Aflatoxin production-inhibiting bacterium, aflatoxin production inhibitor produced by aflatoxin production-inhibiting bacterium and method for producing the same, and method for controlling aflatoxin contamination using the bacterium or the inhibitor

Abstract

To provide a novel strain of aflatoxin production-inhibiting bacteria which is a novel microorganism having a high aflatoxin production-inhibiting activity; a method for efficiently producing inhibitors that include an aflatoxin production inhibitor produced by aflatoxin production-inhibiting bacteria including the novel strain and a drug that enhances the effect of the inhibitor; and a method for controlling aflatoxin contamination using the aflatoxin production-inhibiting bacteria or the aflatoxin production inhibitor.SOLUTION: The aflatoxin production inhibitor comprises a culture product of Klebsiella sp. or Raoultella sp. or a compound represented by general formula (1) (wherein R represents a linear or branched C1-4 alkyl group).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a novel strain of aflatoxin production-inhibiting bacteria, which is a novel microorganism, an aflatoxin production inhibitor produced by aflatoxin production-inhibiting bacteria including the novel strain, a method for producing the same, and a method for controlling aflatoxin contamination using the same. [Background technology]

[0002] Among the toxic compounds called mycotoxins, which are secondary metabolites of mold, aflatoxin is the most serious problem.

[0003] As microorganisms that inhibit aflatoxin production, microorganisms that suppress aflatoxin production by inhibiting the growth of aflatoxin-producing bacteria, microorganisms that can suppress aflatoxin production without inhibiting growth, microorganisms that have aflatoxin decomposition activity, and further microorganisms that adsorb aflatoxin to their bodies have been found. Bacillus subtilis , Nannocystis exedens , Bacillus pumilus , Pseudomonas syringae , Ralstonia paucula or Burkholderia cepacia It has been found that bacteria such as these have the effect of suppressing the growth of aflatoxin-producing bacteria as well as the production of aflatoxin. Streptococcus lactis It suppresses aflatoxin production without affecting the growth of aflatoxin-producing bacteria. Achromobacter xylosoxidans It was found that norsolorinic acid production, which is a biosynthetic precursor of aflatoxin, is inhibited in norsolorinic acid-producing strains. Kluyveromyces genus, Candida Genus and Pichia It has been reported that yeasts of the genus inhibit aflatoxin production. Flavobacterium aurantiacum , Mycobacterium fluoranthenivorans , Rhodococcuserythropolis and Myxococcus fulvus It has been shown that bacteria such as Bacillus subtilis decompose aflatoxins. Lactobacillus rhamnosus Lactic acid bacteria such as the above have been found (Non-Patent Document 1). In addition, Non-Patent Document 2 states: Klebsiella sp. and Klebsiella pneumonia It has been reported that the culture supernatant of this fungus decomposes aflatoxins.

[0004] In addition, some diketopiperazines are known as compounds that inhibit aflatoxin production. For example, Non-Patent Document 3 states: Achromobacter xylosoxidans cyclo(L-Leu-L-Pro) produced by Stenotrophomonas Cyclo(L-Ala-L-Pro) and cyclo(L-Val-L-Pro) produced by sp. have been described. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Sakuta, S.: Prevention of aflatoxin contamination by biocontrol. Mycotoxins, 63(2), 217-224 2013) [Non-Patent Document 2] Ning, M., Zhang, S., Xie, Y., Wang, W., Gao, Y.: Aflatoxin B1 removal by three bacterial strains and optimization of fermentation process parameters. Biotechnology and Applied Biochemistry, 66(6), 930-938 (2019) [Non-Patent Document 3] Yan, PS, Song, Y., Sakuno, E., Nakajima, H., Nakagawa, H., Yabe, K.: Cyclo (L-Leucyl-L-Prolyl) produced by Achromobacter xylosoxidans inhibits aflatoxin production by Aspergillus parasiticus. Appl Environ 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 soil bacterium of Stenotrophomonas sp. that produces aflatoxin production inhibitors. Microbiology, 159, 902-912 (2013) Summary of the Invention [Problem to be solved by the invention]

[0006] The object of the present invention is to provide a new strain of aflatoxin production-inhibiting bacteria, which is a new microorganism with high aflatoxin production-inhibiting activity, an efficient method for producing aflatoxin production inhibitors produced by aflatoxin production-inhibiting bacteria including the novel strain and inhibitors including agents with enhanced effects, and a method for controlling aflatoxin contamination using the aflatoxin production-inhibiting bacteria or aflatoxin production inhibitors. [Means for solving the problem]

