Method for inhibiting aspergillus flavus by using methylmalonic acid

Abstract

The invention relates to the technical field of fungus inhibition, and discloses a method for inhibiting aspergillus flavus by using methylmalonic acid. The method for inhibiting aspergillus flavus by using methylmalonic acid comprises the following steps: S1, activating an aspergillus flavus strain; s2, preparing a culture medium for culturing aspergillus flavus, wherein the culture medium comprises a potato dextrose agar culture medium, namely a PDA culture medium, a potato dextrose broth culture medium, namely a PDB culture medium, a 1640 culture medium, a minimum salt culture medium and a Shaw liquid culture medium; s3, inoculating bacillus amyloliquefaciens and aspergillus flavus spores into the PDA culture medium, and exploring the inhibition effect of the bacillus amyloliquefaciens on the aspergillus flavus; and S4, inoculating aspergillus flavus by using a 1640 culture medium. According to the method for inhibiting the aspergillus flavus by using the methylmalonic acid provided by the invention, the methylmalonic acid can inhibit the growth of the aspergillus flavus, and meanwhile, the methylmalonic acid also can obviously inhibit the toxin production of the aspergillus flavus, so that a new selectivity is provided for the prevention and control of the aspergillus flavus.

Description

Technical Field

[0001] The invention relates to the technical field of fungus inhibition, and in particular to a method for inhibiting Aspergillus flavus by utilizing methylmalonic acid. Background Art

[0002] Peanut is an important oil crop and cash crop in my country. It ranks third in terms of planting area among oil crops in my country. It has a wide planting area and its annual output has always ranked first in the world, reaching 18 million tons, accounting for 40% of the world's total output. However, peanuts are susceptible to infection by Aspergillus flavus (A.flavus) and Aspergillus parasiticus. The aflatoxin produced is highly toxic, mutagenic, and highly carcinogenic. It has a strong destructive effect on human and animal liver tissues and can induce diseases such as primary liver cancer. In recent years, the aflatoxin pollution of peanuts in my country has generally shown an aggravating trend, seriously threatening consumer safety, restricting industrial development and export trade, and causing huge economic losses.

[0003] Many naturally occurring microorganisms in nature have an inhibitory effect on the growth and toxin production of Aspergillus flavus, including bacteria such as Lactobacillus, Bacillus, Pseudomonas, and Burkholderia, and fungi such as non-toxigenic Aspergillus, Trichoderma, Aspergillus niger, and Penicillium. At present, the use of antagonistic microorganisms for biological control is developing rapidly. It can inhibit the growth and reproduction of toxin-producing fungi such as Aspergillus flavus from the source in the field and reduce the risk of aflatoxin contamination in peanuts after harvest. Most microorganisms also have the function of promoting plant growth and improving the nutritional quality of agricultural products. It is a safe, efficient, and environmentally friendly way to prevent and control aflatoxin contamination.

[0004] There are three main modes of biological control of Aspergillus flavus: one is that antagonists grow rapidly, occupy ecological niches, compete for nutrients and living space, and thus replace pathogens; the other is to inhibit fungal growth and spore germination, thereby reducing fungal colonization and infection; the third is to inhibit the biosynthesis of aflatoxin. For antagonistic microorganisms, their metabolites play a major role in inhibiting the growth and toxin production of Aspergillus flavus.

[0005] The metabolites of antagonistic microorganisms play a major role in inhibiting the growth and toxin production of Aspergillus flavus. Currently, about 30 different compounds have been found to have biological activity against Aspergillus flavus, but the types of inhibitory compounds are still limited, mainly including lipopeptides, organic matter, organic acids, antibiotics and enzymes, etc. They play an important role in the prevention and control of Aspergillus flavus, but there are still a large number of unknown substances that have not yet been discovered, and research on their mechanism of action is also relatively lacking.

[0006] Therefore, exploring and developing new aflatoxin inhibitors and conducting in-depth research on their mechanisms of action are of great significance for improving the effectiveness of biological control technology, ensuring food safety, and promoting the sustainable development of the peanut industry. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a method for inhibiting Aspergillus flavus by using methylmalonic acid.

