A novel non-ribosomal antifungal peptide MaAFP1 and its preparation method and application

Abstract

The present invention discloses a novel non-ribosomal antifungal peptide MaAFP1 and its preparation method and application. The antifungal peptide MaAFP1 of the present invention is obtained by separating and purifying the cell-free supernatant fermentation liquid of Metarhizium anisopliae CQMa421, and is a novel non-ribosomal antifungal heptapeptide that inhibits the growth of various animal and plant pathogenic fungi. The molecular formula of MaAFP1 is C 41 H 74 N 18 O 12 , the amino acid sequence from N-terminus to C-terminus is: (D)Arg‑(L)Arg(OH)‑(D)Orn‑(L)Thr‑(D)Orn‑(L)Arg(OH)‑(D)Tyr. Shake flask fermentation was carried out using Metarhizium anisopliae, and the fermentation broth was collected. The antifungal peptide MaAFP1 was obtained by ion exchange chromatography and high performance liquid chromatography separation and purification. The structure of MaAFP1 was analyzed by a combination of Edman degradation method, mass spectrometry, infrared spectroscopy, nuclear magnetic resonance spectroscopy and circular dichroism spectroscopy. In vitro antibacterial tests found that MaAFP1 has a good inhibitory effect on a variety of animal and plant pathogenic fungi, has value in the development of new antibacterial drugs, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a novel non-ribosomal antifungal peptide MaAFP1 and a preparation method and application thereof. Background Art

[0002] Pathogenic fungi or opportunistic fungi can infect human skin and deep organs, cause plant diseases, contaminate food and feed, and seriously affect human health and production and life. People with low immunity or immunodeficiency due to AIDS, organ transplantation, etc. are extremely susceptible to pathogenic fungi, and the mortality rate is extremely high. Crop diseases caused by fungi can lead to a decrease in crop yields. When food and feed are contaminated by fungi, they will cause food and feed to spoil, and some strains will also produce mycotoxins, which will harm the health of humans and livestock. Currently, there are few medical or agricultural antifungal drugs available, and the antibacterial range is narrow. Long-term use of most antifungal drugs leads to increased drug resistance of pathogens, and the dosage used is constantly increasing. In addition, many antifungal drugs have strong toxic side effects. Therefore, there is an urgent need for new safe, efficient and broad-spectrum antifungal drugs.

[0003] Insect pathogenic fungi can produce a large number of natural products with antibacterial, insecticidal, anticancer, and immune-regulating activities that act on insects, plants, and other microorganisms to better adapt to environmental changes and occupy habitats, such as beauvericin and terpenoid compounds fumosorinone. Whole genome sequencing and analysis found that many types of Metarhizium in the model insect pathogenic fungus Metarhizium, such as Metarhizium scarabensis and Metarhizium robertsii, have more than 60 secondary metabolic biosynthetic clusters. However, only a few bioactive substances have been found in Metarhizium, indicating that the natural product resources of insect pathogenic fungi are rich and worthy of study, and are important microbial resources for the development of new drug molecules.

[0004] The Metarhizium anisopliae CQMa421 used in the present invention has been used on a large scale as a microbial insecticide for agricultural pest control. It was found in the application that it has the effect of inhibiting the growth of pathogenic fungi on plant leaves, and a non-ribosomal antifungal peptide MaAFP1 was further separated and purified from its fermentation broth. After testing, the non-ribosomal peptide exhibited a broad spectrum of activity in inhibiting fungal germination and growth. Summary of the invention

[0005] In view of this, one of the purposes of the present invention is to provide a novel non-ribosomal antifungal peptide MaAFP1, with a molecular formula of C 41 H 74 N 18 O 12, molecular weight 1010.575Da, amino acid sequence from N-terminus to C-terminus: (D)Arg-(L)Arg(OH)-(D)Orn-(L)Thr-(D)Orn-(L)Arg(OH)-(D)Tyr, where (L)Arg(OH) is L-type γ-hydroxyarginine, (D)Arg is D-type arginine, (D)Orn is D-type ornithine, (L)Thr is L-type threonine, and (D)Tyr is D-type tyrosine. MaAFP1 is a linear molecule (with free amino and carboxyl termini) with two unnatural amino acids ((L)γ-hydroxyarginine and (D)ornithine) and unnatural D-type configurations of natural amino acids ((D)arginine and (D)tyrosine), with D-type and L-type amino acids arranged alternately. MaAFP1 is a white powder, which gradually turns into dew-like colorless transparent droplets after being exposed to the air for several minutes. It is stable in acidic and alkaline solutions, and has no loss of activity after digestion with α-chymotrypsin and proteinase K. There is no obvious loss of activity after boiling at 100°C for 30 minutes. It is easily soluble in water and methanol, but insoluble in organic solvents such as ethyl acetate, acetone and dichloromethane. MaAFP1 powder can be stored at room temperature, sealed, dry and away from light.