[0007] The above problems can be solved by the present invention as follows: [1] Klebsiella species ( KlebsiellaAn aflatoxin production inhibitor comprising a culture of Raoultella sp. or Raoultella sp. [2] The following general formula (1): [ka] (wherein R is a linear or branched alkyl group having 1 to 4 carbon atoms). An aflatoxin production inhibitor comprising a compound represented by the formula: [3] Klebsiella species ( Klebsiella A method for producing an aflatoxin production inhibitor, comprising a step of culturing Raoultella sp. or Raoultella sp. [4] The culture obtained in the culture step is subjected to the following general formula (1): [ka] (wherein R is a linear or branched alkyl group having 1 to 4 carbon atoms). A process for purifying a compound represented by the formula: The manufacturing method of [3] further comprises: [5] A method for inhibiting aflatoxin production, comprising using an aflatoxin production inhibitor according to [1] or [2]. [6] A method for preventing aflatoxin contamination, comprising inhibiting aflatoxin production by aflatoxin-producing bacteria using the aflatoxin production inhibitor of [1] or [2]. [7] The following general formula (1): [ka] (wherein R is a methyl group). A compound represented by the formula: [8] Klebsiella aerogenes (accession number NITE BP-03899) Klebsiella aerogenes ). Effect of the Invention

[0008] According to the present invention, it is possible to provide a new strain of aflatoxin production-inhibiting bacteria that is a more effective new microorganism, a more active aflatoxin production inhibitor produced by aflatoxin production-inhibiting bacteria including the novel strain, an efficient method for producing them, and a method for controlling aflatoxin contamination using the aflatoxin production-inhibiting bacteria or aflatoxin production inhibitor. [Brief description of the drawings]

[0009]

Figure 1

Figure 2

Figure 3

[0010] (A new strain of aflatoxin-inhibiting bacteria) The novel aflatoxin production inhibitor of the present invention is Klebsiella aerogenes ( Klebsiella aerogenes ) KTTM strain (Accession No. NITE BP-03899). A culture of the aflatoxin production inhibitor (for example, a culture liquid, a culture supernatant, cultured cells, or a disrupted product of cultured cells, or an extract of these cultures) has an aflatoxin production inhibitory activity. Therefore, it can be used as a producer of an aflatoxin production inhibitor.

[0011] The mycological properties of the KTTM strain are as follows: The colony morphology on nutrient agar is cream-colored, round, lenticular, entire, smooth, opaque, and buttery. 16S rDNA sequence analysis revealed that Klebsiella aerogenes It is a motile gram-negative rod-shaped bacterium that is positive for catalase and negative for oxidase, ferments glucose and produces gas. It exhibits β-galactosidase, lysine decarboxylase and ornithine decarboxylase activities, but does not exhibit arginine dihydrolase, urease or gelatinase activities, and oxidizes each sugar. It can grow at 37℃ and 45℃.

[0012] (Aflatoxin production inhibitor) In the present invention, for example, for the purpose of inhibiting aflatoxin production, preventing aflatoxin contamination, and producing an aflatoxin production inhibitor, microorganisms belonging to the genus Klebsiella (Klebsiella species) and the genus Raoultella (Raoultella species) can be used in addition to the KTTM strain. Examples of microorganisms belonging to the genus Klebsiella include Klebsiella aerogenes , Klebsiella pneumoniae , Klebsiella oxytoca Examples of microorganisms belonging to the genus Raoultella include Raoultella planticola , Raoultella terrigena , Raoultella ornithinolytica Examples include:

[0013] (Aflatoxin production inhibitor) The aflatoxin production inhibitor of the present invention is a culture of Klebsiella species or Raoultella species, and / or a compound represented by the following general formula (1): [ka] (wherein R is a linear or branched alkyl group having 1 to 4 carbon atoms). and, if necessary, further contains other components. The aflatoxin production inhibitor of the present invention may comprise (i) a culture of Klebsiella species or Raoultella species alone, (ii) one or more compounds represented by general formula (1) alone, or (iii) a combination of a culture of Klebsiella species or Raoultella species and one or more compounds represented by general formula (1). The method for producing the aflatoxin production inhibitor is not particularly limited and can be appropriately selected depending on the purpose, but it is preferably produced by the method for producing the aflatoxin production inhibitor of the present invention described below.

[0014] The culture in the aflatoxin production inhibitor of the present invention is not particularly limited as long as it is a culture of Klebsiella species or Raoultella species, and can be appropriately selected depending on the purpose. Examples of the culture include a culture medium, a culture supernatant, cultured bacterial cells, and a disruption of cultured bacterial cells. The extract may also be an extract of the culture. Examples of the extract of the culture include an extract supernatant obtained by adding an appropriate organic solvent (e.g., ethanol, methanol, acetone, etc.) to the culture, suspending the culture, and then separating the culture from the bacterial cells by centrifugation, filtration membrane separation, etc., and an extract supernatant obtained by subjecting the extract supernatant to a commonly used isolation and purification treatment. These may be used alone or in combination of two or more. Among these, the culture medium, culture supernatant, or cultured bacterial cells are preferred. The Klebsiella species or Raoultella species may be treated by irradiation or other mutation treatments to enhance the aflatoxin production inhibitory activity.

