CN111011388B - Application of dimethyl flavopenicillin in resisting plant pathogenic fungi - Google Patents
Application of dimethyl flavopenicillin in resisting plant pathogenic fungi Download PDFInfo
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- CN111011388B CN111011388B CN201911304397.2A CN201911304397A CN111011388B CN 111011388 B CN111011388 B CN 111011388B CN 201911304397 A CN201911304397 A CN 201911304397A CN 111011388 B CN111011388 B CN 111011388B
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
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- Life Sciences & Earth Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to the field of compound preparation, and in particular relates to application of dimethyl flavopenicillin in resisting plant pathogenic fungi. Wherein, the dimethyl flavopenicillin is applied to resisting plant pathogenic fungi. The dimethyl xanthil has activity of resisting plant pathogenic fungi, and can be applied to resisting the plant pathogenic fungi, so that the growth of the plant pathogenic fungi in crops or ecological environment is inhibited, and the harm of plant diseases to the crops and the ecological environment is further solved.
Description
Technical Field
The invention relates to the field of compound preparation, and in particular relates to application of dimethyl flavopenicillin in resisting plant pathogenic fungi.
Background
Phytopathogenic fungi, which refer to fungi that can parasitize on plants and cause plant diseases, are important sources of infestation of plant diseases. Nearly eighty percent or so of plant invasive diseases are caused by fungi, and each plant can be harmed by several or even dozens of fungi. Such as wheat rust, rice blast, vegetable soft rot, apple tree rot and the like are all fungal diseases.
The fungal diseases can cause crop yield reduction and agricultural product quality reduction, so that serious economic loss is caused to the production of agricultural products, some fungal diseases can even cause the agricultural products to generate toxins, which endanger the health of people and livestock, for example, wheat grains infected with scab generate vomitoxin and similar female toxins, and poisoning symptoms such as vomit, diarrhea and the like are caused after people and livestock eat the agricultural products; the sweet potato with the black spot disease contains sweet potato black spot mildew ketone and sweet potato black spot mildew diketone, and causes dizziness after being eaten by people and asthma after being eaten by cattle. Therefore, controlling plant fungal diseases has long been a challenge for scientists to struggle with.
The traditional method for preventing and treating plant fungal diseases is to use chemical pesticides, but the problems of environmental pollution, increase of pathogenic bacteria drug resistance, unbalance of microbial flora in plants and the like can be caused by long-term use of chemical pesticides, and due to the increasingly prominent side effects of chemical pesticides and the improvement of the requirements of people on environment-friendly and green foods, the research and development of biopesticides meeting the concepts of environment protection, health and sustainable development becomes urgent.
Agricultural antibiotics are regarded more seriously due to the advantages of high efficiency, easy decomposition, no residue, compatibility with the environment and the like, but because the discovery of the agricultural antibiotics is difficult, the agricultural antibiotics account for only 9 percent in the pesticide market at present. At present, the discovery of new antibiotics through new microbial resources is one of the main solutions.
Dimethylxanthicillin was first isolated from the fermentation broth of the soil fungus Aspergillus sp. in 1968 by Takatsuki et alIsolated and found to have antiviral activity[1]The discovery by Japanese scientist Tsutomu et al that dimeglufosinate has good antitumor activity and low toxicity[2]Sakai et al isolated from the marine fungus Basipeetospora sp and found that it has activity as a thrombopoietic receptor activator[3]. In addition, Dimethylxanthicillin was found to be active against the human pathogenic fungus Candida albicans[4]. The sulfonic acid group-containing homologue BU-4707 (methylsulfonyl xanthylcycline) of the flavopenicillin family compound has been reported to have various antibacterial activities, such as Staphylococcus aureus (Staphylococcus aureus FDA 209P JC-1), drug-resistant Staphylococcus aureus (S.aureus), Bacillus subtilis ATCC 6633, Escherichia coli (Escherichia coli Juhl), Klebsiella pneumoniae (Pseudomonas aeruginosa A9843A), Cryptococcus neoformans (Cryptococcus neoformans D49, human pathogenic fungi), Trichophyton mentagrophytes (Trichophyton mentaphytes No.4329, human pathogenic fungi), etc[5]. However, there is no report on the activity of flavopenicillins against phytopathogenic fungi.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to overcome the problem of crop diseases caused by phytopathogenic fungi in the prior art, and provide the application of the dimethyl xanthil in resisting the phytopathogenic fungi.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
an application of dimethyl xanthocillin in resisting plant pathogenic fungi is disclosed.
