CN113151013B - Endophytic fusarium of high-yield cyclosporine plant and application thereof - Google Patents

Abstract

The invention relates to a plant endophytic Fusarium of high-yield cyclosporine and application thereof, wherein the plant endophytic Fusarium of the high-yield cyclosporine is Fusarium (Fusarium sp.) HU0298, a fermentation product cyclosporine derivative is obtained by fermenting, culturing, extracting and separating a Fusarium (Fusarium sp.) HU0298 strain, and the fermentation culture conditions of the Fusarium (Fusarium sp.) HU0298 are optimized by adopting two modes of liquid fermentation and solid fermentation.

Description

Endophytic fusarium of high-yield cyclosporine plant and application thereof

Technical Field

The invention relates to the technical field of bioengineering, relates to microorganisms and application thereof, and particularly relates to high-yield cyclosporine endophytic fusarium and application thereof.

Background

Cyclosporine (Cyclosporine) is a structurally unique lipophilic cyclic polypeptide consisting of 11 amino acid residues, for a total of 30 cyclosporines obtained by natural product isolation and semi-synthesis to date. Among them, cyclosporin A has a strong immunosuppressive activity, a high selectivity, and almost no inhibitory effect on bone marrow and macrophages, and has been widely used in organ and bone marrow transplantation, such as kidney, liver, heart, etc., as one of the most major clinical first-line anti-rejection drugs for decades of clinical kidney transplantation since it was approved in 1983. With the development of organ transplantation technology, the success rate of organ transplantation is higher and higher, patients are more and more common, and the transplanted patients need to be taken for years or even for life.

The cyclosporine A has various biological activities such as antifungal activity, insect resistance, anti-inflammatory activity, antiviral activity and the like besides immunosuppressive activity, and is also commonly used for treating diseases such as various intractable skin diseases, autoimmune blood diseases and the like, the cyclosporine A is an important drug molecule, also shows obvious application potential in the fields of agriculture, breeding and the like, the existing raw material demand and potential demand are large, and the discovery and preparation process research of high-yield strains of the cyclosporine A are always concerned.

However, cyclosporin a is a cyclic undecapeptide compound with a large molecular weight, in which a plurality of amino acid residues are very common amino acids and have 12 chiral centers, and is difficult to artificially synthesize. Heretofore, the source of cyclosporine A is obtained by means of microbial fermentation, separation and purification, the cyclosporine yield of the existing wild fungus strain is below 1.0g/L, the components of the production culture medium are complex, part of raw materials are high in price, the fermentation culture process is complicated, large-scale equipment investment is required, the production cost is high, and the search for a cyclosporine A high-yield strain and a simple, efficient and low-cost fermentation culture and preparation method is always an important concern of related pharmaceutical enterprises.

Disclosure of Invention

Based on this, there is a need for a highly productive Fusarium endophytic bacterium and its use for the simple, efficient, and cost-effective production of cyclosporin A.

The technical scheme for solving the technical problems is as follows:

a high-cyclosporine-yield plant endophytic Fusarium is Fusarium (Fusarium sp.) HU0298, which is separated from plant Rumex acetosa of south Australian island beach, Shantou City, Guangdong province.

The preservation information of the strain (biological material) is:

name: fusarium (Fusarium sp.) HU 0298;

the preservation number is: GDMCC No. 61466;

the preservation unit: guangdong province culture Collection of microorganisms (GDMCC);

the preservation address is as follows: the city of Guangzhou, Virginian district, first furious Zhonglu No. 100, microbial research institute of Guangdong province, zip code: 510075;

preservation time: 26/1/2021.

A preservation method of Fusarium (Fusarium sp.) HU0298 comprises the following steps: and storing by adopting a PDA culture medium. PDA culture medium: 200g of potato, 20g of glucose, 20g of agar and 1000mL of distilled water, and the pH is adjusted to 7.0.

The DNA of a Fusarium (Fusarium sp.) HU0298 strain is extracted by a CTAB method, and the classification of the HU0298 strain is identified by an ITS gene sequence, wherein an ITS gene PCR amplification primer is as follows: ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3') and ITS5 (5'-GGA AGT AAA AGT CGT AAC AAG G-3'). The gene sequence is compared with related strain sequences in a Genbank database by BLAST, the ITS sequence homology is the highest with Fusarium falciforme and Fusarium solani, both of which are 99 percent, and the HU0298 strain and Fusarium sp belong to the same branch, so that the HU0298 strain is named as Fusarium (Fusarium sp.) HU 0298.

Another object of the present invention is to provide the use of high cyclosporin production by Fusarium endophytic in the preparation of cyclosporin A.

In one embodiment, there is provided a fermentation process of high yield cyclosporin in plants with fusarium sp comprising the steps of:

inoculating Fusarium (Fusarium sp.) HU0298 into a PDA culture medium for activation culture to obtain an activated Fusarium (Fusarium sp.) HU0298 strain, wherein the culture temperature is 20-30 ℃, and the activation culture time is 3-5 days;

inoculating the activated Fusarium (Fusarium sp) HU0298 strain into a PDB culture medium for shake culture to obtain a primary seed solution, wherein the culture temperature is 20-30 ℃, and the shake culture time is 1-3 days;

inoculating the primary seed liquid to a liquid culture medium for liquid fermentation culture to obtain liquid fermentation liquid;

or inoculating the primary seed liquid into a PDB culture medium for culturing for 1-3 days to obtain a secondary seed liquid, then inoculating the secondary seed liquid into a solid culture medium for solid fermentation culture to obtain a solid fermentation product, wherein the culture temperature of liquid fermentation and solid fermentation is 20-30 ℃.

In one embodiment, the liquid medium comprises the following components:

fructose 30g/L, KH₂PO₄ 6.00g/L、CaCl₂ 0.90g/L、MgSO₄0.90g/L, KCl 0.30.30 g/L, 5.0g/L, MnSO g yeast extract₄·4H₂O 6.70mg/L、ZnSO₄·7H₂O 8.30mg/L、FeSO₄·7H₂O 15.00mg/L、CuSO₄ 1.50mg/L、CoCl₂11.00mg/L, pH adjusted to 5.4.