[0006] Preferably, the antifungal peptide MaAFP1 is isolated and purified from the fermentation broth of Metarhizium anisopliae CQMa421 strain, and the CQMa421 strain is deposited in China General Microbiological Culture Collection Center (CGMCC) with the strain collection number CGMCC NO.4609.

[0007] Preferably, the separation and purification method comprises subjecting the fermentation broth to centrifugation, ion exchange chromatography and high performance liquid chromatography (HPLC), and performing step-by-step separation and purification at the ultraviolet absorption peak at 280 nm.

[0008] The second object of the present invention is to provide an antibacterial agent, which contains the antifungal peptide MaAFP1.

[0009] The third object of the present invention is to provide a method for preparing the antifungal peptide MaAFP1, comprising the following steps:

[0010] 1) Preparation of fermentation broth: A spore suspension of Metarhizium anisopliae CQMa421 was inoculated into a 1 / 4SDAY liquid medium with a pH of 6.5 to a final concentration of 5×10 6 spores / ml, and cultured at 28°C and 200rpm for 36 hours to obtain the fermentation liquid; the preservation number of the green muscardine fungus CQMa421 is: CGMCC NO.4609; Alternaria alternata is used as the indicator bacteria for fermentation; note that the liquid volume should be 20%.

[0011] 2) Preparation of crude antifungal peptides: centrifuging the fermentation broth obtained in step 1) and collecting the supernatant of the fermentation broth; adding a pH 5.6 citric acid-sodium phosphate buffer to the supernatant to a concentration of 20 mM, incubating in a boiling water bath for 30 minutes, transferring to 4°C and standing overnight, filtering using filter paper and a Φ=0.22 μm filter membrane in turn to obtain a filtrate; separating and purifying the filtrate using ion exchange chromatography, the separation and purification method comprising: connecting a chromatography column of SP Sepharose resin to a column having a concentration of 20 mM; The detector wavelength was set to 280 nm, and the resin was equilibrated with a 20 mM, pH 5.6, citric acid-sodium phosphate buffer. The filtrate was circulated and loaded at a flow rate of 0.5 mL / min. After loading, the resin was continuously washed with the equilibration buffer until the ultraviolet absorbance value remained constant. Then, a 20 mM, pH 5.6 citric acid-sodium phosphate buffer containing 1 M sodium chloride was used as an eluent. The parameters of the mixed eluent and the equilibration solution were adjusted so that the proportion of the eluent increased from 0% to 100% within 60 column bed volumes, and the component with an elution time of 139.1 min was collected. The crude antimicrobial peptide was obtained by freeze drying.

[0012] 3) Preparation of pure antifungal peptide: Using high performance liquid chromatography to separate and purify the crude antifungal peptide obtained in step 2), the separation and purification method includes: using ultrapure water and methanol containing 0.1% TFA as mobile phases, and a C18 reverse phase chromatography column as a stationary phase; setting the program to 15% methanol at 0 min, 17% methanol at 5 min, 27% methanol at 45 min, and 100% methanol at 50 min; injecting 500 μL each time, with a flow rate of 2 mL / min; the detection wavelength is 280 nm; collecting the component with a retention time of 29.4 min; and freeze-drying to obtain the pure antifungal peptide MaAFP1.

[0013] A fourth object of the present invention is to provide the use of the antifungal peptide MaAFP1 in the preparation of antifungal products.

[0014] Preferably, the product comprises medicine, cosmetics, toiletries, food packaging, antimicrobial coating or pesticide.

