Composition for inhibiting aflatoxin production

Abstract

The objective of this invention is to provide a novel active ingredient useful for inhibiting aflatoxin production.　With this invention, production of aflatoxin by aflatoxin-producing fungi can be inhibited by means of a primary alcohol represented by formula (1): CH3-(CH2)n-OH (1) (where n is 0 or 2-4).

Description

[Technical Field]

[0001] The present invention relates to a composition for inhibiting aflatoxin production and a method for inhibiting aflatoxin production, and in particular to a composition containing a specific primary alcohol as an active ingredient. [Background technology]

[0002] Aflatoxin is a highly toxic mycotoxin with carcinogenic properties. Aflatoxin contamination of agricultural crops, including grains, is a major global problem, and the disposal of contaminated food and feedstuffs results in significant economic losses. Aflatoxin contamination originates from aflatoxin-producing bacteria that are normally present in agricultural soil, and there is a need to develop technologies to control these bacteria.

[0003] Patent Document 1 describes that dioctatin can be used to inhibit aflatoxin production and to control aflatoxin contamination, and Non-Patent Document 1 also describes that another compound has antibacterial activity against aflatoxin-producing bacteria and aflatoxin production inhibitory activity. Non-Patent Document 2 suggests that ethanol inhibits the biosynthesis of aflatoxin B1 by enhancing the oxidative stress response of Aspergillus flavus. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2019-94261 [Non-patent literature]

[0005] [Non-Patent Document 1] JSM Mycotoxins (2019), 69(2), 81-83 [Non-patent document 2] Frontiers in Microbiology (2020), 10, Article 2946, doi:10.3389 / fmicb.2019.02946 Summary of the Invention [Problem to be solved by the invention]

[0006] Various fungicides are used as postharvest pesticides, but their residual properties and toxicity have become a problem, and safer materials are needed. Therefore, an object of the present invention is to provide a novel active ingredient that is useful for inhibiting aflatoxin production. [Means for solving the problem]

[0007] As a result of extensive research aimed at solving the above problems, the present inventors have found that primary alcohols other than ethanol have the effect of inhibiting aflatoxin production, and have thus completed the present invention. That is, the present invention provides a composition for inhibiting aflatoxin production and a method for inhibiting aflatoxin production, as shown below. [1] Formula (1): CH3-(CH2) n -OH (1) (n is 0 or 2 to 4) A composition for inhibiting aflatoxin production by aflatoxin-producing bacteria, comprising a primary alcohol represented by the formula: [2] The composition according to [1] above, wherein the concentration of the primary alcohol represented by the formula (1) is 0.1 to 5 v / v %. [3] The composition according to [1] or [2], wherein the primary alcohol represented by the formula (1) is at least one selected from the group consisting of 1-propanol, methanol, and 1-butanol. [4] The composition according to any one of [1] to [3] above, wherein the aflatoxin-producing fungus is a fungus of the genus Aspergillus. [5] A method for inhibiting aflatoxin production, comprising: Formula (1): CH3-(CH2) n -OH (1) (n is 0 or 2 to 5) The method comprises applying a composition comprising a primary alcohol represented by the formula: [6] The method according to [5] above, wherein the concentration of the primary alcohol represented by the formula (1) is 0.1 to 5 v / v %. [7] The method according to [5] or [6] above, wherein the primary alcohol represented by the formula (1) is at least one selected from the group consisting of 1-propanol, methanol, and 1-butanol. [8] The method according to any one of [5] to [7] above, wherein the aflatoxin-producing fungus is a fungus of the genus Aspergillus. [Effects of the Invention]

[0008] According to the present invention, the primary alcohol represented by the above formula (1) can suppress aflatoxin production by aflatoxin-producing bacteria, thereby making it possible to provide a safer material that is less susceptible to problems of residual properties and toxicity. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention will now be described in further detail. The composition of the present invention is used to inhibit aflatoxin production by aflatoxin-producing bacteria, Formula (1): CH3-(CH2) n -OH (1) (n is 0 or 2 to 4) The primary alcohol represented by formula (1) is specifically methanol, 1-propanol, 1-butanol, and / or 1-pentanol, and preferably at least one selected from the group consisting of 1-propanol, methanol, and 1-butanol.

[0010] The concentration of the primary alcohol represented by formula (1) is not particularly limited as long as it can suppress aflatoxin production by aflatoxin-producing bacteria, and may be, for example, about 0.1 v / v% or more, about 0.2 v / v% or more, or about 0.5 v / v% or more, or about 5 v / v% or less, about 1.5 v / v% or less, or about 0.7 v / v% or less, relative to the composition of the present invention. In other words, the primary alcohol represented by formula (1) can be effective even at a low concentration. In a preferred embodiment, when the primary alcohol represented by formula (1) is methanol, its concentration may be about 0.1 to about 5 v / v % or about 0.8 to about 5 v / v %; when the primary alcohol represented by formula (1) is 1-propanol, its concentration may be about 0.1 to about 1.5% or about 0.5 to about 1.5 v / v %; and when the primary alcohol represented by formula (1) is 1-butanol or 1-pentanol, its concentration may be about 0.1 to about 0.7% or about 0.5 to about 0.7 v / v %.