In one embodiment, the liquid medium comprises the following components:

glucose 30g/L, ammonium sulfate 10g/L, K₂HPO₄7.5g/L and 1mL/L of trace element solution, and adjusting the pH to 5.5, wherein the trace element solution comprises: ZnSO₄ 4.4g/L、FeSO₄ 5g/L、MnCl₂ 180mg/L、Na₂MO₄ 25mg/L、CuSO₄80mg/L and H₂SO₄ 2mL。

In one embodiment, the liquid medium comprises the following components:

malt extract 2% and yeast extract 0.4%, pH adjusted to 5.4;

or 200g/L potato, 20g/L agar and 20g/L glucose.

In one embodiment, the solid medium comprises: the component A and the distilled water are mixed according to a ratio of 1: 1-5, and the component A is any one of corn, rice, wheat bran and coconut oil cake.

In one embodiment, the solid medium comprises: the component B, the component C and the distilled water are mixed according to a ratio of 1:1: 1-5, the component B is corn or wheat bran, and the component C is any one of wheat, peanut oil cake, soybean oil cake, wheat bran and coconut oil cake.

In one embodiment, the solid medium comprises: the corn-soybean milk comprises corn, distilled water and a component D, wherein the component D is any one of glycerol, DL-alpha-aminobutyric acid, malt extract, soybean peptone, yeast extract and casein peptone, and the feed-liquid ratio of the corn, the distilled water and the component D is 1:1: 0.01-0.05.

In one embodiment, there is provided a method for preparing cyclosporin a from high cyclosporin production by endophytic fusarium, comprising the steps of:

carrying out fermentation culture on Fusarium (Fusarium sp.) HU0298 to obtain a fermentation culture;

leaching the fermentation culture with leaching liquor, filtering, and concentrating to obtain extract, wherein the leaching liquor is 95% ethanol or ethyl acetate;

performing silica gel column chromatography on the extract, performing gradient elution by using a dichloromethane-methanol mixed solvent with a volume ratio of 100: 0-80: 20, collecting fractions with a specific shift value of 0.4-0.6 when the dichloromethane-methanol mixed solvent with a volume ratio of 95:5 is expanded in silica gel plate thin layer chromatography, detecting positive fractions by using an acid hydrolysis-ninhydrin color method, and combining to obtain total cyclosporine;

the total cyclosporin was subjected to reverse phase chromatography and eluted with 80% volume fraction of methanol to give cyclosporin A.

The invention has the beneficial effects that: the fermentation product cyclosporine derivative is obtained by fermentation culture, extraction and separation of Fusarium sp HU0298 strain, wherein the yield of the cyclosporine A can reach 5.63g/kg, and the fermentation product cyclosporine derivative has the advantages of lower culture medium cost, relatively simple production process, easiness in large-scale fermentation production and the like, can greatly improve the unit yield of the cyclosporine A, reduce the yield of waste, reduce the production cost, and has good application prospect in the preparation of the cyclosporine A.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.

FIG. 1 is a schematic flow diagram of a fermentation process for high yield production of cyclosporin in Fusarium endophytic bacteria of the present invention;

FIG. 2 is a schematic flow chart of a process for preparing cyclosporin A from high yield of cyclosporin in accordance with an embodiment of the present invention using Fusarium endophytic bacteria;

FIG. 3 shows the colony morphology of Fusarium sp HU0298 in accordance with one embodiment of the present invention;

FIG. 4 shows the hyphal and spore morphology of Fusarium sp HU0298 in accordance with one embodiment of the present invention;

FIG. 5 shows an ITS-based construction of a neighboring tree of Fusarium sp HU0298 according to one embodiment of the present invention.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

In one embodiment, the high cyclosporin production plant endophytic Fusarium is Fusarium (Fusarium sp.) HU0298, which was deposited at the guangdong province collection of microorganisms at 26 months 1/2021 with deposit number GDMCC No. 61466.

In this example, Fusarium (Fusarium sp.) HU0298 strain was isolated from Rumex acetosa (Rumex acetosa) plant of the beach near south Australian island, Shansho, Guangdong province by tissue culture. Respectively soaking stems and leaves of the collected Rumex acetosa plants in 75% ethanol and 1% sodium hypochlorite solution for 30s, washing with sterile water for 2 times, air-drying, shearing the tissues, inoculating the tissues onto the surface of a PDA culture medium, culturing in dark at 25 +/-5 ℃ for 3d, selecting the obtained colonies, inoculating the colonies into a new PDA culture medium, and further purifying to obtain the white filamentous fungi.

The PDA culture medium is prepared by adding appropriate amount of pure water into 200g potato, boiling for 30min, filtering to obtain filtrate, adding 20g glucose and 20g agar, diluting to 1L with pure water, and sterilizing at 121 deg.C for 30 min.

In this embodiment, the colony, hyphae and spore morphology of Fusarium (Fusarium sp.) HU0298 is shown in FIGS. 3 and 4, and the hyphae have a white flocculent shape with a diameter of about 2.0-3.0 μm; the small conidia are oval, 0-1 interval and 3.7-12.5 microns multiplied by 2.0-3.0 microns in size; the large conidia are sickle-shaped, have 1-4 intervals and have the size of 7.8-33.8 mu m multiplied by 2.3-3.5 mu m.

Further, the DNA sequence determination results obtained by sequencing and splicing Fusarium (Fusarium sp.) HU0298 strain are shown in the sequence listing, and a neighbor tree is constructed based on ITS, as shown in fig. 5, the ITS sequence homology is the highest with Fusarium falciforme and Fusarium solani, both 99%, and HU0298 strain belongs to the same branch as Fusarium sp.

In conclusion, the strain is identified as high-yield cyclosporin-producing endophytic Fusarium sp, which is currently deposited in Guangdong province microbial strain collection center (GDMCC, address: Youqian Zhonglu 100, Guangdong province institute of microorganisms), and the date of deposit is as follows: 26/1/2021, the strain having the accession number GDMCC No. 61466.

In one embodiment, a high cyclosporine producing strain of endophytic Fusarium (Fusarium sp.) HU0298 is fermentatively cultured as shown in FIG. 1.

Step 110, inoculating Fusarium (Fusarium sp.) HU0298 into a PDA culture medium for activation culture to obtain an activated Fusarium (Fusarium sp.) HU0298 strain, wherein the culture temperature is 20-30 ℃, and the activation culture time is 3-5 days.