Furthermore, the plant pathogenic fungi are corn microsporum pathogenic bacteria or tomato early blight pathogenic bacteria.
Furthermore, the concentration of the dimethyl xanthil is more than or equal to 12.5 mu g/ml.
Further, the dimethyl xanthinin is isolated from Aspergillus sp.LW1 CCTCC No. M2019453.
Further, the method for separating the dimethyl flavopenicillin comprises the following steps:
inoculating Aspergillus sp.LW1 into a fermentation culture medium, and fermenting and culturing at 20-40 deg.C at 100-300rpm for 4-14 days to obtain thallus;
and (3) extracting and separating the thalli by using an organic solvent to obtain the dimethyl flavopenicillin.
Further, the organic solvent is at least one of ethyl acetate or acetone.
Further, the organic solvent is acetone, and the extraction step comprises:
soaking thallus in acetone, ultrasonic treating for 5-60min, standing, and filtering to obtain acetone extractive solution;
concentrating the acetone extract at 30-50 deg.C under reduced pressure to obtain crude extract of thallus;
and carrying out chromatographic separation on the crude thalli extract, and carrying out reverse HPLC separation and purification on the fraction after the chromatographic separation to obtain the dimethyl flavopenicillin.
Further, the chromatographic separation step comprises:
dissolving the crude thalli extract with methanol, loading, and drying to obtain an elution sample;
eluting the elution sample by sequentially utilizing water, 50% methanol solution and methanol, respectively collecting eluates, and concentrating the eluates under reduced pressure at 30-50 ℃ until the eluates are dried to obtain corresponding fractions;
the fraction obtained when methanol was used as the eluent was the target fraction for separation and purification by reverse phase HPLC.
Further, the reverse phase HPLC separation and purification step comprises:
and (3) redissolving the target fraction by using methanol, then eluting and separating by using 100% acetonitrile, setting the detection wavelength to be 210nm, collecting a liquid phase peak at 5.0min, and drying the collected fraction to obtain the dimethyl flavopenicillin.
Further, the fermentation medium comprises the following components:
20g/L of glucose, 5g/L of yeast powder, 10g/L of soybean peptone and 30g/L of sea salt.
Further, the method comprises the step of inoculating the Aspergillus sp.lw1 into a plate culture medium, and culturing at 20-40 ℃ for resuscitation before the step of inoculating the Aspergillus sp.lw1 into a fermentation culture medium.
The technical scheme of the invention has the following advantages:
1. the invention discovers for the first time that the dimethyl xanthocillin has the activity of resisting plant pathogenic bacteria, and can be applied to resisting plant pathogenic bacteria, in particular to corn microsporum maydis and tomato early blight (Altemaria solani), thereby inhibiting the plant pathogenic bacteria in crops or ecological environment, and further solving the problem of the harm of plant fungal diseases to the crops and the ecological environment.
2. The dimethyl xanthil is separated from Aspergillus sp.LW1 CCTCC No.M2019453, and the activity of the dimethyl xanthil against plant pathogenic fungi is measured, so that the growth of two plant pathogenic fungi, namely tomato early blight and corn small spot pathogen, can be obviously inhibited when the application concentration is 100 ug/m.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
The invention provides an application of dimethyl xanthatin in resisting plant pathogenic fungi, and the dimethyl xanthatin is applied in resisting plant pathogenic fungi. Wherein the plant pathogenic fungi is corn microsporum pathogenic bacteria or tomato early blight pathogenic bacteria. The amount of the dimethyl xantham penicillin is preferably 100 ug/ml.
