CN111471721B - Application of alternaria brassicae metabolite in preparation of aspergillus flavus bacteriostatic agent - Google Patents
Application of alternaria brassicae metabolite in preparation of aspergillus flavus bacteriostatic agent Download PDFInfo
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Abstract
The invention discloses an application of alternaria brassicae metabolite in preparation of an aspergillus flavus bacteriostatic agent, wherein the metabolite is obtained by ultrasonically crushing alternaria brassicae fermentation liquor, filtering, extracting filtrate by using ethyl acetate after concentrating, and concentrating an ethyl acetate phase to constant weight. The invention provides application of alternaria brassicae metabolite to aspergillus flavus bacteriostatic activity and a grain storage bacteriostatic agent. The traditional mould inhibition preparation is antibiotics such as nystatin and the like, has larger residue harm, and the natural bacteriostatic agent tends to be the trend of the future market.
Description
(I) technical field
The invention relates to an aspergillus flavus bacteriostatic agent, in particular to application of alternaria brassicae metabolite in preparation of the aspergillus flavus bacteriostatic agent.
(II) background of the invention
Pollution events caused by aflatoxin occur frequently, and the food safety problem is emphasized by consumers. In this regard, many countries have limited settings for the aflatoxin content of foodstuffs and foodstuffs. The European Union region strengthens the control of the aflatoxin content in the food, and internationally makes a strict and standard limit standard of the aflatoxin content in the food. In recent years, along with the rapid development of economic society of China, the attention degree of people on food safety is continuously improved, but the problem of aflatoxin often occurs in the process of food storage, and the safety of food products is seriously influenced. The development of a biological prevention and control method for aflatoxin, particularly aflatoxin in the grain storage process, is favorable for effectively preventing and controlling aflatoxin, ensures grain safety, and provides a higher-quality grain product for Chinese people. Through research, aflatoxins belong to class I carcinogens, and generally speaking, animals and humans also take certain aflatoxins mainly by means of corresponding dietary channels. When the toxin enters the human body and the animal body, the toxin has strong liver-friendly property, and is very easy to cause liver bleeding, bile duct hyperplasia, fatty degeneration and the like, thereby further causing liver cancer.
Disclosure of the invention
The invention aims to provide application of alternaria brassicae metabolite in preparation of aspergillus flavus bacteriostatic agent, which can inhibit growth of aspergillus flavus and control generation of aflatoxin in a rice storage process.
The technical scheme adopted by the invention is as follows:
the invention provides an application of alternaria brassicae metabolite in preparation of an aspergillus flavus bacteriostatic agent, wherein the metabolite is obtained by performing ultrasonic treatment on alternaria brassicae fermentation liquor to crush cells, filtering, concentrating filtrate, extracting with ethyl acetate, and concentrating an ethyl acetate phase to constant weight.
Further, the alternaria brassicae metabolite is prepared by the following method: inoculating alternaria brassicae into a 500mL conical flask containing 200mL of fermentation medium, and culturing at 28 ℃ and 180r/min for 7d to obtain fermentation liquor; carrying out suction filtration on fermentation liquor after ultrasonic crushing, filtering filtrate by using a microporous filter membrane with the aperture of 0.45 mu m, concentrating the micro-filtrate to 1/4 of the original volume by using a rotary evaporator, extracting by using ethyl acetate with the volume of 1 to ensure that an ethyl acetate phase has no obvious color change by visual observation, combining the ethyl acetate extract phases, and concentrating by using the rotary evaporator again to constant weight to obtain the alternaria brassicae metabolite; the fermentation medium comprises the following components: 3g/L of sodium nitrate, 1g/L of dipotassium phosphate, magnesium sulfate (MgSO) 4 ·7H 2 O) 0.5g/L, potassium chloride 0.5g/L, sulfite0.01g/L of iron, 30g/L of cane sugar and distilled water as a solvent, and the pH value is 7.0-7.2.
Further, the alternaria brassicae (Altemaria brassicae) Gb.PY-F2 is preserved in China center for type culture collection with the preservation number as follows: CCTCC M219126, the preservation date is 3 and 6 months in 2019, and the preservation address is Wuhan university in Wuhan, china, zip code: 430072, disclosed in patent application 201910174849.