Specifically, Fusarium (Fusarium sp.) HU0298 blocks stored in a freezing storage tube are picked by using a sterile inoculating needle, inoculated on a PDA culture medium, activated, placed in a constant-temperature incubator at 28 ℃ and cultured under the dark condition, the PDA culture medium is prepared by cutting 200g of potatoes into blocks, adding water, boiling for 30min, filtering, adding 20g of glucose and 20g of agar, fixing the volume to 1L by pure water, and performing moist heat sterilization at 121 ℃ for 30 min.

Step 120, inoculating the activated Fusarium (Fusarium sp) HU0298 strain into a PDB culture medium for shake culture to obtain a primary seed solution, wherein the culture temperature is 20-30 ℃, and the shake culture time is 1-3 days.

Specifically, the PDB culture medium is prepared by adding water into 200g of potatoes, boiling for 30min, filtering to obtain a filtrate, adding 20g of glucose, diluting to 1L with pure water, and performing moist heat sterilization at 121 ℃ for 30 min.

And 130, inoculating the primary seed liquid to a liquid culture medium for liquid fermentation culture to obtain liquid fermentation liquid.

Specifically, a well activated Fusarium (Fusarium sp.) HU0298 block is selected and inoculated into a conical flask filled with 30ml of PDB culture medium, the conical flask is placed in a constant-temperature shaking incubator at 150rpm and is subjected to shaking culture for 2d under a dark condition, the culture temperature is 20-30 ℃, and the liquid culture medium comprises the following components: fructose 30g/L, KH₂PO₄ 6.00g/L、CaCl₂ 0.90g/L、MgSO₄0.90g/L, KCl 0.30.30 g/L, 5.0g/L, MnSO g yeast extract₄·4H₂O 6.70mg/L、ZnSO₄·7H₂O 8.30mg/L、FeSO₄·7H₂O 15.00mg/L、CuSO₄1.50mg/L and CoCl₂11.00mg/L, pH adjusted to 5.4.

In another embodiment, the liquid medium comprises the following components: glucose 30g/L, ammonium sulfate 10g/L, K₂HPO₄7.5g/L and 1mL/L of trace element solution, and adjusting the pH to 5.5, wherein the trace element solution comprises: ZnSO₄ 4.4g/L、FeSO₄ 5g/L、MnCl₂ 180mg/L、Na₂MO₄ 25mg/L、CuSO₄80mg/L and H₂SO₄ 2mL。

In yet another embodiment, the liquid medium has the composition: malt extract 2% and yeast extract 0.4%, pH adjusted to 5.4;

in yet another embodiment, the liquid culture medium is a PDB liquid culture medium, which comprises 200g/L of potatoes, 20g/L of agar and 20g/L of glucose, and is prepared by boiling 200g of potatoes in water for 30min, filtering to obtain a filtrate, adding 20g of glucose, diluting to 1L with pure water, and performing moist heat sterilization at 121 ℃ for 30 min.

In another fermentation culture mode, the primary seed liquid is inoculated into a PDB culture medium to be cultured for 1-3 days to obtain a secondary seed liquid, and then the secondary seed liquid is inoculated into a solid culture medium to be subjected to solid fermentation culture to obtain a solid fermentation product.

In this example, a Pasteur pipette is used to suck 1ml of the primary seed solution into a conical flask containing 200ml of PDB medium, the conical flask is placed in a constant-temperature shaking incubator at 150rpm under dark condition for shaking culture for 2d to obtain a secondary seed solution, then a Pasteur pipette is used to suck 5ml of the secondary seed solution into a fermentation tank containing a fixed medium for uniform inoculation, and the conical flask is placed in dark for standing culture for 35 d. Wherein the culture temperature for inoculating the first-stage seed liquid into a PDB culture medium for culture and inoculating the second-stage seed liquid into a solid culture medium for solid fermentation culture is 20-30 ℃.

In this embodiment, the solid medium includes: the component A and the distilled water are mixed according to a ratio of 1: 1-5, and the component A is any one of corn, rice, wheat bran and coconut oil cake.

In one embodiment, the solid medium components are shown in table 1.

TABLE 1 solid Medium composition

In another embodiment, the composition of the solid medium is: the component B, the component C and the distilled water are mixed according to a ratio of 1:1: 1-5, the component B is corn or wheat bran, and the component C is any one of wheat, peanut oil cake, soybean oil cake, wheat bran and coconut oil cake.

In one embodiment, the solid medium components are shown in table 2.

TABLE 2 solid Medium composition

In yet another embodiment, the solid medium comprises: the feed liquid comprises corn, distilled water and a component D, wherein the component D is any one of glycerol, DL-alpha-aminobutyric acid, yeast extract and casein peptone, and the feed liquid ratio of the corn, the distilled water and the component D is 1:1: 0.01-0.05.

In one embodiment, the highly cyclosporin-producing endophytic Fusarium (Fusarium sp.) HU0298 of the above embodiments is used to prepare cyclosporin a. Specifically, the fermentation culture is extracted by an organic solvent to obtain an extract which is total cyclosporin, wherein the content of cyclosporin A is up to 5.5% or more, and the extract is separated to obtain cyclosporin A. The total cyclosporine includes cyclosporine A, cyclosporine B and cyclosporine C, and the chemical structural formula of the total cyclosporine is as follows:

wherein, in the chemical structural formula of cyclosporine A, R₁＝CH₃，R₂H, in the chemical formula of cyclosporin B, R₁＝R₂H, in the chemical formula of cyclosporin C, R₁＝CH₃，R₂＝OH。

Wherein, the chemical structural formula of the cyclosporine A is as follows:

the fermentation product cyclosporine derivative is obtained by culturing Fusarium (Fusarium sp.) HU0298 strain in two modes of liquid fermentation and solid fermentation under a proper condition, and the output of cyclosporine A in the obtained fermentation product can reach 1g/kg to 5.10 g/kg. The production process is relatively simple and is easy for large-scale fermentation production.

In one embodiment, as shown in FIG. 2, there is provided a process for preparing cyclosporin A from high yield cyclosporin-producing endophytic Fusarium comprising the steps of:

step 210, carrying out fermentation culture on Fusarium (Fusarium sp.) HU0298, and culturing to obtain a fermentation culture.