[0015] The content of the culture in the aflatoxin production inhibitor is not particularly limited and may be appropriately selected depending on the purpose. The aflatoxin production inhibitor may be the culture itself.

[0016] The compound represented by the general formula (1) in the aflatoxin production inhibitor of the present invention is not particularly limited as long as R is a linear or branched alkyl group having 1 to 4 carbon atoms, and examples thereof include cyclo(L-Ala-Gly) in which R is a methyl group, cyclo(L-Abu(2)-Gly) in which R is an ethyl group, cyclo(L-Val-Gly) in which R is an i-propyl group, cyclo(L-Nva-Gly) in which R is an n-propyl group, cyclo(L-NorLeu-Gly) in which R is an n-butyl group, cyclo(L-Leu-Gly) in which R is an i-butyl group, cyclo(L-Ile-Gly) in which R is a sec-butyl group, and cyclo(L-tert-Leu-Gly) in which R is a tert-butyl group. R is more preferably a methyl group, an ethyl group, an i-propyl group, an n-propyl group, or an i-butyl group, more preferably a methyl group, an ethyl group, an i-propyl group, or an n-propyl group, still more preferably an ethyl group, an i-propyl group, or an n-propyl group, still more preferably an ethyl group or an i-propyl group, and particularly preferably an ethyl group.

[0017] The content of the compound represented by the general formula (1) in the aflatoxin production inhibitor is not particularly limited and can be appropriately selected depending on the purpose. The aflatoxin production inhibitor may be the compound represented by the general formula (1) itself.

[0018] The compound represented by the general formula (1) can be appropriately selected depending on the purpose, and may be produced by the method for producing an aflatoxin production inhibitor of the present invention, which includes a culture step and a purification step, as described below, or may be produced by chemical synthesis.

[0019] The method for producing by chemical synthesis is not particularly limited and can be appropriately 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 mentioned.

[0020] Specifically, one of the methyl esters of the amino acids constituting the compound represented by general formula (1) (e.g., L-Ala-OCH3 and Boc-Gly) is coupled using a dehydrating agent to obtain a dipeptide protected by a Boc group (tert-butoxycarbonyl group) and a methyl ester group, and then these protecting groups are deprotected and cyclized to synthesize the compound represented by general formula (1) (e.g., cyclo(L-Ala-Gly)).

[0021] The other components in the aflatoxin production inhibitor are not particularly limited as long as they do not impair the effects of the present invention, and examples thereof include any pesticide components and pesticide adjuvants. The content of the other components in the aflatoxin production inhibitor is not particularly limited and can be appropriately selected depending on the purpose.

[0022] The pesticide component is not particularly limited and can be appropriately selected depending on the purpose. Examples of the pesticide component include the following active ingredients of pesticides. Examples of the agricultural chemicals include fungicides, bactericides, antiviral agents, plant resistance inducers, insecticides, acaricides, nematicides, insect growth regulators, insect attractants, herbicides, plant growth regulators, synergists, phytotoxicity reducers, bird repellents, fertilizers, soil conditioners, etc. These may be used alone or in combination of two or more.

[0023] The agricultural chemical adjuvant contains a carrier, a surfactant, and other adjuvants, and further contains other components as necessary.

[0024] The carrier is not particularly limited as long as it can be used for agricultural and horticultural purposes, and can be appropriately selected depending on the purpose, and examples thereof include liquid carriers, solid carriers, etc. These may be used alone or in combination of two or more kinds.

[0025] Examples of the liquid carrier include water; alcohols such as isopropyl alcohol and ethylene glycol; cyclohexanone; ketones such as methyl ethyl ketone; propylene glycol; Examples of suitable ethers include ethers such as coal monomethyl ether and diethylene glycol mono-n-butyl 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 glycerin esters of fatty acids; and vegetable oils such as soybean oil and rapeseed oil.

[0026] Examples of the solid carrier include animal and vegetable 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.

[0027] The surfactant is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include nonionic surfactants, anionic surfactants, cationic surfactants, amphoteric surfactants, etc. These surfactants may be used alone or in combination of two or more kinds.

[0028] Examples of the nonionic surfactant include polyoxyethylene alkyl ethers, polyoxyethylene alkylaryl ethers, polyoxyethylene styryl phenyl ethers, polyoxyethylene alkyl esters, polyoxyethylene sorbitan alkylates, polyoxyethylene phenyl ether polymers, polyoxyethylene alkylene aryl phenyl ethers, polyoxyethylene alkylene glycols, and polyoxyethylene polyoxypropylene block polymers.