Further, the ultrasonication conditions are: 300 cycles were performed at 405W for 3s of service, 4s of batch.
Further, the alternaria brassicae is inoculated in a PDA culture medium before fermentation, and is placed in a constant-temperature incubator at 28 ℃ for culture for 7d; then, a sterile puncher is used for punching a fungus cake with the diameter of 5mm on the edge of a colony of the alternaria brassicae, and the fungus cake is inoculated to a fermentation culture medium; PDA culture medium composition: 200g/L of potato, 20g/L of glucose, 15-20 g/L of agar, distilled water as a solvent and natural pH.
Further, the bacteriostatic agent is prepared by dissolving metabolite of alternaria brassicae in acetone to obtain bacteriostatic agent solution; the volume dosage of the acetone is 10-20mL/g calculated by the weight of the alternaria brassicae metabolite.
Further, the Aspergillus flavus is Aspergillus flavus (CGMCC 3.4408).
Further, the bacteriostatic agent is a grain storage bacteriostatic agent, the stored grain is preferably paddy, the grain storage bacteriostatic agent is 0.1-0.5g/mL of Alcoholic solution of metabolites of alternaria brassicae, and the application amount is 0.5L/ton.
Compared with the prior art, the invention has the following beneficial effects: the invention provides a bacteriostatic activity of alternaria brassicae metabolite on aspergillus flavus (CGMCC 3.4408) and application of the bacteriostatic agent in grain storage. The traditional mould inhibitor is antibiotic such as nystatin and the like, the residual hazard is large, the natural bacteriostatic agent tends to be the trend of the future market, and the bacteriostatic agent has large yield, simple process, higher safety and environmental protection.
(IV) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1 Alternaria brassicae CCTCC M2019126 metabolite
1. Inoculating alternaria brassicae CCTCC M2019126 stored in a refrigerator at 4 ℃ into a PDA culture medium, and culturing in a constant-temperature incubator at 28 ℃ for 7d; PDA culture medium composition: 200g of potatoes, 20g of glucose, 15-20 g of agar, 1000mL of distilled water and natural pH.
2. In an ultra-clean workbench, punching a fungus cake with the diameter of 5mm on the edge of a bacterial colony of the alternaria brassicae CCTCC M2019126 in the step 1 by using an aseptic puncher, inoculating the fungus cake into a 500mL conical flask containing 200mL of fermentation medium, and culturing for 7d at 28 ℃ and 180r/min to obtain fermentation liquor; and (3) performing 300 times of circulating ultrasonic crushing on the fermentation liquor under the condition of 405W for 3s per work and 4s at intervals, performing suction filtration, filtering the filtrate by using a microporous filter membrane with the aperture of 0.45 mu M, concentrating the microfiltrate to 1/4 of the original volume by using a rotary evaporator, extracting the microfiltrate by using 1 volume of ethyl acetate until no obvious color change is observed in an ethyl acetate phase by naked eyes, combining the ethyl acetate extract phases, and concentrating to constant weight by using the rotary evaporator again to obtain 2.7g of a metabolite of the alternaria brassicae CCTCC M2019126.
The fermentation medium comprises the following components: 3g/L of sodium nitrate, 1g/L of dipotassium phosphate, magnesium sulfate (MgSO) 4 ·7H 2 O) 0.5g/L, potassium chloride 0.5g/L, ferrous sulfate 0.01g/L, sucrose 30g/L, distilled water as solvent, pH7.0-7.2; the mixture was sterilized by using a high-pressure steam sterilizer at 121 ℃ for 20 minutes.
Example 2 Aspergillus flavus spore suspension
Inoculating Aspergillus flavus (CGMCC 3.4408 (purchased from China general microbiological culture Collection center) in PDA slant culture medium, culturing at 28 deg.C for 10d, picking spores on the slant with an inoculating needle, adding 10mL of 0.2mol/L sterile phosphate buffer solution with 0.05% Tween-80 volume concentration and pH6.0, filtering with gauze to remove mycelium residues, and adjusting the spore concentration to 1 × 10 with 0.2mol/L phosphate buffer solution with pH6.0 7 And CFU/mL is the aspergillus flavus spore suspension.