[0011] The "aflatoxin" described herein is a type of mycotoxin, and known types include B1, B2, G1, and G2. These aflatoxins are also known to be biosynthesized via a common pathway. It is also known that fungi of the genus Aspergillus include fungi that produce the aflatoxins, i.e., "aflatoxin-producing fungi." Examples of aflatoxin-producing fungi of the genus Aspergillus include A. flavus, A. parasiticus, A. nomius, A. pseudotamarii, A. bombycis, and A. parvisclerotigenus.

[0012] Generally, when a chemical substance that inhibits the growth of aflatoxin-producing bacteria is used, there is a concern that if resistant bacteria emerge, the resistant bacteria will preferentially grow and spread. However, the primary alcohols represented by formula (1) have the characteristic effect of inhibiting aflatoxin production even at low concentrations where the inhibitory effect on the growth of aflatoxin-producing bacteria is not exerted. Therefore, according to the present invention, it is expected that the problem of the spread of resistant bacteria can be avoided while preventing aflatoxin contamination of the target. On the other hand, ethanol, 2-propanol, and 2-methyl-2-propanol, which are not included in formula (1), do not have this effect and actually increase aflatoxin production at low concentrations where the inhibitory effect on the growth of aflatoxin-producing bacteria is not exerted. Without being bound by any particular theory, it is believed that the primary alcohols represented by formula (1) can be used preferably because they are not metabolized by aflatoxin-producing bacteria.

[0013] The composition of the present invention may further contain any solvent and / or additive commonly used in the art, as long as it does not impair the objectives of the present invention, and may further contain other components effective for inhibiting aflatoxin production or for antibacterial purposes against aflatoxin-producing bacteria. The solvent may be any solvent commonly used in the art without any particular limitation, and may include, for example, water, a buffer solution, or a mixture thereof.

[0014] The composition of the present invention can be applied to a location where aflatoxin-producing fungi are growing or where aflatoxin-producing fungi are suspected to be growing. Specific target objects to which the composition is applied are not particularly limited, and may be, for example, seeds, plants, harvested crops, or farm fields.

[0015] In another aspect, the present invention also relates to a method for inhibiting aflatoxin production, comprising: Formula (1): CH3-(CH2) n -OH (1) (n is 0 or 2 to 5) The method includes applying a composition comprising a primary alcohol represented by the formula: to aflatoxin-producing fungi. The composition is as described above in relation to the composition of the present invention, and can be used in the method of the present invention in the form of the composition. In one embodiment, the applying step may include applying the composition to a location where aflatoxin-producing fungi are growing and / or where aflatoxin-producing fungi are suspected to be growing.

[0016] The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited to these examples. [Example]

[0017] [Test Example 1] A. flavus IFM47798 strain was inoculated onto potato dextrose agar medium and cultured in the dark at 28°C for 1 week. Spores formed on the medium were scraped off with a platinum loop, suspended in a 30% glycerol solution, and stored at -80°C.

[0018] The spore suspension was diluted to a spore concentration of 2.5 x 10 4 The mixture was added to potato dextrose liquid medium so that the concentration was 0.01 spores / mL, and 2 mL of this mixture was added to each well of a 12-well plate. Various alcohols were then added, and the mixture was left to stand in the dark at 28°C for 48 hours. After the culture was completed, the bacteria were separated by centrifugation, and the supernatant was collected in a separate tube.

[0019] 0.5 mL of chloroform was added to 0.5 mL of the collected supernatant, mixed using a tube mixer, and then centrifuged. The lower chloroform layer was collected in a separate tube and volatilized by air-drying in a draft chamber. The residue was dissolved in 1 mL of 90% aqueous acetonitrile, and the aflatoxins B1, B2, G1, and G2 in the solution were quantified using liquid chromatography-mass spectrometry (LC-MS). The sum of these values ​​was used as the total aflatoxin production (total AF). The total AF in the untreated control group, where no alcohol was added, was set at 100, and the relative value was calculated. The results are shown in Table 1 below.

[0020] The isolated and collected cells were frozen in a -80°C freezer in a tube whose weight had been measured beforehand, and then dried in a freeze dryer. The total weight was measured, and the tube weight was subtracted to determine the dry cell weight. The dry cell weight was then calculated relative to the dry cell weight of the control group, where no alcohol was added, set at 100. The results are shown in Table 1.

[0021] [Table 1]

[0022] Ethanol, known to have antibacterial properties, inhibited A. flavus growth and aflatoxin production at concentrations of 4% v / v or higher. However, when ethanol was applied at lower concentrations, it not only failed to inhibit A. flavus growth but actually increased aflatoxin production by A. flavus. A similar trend was observed with 2-propanol and 2-methyl-2-propanol. In contrast, when primary alcohols other than ethanol were used at low concentrations, aflatoxin production by A. flavus was concentration-dependently inhibited. Interestingly, even at low concentrations where A. flavus growth was still possible, primary alcohols other than ethanol inhibited aflatoxin production by A. flavus.