[0029] Examples of the anionic surfactant include lignin sulfonate, alkylaryl sulfonate, dialkyl sulfosuccinate, polyoxyethylene alkylaryl ether sulfate, alkylnaphthalene sulfonate, and polyoxyethylene styryl phenyl ether sulfate.

[0030] Examples of the cationic surfactant include alkylamine salts. Examples of the amphoteric surfactant include quaternary ammonium salts of alkyl betaines and amine oxides.

[0031] The other auxiliary agents are not particularly limited and can be appropriately selected depending on the purpose. Examples of the other auxiliary agents include binders, thickeners, fixing agents, preservatives and antifungal agents, solvents, stabilizers for agricultural chemical active ingredients, antioxidants, ultraviolet protection agents, crystal precipitation inhibitors, antifoaming agents, physical property improvers, and colorants. The binder, thickener, and adhesive are not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include dextrin, cellulose, methyl cellulose, ethyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, 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.

[0032] The dosage form of the aflatoxin production inhibitor is not particularly limited and may be appropriately selected depending on the purpose, and examples thereof include emulsions, suspensions, wettable powders, water-soluble powders, liquids, sols (flowables), wettable powders, powders, fine granules, granules, tablets, oils, sprays, aerosols, pastes, etc. Among these, liquids are preferred. The method for producing each of the above agents is not particularly limited, and they can be produced by known methods.

[0033] The aflatoxin production inhibitor can also be mixed with or used in combination with other fungicides (fungicides, bactericides, antiviral agents, plant resistance inducers, etc.), insecticides, miticides, nematicides, insect growth regulators, insect attractants, herbicides, plant growth regulators, synergists, chemical damage reducers, bird repellents, fertilizers, soil conditioners, etc.

[0034] (Method of producing an aflatoxin production inhibitor) Among the methods for producing an aflatoxin production inhibitor of the present invention, the method for producing an aflatoxin production inhibitor comprising a culture of Klebsiella species (preferably the KTTM strain) includes at least a step of culturing Klebsiella species or Raoultella species (culture step), and may further include other steps as necessary. Furthermore, among the methods for producing an aflatoxin production inhibitor of the present invention, the method for producing an aflatoxin production inhibitor containing a compound represented by the general formula (1) (particularly cyclo(L-Ala-Gly)) comprises at least a step of culturing Klebsiella species (preferably the KTTM strain) or Raoultella species (cultivation step) and a step of purifying the compound represented by the general formula (1) from the culture obtained in the culture step (purification step), and may further comprise other steps as necessary.

[0035] The culture in the culture step is carried out by inoculating Klebsiella species (preferably the KTTM strain) or Raoultella species into a nutrient medium (hereinafter, sometimes simply referred to as "medium") and culturing at a temperature suitable for the production of the aflatoxin production inhibitor.

[0036] The nutrient medium is not particularly limited and can be appropriately selected depending on the purpose. For example, a known medium that has been conventionally used for culturing Klebsiella species (preferably Klebsiella aerogenes) or Raoultella species can be used. The nutrient source to be added to the nutrient medium is not particularly limited and can be appropriately selected depending on the purpose. Examples of the nutrient source include commercially available peptone, yeast extract, meat extract, corn steep liquor, cottonseed flour, peanut flour, soybean flour, NZ-amine, casein, sodium nitrate, ammonium nitrate, ammonium sulfate, and other nitrogen sources; tomato paste, glycerin, sucrose, starch, glucose, galactose, mannose, dextrin, molasses, fat, and other carbon sources; and inorganic salts such as salt, phosphates, calcium carbonate, magnesium sulfate, and the like. In addition to the nutrient sources, for example, trace amounts of metal salts, and animal oils, vegetable oils, mineral oils, and the like as antifoaming agents can also be added as components to the medium. These materials may be any materials that are utilized by Klebsiella species (preferably the above-mentioned KTTM strain) or Raoultella species and are useful for producing an aflatoxin production inhibitor, and any known culture materials may be used.

[0037] The seed stock for producing the aflatoxin production inhibitor is not particularly limited and can be appropriately selected depending on the purpose. For example, the seed stock can be a growth of Klebsiella species (preferably the above-mentioned KTTM strain) or Raoultella species cultured on an agar medium.

[0038] The culture conditions are not particularly limited and can be appropriately selected depending on the purpose, but it is preferable to culture under aerobic conditions.

[0039] The culture method may be either solid (agar) culture such as slant culture or plate culture, or liquid culture, but liquid culture is preferred from the viewpoint of mass production of the aflatoxin production inhibitor. The liquid culture may be any of shaking culture, stationary culture, and agitation culture, but shaking culture is preferred, and rotary shaking culture is more preferred. When performing mass culture, the culture may be performed using a fermenter or the like.