The dimethyl xanthinin can be isolated from Aspergillus sp.LW1 CCTCC No. M2019453. The fungus is preserved in China center for type culture Collection (CCTCC China, Wuhan university), the preservation number is CCTCC No. M2019453, the preservation date is as follows: 13/6/2019, the fungus is hereinafter referred to as Aspergillus sp.lw1.
Specifically, the method for separating the dimethyl flavopenicillin comprises the following steps:
inoculating Aspergillus sp.LW1 into a fermentation culture medium, and fermenting and culturing at 20-40 deg.C at 100-300rpm for 4-14 days to obtain thallus; and then, extracting and separating the obtained thalli by using an organic solvent to obtain the dimethyl flavopenicillin.
Among them, as a preferable example, Aspergillus sp.LW1 is inoculated into a fermentation medium, and fermentation-cultured at 180rpm at 28 ℃ for 7 days to obtain a cell body. As an alternative example, the organic solvent can be at least one of ethyl acetate or acetone, and the fermentation medium comprises the following raw materials: 20g/L of glucose, 5g/L of yeast powder, 10g/L of soybean peptone and 30g/L of sea salt. As an alternative example, the fermentation medium is formulated according to the following steps: dissolving glucose 20g, yeast powder 5g, soybean peptone 10g, and sea salt 30g in 1l deionized water, adjusting pH to 7.5 with 1M HCl or 1M NaOH, and autoclaving at 121 deg.C for 30 min. The fermentation is carried out by adopting 1000ml conical flasks, each flask is used for fermenting 400ml, and the total fermentation amount is 25 flasks, and the fermentation amount is 10L.
The extraction step comprises soaking thallus in acetone, ultrasonic treating for 30min, standing, and filtering to obtain acetone extract; concentrating the acetone extractive solution at 30-50 deg.C under reduced pressure to obtain crude extract of thallus; and carrying out chromatographic separation on the crude thalli extract, and carrying out reverse HPLC separation and purification on fractions after the chromatographic separation to obtain the dimethyl flavopenicillin.
As an alternative example, standing is carried out until the thallus is settled, then filtering is carried out by gauze, so as to obtain acetone extract, and the reduced pressure concentration mode is rotary evaporation to dryness, wherein the preferable temperature for reduced pressure concentration is 45 ℃.
The chromatographic separation step comprises: dissolving the crude thalli extract with methanol, loading, and drying to obtain an elution sample; continuously eluting the elution sample by using water, 50% methanol solution and methanol, respectively collecting eluates, and concentrating the eluates under reduced pressure at 30-50 ℃; the fraction obtained when methanol was used as the eluent was the target fraction for separation and purification by reverse phase HPLC.
As an alternative example, the chromatographic separation is carried out by using a macroporous adsorbent resin column (HP-20) with a diameter of 10cm and a length of 100cm and a piston at the bottom for controlling the flow rate. The sample loading step comprises: dissolving the crude thalli extract into methanol, and then according to the mass of the crude thalli extract: stirring the macroporous adsorption resin according to the mass ratio of 10:1, and then placing the stirred sample in a fume hood for natural evaporation to obtain an elution sample. The elution step comprises: after obtaining an elution sample, vertically fixing the chromatographic column by using an iron support, slowly filling the elution sample, namely the loaded macroporous adsorption resin into the glass chromatographic column, and lightly knocking the outer wall of the chromatographic column by using an aurilave in the process, so that the macroporous adsorption resin is uniformly and compactly settled; then, water, 50% methanol solution and methanol are selected as eluent to carry out continuous elution, and the eluents are respectively collected and eluted by 1000ml per volume; each fraction recovered was concentrated under reduced pressure (temperature: 35-45 ℃ C.) using a rotary evaporator. The obtained samples are named as H-1, H-2 and H-3 according to water, 50% methanol solution and 3 eluents of methanol.