[0008] The method for inhibiting Aspergillus flavus by using methylmalonic acid provided by the present invention comprises the following steps:

[0009] S1. Activating the Aspergillus flavus strain;

[0010] S2. Prepare culture media for culturing Aspergillus flavus, including potato dextrose agar medium (PDA medium), potato dextrose broth medium (PDB medium), 1640 medium, minimal salt medium, and Sabouraud liquid medium;

[0011] S3, inoculating Bacillus amyloliquefaciens and Aspergillus flavus spores into PDA medium to explore the inhibitory effect of Bacillus amyloliquefaciens on Aspergillus flavus;

[0012] S4. Use 1640 medium to inoculate Aspergillus flavus, and add fermentation broth of Bacillus amyloliquefaciens at different fermentation times to explore the inhibitory effect of Bacillus amyloliquefaciens fermentation broth on Aspergillus flavus;

[0013] S5, using minimal salt medium to inoculate Bacillus amyloliquefaciens for non-target metabolome detection;

[0014] S6. Add methylmalonic acid and succinic acid to PDA medium respectively, and inoculate Aspergillus flavus spores to explore the inhibitory effects of methylmalonic acid and succinic acid on Aspergillus flavus spores;

[0015] S7. Use Sabouraud liquid medium and inoculate Aspergillus flavus for cultivation, use high performance liquid chromatography (HPLC) to determine aflatoxin AFB1, and use an electron microscope to observe the mycelial cell structure.

[0016] Preferably, in S1, the activation of the Aspergillus flavus strain comprises the steps of:

[0017] 2uL of Aspergillus flavus spores frozen in 25% glycerol were taken onto a PDA culture dish, incubated upright for 12h at 28℃ and 90% humidity, and then inverted for 4 days (the same below). The spores were washed with sterile 0.01% Tween 80 (121℃, 20min), and finally counted with a hemocytometer. The spore concentration was 3.5×10 7 cfu / mL (the same below).

[0018] Preferably, the bacterial solution concentration of Bacillus amyloliquefaciens is OD 600 =1.0.

[0019] Preferably, the specific steps of S3 are:

[0020] Tryptone (5 g / L) was added to the PDA medium, and Aspergillus flavus and Bacillus amyloliquefaciens were inoculated. The inoculation amount was 2 uL, and the distance between Aspergillus flavus and Bacillus amyloliquefaciens was 2 cm. The medium was placed in a 28° C. constant temperature incubator and cultured for 4 days.

[0021] Preferably, in S4, the culture medium as a whole adopts 1640 culture medium, the total system is 200uL, divided into four groups, and 10% LB broth culture medium, 10% 1640 culture medium, 10% Bacillus amyloliquefaciens 24h fermentation broth, and 10% Bacillus amyloliquefaciens 48h fermentation broth are added respectively. The LB broth culture medium is a PDB culture medium added with yeast powder peptone (5g / L).

[0022] Preferably, in S4, the fermentation culture conditions of Bacillus amyloliquefaciens are: using LB broth medium, inoculating 200uL of Bacillus amyloliquefaciens bacterial solution, 28°C, 180r / min;

[0023] The fermentation broth was collected by pouring the fermentation broth into a 50 mL sterile centrifuge tube, centrifuging at 5000 r / min for 10 min, and filtering the supernatant through a 0.22 um sterile filter membrane.

[0024] Preferably, in S5, the minimum salt medium is MM medium, and the preparation steps are: dissolving 0.5g glucose, 0.5g tryptone, 0.5g sodium glutamate, 0.5g yeast extract, 0.44g dimethyl phosphate, 0.1g ammonium sulfate, and 0.1g magnesium sulfate in 1L pure water, and adjusting the pH of the culture solution to 7 using hydrochloric acid and sodium hydroxide.

[0025] Preferably, the specific steps of S5 are: inoculating 200uL of Bacillus amyloliquefaciens into 100mL MM culture medium for cultivation, taking samples at 24h and 48h respectively, that is, pouring the fermentation broth into a 50mL sterile centrifuge tube, 5000r / min, 10min, taking the supernatant into a 2mL ep tube, immediately putting it into liquid nitrogen, freezing at -80°C, and performing non-target metabolome detection.

[0026] Preferably, the specific steps of S6 are: adding methylmalonic acid and succinic acid to the PDA culture medium in amounts of 5 mM and 10 mM respectively, at 121° C. for 20 min, to prepare a culture dish; taking 2 uL of Aspergillus flavus spores in the culture dish and culturing for 2 days.