[0015] Preferably, the fungi in the antifungal agent include Aspergillus spp., Fusarium spp., Mucor racemosus, Candida albicans, Cryptococcus neoformans, Alternaria spp., Trichophytonmentagrophytes, Sclerotinia sclerotiorum, Botrytis cinerea, Botryosphaeria dothidea, Colletotrichumfioriniae, Pestalotiopsis microspora and Magnaporthe oryzae.

[0016] The antifungal peptide MaAFP1 of the present invention is a novel non-ribosomal antifungal heptapeptide obtained by separation and purification from the cell-free supernatant fermentation broth of Metarhizium anisopliae CQMa421, which can inhibit the growth of various animal and plant pathogenic fungi. The molecular formula of MaAFP1 is C 41 H 74 N 18 O 12The amino acid sequence from N-terminus to C-terminus is: (D)Arg-(L)Arg(OH)-(D)Orn-(L)Thr-(D)Orn-(L)Arg(OH)-(D)Tyr. The antifungal peptide MaAFP1 was obtained by shake flask fermentation using Metarhizium anisopliae, and the fermentation broth was collected and purified by ion exchange chromatography and high performance liquid chromatography. The structure of MaAFP1 was analyzed by comprehensive use of Edman degradation method, mass spectrometry, infrared spectroscopy, nuclear magnetic resonance spectroscopy and circular dichroism spectroscopy. The in vitro antibacterial test showed that MaAFP1 had a good inhibitory effect on a variety of animal and plant pathogenic fungi, especially on the spore germination and hyphae growth of animal pathogenic fungi such as Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Mucor racemosus, Trichophytonmentagrophytes, Fusarium verticillioides, Candida albicans and Cryptococcusneoformans; and had a strong inhibitory effect on Alternariaalternata, Alternaria solani, Alternaria tenuissima, Botrytis cinerea, Botryosphaeria dothidea, Colletotrichum fioriniae, Fusarium fujikuroi, Fusariumgraminearum, Fusarium oxysporum, Pestalotiopsis microspora, Magnaporthe oryzae and Sclerotinia sclerotiorum and other plant pathogenic fungi have a strong inhibitory effect on spore germination and hyphae growth; do not inhibit yeasts such as Pichia pastoris and Saccharomyces cerevisiae and bacteria such as Escherichia coli, Erwinia carotovora, Pseudomonas syringae and Bacillus subtilis; in vitro, it has no hemolytic effect on mouse and human blood cells. The antifungal peptide MaAFP1 of the present invention exhibits a broad spectrum of fungal growth inhibition activity, has significant advantages, has the value of new antifungal drug development, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1The ultraviolet absorption peak diagram of the separation and purification process of the antifungal peptide MaAFP1, wherein A is the ultraviolet absorption peak diagram of ion exchange chromatography, and B is the ultraviolet absorption peak diagram of high performance liquid chromatography;

[0018] Figure 2 The spectrum of MaAFP1 analyzed by mass spectrometry, infrared spectrum and Edman, wherein A, analytical liquid chromatography is used to detect the purity of purified MaAFP1, B, mass spectrometry is used to determine the molecular weight of MaAFP1, C, infrared spectrum is used to determine the functional groups of MaAFP1, and D, Edman degradation method is used to analyze the peptide sequence of MaAFP1;

[0019] Figure 3 2D NMR 1 H, 1 H-COSY spectrum;

[0020] Figure 4 2D NMR 1 H, 13 C-HSQC spectrum;

[0021] Figure 5 2D NMR 1 H, 13 C-HMBC spectrum;

[0022] Figure 6 Determination of amino acid configuration diagrams by circular dichroism spectroscopy;

[0023] Figure 7 This is the secondary mass spectrum of MaAFP1. DETAILED DESCRIPTION

[0024] The present invention will be described in detail below in conjunction with examples, which are merely illustrative and are not intended to limit the scope of application of the present invention. The present invention is not limited to the following embodiments or examples, and any modifications and variations made without violating the spirit of the present invention should be included within the scope of the present invention. The reagents or materials described in the examples are commercially available unless otherwise specified.

[0025] Experimental Example 1: Isolation and purification of antifungal peptide MaAFP1

[0026] 1. Liquid fermentation culture of CQMa421 to produce MaAFP1

[0027] Bacterial species: Metarhizium anisopliae CQMa421, which has been deposited in the China General Microbiological Culture Collection Center (CGMCC), and the strain collection number is CGMCC NO.4609; this strain has an authorized patent and is used to control the rice pest Rice leaf folder (patent number: CN201110105462.6).