The reverse phase HPLC separation and purification steps comprise: and (3) redissolving the target fraction by using methanol, then eluting and separating, setting the detection wavelength to be 210nm, collecting a liquid phase peak at 5.0min, and drying the collected fraction to obtain the dimethyl flavopenicillin.
As an alternative example, the reversed phase HPLC separation and purification employs YMC-Pack ODS-A column, and after concentrating the H-3 fraction under reduced pressure to dryness and redissolving with methanol, elution is carried out with 100% acetonitrile at A flow rate of 4ml/min for 20 min.
As an alternative example, since the Aspergillus sp.lw1 fungus is cryopreserved, the method further comprises the step of inoculating the cryopreserved Aspergillus sp.lw1 fungus into a plate culture medium and culturing at 28 ℃ for resuscitation before the step of inoculating the Aspergillus sp.lw1 fungus into a fermentation culture medium.
Wherein the plate culture medium is a PDA culture medium, and specifically comprises the following components: 6.0g/L of potato powder, 20.0g/L of glucose and 20.0g/L of agar. The plate culture medium is prepared according to the following steps: weighing 6.0g of potato powder, 20.0g of glucose and 20.0g of agar, dissolving in 1L of deionized water, and autoclaving at 121 deg.C for 30 min.
Examples
Firstly, fermenting strains:
the cryopreserved Aspergillus sp.LW1 was revived in a plate medium at 28 ℃ and inoculated with an inoculating loop in a clean bench to a fermentation medium and cultured at 28 ℃ for 7d at 180 rpm.
Wherein, the plate culture medium is prepared according to the following steps: 6.0g of potato powder, 20.0g of glucose and 20.0g of agar are dissolved in 1L of deionized water, and autoclaved at 121 ℃ for 30 min. The fermentation medium is prepared according to the following steps: dissolving glucose 20g, yeast extract powder 5g, soybean peptone 10g, and sea salt 30g in 1l deionized water, adjusting pH to 7.5 with 1M HCl or 1M NaOH, and autoclaving at 121 deg.C for 30 min.
The fermentation is carried out by adopting 1000ml conical flasks, each flask is used for fermenting 400ml, and the total fermentation amount is 25 flasks, and the fermentation amount is 10L.
Separating dimethyl flavopenicillin:
s1, thallus extraction: separating the product obtained by fermentation in a suction filtration mode to obtain thalli and fermentation liquor;
s2, acetone extraction: soaking the thalli obtained in the step S1 in 1500ml of acetone, performing ultrasonic treatment for 30min, standing, filtering with gauze after the thalli are settled to obtain an acetone extract, performing rotary evaporation on the acetone extract at 45 ℃ to obtain acetone extract crude paste, and performing redissolution and transfer with methanol; repeating the steps of soaking, filtering and evaporating for 3 times, combining the extracts obtained by the three times, and evaporating to obtain 10g of crude thalli extract;
s3, chromatographic separation: dissolving the crude thalli extract into methanol, and according to the mass of the crude thalli extract: stirring and loading the macroporous adsorption resin according to the mass ratio of 10:1, and then placing the stirred sample in a fume hood for natural evaporation to obtain an elution sample. Then vertically fixing the chromatographic column by using an iron support, slowly filling the eluted sample, namely the loaded macroporous adsorption resin into a glass chromatographic column (the diameter is 10cm, and the length is 100cm), and lightly knocking the outer wall of the chromatographic column by using an ear washing ball, so that the macroporous adsorption resin is uniformly and compactly settled; then, water, 50% methanol solution, methanol and acetone are selected as eluent to carry out continuous elution, and the eluents are respectively collected to elute 1000ml per volume; each fraction recovered was concentrated under reduced pressure (40 ℃ C.) by a rotary evaporator. The obtained samples are named as H-1, H-2 and H-3 according to water, 50% methanol solution and 3 eluents of methanol.