[0040] The temperature for the culture is not particularly limited as long as it is within a range in which the growth of Klebsiella species (preferably the KTTM strain) or Raoultella species is not substantially inhibited and an aflatoxin production inhibitor can be produced, and can be appropriately selected depending on the producing bacteria used, with a temperature of 20°C to 37°C being preferred.

[0041] The period of the culture is not particularly limited and can be appropriately selected according to the accumulation of the aflatoxin production inhibitor. Usually, the accumulation of the aflatoxin production inhibitor reaches a maximum after 3 to 7 days of culture.

[0042] The purification step is a step of purifying the compound represented by the general formula (1) from the culture obtained in the culture step. The explanation regarding the culture in the aflatoxin production inhibitor of the present invention can be applied to the culture as it is.

[0043] The method for purifying the compound represented by the general formula (1) from the culture is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include solvent extraction, partition or adsorption chromatography, high performance liquid chromatography, reverse phase chromatography, etc. These methods may be used in combination. The purification step may be carried out once, but is preferably carried out multiple times in terms of improving the yield of the compound represented by the general formula (1).

[0044] The method for confirming that the substance obtained in the purification step is the compound represented by the general formula (1) is not particularly limited and can be appropriately selected depending on the purpose. For example, ESI-time-of-flight mass spectrometry (ESI-TOFMS), a method of measuring the optical rotation with a polarimeter, proton nuclear magnetic resonance spectroscopy ( 1 H-NMR), Carbon Nuclear Magnetic Resonance Spectroscopy ( 13 Examples of such methods include analysis using C-NMR.

[0045] (Methods for preventing aflatoxin contamination) The aflatoxin contamination control method of the present invention is a method of inhibiting aflatoxin production by aflatoxin-producing bacteria using the aflatoxin production inhibitor of the present invention, and is not particularly limited as long as it is a method of applying the aflatoxin production inhibitor to an object attached and / or infected by the aflatoxin-producing bacteria or an object in which attachment and / or infection by the aflatoxin-producing bacteria is to be prevented, and can be appropriately selected depending on the purpose. The aflatoxin contamination control method of the present invention can control aflatoxin contamination by inhibiting aflatoxin production by aflatoxin-producing bacteria, although it is not limited thereto.

[0046] The target object is not particularly limited and can be appropriately selected depending on the purpose. Examples of the target object include plants and agricultural crops. Examples of the agricultural crops include grains such as corn, rice, buckwheat, and pearl barley; nuts such as peanuts, pistachio nuts, and Brazil nuts; spices such as nutmeg, chili peppers, and paprika; beans such as coffee beans; sesame seeds; cottonseed; and the like.

[0047] The method of applying the aflatoxin production inhibitor is not particularly limited and can be appropriately selected depending on the purpose. Examples of the method include applying the aflatoxin production inhibitor as is or after diluting it with water or the like (e.g., spraying, misting, atomizing, dusting, granules, water surface application, box application, etc.), applying it to the soil (e.g., mixing, irrigation, etc.), applying it to the surface (e.g., painting, coating, covering, etc.), and immersing.

[0048] The application amount of the aflatoxin production inhibitor is not particularly limited, and can be appropriately selected depending on various conditions such as the concentration of the active ingredient in the aflatoxin production inhibitor, the form of the formulation, the type of target disease or crop, the extent of damage caused by the disease, the application site, application method, application time, the types and amounts of chemicals or fertilizers used together or in combination, and weather.

[0049] The application concentration of the aflatoxin production inhibitor is not particularly limited and can be appropriately selected depending on the purpose. EXAMPLES

[0050] The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited thereto.

[0051] Example 1: Cultivation of aflatoxin production inhibitors In this example, Klebsiella aerogenes ( Klebsiella aerogenes ) A culture medium of the KTTM strain was prepared. A loopful of the colony of the KTTM strain grown on Bennett solid medium (agar 1.5%, glucose 1%, peptone 0.2%, meat extract 0.1%, yeast extract 0.1%, pH 7.2) was taken and inoculated into 7 mL of Bennett liquid medium (glucose 1%, peptone 0.2%, meat extract 0.1%, yeast extract 0.1%, pH 7.2) in a test tube, and cultured at 27.5°C for 2 days with shaking (150 rpm). An 80% aqueous glycerol solution was added to the culture solution to adjust the glycerol concentration to 20%, and the culture was stored at -80°C. 0.21 mL of the 20% glycerol stock solution of the KTTM strain was inoculated into 7 mL of Bennett liquid medium in a test tube, and cultured with shaking (150 rpm) at 27.5° C. for 3 days.