Specifically, Fusarium (Fusarium sp) HU0298 is inoculated in a culture medium, the culture temperature is 20-30 ℃, and the Fusarium sp is subjected to static culture in the dark. In this example, Fusarium (Fusarium sp.) HU0298 was inoculated into the solid medium for solid fermentation culture; in another embodiment, Fusarium sp (Fusarium sp.) HU0298 is inoculated into the liquid medium for liquid fermentation culture.

And step 220, leaching the fermentation culture with a leaching liquor, filtering and concentrating to obtain an extract, wherein the leaching liquor is 95% ethanol or ethyl acetate.

Specifically, the fermentation culture is leached by ethyl acetate, and then the ethyl acetate extract is concentrated to be dry to obtain the ethyl acetate extract, wherein the leaching times are 1-3 times, and each leaching time is 36-60h, and in a preferred embodiment, the leaching time is 48 h.

In one embodiment, the fermentation culture is leached by equal volume of 95% ethanol for 36-60 hours, the ethanol extract is obtained by filtration, then the ethanol extract is concentrated under reduced pressure until the volume is 1/20 of the ethanol extract, a primary concentrate is obtained, the primary concentrate is in an extract state, the primary concentrate is dissolved by ethanol water solution with the volume fraction of 80%, the petroleum ether extract and the residual ethanol water solution are extracted by equal volume, the ethanol in the ethanol water solution is removed, then the ethanol is supplemented with water to the original volume, the equal volume of ethyl acetate is continuously used for extraction, and finally the ethyl acetate extract is obtained by filtration and concentration, wherein the times of petroleum ether extraction and ethyl acetate extraction are 1-3 times.

And step 230, performing silica gel column chromatography on the extract, performing gradient elution by using a dichloromethane-methanol mixed solvent with a volume ratio of 100: 0-80: 20, collecting fractions with a specific displacement value of 0.4-0.6 when the dichloromethane-methanol mixed solvent with a volume ratio of 95:5 is developed in silica gel plate thin layer chromatography, detecting the fractions with positive values by using an acid hydrolysis-ninhydrin color development method, and combining the fractions to obtain the total cyclosporine.

Specifically, the ethyl acetate extract is put into a normal-phase silica gel column, the particle size of the silica gel is 100-200 meshes, when gradient elution is carried out by using a dichloromethane-methanol mixed solvent with the volume ratio of 100: 0-80: 20, similar fractions are detected and combined by combining three color development methods of vanillin sulfate, ninhydrin and acid hydrolysis-ninhydrin, and total cyclosporine with the specific displacement value of 0.4-0.6 when the dichloromethane-methanol volume ratio on a thin-layer plate is 95:5 is expanded is collected.

In step 240, the total cyclosporin is subjected to reverse phase chromatography, eluting with 80% by volume methanol, to give cyclosporin A.

Specifically, the collected total cyclosporine fractions are subjected to high performance liquid chromatography preparation, and isocratic elution is carried out by taking a mobile phase as 80% methanol in volume fraction to obtain cyclosporine A, cyclosporine B and cyclosporine C.

Specifically, the obtained colorless solid, namely a cyclosporine A sample is subjected to structural characterization by adopting mass spectrum and nuclear magnetic resonance spectrum, and the molecular formula is C₆₂H₂₄N₁₁₁O₁₂Positive ion ESIMS M/z 1224[ M + Na ]]⁺Negative ion ESIMS M/z 1200[ M-H ]]^－And 1236[ M + Cl]^－. It is provided with¹H NMR and¹³the C NMR spectrum data are shown in Table 3, which shows that the product isolated is cyclosporin A.

TABLE 3 Cyclosporin A¹H and¹³c NMR spectral data

Example 1

Culturing Fusarium (Fusarium sp.) HU0298 in a seed solution, inoculating the Fusarium (Fusarium sp.) HU0298 into a fermentation tank filled with a solid culture medium, uniformly inoculating, culturing to obtain a fermentation culture, leaching the fermentation culture with a leaching solution, filtering and concentrating to obtain an extract, performing silica gel column chromatography on the extract, performing gradient elution with a dichloromethane-methanol mixed solvent with a volume ratio of 100:0 to 80:20, collecting fractions with a specific displacement value of 0.4-0.6 when the dichloromethane-methanol mixed solvent with a volume ratio of 95:5 in silica gel plate thin layer chromatography is developed, detecting positive fractions by an acid hydrolysis-ninhydrin color development method, combining to obtain total cyclosporine, and finally performing reverse phase chromatography on the total cyclosporine, and eluting with methanol with a volume fraction of 80% to obtain cyclosporine A. Wherein the solid culture medium comprises the following components: corn and distilled water, wherein the feed-liquid ratio is 1: 1.

Example 2

Fusarium (Fusarium sp.) HU0298 is subjected to seed liquid culture, then inoculated into a fermentation tank filled with a solid culture medium for uniform inoculation, a fermentation culture is obtained through culture, the fermentation culture is leached by using leaching liquor, filtered and concentrated to obtain an extract, then the extract is subjected to silica gel column chromatography, gradient elution is carried out by using a dichloromethane-methanol mixed solvent with the volume ratio of 100: 0-80: 20, when the dichloromethane-methanol mixed solvent with the volume ratio of 95:5 in silica gel plate thin layer chromatography is developed, the specific displacement value is 0.4-0.6, and the components which are detected to be positive by an acid hydrolysis-ninhydrin color development method are combined to obtain total cyclosporine, finally the total cyclosporine is subjected to reverse phase chromatography, and elution is carried out by using methanol with the volume fraction of 80%, so as to obtain the cyclosporine A. Wherein the solid medium comprises the following components: rice and distilled water, wherein the feed-liquid ratio is 1: 1.