S4, reverse phase HPLC separation and purification: concentrating the component H-3 under reduced pressure, evaporating to dryness, dissolving with methanol, separating by HPLC (column: YMC-Pack ODS-A), eluting with 100% acetonitrile at flow rate of 4ml/min for 20min, detecting with ultraviolet detector at wavelength of 210nm, collecting liquid phase peak at 5.0min to obtain H-3 fraction, and finally obtaining product 41.2 mg.
Thirdly, H-3 structure identification:
process for preparing compounds1H NMR and13the C NMR data are shown in Table 1. The compound H-3 is yellow needle crystal, ESI-MS gives the molecular quasi-ion peak M/z 317.1[ M + H ]+]。13The C NMR spectrum shows 8C signals, wherein one low field region signal delta is containedC173.4, three aromatic carbon signals deltaC129.9、δC131.9、δC114.6, one connected oxygen aromatic carbon signal deltaC161.2, and an oxygenated aliphatic carbon signal deltaC55.5。1H NMR spectrum shows 4 proton signals deltaH6.98(2H,s)、δHThe data of 6.98(4H, d,8.4Hz), 7.76(4H, d,8.4Hz) and 3.85(6H, s) are consistent with the dimethyl flavopenicillin through literature query and comparison[1]The structure was identified as dimethyl flavopenicillin (xanthocilin X dimethyl ether).
TABLE 1 preparation of compound H3 (DMSO-d)6)1H NMR (400Hz) and13c NMR (300Hz) data sheet
The structure of the compound is as follows:
fourth, determination of anti-plant pathogenic fungi activity
The plant pathogenic fungi used in the embodiment are corn microsporum pathogenic bacteria and tomato early blight pathogenic bacteria, and the used pathogenic bacteria strains are all laboratory existing strains. The activity determination experimental procedure was as follows:
s1, culturing the plant pathogenic fungi: in a clean bench, the cryopreserved plant pathogenic fungi strains were activated onto a plate medium. And (4) standing and culturing at 28 ℃, and observing the growth condition of the fungi. Expanding the newly grown fungus colony to the whole plate, and performing the next activity screening experiment;
s2, preparation of detection medium: weighing 24g of ready-to-use culture medium, dissolving in 1L of deionized water, autoclaving at 121 ℃ for 30min to prepare a PDB culture medium, and cooling in a ventilating shade position for later use after sterilization; samples to be tested for activity were prepared in 100mg/ml solution in dimethyl sulfoxide (DMSO). In a super clean bench, a sterile 24-well plate is taken, 1ml of PDB culture medium is added into each well in the plate, then 1ul of solution prepared by a sample to be tested for activity and DMSO is added into each well, namely the final concentration of the sample is 100 mu g/ml, and a positive control group (added with ketoconazole) and a blank control group (not added with the sample) are set up. Simultaneously, the activity of samples with different concentrations (50 mug/ml, 25 mug/ml, 12.5 mug/ml and 6.25 mug/ml) is measured after samples are diluted by times;
s3, judging whether the sample to be tested has the activity of inhibiting the growth of the plant pathogenic fungi: puncturing plant pathogenic fungi with good growth condition with a 5 mm-diameter nuclear magnetic tube to obtain stipe, taking fungi with high activity at the outermost layer of the fungi ring, transferring the stipe into a 24-pore plate by using an inoculating loop, culturing for 48h, and observing the growth condition of hyphae (the size change of the fungi ring and the length change of the hyphae). The growth of hyphae is shown in Table 2.
Principle of judging activity intensity: based on the difference (delta d) between the diameter of the fungal ring formed after the culture of the stipe and the diameter of the blank group, delta d >3mm is regarded as the activity (++) for strongly inhibiting the growth of the plant pathogenic fungi, 1mm < delta d <3mm is regarded as the activity (++) for moderately inhibiting the growth of the plant pathogenic fungi, and delta d <1mm is regarded as the activity (-) for weakly or not inhibiting the growth of the plant pathogenic fungi.