[0052] Test Example 1: Evaluation of aflatoxin production inhibitory activity (1) (1) Preparation of spore suspension Aspergillus flavus ( Aspergillus flavus IMF47798 (Reference Strain obtained from the International Union of Microbiological Societies) was cultured on a potato dextrose agar medium (PDA medium; Difco, MD, USA) plate at 27°C for 14 days, and spores were scraped from the colony with a platinum loop and cultured at 1.1 × 10 5 A spore suspension was prepared by suspending the spores in distilled water containing 0.1% by mass of Tween 20 to give a CFU / μL concentration.

[0053] (2) Evaluation of aflatoxin production inhibitory activity 10 mL of distilled water was added to a beaker (100 mL capacity) containing 100 dried peanuts (approximately 0.5 g / piece, Chiba Hantachi Peanut, Midorikawa Shoten, Chiba) with the shells and skins removed, and the peanuts were autoclaved for 20 minutes at 120° C. The autoclaved peanuts were immersed in a solution prepared by serially diluting the culture solution prepared in Example 1 with sterilized Millipore water, and then transferred to a 24-well microplate, one peanut per well. As a control, peanuts were immersed in sterilized Millipore water instead of the diluted culture medium in Example 1, and then transferred to a 24-well microplate at one piece per well. The peanuts in these wells were inoculated with 5 μL / well of the spore suspension prepared in Test Example 1(1), and cultured statically at 25° C. for 14 days.

[0054] The mold-grown peanuts were transferred one by one to a Falcon tube (15 mL), and 5.0 mL of 90% acetonitrile aqueous solution was added and crushed with a spatula for extraction. 2.0 mL of the resulting crushed extract was applied to an aflatoxin purification column (Autoprep MF-A 1000, Resonac, Tokyo), and 1.0 mL of the column effluent was taken in a Falcon tube (15 mL) and freeze-dried. The residue after freeze-drying was dissolved in 0.5 mL of 10% acetonitrile aqueous solution, and the solution was passed through a filter (Minisart RC4, Sartorius, Gottingen, Germany), and the mass of aflatoxin B1 was measured by high-performance liquid chromatography under the following measurement conditions.

[0055] [HPLC measurement conditions] -HPLC- Equipment: SHIMADZU 20A HPLC system (pump: LC20AD, autosampler: SIL-20AC, oven: CTO-20AC, fluorescence detector: RF-20A xs) Column: Capcell-pak C 18 Column UG120 (diameter 4.6 mm, length 250 mm, manufactured by Osaka Soda) Elution: Acetonitrile:Methanol:Water (1:3:6, V / V / V) Isocratic elution Flow rate: 1.0mL / min Retention time: Aflatoxin B1 (12.5 minutes) Detection: Fluorescence at 450 nm (excitation wavelength: 365 nm)

[0056] The results are shown in Figure 1. The culture medium of the KTTM strain (cell count: 2.3 × 10 9Peanuts were soaked in 10-fold diluted solutions of aflatoxin-producing bacteria (100,000 cells / mL), and then cultured. The aflatoxin concentration in the peanuts was then analyzed. 5 Even in the case of peanuts soaked in water, the amount of aflatoxin produced was more strongly suppressed than in the control (soaked in water). The "○" (white circle) in the figure represents the amount of aflatoxin in each peanut, but this experiment is subject to large variations in the amount of aflatoxin produced between individual peanuts due to differences in the shape of the peanuts, etc. It was separately confirmed that the culture medium of Klebsiella aerogenes does not decompose aflatoxin.

[0057] Test Example 2: Evaluation of aflatoxin production inhibitory activity (2) Klebsiella pneumoniae NBRC 3319, Klebsiella aerogenes NBRC 12010, Klebsiella oxytoca NBRC 102593, Raoultella planticola NBRC 14939, Raoultella terrigena NBRC 14941, Raoultella ornithinolytica NBRC 105727 was cultured in the same manner as in Example 1. The aflatoxin production inhibitory activity was evaluated in the same manner as in Test Example 1.

[0058] The results are shown in Figure 2. Klebsiella pneumoniae NBRC 3319, Klebsiella aerogenes NBRC 12010, Klebsiella oxytoca NBRC 102593, Raoultella planticola NBRC 14939, Raoultella terrigena NBRC 14941, Raoultella ornithinolyticaNBRC 105727 The culture solution of each strain was diluted 10-fold up to 1000-fold, and the peanuts were soaked in the diluted solution. The aflatoxin-producing bacteria were then inoculated and cultured on the peanuts. The aflatoxin concentration in the peanuts was then analyzed. 3 The aflatoxin production was more suppressed in the case of soaking in water than in the control (water soaking). The control shows the average value for 12 peanuts, and the others show the average value for 2 peanuts. In this test example, the same KTTM strain as that evaluated in Test Example 1 was used. Klebsiella aerogenes (NBRC 12010 strain) was used, but the KTTM strain showed stronger aflatoxin production inhibitory activity than the NBRC 12010 strain.