Example 3

Culturing Fusarium (Fusarium sp.) HU0298 in a seed solution, inoculating the Fusarium (Fusarium sp.) HU0298 into a fermentation tank filled with a solid culture medium, uniformly inoculating, culturing to obtain a fermentation culture, leaching the fermentation culture with a leaching solution, filtering and concentrating to obtain an extract, performing silica gel column chromatography on the extract, performing gradient elution with a dichloromethane-methanol mixed solvent with a volume ratio of 100:0 to 80:20, collecting fractions with a specific displacement value of 0.4-0.6 when the dichloromethane-methanol mixed solvent with a volume ratio of 95:5 in silica gel plate thin layer chromatography is developed, detecting positive fractions by an acid hydrolysis-ninhydrin color development method, combining to obtain total cyclosporine, and finally performing reverse phase chromatography on the total cyclosporine, and eluting with methanol with a volume fraction of 80% to obtain cyclosporine A. Wherein the solid culture medium comprises the following components: wheat and distilled water, wherein the feed-liquid ratio is 1: 1.

Example 4

Culturing Fusarium (Fusarium sp.) HU0298 in a seed solution, inoculating the Fusarium (Fusarium sp.) HU0298 into a fermentation tank filled with a solid culture medium, uniformly inoculating, culturing to obtain a fermentation culture, leaching the fermentation culture with a leaching solution, filtering and concentrating to obtain an extract, performing silica gel column chromatography on the extract, performing gradient elution with a dichloromethane-methanol mixed solvent with a volume ratio of 100:0 to 80:20, collecting fractions with a specific displacement value of 0.4-0.6 when the dichloromethane-methanol mixed solvent with a volume ratio of 95:5 in silica gel plate thin layer chromatography is developed, detecting positive fractions by an acid hydrolysis-ninhydrin color development method, combining to obtain total cyclosporine, and finally performing reverse phase chromatography on the total cyclosporine, and eluting with methanol with a volume fraction of 80% to obtain cyclosporine A. Wherein the solid culture medium comprises the following components: wheat bran and distilled water, wherein the feed-liquid ratio is 1:2.

Example 5

Culturing Fusarium (Fusarium sp.) HU0298 in a seed solution, inoculating the Fusarium (Fusarium sp.) HU0298 into a fermentation tank filled with a solid culture medium, uniformly inoculating, culturing to obtain a fermentation culture, leaching the fermentation culture with a leaching solution, filtering and concentrating to obtain an extract, performing silica gel column chromatography on the extract, performing gradient elution with a dichloromethane-methanol mixed solvent with a volume ratio of 100:0 to 80:20, collecting fractions with a specific displacement value of 0.4-0.6 when the dichloromethane-methanol mixed solvent with a volume ratio of 95:5 in silica gel plate thin layer chromatography is developed, detecting positive fractions by an acid hydrolysis-ninhydrin color development method, combining to obtain total cyclosporine, and finally performing reverse phase chromatography on the total cyclosporine, and eluting with methanol with a volume fraction of 80% to obtain cyclosporine A. Wherein the solid culture medium comprises the following components: coconut oil cake and distilled water, wherein the material-liquid ratio is 1: 3.

Example 6

Culturing Fusarium (Fusarium sp.) HU0298 in a seed solution, inoculating the Fusarium (Fusarium sp.) HU0298 into a fermentation tank filled with a solid culture medium, uniformly inoculating, culturing to obtain a fermentation culture, leaching the fermentation culture with a leaching solution, filtering and concentrating to obtain an extract, performing silica gel column chromatography on the extract, performing gradient elution with a dichloromethane-methanol mixed solvent with a volume ratio of 100:0 to 80:20, collecting fractions with a specific displacement value of 0.4-0.6 when the dichloromethane-methanol mixed solvent with a volume ratio of 95:5 in silica gel plate thin layer chromatography is developed, detecting positive fractions by an acid hydrolysis-ninhydrin color development method, combining to obtain total cyclosporine, and finally performing reverse phase chromatography on the total cyclosporine, and eluting with methanol with a volume fraction of 80% to obtain cyclosporine A. Wherein the solid culture medium comprises the following components: corn, wheat and distilled water, wherein the ratio of material to liquid is 1:1:2.

Example 7

Culturing Fusarium (Fusarium sp.) HU0298 in a seed solution, inoculating the Fusarium (Fusarium sp.) HU0298 into a fermentation tank filled with a solid culture medium, uniformly inoculating, culturing to obtain a fermentation culture, leaching the fermentation culture with a leaching solution, filtering and concentrating to obtain an extract, performing silica gel column chromatography on the extract, performing gradient elution with a dichloromethane-methanol mixed solvent with a volume ratio of 100:0 to 80:20, collecting fractions with a specific displacement value of 0.4-0.6 when the dichloromethane-methanol mixed solvent with a volume ratio of 95:5 in silica gel plate thin layer chromatography is developed, detecting positive fractions by an acid hydrolysis-ninhydrin color development method, combining to obtain total cyclosporine, and finally performing reverse phase chromatography on the total cyclosporine, and eluting with methanol with a volume fraction of 80% to obtain cyclosporine A. Wherein the solid culture medium comprises the following components: corn, peanut oil cake and distilled water, wherein the feed-liquid ratio is 1:1: 2.5.

Example 8

Culturing Fusarium (Fusarium sp.) HU0298 in a seed solution, inoculating the Fusarium (Fusarium sp.) HU0298 into a fermentation tank filled with a solid culture medium, uniformly inoculating, culturing to obtain a fermentation culture, leaching the fermentation culture with a leaching solution, filtering and concentrating to obtain an extract, performing silica gel column chromatography on the extract, performing gradient elution with a dichloromethane-methanol mixed solvent with a volume ratio of 100:0 to 80:20, collecting fractions with a specific displacement value of 0.4-0.6 when the dichloromethane-methanol mixed solvent with a volume ratio of 95:5 in silica gel plate thin layer chromatography is developed, detecting positive fractions by an acid hydrolysis-ninhydrin color development method, combining to obtain total cyclosporine, and finally performing reverse phase chromatography on the total cyclosporine, and eluting with methanol with a volume fraction of 80% to obtain cyclosporine A. Wherein the solid medium comprises the following components: corn, soybean oil cake and distilled water, wherein the feed-liquid ratio is 1:1: 2.5.