[0028] Methods: A final concentration of 5×10 6 The spore suspension of Metarhizium anisopliae CQMa421 with a concentration of 100 / ml was filled with 20% of the liquid, and then cultured at 28°C and 200 rpm for 36 hours to obtain the fermentation liquid. Note: Alternaria alternata was used as the indicator bacteria.

[0029] 2. Ion exchange chromatography to separate active substances

[0030] The fermentation broth was centrifuged at 10000rpm for 10min and the supernatant of the fermentation broth was collected. Citric acid-sodium phosphate buffer (pH5.6) was added to the supernatant of the fermentation broth to a concentration of 20mM, and then placed in a boiling water bath and incubated for 30 minutes. The supernatant of the fermentation broth was then transferred to 4°C and allowed to stand overnight, and then filtered using filter paper and filter membrane (Φ=0.22μm) to obtain the filtrate. The supernatant of the fermentation broth after purification using cation exchange resin (SP Sepharose) was the filtrate. A chromatography column containing 5 ml of SP Sepharose resin was connected to plus, use 20mM citric acid-sodium phosphate buffer (pH5.6) to equilibrate the resin. Circulate 1 liter of culture medium at a flow rate of 0.5mL / min. After loading, continue to wash the resin with the equilibration buffer until the UV absorbance value remains unchanged. Use 20mM, pH5.6 citric acid-sodium phosphate buffer (containing 1M sodium chloride) as the eluent, adjust the parameters of the mixed eluent and the equilibration liquid so that the proportion of the eluent increases from 0% to 100% within 60 column bed volumes. Collect one tube for every 2mL of eluent until the UV detection curve returns to the baseline level. The UV absorption peak during the separation process is as follows Figure 1 As shown in A, the arrow-marked MaAFP1 is the active peak, and the elution time is 139.1 min. All the collected solutions under this peak are combined and freeze-dried using a freeze dryer to obtain a crude antimicrobial peptide.

[0031] 3. Separation of target substance MaAFP1 by HPLC

[0032] The crude antimicrobial peptide obtained by ion exchange was purified using high performance liquid chromatography. Ultrapure water (containing 0.1% TFA) and methanol (containing 0.1% TFA) were used as mobile phases, and a C18 reverse phase column (10mm×250mm) was used as the stationary phase. The detection wavelength was set to 280nm. The program was set as follows: 0min, 15% methanol; 5min, 17% methanol; 45min, 27% methanol; 50min, 100% methanol; 500μL was injected each time; the flow rate was maintained at 2mL / min. The UV absorption peak diagram during the separation and purification process is shown in the figure below. Figure 1 As shown in B, the arrow indicates the active peak with a retention time of 29.4 min. The active peak components were collected and then freeze-dried to obtain MaAFP1 powder.

[0033] Experimental Example 2: Structural identification of MaAFP1

[0034] 1. Determination of the exact molecular weight of MaAFP1 by mass spectrometry

[0035] The purity of MaAFP1 obtained by the above separation and purification was 99% ( Figure 2 A) The molecular weight of MaAFP1 was determined using an orbitrap mass spectrometer Q Exactive Plus. Figure 2 As shown in Figure B, four main peaks were observed in the positive ion mode, with m / z of 253.651, 337.866, 506.296, and 1011.583, which were calculated to be the same molecules with 4, 3, 2, and 1 charges, respectively, indicating that MaAFP1 has at least 4 primary amino groups. The exact molecular weight of MaAFP1, 1010.575 Da, was obtained by deducting the mass of hydrogen ions from 1011.583.

[0036] 2. Sequence analysis by infrared spectroscopy and Edman degradation

[0037] The main functional groups of MaAFP1 were determined using an infrared spectrometer. 1 mg of MaAFP1 powder was taken, mixed with MaAFP1 and potassium bromide, pressed into tablets, and then dried under an infrared lamp. The test results are as follows: Figure 2 C, respectively at 617.72, 839.38, 1384.24, 1636.57, 2850.80, 2919.26, 3446.01cm -1 There are absorption peaks at 3446.01 and 1636.57 cm -1 The strongest absorption peaks are at 3446.01cm, which are speculated to be the secondary amine and carbonyl groups in the amide bond. -1 It may also contain primary amino groups.

