Aspergillus flavus selective culture medium, preparation method and application thereof, and separation method and culture method of aspergillus flavus
By using selective culture media composed of corn flour, peanut flour and organic acids, the problem of low separation and culture efficiency of Aspergillus aflatoxin is solved, and efficient separation and growth of Aspergillus aflatoxin is achieved, which is suitable for rapid screening and public health prevention and control.
Patent Information
- Application Number
- CN202510802948.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-16
- Publication Date
- 2025-08-29
AI Technical Summary
The prior art is difficult to efficiently isolate and cultivate Aspergillus aflatoxin, especially in drug-resistant subpopulations in environmental samples, resulting in failure of antifungal treatment and spread of contamination.
A selective culture medium containing corn flour, peanut powder, organic acids and complex antibiotics was used to adjust the pH to 5.5-6.5, and was used for the isolation and culture of Aspergillus aflatoxin to improve its growth efficiency in corn and peanuts.
It significantly improves the separation efficiency and growth rate of Aspergillus aflatoxin, is suitable for rapid screening and large-scale epidemiological investigations, and provides targeted antifungal treatment and public health prevention and control support.
Smart Images
Figure CN120555210A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, in particular to a flavus selective culture medium and a preparation method and application thereof, and a flavus isolation method and culture method. Background Art
[0002] Aspergillus flavus is an important filamentous fungus widely distributed in nature, belonging to the genus Aspergillus of the phylum Ascomycota. As the second most common opportunistic pathogen of human aspergillosis, A. flavus can cause invasive and non-invasive aspergillosis, allergic sinusitis, and other diseases. It is also a plant pathogen and a contaminant of agricultural products (particularly peanuts, corn, and grains), posing a dual threat in both medicine and agriculture. Its metabolic activities are closely related to human production and life. On the one hand, A. flavus secretes a variety of enzymes and organic acids, which have potential applications in food fermentation and biotransformation. On the other hand, the aflatoxins (particularly type B1) it produces are highly carcinogenic and teratogenic, posing a threat to agricultural product safety, food processing, and damage to the livers of humans and animals. In severe cases, they can cause poisoning, liver cancer, and even death.
[0003] Environmental monitoring of Aspergillus flavus is a key component in controlling agricultural contamination and the spread of drug resistance, but existing isolation technologies struggle to meet the demands of precise research. Aspergillus flavus not only contaminates crops such as corn and peanuts by secreting highly carcinogenic aflatoxins, but its rapidly evolving resistance to azoles in the environment in recent years has also raised public health concerns. Studies have demonstrated that residual azole fungicides (e.g., tebuconazole and fluconazole) in agricultural soils can drive the accumulation and spread of drug-resistant genes (e.g., CYP51A mutants) in Aspergillus flavus through sustained selective pressure. This "environmental resistance reservoir" may be transferred to isolated strains via spore aerosols or crop symbiosis, leading to the failure of antifungal treatment. Therefore, efficient isolation of Aspergillus flavus from soil and environmental samples, particularly drug-resistant subpopulations, is a key prerequisite for interrupting the chain of contamination and the spread of drug resistance. However, environmental isolation methods using traditional culture media have significant limitations, such as low selection efficiency, insufficient recovery rates, and the lack of screening for drug-resistant phenotypes. Therefore, it is necessary to find a new culture medium to address the "high attrition and low resolution" dilemma in environmental strain isolation.
[0004] In view of this, the present invention is proposed. Summary of the Invention
[0005] The first object of the present invention is to provide a flavus selective culture medium to solve the above technical problems.
[0006] The second object of the present invention is to provide a method for preparing the above-mentioned Aspergillus flavus selective culture medium.
[0007] The third object of the present invention is to provide the use of the above-mentioned Aspergillus flavus selective culture medium in the isolation of Aspergillus flavus.
[0008] The fourth object of the present invention is to provide a method for isolating Aspergillus flavus.
[0009] The fifth object of the present invention is to provide a method for culturing Aspergillus flavus.