[0059] Example 2: Production of aflatoxin production inhibitor (2) In this example, Klebsiella aerogenes ( Klebsiella aerogenes ) The active substance, cyclo(L-Ala-Gly), was purified from the culture medium of the KTTM strain, and an aflatoxin production inhibitor containing the active substance was prepared.

[0060] (1)Culture process 0.21 mL of the 20% glycerol stock solution of the KTTM strain prepared in Example 1 was inoculated into 7 mL of Bennett liquid medium in a test tube and cultured with shaking (150 rpm) at 27.5° C. for 2 days to obtain a preculture solution. For the main culture, 3 mL of the preculture solution was inoculated into 100 mL of Bennett liquid medium in a 500 mL Erlenmeyer flask and cultured with shaking (150 rpm) at 27.5° C. for 5 days.

[0061] (2) Purification process The culture supernatant obtained by removing the bacterial cells from 3 L of the culture solution obtained in the above culture step by centrifugation (5,000×g, 10 minutes) was passed through an activated charcoal column (Activated Charcoal, 100 g, FUJIFILM Wako Chemicals) packed with water as a solvent. Next, after washing with 300 mL of water, elution was performed with 500 mL of 10% ethanol by volume. The eluted fraction of the 10% ethanol by volume obtained was concentrated to obtain a concentrate (18.5 mL), which was subjected to reverse phase high performance liquid chromatography (RP-HPLC) three times under the following measurement conditions to purify the active substance.

[0062] [RP-HPLC measurement conditions: 1st time] Column: Capcellpak C 18 Column (length 250mm, diameter 10mm, made by Osaka Soda): Eluent: A 0.1% by volume trifluoroacetic acid aqueous solution / B 100% by volume acetonitrile Elution conditions: Linear gradient elution from A:B=100:0 to A:B=0:100 for 15 minutes Flow rate: 2.5mL / min Detection: UV300nm Active fraction yield: 40.6mg [RP-HPLC measurement conditions: 2nd time] Column: Capcellpak C 18 Column (length 250 mm, diameter 4.6 mm, made by Osaka Soda) Eluent: A 0.1% by volume trifluoroacetic acid aqueous solution / B 100% by volume acetonitrile Elution conditions: Linear gradient elution from A:B=100:0 to A:B=0:100 for 15 minutes Flow rate: 1.0mL / min Detection: UV220nm Active fraction yield: 4.0mg [RP-HPLC measurement conditions: 3rd time] Column: Senshu Pak DOCOSIL SP-100 column (length 250 mm, diameter 4.6 mm, Senshu Scientific) Elution: Isocratic elution (aqueous solution containing 10mmol / L ammonium acetate) Flow rate: 1.0mL / min Detection: UV210nm Active substance retention time: 5.2 minutes Active substance yield: 1.15mg

[0063] (3) Identification process The obtained active substance was identified as cyclo(L-Ala-Gly) based on the retention time in the RP-HPLC and comparison with a standard specimen of the following physicochemical properties.

[0064] (a) Experimental value by ESI-time of flight mass spectrometry (ESI-TOFMS: positive ion mode): m / z 129.0666 (M+H) + (calcd for C5H9N2O2, 129.0664). (b) Specific rotation is, [α] 27 D =-13.8 (c=0.06, H2O). (c) Nuclear magnetic resonance spectroscopy measured at 500 MHz in deuterated dimethyl sulfoxide (DMSO-d6) at 25°C. 1 H and 13 The C nuclear magnetic resonance spectrum was as follows: δ H (DMSO-d6, 500 MHz): 8.16 (1H, br.s, NH), 7.97 (1H, br.s, NH), 3.84(1H, q, J = 7 Hz), 2.72 (2H, m), 1.25 (3H, d, J = 7 Hz); δ C (DMSO-d6, 125 MHz): 168.9, 166.3, 49.7, 44.5, 18.7.

[0065] Test Example 3: Evaluation of aflatoxin production inhibitory activity (2) As evaluation samples, cyclo(L-Ala-Gly) and its related compounds shown in FIG. 3 were used. Cyclo(L-Ala-Gly) was a commercially available product (Bachem, Budendorf, Switzerland). Cyclo(L-Ala-L-Pro) synthesized in Non-Patent Document 2 was used. 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) were synthesized by the following method and used.