Example 3 bacteriostatic test of Alternaria brassicae CCTCC M2019126 metabolite on Aspergillus flavus
1. Taking 1.0g of the metabolite of the alternaria brassicae CCTCC M2019126 prepared by the method in the embodiment 1, and dissolving the metabolite in 10mL of acetone to obtain the bacteriostatic agent solution. Adding 1mL of bacteriostatic agent solution into a plate, pouring 15mL of LPDA culture medium, mixing uniformly, dissolving 3 small holes with the diameter of 2.5mm on each plate by using an inoculating stick after solidification, adding 0.01mL of aspergillus flavus spore suspension prepared in example 2 into each small hole, culturing for 5 days at 28 ℃, and observing the diameter of a bacteriostatic circle, wherein the results are shown in Table 1. Acetone was used as a blank in place of the bacteriostatic solution under the same conditions.
TABLE 1 diameter of zone of inhibition (mm)
2. Dissolving 1.0g of streptomyces brassicae CCTCC M2019126 bacteriostatic agent in 4mL of absolute ethanol to obtain a bacteriostatic agent ethanol solution. Uniformly spraying the mixture on the surface of the paddy according to the application amount of 0.5 liter/ton, uniformly mixing, and storing at 20 ℃. One year later, an aflatoxin enzyme linked immunosorbent assay kit (Shenzhen, boaotongke, bio-products, ltd.) is used for detecting the aflatoxin content in the rice, the aflatoxin is not detected, and a control group without adding a bacteriostatic agent is used for detecting the aflatoxin content of 8.5 mug/kg.
Example 4 bacteriostatic test of Alternaria brassicae CCTCC M2019126 metabolite on Aspergillus flavus
1. Taking 1.0g of the metabolite of the alternaria brassicae CCTCC M2019126 prepared by the method in the embodiment 1, and dissolving the metabolite in 15mL of acetone to obtain the bacteriostatic agent solution. Adding 1mL of bacteriostatic agent solution into a plate, pouring 15mLPDA culture medium, mixing uniformly, dissolving 3 small holes with the diameter of 2.5mm on each plate after solidification by using an inoculating rod, adding 0.01mL of the aspergillus flavus spore suspension prepared in the example 2 into each small hole, culturing for 5 days at 28 ℃, and observing the diameter of a bacteriostatic ring, wherein the results are shown in a table 2. Acetone was used as a blank in place of the bacteriostatic solution under the same conditions.
TABLE 2 diameter of zone of inhibition (mm)
2. Dissolving 1.0g of streptomyces brassicae CCTCC M2019126 bacteriostatic agent in 5mL of absolute ethanol to obtain a bacteriostatic agent ethanol solution. Uniformly spraying the mixture on the surface of the paddy according to the application amount of 0.5 liter/ton, uniformly mixing, and storing at 20 ℃. After one year, the aflatoxin content in the rice was detected by using an aflatoxin enzyme linked immunosorbent assay kit (Boaotongke, ltd., shenzhen), the aflatoxin was not detected, and the aflatoxin content was detected by using a control group without adding a bacteriostatic agent to obtain 8.5. Mu.g/kg.
Example 5 bacteriostatic test of Alternaria brassicae CCTCC M2019126 metabolite on Aspergillus flavus
1. Taking 1.0g of the metabolite of the alternaria brassicae CCTCC M2019125 prepared by the method in the embodiment 1, and dissolving the metabolite in 20mL of acetone to obtain the bacteriostatic agent solution. Adding 1mL of bacteriostatic agent solution into a plate, pouring 15mL of PDA culture medium, mixing uniformly, dissolving 3 small holes with the diameter of 2.5mm on each plate after solidification by using an inoculating rod, adding 0.01mL of the aspergillus flavus spore suspension prepared in the example 2 into each small hole, culturing for 5 days at 28 ℃, and observing the diameter of a bacteriostatic ring, wherein the results are shown in a table 3. Acetone was used as a blank in place of the bacteriostatic solution under the same conditions.