[0010] To achieve the above objectives, the following technical solutions are adopted:
[0011] In a first aspect, the present invention provides a flavus selective culture medium comprising the following components:
[0012] Corn flour 50–100 g / L, peanut meal 50–100 g / L, citric acid 1–5 g / L, malic acid 1–5 g / L, agar powder 15–20 g / L, glucose 10–20 g / L, chloramphenicol 50–100 mg / L, cycloheximide 100–200 mg / L, sodium chloride 5–10 g / L, dichloronaphthalene rose bengal chloramphenicol agar 15–20 g / L, yeast extract 2–5 g / L, peptone 5–10 g / L, magnesium sulfate 0.5 g / L, and potassium dihydrogen phosphate 1 g / L;
[0013] The pH of the Aspergillus flavus selective culture medium is 5.5-6.5.
[0014] As a further technical solution, the Aspergillus flavus selective culture medium includes the following components:
[0015] Corn flour 50g / L, peanut powder 50g / L, citric acid 5g / L, malic acid 2g / L, agar powder 17g / L, glucose 15g / L, chloramphenicol 60mg / L, cycloheximide 120mg / L, sodium chloride 10g / L, dichloronaphthalene rose bengal chloramphenicol agar 15g / L, yeast extract 2g / L, peptone 8g / L, magnesium sulfate 0.5g / L and potassium dihydrogen phosphate 1g / L.
[0016] As a further technical solution, the pH of the Aspergillus flavus selective culture medium is 6.0.
[0017] In a second aspect, the present invention provides a method for preparing the aforementioned Aspergillus flavus selective culture medium, comprising: mixing various raw materials in water according to a formula, and adjusting the pH to obtain the Aspergillus flavus selective culture medium.
[0018] In a third aspect, the present invention provides the use of the above-mentioned Aspergillus flavus selective culture medium in the isolation of Aspergillus flavus.
[0019] In a fourth aspect, the present invention provides a method for isolating Aspergillus flavus, comprising the following steps:
[0020] The sample is inoculated into the Aspergillus flavus selective culture medium for cultivation, and a culture rich in Aspergillus flavus is obtained after cultivation.
[0021] As a further technical solution, the culture temperature is 35-38° C., and the culture time is 4-6 days.
[0022] As a further technical solution, the culture temperature is 37° C. and the culture time is 5 days.
[0023] In a fifth aspect, the present invention provides a method for culturing Aspergillus flavus, comprising the following steps:
[0024] The sample is inoculated into the Aspergillus flavus selective culture medium for culture.
[0025] As a further technical solution, the culture temperature is 35-38°C.
[0026] Compared with the prior art, the present invention has the following beneficial effects:
[0027] Aspergillus flavus primarily grows in corn and peanuts. In addition, appropriate amounts of organic acids, such as citric acid and malic acid, can serve as carbon sources and promote the growth of Aspergillus flavus through the tricarboxylic acid cycle. Based on the principle of host matrix preference and the energy metabolism of Aspergillus flavus, the present invention adds peanuts, corn extract, citric acid, and malic acid to a basal culture medium, along with a compound antibiotic, to prepare a selective culture medium. This provides a highly efficient culture medium for Aspergillus flavus that significantly improves the efficiency of Aspergillus flavus isolation compared to traditional PDA and SDA culture media. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.
[0029] Figure 1 :Effects of different culture media on the separation of strains in samples (separation effects of some samples); A. New culture medium A; B. New culture medium B; C. PDA culture medium; D. SDA culture medium;
[0030] Figure 2 :Microscopic morphological observation of Aspergillus flavus strains; A. Cylindrical molecular spore head; B. Spherical conidial head; C. Sporulation structure (400X); D. Conidia (400X);
[0031] Figure 3:Colony morphology of Aspergillus flavus strains in different culture media; A. New culture medium A; B. New culture medium B; C. PDA culture medium; D. SDA culture medium;
[0032] Figure 4 : ITS gene PCR amplification; Note: M: Maker 2000; 1-14: bacterial species PCR products;
[0033] Figure 5 Evaluation of the growth rate of 14 Aspergillus flavus strains using different culture media; A. New culture medium A; B. New culture medium B; C. PDA culture medium; D. SDA culture medium; Note: ****, p<0.0001, extremely significant difference. DETAILED DESCRIPTION
[0034] Below in conjunction with embodiment and example, embodiment of the present invention is described in detail, but those skilled in the art will appreciate that the following embodiment and example are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise all fall within the scope of protection of the present invention. Unspecified conditions are carried out according to the conditions of normal conditions or manufacturer's recommendations. Reagents used or instruments not specified by the manufacturer are conventional products that can be purchased commercially.