[0066] D-Ala-OMe-HCl (0.415 g, 3 mmol, Watanabe Chemical Industry, Hiroshima, Japan) was dissolved in 3 mL of dry DMF. To this solution, Boc-Gly-OH (0.526 g, 3 mmol, Watanabe Chemical Industry), 2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU) (1.37 g, 3.6 mmol), and N,N-diisopropylethylamine (DIPEA) (0.38 g, 6.0 mmol) were added, and the mixture was stirred at room temperature overnight. Ethyl acetate (100 mL) was added to the reaction solution, which was then washed with 5% NaHCO3 (120 mL) and 5% NaCl (120 mL), and dried over anhydrous Na2SO4. After evaporation, the residue (0.451 g) was chromatographed on a silica gel column (Silica gel 60 (0.063-0.200 mm), 20 g, Merck, Darmstadt, Germany) and eluted isocratically with n-hexane-EtOAc (1:1) to give the Boc- and OMe-protected dipeptide (0.296 g). The protected dipeptide (0.296 g) was deprotected and cyclized by autoclaving in water (30 mL) at 121 °C for 4 h. 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, CHCl:MeOH (9:1)) to give cyclo(D-Ala-Gly) (42 mg).

[0067] Similarly, 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 Industry), respectively. 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 = 7 Hz), 3.72 (2H, m), 1.25 (3H, d, J = 7 Hz); 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 = 17 Hz), 1.61-1.77 (2H, m), 0.85 (3H, t, J = 7 Hz); δ C (DMSO-d6, 125 MHz): 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, 500 MHz): 8.02 (1H, br.s, NH), 3.70 (2H); δ C (DMSO-d6, 125 MHz): 166.1, 44.3.

[0068] (Evaluation method) The sample solution (100 μL) was added to 1.9 mL of potato dextrose liquid medium in a well of a microplate (24 wells). A spore suspension (5 μL) of Aspergillus flavus was inoculated into the medium and cultured at 25°C for 4 days. A mixture of 100 μL of culture broth and 400 μL of water:acetonitrile (9:1, V / V) was filtered (MinisartRC4, Sartorius, Göttingen, Germany), and the amount of aflatoxin B1 in the filtrate was measured using a Capcell pak C 250 mm × 4.6 mm inner diameter filter. 18 The analysis was performed by HPLC using a UG 120 column (Osaka Soda). The HPLC was performed under the conditions of isoclac elution with acetonitrile:methanol:water (1:3:6, V / V / V), 20 minutes, flow rate of 1.0 mL, and fluorescence detection at 450 nm (excitation wavelength: 365 nm).

[0069] The results are shown in Figure 3. The concentrations shown in Figure 3 are the concentrations (IC 50). The aflatoxin production inhibitor of the present invention can be an active ingredient of the compound represented by the following formula: [ka] (wherein R is a linear or branched alkyl group having 1 to 4 carbon atoms). The diketopiperazines represented by the formula (I) exhibited a higher aflatoxin production inhibitory effect than cyclo(L-Ala-L-Pro), which is known to have aflatoxin production inhibitory activity. In particular, cyclo(L-Abu(2)-Gly), in which the methyl group of the natural product cyclo(L-Ala-Gly) was replaced with an ethyl group, exhibited 75-fold higher activity than the natural product, and exhibited extremely excellent aflatoxin production inhibitory activity. In addition, neither cyclo(D-Ala-Gly), which is a stereoisomer of cyclo(L-Ala-Gly), nor cyclo(Gly-Gly) which does not have the group R (i.e., the group corresponding to R is a hydrogen atom), exhibited sufficient inhibitory activity against aflatoxin production. [Industrial Applicability]

[0070] The aflatoxin production inhibitor of the present invention has excellent aflatoxin production inhibitory activity and can therefore be used in a method for preventing aflatoxin contamination. [Accession number]

[0071] Klebsiella aerogenes ( Klebsiella aerogenes The KTTM strain was deposited internationally on June 8, 2023 at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) under the accession number NITE BP-03899.

Claims

1. An aflatoxin production inhibitor comprising a culture of Klebsiella sp. or Raoultella sp.

2. The following general formula (1): 【Chemistry 1】 (wherein R is a linear or branched alkyl group having 1 to 4 carbon atoms). An aflatoxin production inhibitor comprising a compound represented by the formula:

3. A method for producing an aflatoxin production inhibitor, comprising a step of culturing Klebsiella sp. or Raoultella sp.

4. The culture obtained in the culture step is subjected to the following general formula (1): 【Chemistry 2】 (wherein R is a linear or branched alkyl group having 1 to 4 carbon atoms). A process for purifying a compound represented by the formula: The method of claim 3 further comprising:

5. A method for inhibiting aflatoxin production, comprising using the aflatoxin production inhibitor according to claim 1 or 2.

6. A method for preventing aflatoxin contamination, comprising inhibiting aflatoxin production by aflatoxin-producing bacteria using the aflatoxin production inhibitor according to claim 1 or 2.

7. The following general formula (1): 【Chemistry 3】 (wherein R is a methyl group). A compound represented by the formula:

8. Klebsiella aerogenes, accession number NITE BP-03899.

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