Use of 2-ketobutyric acid
By using 2-ketobutyric acid as an antibacterial agent and anti-aspergillus drug, the problems of growth inhibition of Aspergillus flavus and aflatoxin synthesis were solved, achieving a highly efficient antibacterial effect and a reduction in aflatoxin.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- XIAMEN MEDICAL COLLEGE
- Filing Date
- 2023-07-26
- Publication Date
- 2026-06-02
AI Technical Summary
There is a lack of effective methods in the current technology to inhibit the growth of Aspergillus flavus and reduce the production of aflatoxin.
2-Ketobutyric acid was used as an antibacterial agent and anti-aspergillus drug, with a concentration range of 1 mg/ml-100 mg/ml. It was cultured by direct contact with Aspergillus flavus strains to observe the inhibition zone and growth inhibition effect. Its effect on aflatoxin synthesis was verified by transcriptomics and HPLC analysis.
2-Ketobutyric acid (2-Ketobutyric acid) showed an inhibition rate of over 90% against Aspergillus flavus at a concentration of 2 mg/mL. It significantly downregulated the gene expression of aflatoxin and reduced its synthesis, damaged the fungal cell membrane, and effectively inhibited the production of aflatoxin.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention relates to the application of 2-ketobutyric acid. Background Technology
[0002] Alpha-keto acids have been shown to form in food via transaminases or through chemical reactions between amino acids and lipid-derived carbonyl groups. These compounds contribute significantly to the flavor of food. Among them, 2-ketobutyric acid (alpha-ketobutyric acid), also known as 2-oxobutanoic acid, has a molecular weight of 102.09 Da, a melting point of 30-34℃, a boiling point of 84℃ (20 mm Hg), and is a white powder with a caramel flavor. It is a precursor for the synthesis of many different compounds, including fats, sugars, porphyrins, and amino acids, and can also be used as a food additive. It is also a precursor for some drugs, such as the epilepsy drugs levetiracetam and brivaracetam, and the anti-tuberculosis drug ethambutol (Proc Natl Acad Sci US A.2010,107(14):6234-9). There is no literature on the antifungal properties of 2-ketobutyric acid. This invention discovers that 2-ketobutyric acid can effectively inhibit Aspergillus flavus and reduce its aflatoxin production, thus it can be used as an antifungal agent. Summary of the Invention
[0003] The main objective of this invention is to provide the application of 2-ketobutyric acid in the inhibition of aflatoxin.
[0004] Another object of the present invention is to provide the use of 2-ketobutyric acid in the preparation of aflatoxin inhibitors or anti-aflatoxin drugs.
[0005] Another object of the present invention is to provide an aflatoxin inhibitor comprising 2-ketobutyric acid.
[0006] Furthermore, the aflatoxin inhibitor, 2-ketobutyric acid, has a concentration of 1 mg / ml to 100 mg / ml, preferably 2 mg / ml to 100 mg / ml.
[0007] Another object of the present invention is to provide an anti-aflatoxin drug comprising 2-ketobutyric acid.
[0008] Furthermore, the concentration of 2-ketobutyric acid is 1 mg / ml to 100 mg / ml. Preferably, it is 2 mg / ml to 100 mg / ml.
[0009] Another object of the present invention is to provide the application of 2-ketobutyric acid in inhibiting aflatoxin synthesis in Aspergillus flavus.
[0010] Another object of the present invention is to provide the application of 2-ketobutyric acid in the preparation of aflatoxin synthesis inhibitors.
[0011] Compared with the prior art, this technical solution has the following advantages:
[0012] This invention reveals that 2-ketobutyric acid (2 mg) clearly shows an inhibition zone against Aspergillus flavus on agar plates. After 24 and 48 hours of incubation with 2 mg / mL of 2-ketobutyric acid, Aspergillus flavus showed an effective inhibition rate of over 90%. Propidium iodide (PI) staining showed that 2-ketobutyric acid treatment damaged the fungal cell membrane. Transcriptomic analysis showed that 2-ketobutyric acid downregulated the expression of genes involved in aflatoxin biosynthesis. HPLC analysis also showed a significant decrease in aflatoxin levels in Aspergillus flavus after treatment with 2-ketobutyric acid compared to the control group. Attached Figure Description
[0013] The present invention will be further described below with reference to the accompanying drawings and embodiments.