[0035] In a first aspect, the present invention provides a flavus selective culture medium comprising the following components:
[0036] Corn flour 50–100 g / L, peanut meal 50–100 g / L, citric acid 1–5 g / L, malic acid 1–5 g / L, agar powder 15–20 g / L, glucose 10–20 g / L, chloramphenicol 50–100 mg / L, cycloheximide 100–200 mg / L, sodium chloride 5–10 g / L, dichloronaphthalene rose bengal chloramphenicol agar 15–20 g / L, yeast extract 2–5 g / L, peptone 5–10 g / L, magnesium sulfate 0.5 g / L, and potassium dihydrogen phosphate 1 g / L;
[0037] The pH of the Aspergillus flavus selective culture medium is 5.5-6.5.
[0038] The present invention is based on the principle of host matrix preference, and peanuts, corn extracts, organic acids (citric acid and malic acid) and compound antibiotics are added to the basal culture medium to prepare a selective culture medium. This culture medium can significantly improve the efficiency of aflatoxin separation, and its dual application value is reflected in: in the medical field, it can provide a new method for rapid screening of clinical samples, and in the agricultural field, it lays a technical foundation for establishing an agricultural product pollution early warning system. The separation or culture method provided by the present invention has high sensitivity and good specificity, and is particularly suitable for rapid clinical diagnosis and large-scale epidemiological surveys in resource-limited areas. It can not only accelerate the identification of pathogenic bacteria species, but also provide important technical support for targeted antifungal treatment and public health prevention and control.
[0039] In some optional embodiments, the Aspergillus flavus selective culture medium comprises the following components:
[0040] Corn flour 50g / L, peanut powder 50g / L, citric acid 5g / L, malic acid 2g / L, agar powder 17g / L, glucose 15g / L, chloramphenicol 60mg / L, cycloheximide 120mg / L, sodium chloride 10g / L, dichloronaphthalene rose bengal chloramphenicol agar 15g / L, yeast extract 2g / L, peptone 8g / L, magnesium sulfate 0.5g / L and potassium dihydrogen phosphate 1g / L.
[0041] In some optional embodiments, the pH of the Aspergillus flavus selective culture medium is 6.0.
[0042] By further optimizing and adjusting the concentration and pH of the culture medium components, the separation and culture effects of the culture medium are improved.
[0043] In a second aspect, the present invention provides a method for preparing the aforementioned Aspergillus flavus selective culture medium, comprising: mixing various raw materials in water according to a formula, and adjusting the pH to obtain the Aspergillus flavus selective culture medium.
[0044] The preparation method is simple and convenient, and the prepared culture medium can be used for the isolation and culture of Aspergillus flavus.
[0045] In a third aspect, the present invention provides the use of the aforementioned Aspergillus flavus selective culture medium in the isolation of Aspergillus flavus.
[0046] The culture medium provided by the present invention is suitable for the growth of Aspergillus flavus and a small number of microorganisms, and therefore the culture medium can be used for the preliminary separation of Aspergillus flavus.
[0047] In a fourth aspect, the present invention provides a method for isolating Aspergillus flavus, comprising the following steps:
[0048] The sample is inoculated into the Aspergillus flavus selective culture medium for cultivation, and a culture rich in Aspergillus flavus is obtained after cultivation.
[0049] The culture medium provided by the present invention is suitable for the growth of Aspergillus flavus and a small number of microorganisms, and therefore can be used for the preliminary separation of Aspergillus flavus.
[0050] In some optional embodiments, the culture temperature is 35-38° C., and the culture time is 4-6 days.
[0051] In some preferred embodiments, the culture temperature is 37° C. and the culture time is 5 days.