[0023] [Test Example 2] Except for using A. flavus MAFF111229 strain or A. parasiticus NRRL2999 strain instead of A. flavus IFM47798 strain and using methanol, 1-propanol, or 1-butanol as the alcohol, the total AF and dry cell weight were measured and calculated relative to the untreated control in the same manner as in Test Example 1. The results are shown in Tables 2 and 3.

[0024] [Table 2]

[0025] [Table 3]

[0026] Even when the type of test bacteria was changed to another strain or species, the characteristic effect of inhibiting aflatoxin production was confirmed for primary alcohols other than ethanol, at low concentrations where the growth of aflatoxin-producing bacteria was not completely inhibited. Among these, 1-propanol is particularly useful for preventing aflatoxin contamination of agricultural products, because it is effective at low concentrations and is also used as a food additive.

[0027] [Test Example 3] Ten grams of hulled corn kernels were placed in a petri dish and soaked in a spore suspension of A. flavus MAFF111229 (spore concentration 1 × 10 6 The grains were inoculated with 0.1 mL of 1000 spores / mL of ethanol per dish. Methanol, 1-propanol, or 1-butanol was then sprayed onto the grain surface using a spray vial at a rate of 0.2 mL per dish (20 μL per 1 g of grain). A group that was not sprayed with alcohol was prepared as a positive control, and a group that was not inoculated with the bacteria or sprayed with alcohol was prepared as a negative control. The grains from each test group were placed on an agar medium, the dish was wrapped in parafilm, and left to stand at 28°C for one week.

[0028] The grains from each test group were collected and crushed in a blender, and 40 mL of 90% acetonitrile was added, mixed on a shaker for 30 minutes, and then centrifuged to collect the supernatant. 5 mL of the supernatant was then applied to a solid-phase extraction column, Autoprep. (R) The solution was subjected to an MF-A1000 (Showa Denko K.K.), and the first 2 mL of the solution was collected. After drying by nitrogen spray, the residue was dissolved in 0.5 mL of 90% acetonitrile. Aflatoxins B1, B2, G1, and G2 in this solution were individually quantified by LC-MS, and the sum of these values ​​was used as the total aflatoxin production (total AF). The total AF in the positive control group was set at 100, and the relative value was calculated. The results are shown in Table 4.

[0029] [Table 4]

[0030] It was confirmed that the alcohols that showed aflatoxin production inhibitory action in Test Examples 1 and 2 can actually inhibit aflatoxin production on corn kernels as well.

[0031] From the above, it was found that the primary alcohol represented by the above formula (1) can suppress aflatoxin production by aflatoxin-producing bacteria, making it possible to provide a safer material that is less likely to cause problems with residue and toxicity.

Claims

1. Formula (1): CH 3 -(CH 2 ) n -OH (1) (n is 0) A composition for inhibiting aflatoxin production by aflatoxin-producing bacteria, comprising as an active ingredient a primary alcohol represented by the formula: the primary alcohol represented by the formula (1) is methanol, the concentration of the methanol is 0.3 to 4 v / v %; The composition as described above, wherein the aflatoxin-producing fungus is a fungus of the genus Aspergillus.

2. The composition according to claim 1, wherein the fungus of the genus Aspergillus is Aspergillus flavus or Aspergillus paraciticus.

3. Formula (1): CH 3 -(CH 2 ) n -OH (1) (n is 2) A composition for inhibiting aflatoxin production by aflatoxin-producing bacteria, comprising as an active ingredient a primary alcohol represented by the formula: the primary alcohol represented by the formula (1) is 1-propanol, the concentration of the 1-propanol is 0.3 to 0.6 v / v %; The composition, wherein the aflatoxin-producing fungus is Aspergillus flavus.

4. Formula (1): CH 3 -(CH 2 ) n -OH (1) (n is 0) A method for inhibiting aflatoxin production, comprising the step of applying a composition containing as an active ingredient a primary alcohol represented by the formula: the primary alcohol represented by the formula (1) is methanol, The method as described above, wherein the concentration of the methanol is 0.3 to 4 v / v %; and the aflatoxin-producing fungus is a fungus of the genus Aspergillus.

5. The method according to claim 4, wherein the fungus of the genus Aspergillus is Aspergillus flavus or Aspergillus paraciticus.

6. Formula (1): CH 3 -(CH 2 ) n -OH (1) (n is 2) A method for inhibiting aflatoxin production, comprising the step of applying a composition containing as an active ingredient a primary alcohol represented by the formula: the primary alcohol represented by the formula (1) is 1-propanol, the concentration of the 1-propanol is 0.3 to 0.6 v / v %; The method as described above, wherein the aflatoxin-producing fungus is Aspergillus flavus.

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