion in water, aflatoxin production was also strongly inhibited compared to the control (soaked in water). The "○" (white circle) in the figure represents the amount of aflatoxin in each peanut individual. Due to differences in peanut shape and other factors, the aflatoxin production varies considerably between individuals.

[0088] In addition, it was confirmed that the culture medium of Klebsiella pneumoniae does not decompose aflatoxin.

[0089] Experimental Example 2: Evaluation of Activity in Inhibiting Aflatoxin Production (2) The following bacteria were cultured in the same manner as in Example 1: Klebsiella pneumoniae NBRC 3319, Klebsiella gas-producing bacteria NBRC 12010, Klebsiella acid-producing bacteria NBRC 102593, Raoultella phytogenes NBRC 14939, Raoultella terrestrialis NBRC 14941, and Raoultella ornithine-lysinophilic bacteria NBRC 105727.

[0090] The activity of inhibiting aflatoxin production was evaluated in the same manner as in Experimental Example 1.

[0091] The results are shown in Figure 2 Aflatoxin-producing bacteria were inoculated into peanuts soaked in culture media containing *Klebsiella pneumoniae* NBRC 3319, *Klebsiella pneumoniae* NBRC 12010, *Klebsiella acidogenic* NBRC 102593, *Raoultella phytogenes* NBRC 14939, *Raoultella terrestrialis* NBRC 14941, and *Raoultella ornithine-lysinogen* NBRC 105727, diluted 10-fold serially to 1000-fold. The aflatoxin concentration in the peanuts was then analyzed. Among all six strains, even in the 1000-fold dilution (dilution ratio 10...), the aflatoxin concentration was... 3 In the case of [specific conditions], aflatoxin production was also strongly inhibited in all peanuts compared to the control (soaked in water). The control was the average of 12 peanuts, and the others were the average of 2 peanuts.

[0092] In addition, the same Klebsiella pneumoniae strain (NBRC12010) used in this test case as the KTTM strain evaluated in Test Case 1, but the KTTM strain showed stronger activity in inhibiting aflatoxin production compared with the NBRC 12010 strain.

[0093] Example 2: Preparation of aflatoxin production inhibitor (2) In this embodiment, it is composed of Klebsiella pneumoniae (Klebsiella pneumoniae). Klebsiella aerogenes The culture medium of KTTM strain was used to purify cyclo(L-Ala-Gly), which is the active substance, to prepare an aflatoxin production inhibitor containing the active substance.

[0094] (1) Cultivation process 0.21 mL of the 20% glycerol culture solution of the KTTM strain prepared in Example 1 was inoculated into 7 mL of Bennett liquid medium already filled into a test tube, and cultured with shaking (150 rpm) at 27.5 °C for 2 days to obtain the pre-culture solution. In this culture, 3 mL of the pre-culture solution was inoculated into 100 mL of Bennett liquid medium already filled into a 500 mL Erlenmeyer flask, and cultured with shaking (150 rpm) at 27.5 °C for 5 days.

[0095] (2) Purification process Cells were removed from the culture medium obtained in the 3L culture step by centrifugation (5,000×g, 10 min). The resulting culture supernatant was then purified by chromatographic analysis on a packed activated carbon column (Activated Charcoal, 100g, FUJIFILM Wako Chemicals) using water as the solvent. The supernatant was then washed with 300 mL of water and eluted with 500 mL of 10% ethanol. The concentrated solution (18.5 mL) obtained by concentrating the 10% ethanol eluent was subjected to three reversed-phase high-performance liquid chromatography (RP-HPLC) tests under the following conditions to purify the active substance.

[0096] [RP-HPLC determination conditions: first time] • Chromatographic column: Capcellpak C 18 column (250mm in length, 10mm in diameter, manufactured by Osaka Soda): • Eluent: A 0.1 v / v trifluoroacetic acid aqueous solution / B 100 v / v acetonitrile • Elution conditions: 15-minute linear gradient elution from A:B=100:0 to A:B=0:100 • Flow rate: 2.5 mL / min • Detection; UV300nm • Active ingredient yield: 40.6 mg [RP-HPLC determination conditions: second time] • Chromatographic column: Capcellpak C 18Column (250mm in length, 4.6mm in diameter, manufactured by Osaka Soda) • Eluent: A 0.1 v / v trifluoroacetic acid aqueous solution / B 100 v / v acetonitrile • Elution conditions: 15-minute linear gradient elution from A:B=100:0 to A:B=0:100 • Flow rate: 1.0 mL / min • Detection: UV220nm • Active ingredient yield: 4.0 mg [RP-HPLC determination conditions: third time] • Chromatographic column: Senshu Pak DOCOSIL SP-100 column (250mm length, 4.6mm diameter, manufactured by Senshu Science) • Elution: Isocratic elution (aqueous solution containing 10 mmol / L ammonium acetate) • Flow rate: 1.0 mL / min • Detection: UV210nm • Retention time of active substances: 5.2 minutes • Active substance yield: 1.15 mg (3) Identification process Based on a comparison of the retention time and subsequent physicochemical properties with those of the standard on RP-HPLC, the obtained active substance was identified as cyclo(L-Ala-Gly).