[0027] Preferably, the specific steps in S7 are:

[0028] Sabouraud liquid medium was inoculated with Aspergillus flavus at a final working concentration of 3.5×10 3 cfu / mL, 28°C, 180r / min, culture for 4 days;

[0029] The mycelium cultured in the above Sabouraud liquid culture was filtered through four layers of sterile gauze, collected into a 5 mL cryotube, freeze-dried at -60°C in a vacuum oven, and the dry weight was weighed;

[0030] Weigh 10 mg of vacuum freeze-dried Aspergillus flavus hyphae in a 1.5 mL eppendorf tube, add 1 mL of 80% methanol (HPLC grade methanol, ddH2O), add 4 steel balls with a diameter of 3 mm, and incubate for 45 h. Z Grind for 4 minutes, then centrifuge at 10000r / min for 10 minutes, take the supernatant and pass it through a 0.22uM organic membrane, dilute it 10 times with 80% methanol, and measure the sample by high performance liquid chromatography (HPLC);

[0031] The hyphae were collected using four layers of sterile gauze and placed in 3% glutaraldehyde. The cell structure of the hyphae was observed using an electron microscope. In the same manner, the hyphae cultured in a culture dish supplemented with methylmalonic acid were observed using an electron microscope.

[0032] The invention provides a flavus inhibitor or microbial agent, the active ingredient of which includes methylmalonic acid.

[0033] Preferably, the aflatoxin inhibitor or microbial agent comprises active ingredients of methylmalonic acid-producing microorganisms.

[0034] The present invention also provides an application of the methylmalonic acid and the microorganism producing methylmalonic acid as mentioned above in inhibiting the growth of Aspergillus flavus and the biosynthesis of aflatoxin during the growth and storage of crops.

[0035] Compared with the related art, the method for inhibiting Aspergillus flavus using methylmalonic acid provided by the present invention has the following beneficial effects:

[0036] The present invention performs non-targeted metabolome sequencing on the fermentation broth of a Bacillus amyloliquefaciens strain that significantly inhibits the growth of aflatoxin, understands the types of main metabolites produced by the strain, and finds that the strain produces a large amount of substances in the propionic acid metabolic pathway such as methylmalonic acid and succinic acid. By adding the two to a potato dextrose agar medium (PDA) or a potato dextrose broth medium (PDB) inoculated with Aspergillus flavus spores, it is found that methylmalonic acid can inhibit the growth of Aspergillus flavus. At the same time, the Aspergillus flavus cultured in PDB is tested and it is found that methylmalonic acid also significantly inhibits the toxin production of Aspergillus flavus. Scanning and projection electron microscopy further reveal that methylmalonic acid can shrink the hyphae of Aspergillus flavus and destroy the intracellular structure.

[0037] The invention describes that methylmalonic acid has the ability to inhibit Aspergillus flavus and aflatoxin, and provides new selectivity for the prevention and control of Aspergillus flavus. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1This is a diagram showing the inhibitory effect of Bacillus amyloliquefaciens on Aspergillus flavus;

[0039] Figure 2 This is a diagram showing the inhibitory effect of fermentation metabolites of Bacillus amyloliquefaciens on Aspergillus flavus;

[0040] Figure 3 Schematic diagram of the significant enrichment of methylmalonic acid and succinic acid in the fermentation metabolite after 48 hours;

[0041] Figure 4 This is a graph showing the inhibitory effect of methylmalonic acid and succinic acid on mycelial growth in PDA;

[0042] Figure 5 This is a diagram showing the inhibitory effect of methylmalonic acid on hyphae growth and toxin production in PDB;

[0043] Figure 6 This is the electron microscopy result of Aspergillus flavus hyphae treated with methylmalonic acid. DETAILED DESCRIPTION

[0044] The present invention will be further described below in conjunction with the accompanying drawings and implementation modes.