[0014] Figure 1 The inhibition zone of 2-ketobutyric acid against Aspergillus flavus CCTCC AF 2023038 is shown. The left image represents 24 hours, and the right image represents 48 hours.
[0015] Figure 2 The inhibition rate of 2-ketobutyric acid against Aspergillus flavus CCTCC AF 2023038 is given.
[0016] Figure 3 The left image shows the 2-ketobutyric acid staining analysis of propidium iodide (PI) on Aspergillus flavus CCTCC AF 2023038. The right image shows the concentration of 1 mg / ml and 2 mg / ml, respectively.
[0017] Figure 4 Transcriptomic volcano plot analysis of 2-ketobutyric acid in Aspergillus flavus CCTCC AF 2023038. Up indicates upregulated genes, down indicates downregulated genes.
[0018] Figure 5 Transcriptome (A) GO and (B) KEGG enrichment analysis of 2-ketobutyric acid for Aspergillus flavus CCTCC AF 2023038.
[0019] Figure 6 Analysis of aflatoxin production in the culture medium of Aspergillus flavus CCTCC AF 2023038 after treatment with 2-ketobutyric acid as a control group. Detailed Implementation
[0020] Aspergillus flavus: In the following examples, the Aspergillus flavus strain used in the experiments was Aspergillus flavus CCTCC AF 2023038, which was obtained from the China Center of Industrial Culture Collection (CICC). The Aspergillus flavus was cultured using potato dextrose (PDB) medium.
[0021] Example 1 Antibacterial Analysis
[0022] Fungal antagonism experiments were conducted using the dilution method. 6mm paper discs containing 2-ketobutyric acid (0, 1, 2, and 4 mg) were arranged on agar plates containing Aspergillus flavus CCTCC AF 2023038. The plates were incubated at 30°C for 48 hours, and the inhibition zones were observed.
[0023] Different concentrations of 2-ketobutyric acid (4, 2, 1, 0.5, 0.25, and 0 mg / mL) were added (1 mL) to a culture of Aspergillus flavus CCTCC AF 2023038 containing an OD600 of approximately 0.1–0.2. The culture was incubated at 30°C for 48 hours, and fungal growth was measured at 600 nm using a microplate reader.
[0024] Example 2: Propidium iodide (PI) staining of fungi
[0025] Different concentrations of 2-ketobutyric acid (2, 1, and 0 mg / mL) were added (1 mL) to a culture of Aspergillus flavus CCTCC AF 2023038 containing an OD600 of approximately 0.1–0.2. The culture was incubated at 30°C for 48 hours. The fungi were collected and the hyphae were washed three times with PBS buffer. The fungi were resuspended in PBS buffer and propidium iodide (PI) (100 μg / mL) was added for 5 minutes in the dark. Fluorescence imaging was performed using a fluorescence microscope (Olympus BX43F-R).
[0026] Example 3: Transcriptome analysis of Aspergillus flavus
[0027] Different concentrations of 2-ketobutyric acid (1 and 0 mg / mL) were added to Aspergillus flavus CCTCAF 2023038 bacterial culture containing an OD600 of approximately 0.1–0.2. The culture was incubated at 30°C for 48 hours. Fungal collection was entrusted to Shanghai Meiji Biomedical Technology Co., Ltd. for RNA extraction, RNA-seq, and bioinformatics analysis.
[0028] Example 4: Aflatoxin Analysis
[0029] Aspergillus flavus spores (CCTCC AF 2023038) were added to 50 mL of fresh PDB to achieve an OD600 of approximately 0.1–0.2. 1 mg / mL of 2-ketobutyric acid was added, and the mixture was incubated at 30°C with shaking (150 rpm, THZ-98C, Shanghai Yiheng Scientific Instruments Co., Ltd.) for 14 days. The supernatant was collected, and aflatoxin was detected using an HPLC system (LC-20AD, Shimadzu Experimental Equipment Co., Ltd.) with a C18 column (5 μm, 4.6 × 150 mm) and a fluorescence detector (RF-20A, Shimadzu Experimental Equipment Co., Ltd.). The mobile phase was set to 40% methanol and 60% H2O, with a flow rate of 1 mL / min and an analysis time of 25 minutes. Aflatoxins (B1, B2, G1, G2, M1, and M2) were purchased from Wuxi Guanyi Biotechnology Co., Ltd. and used for standard curve analysis.