[0038] The Edman degradation method was further used to determine the sequence of MaAFP1 from the N-terminus. 2 mg of MaAFP1 was dissolved in water, spotted onto a glass fiber board and fixed thereon, the fiberboard was soaked in a 12% trimethylamine solution, 100 μL of phenyl isothiocyanate was added, nitrogen was introduced to replace the air, and the peptide was allowed to stand at 40°C for 90 minutes to convert the peptide into an phenylaminothiocarboxylate peptide. 200 ml of 100% trifluoroacetic acid was added and kept at 40°C for 15 minutes to cleave the N-terminal residue in the form of 2-phenylaminothiazolinone. The trifluoroacetic acid concentration was diluted to 25% to convert it into a stable phenylhydantoin thiourea derivative (PTH-AA). PTH-AA was then transported to a high performance liquid chromatograph and detected using a Wako-Pack WS-PTH Column II (4.6×250 mm). The results are shown in the figure. Figure 2 As shown in D, it is shown that MaAFP1 is a symmetrical peptide with the sequence of Arg-X-Trp-Thr-Trp-X-Tyr, in which X has no signal and is presumed to be an unnatural amino acid.

[0039] 3. 2D NMR spectroscopy analysis of MaAFP1 sequence

[0040] Combination 1 H, 1 H-COSY, 1 H, 13 C-HSQC and 1 H, 13 Analyze the side chain structure of the residues by two-dimensional NMR spectroscopy such as C-HMBC. Take 50 mg of MaAFP1 powder and dissolve it in 600 μL of deuterated dimethyl sulfoxide. Transfer the solution to an NMR tube and use a 600 MHz NMR spectrometer to measure 1 H, 1 H-COSY, 1 H, 13 C-HSQC and 1 H, 13 C-HMBC. Then add 50 μl of heavy water to the NMR tube, shake and mix, and measure again using a 600 MHz NMR spectrometer. 1 H, 1 H-COSY, to determine the chemical shift of active hydrogen. Figure 3 , Figure 4 , Figure 5 Determination 1 H, 1 H-COSY, 1 H, 13 C-HSQC and 1 H, 13The spectrum of C-HMBC shows that MaAFP1 contains arginine (Arg), ornithine (Orn), threonine (Thr), tyrosine (Tyr) and γ-hydroxyarginine (Arg(OH)), but no tryptophan (Trp). Because the side chain end of Orn has a primary amino group, it can react with phenyl isothiocyanate in the Edman degradation method, thereby increasing the retention time in liquid chromatography, and is therefore misidentified as Trp.

[0041] 4. Circular dichroism spectroscopy analysis of amino acid configuration

[0042] Take a hard glass tube, weigh 1 mg of MaAFP1 into it, and add 1 ml of 6M hydrochloric acid. Pass nitrogen and seal it. Put the glass tube into an oil bath and heat it at 110℃ for 12 hours. Take out the hydrolyzate and add sodium hydroxide to adjust the pH to 8.0. Add potassium borate buffer (pH8.5) with a final concentration of 50mM and 2 times the molar amount of 2,4-dinitrofluorobenzene (DNFB), and place it in a water bath at 60℃ for 40 minutes. Add 50 microliters of 1M hydrochloric acid to acidify the reaction solution to terminate the reaction. At the same time, use the above-mentioned markers to mark the purchased L and D types of arginine, ornithine, tyrosine and threonine respectively. Use a semi-preparative high performance liquid chromatograph to separate the labeled amino acid standard (DNB-AA), and then separate the labeled hydrolyzate; and judge the type of amino acid corresponding to each retention time in the hydrolyzate by retention time. The spectra of each amino acid in the standard amino acid and the hydrolyzed sample in the range of 200-400nm are measured by circular dichroism. By comparing the spectra of samples and standards ( Figure 6 ) It is known that Tyr, Orn and Arg are D-type, and Thr is L-type; there is no standard for Arg(OH), but after comparing its spectrum with that of arginine, it was determined to be L-type.