[0045] The present invention provides a method for inhibiting Aspergillus flavus by using methylmalonic acid, comprising the following steps:

[0046] S1. Activate the Aspergillus flavus strain: Pipette 2uL of Aspergillus flavus spore solution frozen with 25% glycerol onto a PDA culture dish, culture it upright for 12h at 28℃ and 90% humidity, then culture it inverted for 4 days (the same below), wash the spores with sterile 0.01% Tween 80 (121℃, 20min), and finally count them with a hemocytometer. The spore concentration is 3.5×10 7 cfu / mL (the same below);

[0047] S2. Prepare culture media for culturing Aspergillus flavus, including potato dextrose agar medium (PDA medium), potato dextrose broth medium (PDB medium), 1640 medium, minimal salt medium, and Sabouraud liquid medium;

[0048] S3. Add tryptone (5 g / L) to the PDA medium, and inoculate Aspergillus flavus and Bacillus amyloliquefaciens with an inoculation volume of 2 uL. The distance between Aspergillus flavus and Bacillus amyloliquefaciens was 2 cm. Put it in a 28°C constant temperature incubator for 4 days to observe the inhibition of Bacillus amyloliquefaciens on Aspergillus flavus. Here, the bacterial solution concentration of Bacillus amyloliquefaciens is OD 600 =1.0;

[0049] S4. Inoculate Aspergillus flavus with 1640 culture medium to explore the inhibitory effect of Bacillus amyloliquefaciens fermentation broth on Aspergillus flavus: The culture medium as a whole adopts 1640 culture medium, the total system is 200uL, and is divided into four groups, and 10% LB broth culture medium, 10% 1640 culture medium, 10% Bacillus amyloliquefaciens 24h fermentation broth, and 10% Bacillus amyloliquefaciens 48h fermentation broth are added for cultivation, and LB broth culture medium is PDB culture medium added with yeast powder peptone (5g / L); wherein the fermentation culture conditions of Bacillus amyloliquefaciens are: LB broth culture medium is used, 200uL Bacillus amyloliquefaciens bacterial liquid is inoculated, 28°C, 180r / min; the fermentation broth collection method is: pour the fermentation broth into a 50mL sterile centrifuge tube, 5000r / min, 10min, take the supernatant through a 0.22um sterile filter membrane, and observe the inhibitory effect of Bacillus amyloliquefaciens fermentation broth on Aspergillus flavus:

[0050] S5. Use minimal salt medium (MM medium, prepared by dissolving 0.5 g glucose, 0.5 g tryptone, 0.5 g sodium glutamate, 0.5 g yeast extract, 0.44 g dimethyl phosphate, 0.1 g ammonium sulfate, and 0.1 g magnesium sulfate in 1 L pure water, and adjust the pH of the culture solution to 7 using hydrochloric acid and sodium hydroxide), inoculate 200 uL of Bacillus amyloliquefaciens into 100 mL MM medium for culture, take samples at 24 h and 48 h, that is, pour the fermentation broth into a 50 mL sterile centrifuge tube, 5000 r / min, 10 min, take the supernatant into a 2 mL ep tube, immediately put it into liquid nitrogen, and freeze it at -80 ° C for non-target metabolome detection;

[0051] S6. Add methylmalonic acid and succinic acid to the PDA medium in amounts of 5 mM and 10 mM, respectively, at 121° C. for 20 min, prepare a culture dish, and take 2 uL of Aspergillus flavus spores in the culture dish, culture for 2 days, and observe the inhibitory effects of methylmalonic acid and succinic acid on Aspergillus flavus spores;

[0052] S7, Sabouraud liquid medium was inoculated with Aspergillus flavus, with a final working concentration of 3.5×10 3 cfu / mL, 28°C, 180r / min, culture for 4 days;

[0053] The mycelium cultured in the above Sabouraud liquid culture was filtered through four layers of sterile gauze, collected into a 5 mL cryotube, freeze-dried at -60°C in a vacuum oven, and the dry weight was weighed;

[0054] Weigh 10 mg of vacuum freeze-dried Aspergillus flavus hyphae in a 1.5 mL eppendorf tube, add 1 mL of 80% methanol (HPLC grade methanol, ddH2O), add 4 steel balls with a diameter of 3 mm, and incubate for 45 h. ZGrind for 4 minutes, then centrifuge at 10000r / min for 10 minutes, take the supernatant and pass it through a 0.22uM organic membrane, dilute it 10 times with 80% methanol, and measure the sample by high performance liquid chromatography (HPLC);

[0055] The hyphae were collected using four layers of sterile gauze and placed in 3% glutaraldehyde. The cell structure of the hyphae was observed using an electron microscope. In the same manner, the hyphae cultured in a culture dish supplemented with methylmalonic acid were observed using an electron microscope.