[0097] (a) The experimental value based on ESI-TOFMS (positive ion mode) is m / z 129.0666 (M+H). ＋ (calcd for C5H9N2O2, 129.0664).

[0098] (b) Specific rotation is [α] 27 D =-13.8 (c=0.06, H2O).

[0099] (c) Measured by nuclear magnetic resonance spectroscopy at 500 MHz in deuterated dimethyl sulfoxide (DMSO-d6) at 25 °C. 1 H and 13 The C10 NMR spectrum is described below.

[0100] δ H(DMSO-d6,500 MHz):8.16(1H,br.s,NH),7.97(1H,br.s,NH),3.84(1H,q,J=7Hz),2.72(2H,m),1.25(3H,d,J=7Hz);δ C (DMSO-d6,125 MHz):168.9,166.3,49.7,44.5,18.7. Experimental Example 3: Evaluation of Activity in Inhibiting Aflatoxin Production (2) As an evaluation sample, use Figure 3 The cyclo(L-Ala-Gly) and its similar compounds are shown.

[0101] Cyclo (L-Ala-Gly) uses commercially available products (Bachem, Budendorf, Switzerland).

[0102] Cyclo (L-Ala-L-Pro) uses the substance synthesized in Non-Patent Document 2.

[0103] Cyclo(D-Ala-Gly), cyclo(L-Abu(2)-Gly), cyclo(Gly-Gly), cyclo(L-Val-Gly), cyclo(L-Nva-Gly) and cyclo(L-Leu-Gly) are synthesized and used by the following methods.

[0104] D-Ala-OMe-HCl (0.415 g, 3 mmol, Watanabe Chemical Industries, Ltd., Hiroshima, Japan) was dissolved in 3 mL of dry DMF. Boc-Gly-OH (0.526 g, 3 mmol, Watanabe Chemical Industries, Ltd.), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HBTU) (1.37 g, 3.6 mmol), and N,N-diisopropylethylamine (DIPEA) (0.38 g, 6.0 mmol) were added to this solution, and the mixture was stirred overnight at room temperature. Ethyl acetate (100 mL) was added to the reaction mixture, followed by washing with 5% NaHCO3 (120 mL) and 5% NaCl (120 mL), and drying with anhydrous Na2SO4. After evaporation, the residue (0.451 g) was loaded onto a silica gel column (Silica gel 60 (0.063-0.200 mm), 20 g, Merck, Darmstadt, Germany) and eluted isocratically with hexane-based EtOAc (1:1) to obtain a dipeptide (0.296 g) protected by Boc- and OMe-. The protected dipeptide (0.296 g) was placed in water (30 mL) and autoclaved at 121 °C for 4 hours to deprotect and cyclize. The aqueous solution was evaporated, and the residue (0.238 g) was purified by silica gel column chromatography (Silica gel 60 (0.063-0.200 mm), 20 g, CH2Cl2:MeOH (9:1)) to obtain cyclo(D-Ala-Gly) (42 mg).

[0105] In the same manner, 84 mg of cyclo(L-Abu(2)-Gly), 22 mg of cyclo(Gly-Gly), 106 mg of cyclo(L-Val-Gly), 66 mg of cyclo(L-Nva-Gly), and 137 mg of cyclo(L-Leu-Gly) were obtained from 6 mmol of Gly-OMe-HCl and Boc-L-Abu(2)-OH, Gly-OMe-HCl and Boc-Gly-OH, L-Val-OMe-HCl and Boc-Gly-OH, L-Nva-OMe-HCl and Boc-Gly-OH, and L-Leu-OMe-HCl and Boc-Gly-OH (WATANABE CHEMICAL INDUSTRIES,LTD.).