Example 9

Culturing Fusarium (Fusarium sp.) HU0298 in a seed solution, inoculating the Fusarium (Fusarium sp.) HU0298 into a fermentation tank filled with a solid culture medium, uniformly inoculating, culturing to obtain a fermentation culture, leaching the fermentation culture with a leaching solution, filtering and concentrating to obtain an extract, performing silica gel column chromatography on the extract, performing gradient elution with a dichloromethane-methanol mixed solvent with a volume ratio of 100:0 to 80:20, collecting fractions with a specific displacement value of 0.4-0.6 when the dichloromethane-methanol mixed solvent with a volume ratio of 95:5 in silica gel plate thin layer chromatography is developed, detecting positive fractions by an acid hydrolysis-ninhydrin color development method, combining to obtain total cyclosporine, and finally performing reverse phase chromatography on the total cyclosporine, and eluting with methanol with a volume fraction of 80% to obtain cyclosporine A. Wherein the solid culture medium comprises the following components: corn, wheat bran and distilled water, wherein the feed-liquid ratio is 1:1: 3.

Example 10

Fusarium (Fusarium sp.) HU0298 is subjected to seed liquid culture, then inoculated into a fermentation tank filled with a solid culture medium for uniform inoculation, a fermentation culture is obtained through culture, the fermentation culture is leached by using leaching liquor, filtered and concentrated to obtain an extract, then the extract is subjected to silica gel column chromatography, gradient elution is carried out by using a dichloromethane-methanol mixed solvent with the volume ratio of 100: 0-80: 20, when the dichloromethane-methanol mixed solvent with the volume ratio of 95:5 in silica gel plate thin layer chromatography is developed, the specific displacement value is 0.4-0.6, and the components which are detected to be positive by an acid hydrolysis-ninhydrin color development method are combined to obtain total cyclosporine, finally the total cyclosporine is subjected to reverse phase chromatography, and elution is carried out by using methanol with the volume fraction of 80%, so as to obtain the cyclosporine A. Wherein the solid culture medium comprises the following components: corn, coconut oil cake and distilled water, wherein the material-liquid ratio is 1:1: 4.

Example 11

Culturing Fusarium (Fusarium sp.) HU0298 in a seed solution, inoculating the Fusarium (Fusarium sp.) HU0298 into a fermentation tank filled with a solid culture medium, uniformly inoculating, culturing to obtain a fermentation culture, leaching the fermentation culture with a leaching solution, filtering and concentrating to obtain an extract, performing silica gel column chromatography on the extract, performing gradient elution with a dichloromethane-methanol mixed solvent with a volume ratio of 100:0 to 80:20, collecting fractions with a specific displacement value of 0.4-0.6 when the dichloromethane-methanol mixed solvent with a volume ratio of 95:5 in silica gel plate thin layer chromatography is developed, detecting positive fractions by an acid hydrolysis-ninhydrin color development method, combining to obtain total cyclosporine, and finally performing reverse phase chromatography on the total cyclosporine, and eluting with methanol with a volume fraction of 80% to obtain cyclosporine A. Wherein the solid culture medium comprises the following components: wheat bran, coconut oil cake and distilled water, wherein the material-liquid ratio is 1:1: 5.

Example 12

Culturing Fusarium (Fusarium sp.) HU0298 in a seed solution, inoculating the Fusarium (Fusarium sp.) HU0298 into a fermentation tank filled with a solid culture medium, uniformly inoculating, culturing to obtain a fermentation culture, leaching the fermentation culture with a leaching solution, filtering and concentrating to obtain an extract, performing silica gel column chromatography on the extract, performing gradient elution with a dichloromethane-methanol mixed solvent with a volume ratio of 100:0 to 80:20, collecting fractions with a specific displacement value of 0.4-0.6 when the dichloromethane-methanol mixed solvent with a volume ratio of 95:5 in silica gel plate thin layer chromatography is developed, detecting positive fractions by an acid hydrolysis-ninhydrin color development method, combining to obtain total cyclosporine, and finally performing reverse phase chromatography on the total cyclosporine, and eluting with methanol with a volume fraction of 80% to obtain cyclosporine A. Wherein the solid culture medium comprises the following components: corn and 2% glycerol, wherein the feed-liquid ratio is 1: 1.

Example 13

Culturing Fusarium (Fusarium sp.) HU0298 in a seed solution, inoculating the Fusarium (Fusarium sp.) HU0298 into a fermentation tank filled with a solid culture medium, uniformly inoculating, culturing to obtain a fermentation culture, leaching the fermentation culture with a leaching solution, filtering and concentrating to obtain an extract, performing silica gel column chromatography on the extract, performing gradient elution with a dichloromethane-methanol mixed solvent with a volume ratio of 100:0 to 80:20, collecting fractions with a specific displacement value of 0.4-0.6 when the dichloromethane-methanol mixed solvent with a volume ratio of 95:5 in silica gel plate thin layer chromatography is developed, detecting positive fractions by an acid hydrolysis-ninhydrin color development method, combining to obtain total cyclosporine, and finally performing reverse phase chromatography on the total cyclosporine, and eluting with methanol with a volume fraction of 80% to obtain cyclosporine A. Wherein the solid culture medium comprises the following components: corn and 0.5% malt extract, the feed-liquid ratio is 1: 1.

Example 14

Culturing Fusarium (Fusarium sp.) HU0298 in a seed solution, inoculating the Fusarium (Fusarium sp.) HU0298 into a fermentation tank filled with a solid culture medium, uniformly inoculating, culturing to obtain a fermentation culture, leaching the fermentation culture with a leaching solution, filtering and concentrating to obtain an extract, performing silica gel column chromatography on the extract, performing gradient elution with a dichloromethane-methanol mixed solvent with a volume ratio of 100:0 to 80:20, collecting fractions with a specific displacement value of 0.4-0.6 when the dichloromethane-methanol mixed solvent with a volume ratio of 95:5 in silica gel plate thin layer chromatography is developed, detecting positive fractions by an acid hydrolysis-ninhydrin color development method, combining to obtain total cyclosporine, and finally performing reverse phase chromatography on the total cyclosporine, and eluting with methanol with a volume fraction of 80% to obtain cyclosporine A. Wherein the solid medium comprises the following components: corn and 0.1% soybean peptone, the feed-liquid ratio is 1: 1.