[0056] Through the method for inhibiting Aspergillus flavus using methylmalonic acid provided in the present invention, it is found that:

[0057] By using Bacillus spores with antibacterial activity against Aspergillus flavus (such as Figure 1 The antibacterial activity of the fermentation broth of the yeast powder peptone medium was tested and it was found that the fermentation broth of the bacteria cultured at 28°C and 180 r / min for 24 h and 48 h inhibited the germination of Aspergillus flavus spores to varying degrees. 6 CFU / mL, the culture medium is 1640. Compared with the 24h fermentation broth, the 48h fermentation product can significantly inhibit the growth of Aspergillus flavus. The inhibition rate of the 24h fermentation broth on Aspergillus flavus is 9.45%, and the inhibition rate of the 48h fermentation broth on Aspergillus flavus is 53.82%. Figure 2 As shown in the figure, the row LB represents the addition of 10% LB broth medium, 1640 represents the addition of 10% 1640 medium, 24h represents the addition of 10% Bacillus amyloliquefaciens fermentation broth for 24 hours, 48h represents the addition of 10% Bacillus amyloliquefaciens fermentation broth for 48 hours, and the vertical column represents the working concentration of Aspergillus flavus spores, which are 3.5×10 6 , 10 5 , 10 4 , 10 3 , 10 2 cfu / mL, 0 (blank control).

[0058] By performing non-targeted metabolomic analysis on the fermentation broth of the bacteria after 24h and 48h of culture, it was found that the main metabolite components changed significantly with the increase of fermentation time. Compared with the metabolites of 24h, 416 metabolites increased significantly and 545 metabolites decreased significantly after 48h. KEGG enrichment analysis of the main differential metabolite pathways (such as Figure 3 As shown in Figure 2, with the increase of fermentation time, the correlation between nucleotide metabolism, citric acid cycle and pyruvate metabolism in the fermentation broth increased significantly. Among them, the metabolites that increased significantly at 48h included methylmalonic acid, succinic acid, and surfactin.

[0059] The effects of methylmalonic acid and succinic acid on the growth of Aspergillus flavus were verified in vitro. When 5mM and 10mM methylmalonic acid and succinic acid were added to potato dextrose agar (PDA) medium, respectively, it was found that methylmalonic acid significantly inhibited the growth of Aspergillus flavus with increasing concentration, while succinic acid had a weaker inhibitory effect on Aspergillus flavus (e.g. Figure 4 As shown). Similarly, in liquid Sabouraud liquid culture medium, under the treatment of 10mM, the growth of mycelium was abnormal, 20mM methylmalonic acid significantly inhibited the germination and spore production of Aspergillus flavus, and the spores did not germinate at all under the treatment of 50mM. The inhibition rate of Aspergillus flavus biomass (dry weight) was 61.60% under the 10mM treatment, and the inhibition rate was 95.42% at 20mM. The cultured mycelium was collected and aflatoxin AFB1 was detected by liquid phase. It was found that the inhibition rate of 10mM methylmalonic acid on aflatoxin AFB1 was 80.28% (as shown). Figure 5 shown).

[0060] By collecting control hyphae and hyphae treated with methylmalonic acid grown in Sabouraud liquid medium for 4 days, scanning electron microscopy and transmission electron microscopy were used to detect the hyphae. It was found that the hyphae of Aspergillus flavus treated with methylmalonic acid shrank, the cell wall and fiber layer became thinner, and the function of protecting cells was lost. Mitochondria also lysed (such as Figure 6 shown).

[0061] Through the above exploration, the present invention illustrates that methylmalonic acid has the ability to inhibit Aspergillus flavus and aflatoxin, providing a new selectivity for the prevention and control of Aspergillus flavus.

[0062] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for inhibiting Aspergillus flavus using methylmalonic acid, characterized in that: The steps include: S1. Activating the Aspergillus flavus strain; S2. Prepare culture media for culturing Aspergillus flavus, including potato dextrose agar medium (PDA medium), potato dextrose broth medium (PDB medium), 1640 medium, minimal salt medium, and Sabouraud liquid medium; S3, inoculating Bacillus amyloliquefaciens and Aspergillus flavus spores into PDA medium to explore the inhibitory effect of Bacillus amyloliquefaciens on Aspergillus flavus; S4. Use 1640 medium to inoculate Aspergillus flavus, and add fermentation broth of Bacillus amyloliquefaciens at different fermentation times to explore the inhibitory effect of Bacillus amyloliquefaciens fermentation broth on Aspergillus flavus; S5, using minimal salt medium to inoculate Bacillus amyloliquefaciens for non-target metabolome detection; S6. Add methylmalonic acid and succinic acid to PDA medium respectively, and inoculate Aspergillus flavus spores to explore the inhibitory effects of methylmalonic acid and succinic acid on Aspergillus flavus spores; S7. Use Sabouraud liquid medium and inoculate Aspergillus flavus for cultivation, use high performance liquid chromatography to determine aflatoxin AFB1, and use an electron microscope to observe the mycelial cell structure.