[0052] In a fifth aspect, the present invention provides a method for culturing Aspergillus flavus, comprising the following steps:
[0053] The sample is inoculated into the Aspergillus flavus selective culture medium for culture.
[0054] The culture medium provided by the present invention is suitable for the growth of Aspergillus flavus and a small number of microorganisms, and therefore can be used for the cultivation of Aspergillus flavus.
[0055] In some optional embodiments, the culture temperature is 35-38°C.
[0056] In some preferred embodiments, the culture temperature is 37°C.
[0057] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.
[0058] Example 1: Isolation and identification of Aspergillus flavus strains in environmental samples
[0059] (1) Evaluation of the effectiveness of selective culture medium in isolating Aspergillus flavus strains from environmental samples
[0060] Nine environmental samples (as shown in Table 1) were cultured at 37°C for 5 days using novel culture medium A, novel culture medium B, PDA and SDA, respectively.
[0061] Table 1 Experimental samples used for separation and their sources
[0062]
[0063] The results are shown in Table 2 and Figure 1As shown, by comparing the traditional culture medium and the new selective culture medium (the new culture medium A ratio is: corn flour 50g / L, peanut powder 50g / L, citric acid 5g / L, malic acid 2g / L, agar powder 17g / L, glucose 15g / L, chloramphenicol 60mg / L, cycloheximide 120mg / L, sodium chloride 10g / L, dichloronaphthalene rose red chloramphenicol agar 15g / L, yeast extract 2g / L, peptone 8g / L, magnesium sulfate 0.5g / L, potassium dihydrogen phosphate 1g / L, pH=6.0. The new culture medium B ratio is corn flour 50g / L, L, peanut powder 50g / L, citric acid 3g / L, malic acid 2g / L, agar powder 17g / L, glucose 15g / L, chloramphenicol 60mg / L, cycloheximide 120mg / L, sodium chloride 10g / L, dichloronaphthalene rose bengal chloramphenicol agar 15g / L, yeast extract 2g / L, peptone 8g / L, magnesium sulfate 0.5g / L, potassium dihydrogen phosphate 1g / L, pH = 6.0) to separate environmental samples. The results showed that at the same environmental sample dilution multiple, the new medium was significantly more effective than the traditional medium in separating Aspergillus flavus strains from 9 samples. The number of colony morphological types in the new medium was 1-3, while the number in the traditional medium was 8-14. Therefore, the microbial diversity in the new medium was significantly lower than that in the traditional medium (P < 0.05). The second highest value was the total colony count. At the same dilution concentration, the total colony count in the traditional medium was often uncountable, while the total colony count in the new medium was 3×10 2 -2.4×10 3 In addition, the total number of Aspergillus flavus colonies in the new culture medium accounts for as high as 86%-100%, while the number of Aspergillus flavus colonies in the traditional culture medium is less or negative.
[0064] Table 2 Effects of different culture media on the isolation of aflatoxin strains from environmental samples
[0065]
[0066] (2) Colony morphology observation
[0067] Two different types of Aspergillus flavus cultured on the new medium A were observed under a microscope. Figure 2 Figures A and B show different types of conidial heads from Aspergillus flavus strains. A is a cylindrical conidiophore, while B is a spherical conidial head. C shows the conidiophore and its structure: the conidiophore is upright, with a bulge at the apex forming an apicocapsule. The apicocapsule is covered with radially arranged phialides, on which conidia are produced in chains (resembling beads). D shows conidia.
[0068] Select a strain of Aspergillus flavus and culture it in the above four culture media for 5 days. Figure 3 As shown in the figure, when the same Aspergillus flavus strain was cultured on different culture media, its morphology and growth rate showed diversity. Figure 3 In the figure, A, B, C, and D represent novel medium A, novel medium B, PDA medium, and SDA medium, respectively. When the Aspergillus flavus strain was inoculated on novel medium A and novel medium B, it grew rapidly, covering the entire plate within 5 days. However, the conventional culture grew more slowly and had a weaker sporulation ability on PDA medium.