Example 15

Culturing Fusarium (Fusarium sp.) HU0298 in a seed solution, inoculating the Fusarium (Fusarium sp.) HU0298 into a fermentation tank filled with a solid culture medium, uniformly inoculating, culturing to obtain a fermentation culture, leaching the fermentation culture with a leaching solution, filtering and concentrating to obtain an extract, performing silica gel column chromatography on the extract, performing gradient elution with a dichloromethane-methanol mixed solvent with a volume ratio of 100:0 to 80:20, collecting fractions with a specific displacement value of 0.4-0.6 when the dichloromethane-methanol mixed solvent with a volume ratio of 95:5 in silica gel plate thin layer chromatography is developed, detecting positive fractions by an acid hydrolysis-ninhydrin color development method, combining to obtain total cyclosporine, and finally performing reverse phase chromatography on the total cyclosporine, and eluting with methanol with a volume fraction of 80% to obtain cyclosporine A. Wherein the solid culture medium comprises the following components: corn and 0.2% yeast extract, the feed-liquid ratio is 1: 1.

Example 16

Culturing Fusarium (Fusarium sp.) HU0298 in a seed solution, inoculating the Fusarium (Fusarium sp.) HU0298 into a fermentation tank filled with a solid culture medium, uniformly inoculating, culturing to obtain a fermentation culture, leaching the fermentation culture with a leaching solution, filtering and concentrating to obtain an extract, performing silica gel column chromatography on the extract, performing gradient elution with a dichloromethane-methanol mixed solvent with a volume ratio of 100:0 to 80:20, collecting fractions with a specific displacement value of 0.4-0.6 when the dichloromethane-methanol mixed solvent with a volume ratio of 95:5 in silica gel plate thin layer chromatography is developed, detecting positive fractions by an acid hydrolysis-ninhydrin color development method, combining to obtain total cyclosporine, and finally performing reverse phase chromatography on the total cyclosporine, and eluting with methanol with a volume fraction of 80% to obtain cyclosporine A. Wherein the solid culture medium comprises the following components: corn and 0.2% casein peptone, the feed-liquid ratio is 1: 1.

Example 17

Culturing Fusarium (Fusarium sp.) HU0298 in a seed solution, inoculating the Fusarium (Fusarium sp.) HU0298 into a fermentation tank filled with a liquid culture medium, uniformly inoculating the Fusarium (Fusarium sp.) HU0298, culturing to obtain a fermentation culture, leaching the fermentation culture with a leaching solution, filtering and concentrating to obtain an extract, performing silica gel column chromatography on the extract, performing gradient elution with a dichloromethane-methanol mixed solvent with a volume ratio of 100:0 to 80:20, collecting fractions with a specific displacement value of 0.4-0.6 when the dichloromethane-methanol mixed solvent with a volume ratio of 95:5 in silica gel plate thin layer chromatography is developed, detecting positive fractions by an acid hydrolysis-ninhydrin color development method, combining the fractions to obtain total cyclosporine, performing reverse phase chromatography on the total cyclosporine, and washing with 80% methanol by volume fractionRemoving to obtain cyclosporine A. Wherein the liquid culture medium comprises the following components: fructose 30g/L, KH₂PO₄ 6.00g/L、CaCl₂ 0.90g/L、MgSO₄0.90g/L, KCl 0.30.30 g/L, 5.0g/L, MnSO g yeast extract₄·4H₂O 6.70mg/L、ZnSO₄·7H₂O 8.30mg/L、FeSO₄·7H₂O 15.00mg/L、CuSO₄1.50mg/L and CoCl₂11.00mg/L, pH adjusted to 5.4.

Example 18

Carrying out seed liquid culture on Fusarium (Fusarium sp.) HU0298, then inoculating the Fusarium (Fusarium sp.) HU0298 into a fermentation tank filled with a liquid culture medium for uniform inoculation, obtaining a fermentation culture through culture, leaching the fermentation culture with a leaching solution, filtering and concentrating to obtain an extract, then carrying out silica gel column chromatography on the extract, carrying out gradient elution with a dichloromethane-methanol mixed solvent with a volume ratio of 100: 0-80: 20, collecting fractions with a specific displacement value of 0.4-0.6 when the dichloromethane-methanol mixed solvent with a volume ratio of 95:5 is developed in silica gel plate thin layer chromatography, detecting positive fractions through an acid hydrolysis-ninhydrin color development method, combining to obtain total cyclosporine, and finally carrying out reverse phase chromatography on the total cyclosporine, and eluting with methanol with a volume fraction of 80% to obtain cyclosporine A. Wherein the liquid culture medium comprises the following components: 30g/L glucose and 10g/L, K ammonium sulfate₂HPO₄7.5g/L and 1mL/L of trace element solution, and adjusting the pH to 5.5, wherein the trace element solution comprises: ZnSO₄ 4.4g/L、FeSO₄ 5g/L、MnCl₂ 180mg/L、Na₂MO₄25mg/L、CuSO₄80mg/L and H₂SO₄ 2mL。

Example 19

After Fusarium (Fusarium sp.) HU0298 is subjected to seed liquid culture, the Fusarium sp HU0298 is inoculated into a fermentation tank filled with a liquid culture medium to be uniformly inoculated, a fermentation culture is obtained through culture, the fermentation culture is leached by using leaching liquor, filtered and concentrated to obtain extract, then the extract is subjected to silica gel column chromatography, dichloromethane-methanol mixed solvent with the volume ratio of 100:0 to 80:20 is subjected to gradient elution, when dichloromethane-methanol mixed solvent with the volume ratio of 95:5 in silica gel plate thin layer chromatography is collected and developed, the specific displacement value is 0.4-0.6, and the components which are detected to be positive by an acid hydrolysis-ninhydrin color development method are combined to obtain total cyclosporine, finally the total cyclosporine is subjected to reverse phase chromatography, and is eluted by using methanol with the volume fraction of 80%, and cyclosporine A is obtained. Wherein the liquid culture medium comprises the following components: malt extract 2% and yeast extract 0.4%, pH adjusted to 5.4.