TABLE 2 growth of the hyphae of each group
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Reference documents:
[1]Takatsuki A，Suzuki S，Ando K，et al.Kunio Ando et al.New antiviral antibiotics:xanthocillin mono and dimethylether and methoxyxanthocillin X dimethylether.Isolation and charac-terixation.J Antibiot，1968，21:671-675.
[2]Kurihara H,et al.Method of inhibiting tumor growth using Xanthocillin X dimethylether.U.S.5,210,097,May 11(1993).
[3]Sakai R,et al.Xanthocillin as thrombopoietin mimetic small molecules.Bioorg Med Chem 2005(13):6388-6395.
[4]Morimo T,et al.NK372135s,novel antifungal agents produced by Neosartoria fischeri.J Antibiot.1994,47(12):1546-8.
[5]Tsunakawa M,et al.BU-4704,a new member of the xanthocillin class.J Antibiot.1993,46(4):687-8.
Claims (10)
1. an application of dimethyl xanthocillin in resisting plant pathogenic fungi, wherein the plant pathogenic fungi are corn microsporum pathogenic bacteria or tomato early blight pathogenic bacteria.
2. The use of claim 1, wherein the concentration of the dimehtyl xantham penicillin administered is 12.5 μ g/ml or greater.
3. Use according to any one of claims 1-2, wherein the dimethylaluminixin is isolated from the Aspergillus sp.lw1 CCTCC No. m 2019453.
4. Use according to claim 3, characterized in that the isolation of dimethyl xanthenicillin comprises the following steps:
inoculating Aspergillus sp.LW1 CCTCC No.M2019453 into a fermentation culture medium, and performing fermentation culture at 20-40 deg.C at 100-300rpm for 4-14 days to obtain thallus;
and (3) extracting and separating the thalli by using an organic solvent to obtain the dimethyl flavopenicillin.
5. The use according to claim 4, wherein the organic solvent is at least one of ethyl acetate or acetone.
6. Use according to claim 5, wherein the organic solvent is acetone and the extraction step comprises:
soaking thallus in acetone, ultrasonic treating for 5-60min, standing, and filtering to obtain acetone extractive solution;
concentrating the acetone extract at 30-50 deg.C under reduced pressure to obtain crude extract of thallus;
and carrying out chromatographic separation on the crude thalli extract, and carrying out reverse HPLC separation and purification on the fraction after the chromatographic separation to obtain the dimethyl flavopenicillin.
7. Use according to claim 6, wherein the chromatographic separation step comprises:
dissolving the crude thalli extract with methanol, loading, and drying to obtain an elution sample;
eluting the elution sample by sequentially utilizing water, 50% methanol solution and methanol, respectively collecting eluates, and concentrating the eluates under reduced pressure at 30-50 ℃ until the eluates are dried to obtain corresponding fractions;
the fraction obtained when methanol was used as the eluent was the target fraction for separation and purification by reverse phase HPLC.
8. Use according to claim 7, wherein the reverse phase HPLC separation purification step comprises:
and (3) redissolving the target fraction by using methanol, then eluting and separating by using 100% acetonitrile, setting the detection wavelength to be 210nm, collecting a liquid phase peak at 5.0min, and drying the collected fraction to obtain the dimethyl flavopenicillin.
9. Use according to any one of claims 4 to 8, wherein the fermentation medium comprises the following components:
20g/L of glucose, 5g/L of yeast powder, 10g/L of soybean peptone and 30g/L of sea salt.
10. The use according to any one of claims 4 to 8, further comprising the step of inoculating the cryopreserved Aspergillus sp.LW1 CCTCC No. M2019453 into a plating medium and incubating at 20-40 ℃ for resuscitation prior to the step of inoculating the Aspergillus sp.LW1 CCTCC No. M2019453 into a fermentation medium.
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