TABLE 3 antibacterial circle diameter (mm)
2. Dissolving 1.0g of metabolite of alternaria brassicae CCTCC M2019126 in 6mL of absolute ethanol to obtain an ethanol solution of the bacteriostatic agent. Uniformly spraying the mixture on the surface of the paddy according to the application amount of 0.5 liter/ton, uniformly mixing, and storing at 20 ℃. One year later, an aflatoxin enzyme linked immunosorbent assay kit (Shenzhen, boaotongke, bio-products, ltd.) is used for detecting the aflatoxin content in the rice, the aflatoxin is not detected, and a control group without adding a bacteriostatic agent is used for detecting the aflatoxin content of 8.5 mug/kg.
Claims (6)
1. A metabolite of Alternaria brassicaeThe application of the aspergillus flavus bacteriostatic agent in preparation is characterized in that the alternaria brassicae metabolite is prepared according to the following method: inoculating alternaria brassicae in a fermentation medium, and culturing at 28 ℃ and 180r/min for 7d to obtain a fermentation liquid; carrying out ultrasonic crushing on the fermentation liquor, carrying out suction filtration, filtering the filtrate by using a microporous filter membrane with the aperture of 0.45 mu m, concentrating the micro-filtrate to 1/4 of the original volume, extracting the micro-filtrate by using ethyl acetate until the ethyl acetate phase has no obvious color change when being observed by naked eyes, combining the ethyl acetate extract phases, and concentrating the mixed phases again to constant weight to obtain the alternaria brassicae metabolite; the fermentation medium comprises the following components: 3g/L of sodium nitrate, 1g/L of dipotassium phosphate, 0.5g/L of magnesium sulfate, 0.5g/L of potassium chloride, 0.01g/L of ferrous sulfate, 30g/L of cane sugar, distilled water as a solvent and pH of 7.0-7.2; the Alternaria brassicae is Alternaria brassicae (S. Brassicae) ((S. Brassicae))Altemaria brassicae）CCTCC M 219126。
2. Use according to claim 1, characterized in that the ultrasonication conditions are: 300 cycles were performed at 405W for 3s of service, 4s of batch.
3. The use of claim 1, wherein the alternaria brassicae is inoculated in a PDA culture medium before fermentation, and is cultured in a constant temperature incubator at 28 ℃ for 7d; then, punching a bacterial cake with the diameter of 5mm on the edge of the colony of the alternaria brassicae by using an aseptic puncher, and inoculating the bacterial cake to a fermentation culture medium; PDA culture medium composition: 200g/L of potato, 20g/L of glucose, 15 to 20g/L of agar, distilled water as a solvent and natural pH.
4. The use of claim 1, wherein the bacteriostatic agent is a bacteriostatic agent solution prepared by dissolving a metabolite of alternaria brassicae in acetone; the volume dosage of the acetone is 10-20mL/g calculated by the weight of the metabolite of the alternaria brassicae.
5. The use according to claim 1, wherein, characterized in that the aspergillus flavus is aspergillus flavus (A. Flavus)Aspergillus flavus）CGMCC 3.4408。
6. The use of claim 1, wherein the bacteriostatic agent is a stored grain bacteriostatic agent; the stored grain is paddy, and the stored grain bacteriostatic agent is 0.1-0.5g/mL of alternaria brassicae metabolite ethanol solution.
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| CN109897788A (en) * | 2019-03-08 | 2019-06-18 | 浙江工业大学 | A kind of rape rod method and preparing the application in bacteriostatic agent |
| CN110066830A (en) * | 2019-03-08 | 2019-07-30 | 浙江工业大学 | Gingko endogenous fungus metabolite and preparing the application in bacteriostatic agent |
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| CN109897788A (en) * | 2019-03-08 | 2019-06-18 | 浙江工业大学 | A kind of rape rod method and preparing the application in bacteriostatic agent |
| CN110066830A (en) * | 2019-03-08 | 2019-07-30 | 浙江工业大学 | Gingko endogenous fungus metabolite and preparing the application in bacteriostatic agent |
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| Sinalexin, a phytoalexin from white mustard elicited by destruxin B and Alternaria brassicae;M. Soledade等;《Phytochemistry》;ELSEVIER;19971130;第46卷(第5期);第833-837页 * |
| 芸薹链格孢菌孢子萌发及附着胞形成的初步定量研究;马海霞等;《吉林农业大学学报》;维普;20131231(第4期);第393-397页 * |
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