In examples 1 to 19, the yield of cyclosporin A in the fermentation 35 days after fermentation culture of Fusarium (Fusarium sp.) HU0298 is shown in Table 4:

TABLE 4 Fusarium (Fusarium sp.) HU0298 yield of cyclosporin A by solid fermentation for 35 days

TABLE 5 yield of Cyclosporin A by liquid fermentation of Fusarium (Fusarium sp.) HU0298 for 35 days

As can be seen from tables 4 and 5, the yield of cyclosporin A in the liquid fermentation product was low and both exceeded 1g/L, and in the single-component solid medium, the yield of cyclosporin A in the fermented corn extract reached 2.36g/kg, while the content of cyclosporin A in the fermented wheat bran and coconut oil cake extracts was significantly higher than that in the fermented corn extract, and the yields of cyclosporin A reached 3.27g/kg and 2.82g/kg, respectively, which were 1.4 times and 1.2 times that of the fermented corn extract. In a multi-component solid culture medium, the yield of the cyclosporine A can be obviously improved by adding the same amount of wheat, wheat bran and coconut oil cake into a corn culture medium, and the yield of the cyclosporine A can reach 3.62g/kg, 3.34g/kg and 3.31g/kg which are respectively 1.5, 1.4 and 1.4 times of that of a corn fermentation extract without adding other raw materials; in addition, the yield of cyclosporin A in the fermentation extract of the combined culture medium with the same amount of wheat bran and coconut oil cake also reaches 3.00 g/kg. In the results, compared with the corn fermentation culture, the wheat bran and the coconut oil cake are used for carrying out fermentation culture on Fusarium (Fusarium sp) HU0298, or the wheat bran, the wheat bran and the coconut oil cake are added into the corn culture medium, so that the yield of the cyclosporine A can be effectively improved, and the production cost is greatly reduced.

The results also showed that there was no significant difference in the production of cyclosporin A when glycerol was added to the corn solid medium as the second carbon source, but when 0.2% yeast extract and 0.1% soybean peptone were added to the corn solid medium as the second nitrogen source, the production of cyclosporin A reached 4.63 and 5.10g/kg, respectively, which were 2.0-fold and 2.16-fold, respectively, of the corn fermentation. It can be seen that the addition of 0.2% yeast extract and 0.1% soy peptone greatly increased the yield of cyclosporin a in Fusarium (Fusarium sp.) HU0298 fermentation.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above embodiments only express a few embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Huizhou college

<120> high-yield cyclosporine plant endophytic fusarium and application thereof

<130> 1

<160> 1

<170> SIPOSequenceListing 1.0

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<211> 591

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<213> Fusarium (Fusarium sp.HU0298 gene sequence)

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tatacaactc atcaaccctg tgaacatacc tataacgttg cctcggcggg aacagacggc 120

cccgtaacac gggccgcccc cgccagagga ccccctaact ctgtttctat aatgtttctt 180

ctgagtaaac aagcaaataa attaaaactt tcaacaacgg atctcttggc tctggcatcg 240

atctttgaac gcacattgcg cccgccagta ttctggcggg catgcctgtt cgagcgtcat 300

atctttgaac gcacattgcg cccgccagta ttctggcggg catgcctgtt cgagcgtcat 360

tacaaccctc aggcccccgg gcctggcgtt ggggatcggc ggaagccccc tgcgggcaca 420

acgccgtccc ccaaatacag tggcggtccc gccgcagctt ccattgcgta gtagctaaca 480

cctcgcaact ggagagcggc gcggccacgc cgtaaaacac ccaacttctg aatgttgacc 540

tcgaatcagg taggaatacc cgctgaactt aagcatatca ataagcggag g 591

Claims (2)

1. A fermentation method of high-yield cyclosporine plant endophytic fusarium is characterized by comprising the following steps:

fusarium (F.), (Fusarium sp.) HU0298 is inoculated into PDA culture medium for activation culture to obtain activated fusarium (F.) (Fusarium sp.) HU0298 strain, wherein the culture temperature is 20-30 ℃, and the activation culture time is 3-5 days;

activated fusarium (f) ((ii))Fusarium sp.) inoculating HU0298 strain into PDB culture medium, and performing shake culture to obtain primary seed liquid, wherein the culture temperature is 20-30 ℃, and the shake culture time is 1-3 days;

inoculating the first-stage seed liquid into a PDB culture medium to be cultured for 1-3 days to obtain a second-stage seed liquid, and then inoculating the second-stage seed liquid into a solid culture medium to be subjected to solid fermentation culture to obtain a solid fermentation product, wherein the solid fermentation culture temperature is 20-30 ℃;

the composition of the solid medium was set as follows:

the solid medium comprises: the component A and distilled water are mixed according to a feed-liquid ratio of 1: 1-5, and the component A is any one of corn, rice, wheat bran and coconut oil cake;

or, the solid medium comprises: the feed-liquid ratio of the component B to the component C to the distilled water is 1:1: 1-5, when the component B is corn, the component C is any one of wheat, wheat bran and coconut oil cake, and when the component B is wheat bran, the component C is coconut oil cake;

or, the solid medium comprises: the feed liquid comprises corn, distilled water and a component D, wherein the component D is any one of glycerol, malt extract, soybean peptone, yeast extract and casein peptone, and the feed liquid ratio of the corn, the distilled water and the component D is 1:1: 0.01-0.05;

wherein the high-yield cyclosporine plant endophytic fusarium is fusarium (F.), (Fusarium sp.) HU0298, and the high-yield cyclosporin-producing plant endophytic fusarium is deposited at 26.1.2021 in Guangdong province, with the deposit number being GDMCC No. 61466.

2. A method for preparing cyclosporine A by high-yield cyclosporine-producing plant endophytic fusarium is characterized by comprising the following steps:

performing fermentation culture by using the fermentation method of the high-yield cyclosporine plant endophytic fusarium of claim 1 to obtain a fermentation culture;

leaching the fermentation culture with a leaching liquor, filtering, and concentrating to obtain an extract, wherein the leaching liquor is 95% ethanol or ethyl acetate;

performing silica gel column chromatography on the extract, performing gradient elution by using a dichloromethane-methanol mixed solvent with a volume ratio of 100: 0-80: 20, collecting fractions with a specific shift value of 0.4-0.6 when the dichloromethane-methanol mixed solvent with a volume ratio of 95:5 is expanded in silica gel plate thin layer chromatography, detecting positive fractions by using an acid hydrolysis-ninhydrin color method, and combining to obtain total cyclosporine;

the total cyclosporin was subjected to reverse phase chromatography and eluted with 80% volume fraction of methanol to give cyclosporin A.

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