2. The method for inhibiting Aspergillus flavus using methylmalonic acid as claimed in claim 1, characterized in that: In the S4, the culture medium as a whole adopts 1640 culture medium, the total system is 200uL, divided into four groups, and 10% LB broth culture medium, 10% 1640 culture medium, 10% Bacillus amyloliquefaciens 24h fermentation broth, and 10% Bacillus amyloliquefaciens 48h fermentation broth are added respectively. The LB broth culture medium is the PDB culture medium added with yeast powder peptone.

3. The method for inhibiting Aspergillus flavus using methylmalonic acid as claimed in claim 1, characterized in that: In the S5, the minimum salt medium is the MM medium, and the preparation steps are as follows: 0.5g glucose, 0.5g tryptone, 0.5g sodium glutamate, 0.5g yeast extract, 0.44g dimethyl phosphate, 0.1g ammonium sulfate, and 0.1g magnesium sulfate are dissolved in 1L pure water, and the pH of the culture solution is adjusted to 7 using hydrochloric acid and sodium hydroxide.

4. The method for inhibiting Aspergillus flavus using methylmalonic acid as claimed in claim 1, characterized in that: The specific steps of S5 are as follows: 200uL of Bacillus amyloliquefaciens is inoculated into 100mL MM culture medium for cultivation, and samples are taken at 24h and 48h, that is, the fermentation liquid is poured into a 50mL sterile centrifuge tube, 5000r / min, 10min, and the supernatant is taken into a 2mL ep tube, immediately put into liquid nitrogen, and frozen at -80°C for non-target metabolome detection.

5. The method for inhibiting Aspergillus flavus using methylmalonic acid as claimed in claim 1, characterized in that: The specific steps of S6 are: adding methylmalonic acid and succinic acid to the PDA culture medium in amounts of 5 mM and 10 mM respectively, at 121° C. for 20 min, to prepare a culture dish; taking 2 uL of Aspergillus flavus spores in the culture dish and culturing for 2 days.

6. The method for inhibiting Aspergillus flavus using methylmalonic acid as claimed in claim 1, characterized in that: The specific steps in S7 are: Sabouraud liquid medium was inoculated with Aspergillus flavus at a final working concentration of 3.5×10 3 cfu / mL, 28°C, 180r / min, culture for 4 days; The mycelium cultured in the above Sabouraud liquid culture was filtered through four layers of sterile gauze, collected into a 5 mL cryotube, freeze-dried at -60°C in a vacuum oven, and the dry weight was weighed; Weigh 10 mg of vacuum freeze-dried Aspergillus flavus hyphae into a 1.5 mL eppendorf tube, add 1 mL of 80% methanol, add 4 steel balls with a diameter of 3 mm, and incubate for 45 h. Z Grind for 4 minutes, then centrifuge at 10000r / min for 10 minutes, take the supernatant and pass it through a 0.22uM organic membrane, dilute it 10 times with 80% methanol, and measure the sample by high performance liquid chromatography; The hyphae were collected using four layers of sterile gauze and placed in 3% glutaraldehyde. The cell structure of the hyphae was observed using an electron microscope. In the same manner, the hyphae cultured in a culture dish supplemented with methylmalonic acid were observed using an electron microscope.

7. A flavus inhibitor or microbial agent, characterized in that: Its active ingredients include methylmalonic acid.

8. The aflatoxin inhibitor or microbial agent according to claim 7, characterized in that: Its active ingredients include methylmalonic acid-producing microorganisms.

9. Use of methylmalonic acid and methylmalonic acid-producing microorganisms as described in claims 7-8 in inhibiting the growth of Aspergillus flavus and the biosynthesis of aflatoxin during the growth and storage of crops.

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