[0069] (3) Molecular identification
[0070] The PCR products amplified from the ITS gene fragments of the 14 isolated strains (numbers 1-7 were derived from the strains cultured in the novel medium A, and numbers 8-14 were derived from the strains cultured in the novel medium B) were detected by 1% (w / v) agarose gel. The band was 750 bp and there were no other bands (such as Figure 4 The PCR products were sent to Bioengineering for Sanger sequencing. The sequencing results were spliced and BLAST was performed on NCBI. All 14 strains were Aspergillus flavus (as shown in Table 3).
[0071] Table 3 ITS sequence alignment results
[0072]
[0073]
[0074] Example 2: Effects of different culture media on the growth of Aspergillus flavus strains
[0075] The above 14 strains of Aspergillus flavus were cultured in the new medium A, new medium B, PDA medium and SDA medium for the same time. The results are as follows: Figure 5 shown.
[0076] Figure 5 The average colony diameters of 14 A. flavus strains grown on A. peanut-based selective medium; B. corn-based selective medium; C. PDA medium; and D. SDA medium were shown. Comparison of the colony diameters of the 14 A. flavus strains on the four media revealed the following correlation: novel medium B (7.57±0.21-8.27±0.06 cm) > novel medium A (6.73±0.06-7.93±0.12 cm) > SDA medium (5.73±0.06-7.00±0.10 cm) > PDA medium (5.17±0.15-6.57±0.12 cm) (Table 4). Colony diameters of strains grown on peanut and corn media were significantly larger than those on traditional media (P<0.001), and morphological differences were significant. Traditional media, especially PDA, were not conducive to sporulation by A. flavus strains.
[0077] Table 4 Colony diameters (cm) of 14 Aspergillus flavus strains cultured in different culture media
[0078]
[0079]
[0080] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A selective culture medium for Aspergillus flavus, characterized in that Includes the following components: Corn flour 50–100 g / L, peanut meal 50–100 g / L, citric acid 1–5 g / L, malic acid 1–5 g / L, agar powder 15–20 g / L, glucose 10–20 g / L, chloramphenicol 50–100 mg / L, cycloheximide 100–200 mg / L, sodium chloride 5–10 g / L, dichloronaphthalene rose bengal chloramphenicol agar 15–20 g / L, yeast extract 2–5 g / L, peptone 5–10 g / L, magnesium sulfate 0.5 g / L, and potassium dihydrogen phosphate 1 g / L; The pH of the Aspergillus flavus selective culture medium is 5.5-6.
5.
2. The Aspergillus flavus selective culture medium according to claim 1, characterized in that The Aspergillus flavus selective culture medium comprises the following components: Corn flour 50g / L, peanut powder 50g / L, citric acid 5g / L, malic acid 2g / L, agar powder 17g / L, glucose 15g / L, chloramphenicol 60mg / L, cycloheximide 120mg / L, sodium chloride 10g / L, dichloronaphthalene rose bengal chloramphenicol agar 15g / L, yeast extract 2g / L, peptone 8g / L, magnesium sulfate 0.5g / L and potassium dihydrogen phosphate 1g / L.
3. The Aspergillus flavus selective culture medium according to claim 1, characterized in that The pH of the Aspergillus flavus selective culture medium is 6.
0.
4. The method for preparing the Aspergillus flavus selective culture medium according to any one of claims 1 to 3, characterized in that: include: The various raw materials are mixed in water according to the formula, and the pH is adjusted to prepare the Aspergillus flavus selective culture medium.
5. Use of the Aspergillus flavus selective culture medium according to any one of claims 1 to 3 in isolating Aspergillus flavus.
6. A method for isolating Aspergillus flavus, characterized in that: The steps include: The sample is inoculated into the Aspergillus flavus selective culture medium according to any one of claims 1 to 3 and cultured to obtain a culture rich in Aspergillus flavus.
7. The separation method according to claim 6, characterized in that The culture temperature is 35-38° C., and the culture time is 4-6 days.
8. The separation method according to claim 7, characterized in that The culture temperature is 37° C. and the culture time is 5 days.
9. A method for cultivating Aspergillus flavus, characterized in that: The steps include: The sample is inoculated into the Aspergillus flavus selective culture medium according to any one of claims 1 to 3 for culture.
10. The culture method according to claim 9, characterized in that The culture temperature is 35-38°C.