[0106] Cyclo(D-Ala-Gly): ESI-Q / TOFMS m / z 129.0666 (M+H) + (calcd for C5H9N2O2, 129.0664); [α] 27 D +3.7 (c = 0.26, H2O); δ H (DMSO-d6, 500 MHz): 8.16 (1H, br.s, NH), 7.98 (1H, br.s, NH), 3.84 (1H, q, J = 7Hz), 3.72 (2H, m), 1.25 (3H, d, J = 7Hz); δ C (DMSO-d6, 125 MHz): 168.6, 166.3, 49.7, 44.5, 18.7. Cyclo(L-Abu(2)-Gly): ESI-Q / TOFMS m / z 143.0823 (M+H) + (calcd for C6H 11 N2O2, 143.0821); [α] 27 D +21.2 (c = 0.64, H2O); δ H (DMSO-d6, 500 MHz): 8.17 (1H, br.s, NH), 8.00 (1H, br.s, NH), 3.77 (1H, d, J = 17 Hz), 3.72 (1H, m), 3.68 (1H, d, J = 17Hz), 1.61 - 1.77 (2H, m), 0.85 (3H, t, J = 7Hz); δ C (DMSO-d6, 125MHz): 167.8, 166.1, 55.1, 44.3, 25.9, 8.8. Cyclo(Gly-Gly): ESI-Q / TOFMS m / z 115.0506 (M+H) + (calcd for C4H7N2O2, 115.0508); δ H (DMSO-d6, 500MHz): 8.02 (1H, br.s, NH), 3.70 (2H); δ C (DMSO-d6, 125 MHz): 166.1, 44.3. (Evaluation method) Add 100 μL of sample solution to 1.9 mL of potato dextrose liquid medium in the wells of a 24-well microplate. Inoculate the medium with a spore suspension of Aspergillus flavus (5 μL) and incubate at 25°C for 4 days. Filter 100 μL of the culture broth with 400 μL of a water:acetonitrile (9:1, V / V) mixture (Minisart RC4, Sartorius, Göttingen, Germany) using a Capcell plate (250 mm × 4.6 mm inner diameter). 18 The amount of aflatoxin B1 in the filtrate was analyzed by HPLC using a UG 120 column (manufactured by OsakaSoda). The HPLC was performed under the following conditions: isocratic elution with acetonitrile:methanol:water (1:3:6, V / V / V), 20 min, flow rate 1.0 mL, and fluorescence detection at 450 nm (excitation wavelength: 365 nm).

[0107] The results are shown in Figure 3 . Figure 3 The concentrations shown are the concentrations (IC50) of each evaluation sample that inhibited aflatoxin B1 levels to 1 / 2. 50 ).

[0108] Compared with cyclo(L-Ala-L-Pro), which is known to have activity in inhibiting aflatoxin production, diketopiperazines represented by the following formula, which can be used as the active ingredient of the aflatoxin production inhibitor of the present invention, exhibit a higher inhibitory effect on aflatoxin production.

[0109] [Chemical Formula 5] (In formula (1), R is a straight-chain or branched alkyl group with 1 to 4 carbon atoms) In particular, the activity of cyclo(L-Abu(2)-Gly), which replaces the methyl group of the natural cyclo(L-Ala-Gly) with an ethyl group, increased by 75 times compared to the natural product, showing extremely excellent activity in inhibiting aflatoxin production.

[0110] In addition, neither cyclo(D-Ala-Gly), a stereoisomer of cyclo(L-Ala-Gly), nor cyclo(Gly-Gly), which does not have the group R (i.e., the group equivalent to R is a hydrogen atom), showed sufficient activity to inhibit aflatoxin production.

[0111] Industrial applicability The aflatoxin production inhibitor of the present invention has excellent activity in inhibiting the production of aflatoxin, and therefore can be used in methods for preventing aflatoxin contamination.

[0112] [Collection Number] Klebsiella pneumoniae ( Klebsiella aerogenes Strain KTTM was internationally deposited on June 8, 2023, at the National Institute for Technology Evaluation of Japan Patent Microbial Collection Center (NPMD) (Room 122, 2-5-8 Kazusa-Kamazu, Kisarazu City, Chiba Prefecture, Japan 292-0818, Japan) with accession number NITE BP-03899.

Claims

1. An aflatoxin production inhibitor comprising Klebsiella spp. ) or Raoulbacterium genus ( ) culture.

2. An aflatoxin production inhibitor comprising a compound represented by the following general formula (1), [Chemical Formula 1] ， In formula (1), R is a straight-chain or branched alkyl group with 1 to 4 carbon atoms.

3. A method for preparing an aflatoxin production inhibitor, comprising inhibiting Klebsiella spp. ( ) or Raoulbacterium genus ( The process of cultivation.

4. The preparation method according to claim 3, further comprising: A process for purifying a compound represented by the following general formula (1) from the culture obtained in the culture process. [Chemical Formula 2] ， In formula (1), R is a straight-chain or branched alkyl group with 1 to 4 carbon atoms.

5. A method for inhibiting aflatoxin production, characterized in that, Use the aflatoxin production inhibitor as described in claim 1 or 2.

6. A method for preventing aflatoxin contamination, characterized in that, The aflatoxin production inhibitor according to claim 1 or 2 is used to inhibit the production of aflatoxin caused by aflatoxin-producing bacteria.

7. A compound represented by the following general formula (1), [Chemical Formula 3] ， In formula (1), R is a methyl group.

8. A type of gas-producing Klebsiella pneumoniae ( (), its accession number is NITE BP-03899.

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