[0030] Figure 1 The results showed that after 24 hours of culture, Aspergillus flavus showed a clear inhibition zone at 2 mg of 2-ketobutyric acid, and the inhibition zone remained clear even after 48 hours of culture.
[0031] Figure 2 The results showed that after treatment with 2 mg / mL 2-ketobutyric acid for 24 hours and 48 hours, the antifungal rate of Aspergillus flavus could reach over 90%.
[0032] Propidium iodide (PI) staining was performed to verify whether 2-ketobutyric acid (2-ketobutyric acid) affects cell membrane integrity. If the cell membrane is damaged, the PI dye enters the cell and reacts with DNA and RNA, resulting in observed red fluorescence. Results showed that no red fluorescence was observed in the control group without 2-ketobutyric acid treatment, indicating that the cell membrane of *Aspergillus flavus* CCTCC AF 2023038 was intact. However, Figure 3 Red fluorescence was clearly observed in Aspergillus flavus CCTCC AF 2023038 in the presence of 1 mg / mL and 2 mg / mL 2-ketobutyric acid. This indicates that 2-ketobutyric acid treatment damages the fungal cell membrane.
[0033] Transcriptome analysis was performed on Aspergillus flavus CCTCC AF 2023038 treated with 2-ketobutyric acid (1 mg / mL). Volcano plot analysis was used. Figure 4 In Aspergillus flavus CCTCC AF 2023038 treated with 2-ketobutyric acid, 3808 differentially expressed genes (DEGs) were identified, with 1298 upregulated genes and 2510 downregulated genes. According to GO and KEGG enrichment analyses... Figure 5 The 2-ketobutyric acid (2-ketobutyric acid) exhibited high enrichment factor and low p-regulation value in the processes of fungal toxin metabolism, fungal toxin biosynthesis, and aflatoxin biosynthesis, indicating that 2-ketobutyric acid downregulates the gene expression of aflatoxin biosynthesis.
[0034] To determine the effect of 2-ketobutyric acid (2-ketobutyric acid) on aflatoxin production by *Aspergillus flavus* CCTCC AF 2023038, the aflatoxin yield was analyzed after 14 days of culture. The results showed that aflatoxin G1 was detectable in the control group on day 4 and in the bacterial culture supplemented with 1 mg / mL 2-ketobutyric acid on day 8. Figure 6 In the presence of 2-ketobutyric acid, the production of aflatoxin G1 was significantly lower than that in the control group. After 14 days of culture, mycelium was harvested, and the aflatoxin G1 levels in the control group and the 2-ketobutyric acid-treated group were 86.2 μg / g and 49.3 μg / g, respectively.
[0035] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.
Claims
The application of 1,2-ketobutyric acid in the in vitro inhibition of aflatoxin for non-disease diagnostic and therapeutic purposes. Application of 2,2-ketobutyric acid in the preparation of aflatoxin inhibitors.
3. The application as described in claim 2, characterized in that, The aflatoxin inhibitor contains 2-ketobutyric acid at a concentration of 1 mg / ml to 100 mg / ml. 4.2-Ketobutyric acid in the preparation of anti-aspergillus drugs.
5. The application as described in claim 4, characterized in that, In the aforementioned anti-aflatoxin drug, the concentration of 2-ketobutyric acid is 1 mg / ml-100 mg / ml.
6. The application of 2-ketobutyric acid in inhibiting aflatoxin synthesis in Aspergillus flavus in vitro for non-disease diagnosis and treatment purposes.
7. Application of 2-ketobutyric acid as an inhibitor of aflatoxin synthesis in the preparation of Aspergillus flavus.