[0043] 5. Confirmation of amino acid sequence by secondary mass spectrometry

[0044] Take 1 microgram of MaAFP1, dissolve it in ultrapure water, and use the orbital trap mass spectrometer Q Exactive Plus for detection and analysis. Select the parent ion with m / z of 1011.58371 and apply 20 eV voltage for fragmentation. Figure 7 ) were identified, and the sequence of MaAFP1 was confirmed to be: (D)Arg-(L)Arg(OH)-(D)Orn-(L)Thr-(D)Orn-(L)Arg(OH)-(D)Tyr.

[0045] Example 3: Determination of the half inhibitory concentration and minimum inhibitory concentration of MaAFP1 against pathogens

[0046] Use ultrapure water to prepare different concentrations of MaAFP1, mix it with molten PDA medium, and prepare plates. Make a suspension of pathogenic bacteria spores and apply it on the plate, incubate it at 28℃, and measure its germination rate every 1 hour. Also drop the cell suspension on the plate, incubate it at 28℃ for 3 days, and measure its colony diameter. Fit the concentration-diameter / germination rate curve, and calculate the minimum inhibitory concentration (MIC) that inhibits the germination of half of the fungal spores according to the curve equation. 50 ).

[0047] The minimum inhibitory concentration (MIC) of the antifungal peptide MaAFP1 against common animal pathogenic fungi and bacteria 50 ) As shown in Table 1, the minimum inhibitory concentration (MIC) of common plant pathogenic fungi 50 ) are shown in Table 2. The results showed that the antifungal peptide MaAFP1 had a strong inhibitory effect on the spore germination and mycelial growth of animal pathogenic fungi Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Mucor racemosus, Trichophyton mentagrophytes, Fusarium verticillioides, Candida albicans and Cryptococcus neoformans; and on plant pathogenic fungi Alternaria alternata, Alternaria solani, Alternaria tenuissima, Botrytis cinerea, Botryosphaeria dothidea, Colletotrichum fioroniae, Fusarium fujikuroi, Fusarium graminearum, Fusariumoxysporum, Pestalotiopsis microspora, Magnaporthe oryzae, and Sclerotiniasclerotiorum, and had a strong inhibitory effect on the spore germination and hyphae growth; it did not inhibit yeasts, such as Pichiapastoris and Saccharomyces cerevisiae, and bacteria, such as Escherichia coli, Erwiniacarotovora, Pseudomonas syringae, and Bacillus subtilis. The hemolysis experiment also showed that MaAFP1 had no hemolytic effect on mouse and human red blood cells. It can be seen that the antifungal peptide MaAFP1 has a broad-spectrum antibacterial activity against filamentous pathogenic fungi and has broad application prospects.

[0048] Table 1 Minimum inhibitory concentration of MaAFP1 against animal pathogenic fungi and bacteria

[0049]

[0050] Table 2 Minimum inhibitory concentration of MaAFP1 against plant pathogenic fungi

[0051]

[0052]

[0053] The conventional techniques and schemes not described in detail in the above embodiments are well known in the art, so they will not be described in detail here. The above embodiments and / or experimental examples describe the preferred embodiments of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, the technical scheme of the present invention can be subjected to a variety of simple modifications, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A novel non-ribosomal antifungal peptide MaAFP1, characterized in that: The molecular formula is: C 41 H 74 N 18 O 12 The amino acid sequence from N-terminus to C-terminus is: (D)Arg-(L)Arg(OH)-(D)Orn-(L)Thr-(D)Orn-(L)Arg(OH)-(D)Tyr, wherein (L)Arg(OH) is L-type γ-hydroxyarginine.

2. The antifungal peptide MaAFP1 according to claim 1, characterized in that The antifungal peptide MaAFP1 is isolated and purified from the fermentation broth of Metarhizium anisopliae CQMa421 strain, and the CQMa421 strain is preserved in the China General Microbiological Culture Collection Center, and the culture collection number is CGMCC NO.4609.

3. The antifungal peptide MaAFP1 according to claim 2, characterized in that The separation and purification method comprises subjecting the fermentation broth to centrifugation, ion exchange chromatography and high performance liquid chromatography, and performing step-by-step separation and purification at the ultraviolet absorption peak of 280 nm.

4. An antibacterial agent, characterized in that Contains the antifungal peptide MaAFP1 according to claim 1.

5. A method for preparing the antifungal peptide MaAFP1 according to claim 1, characterized in that: The steps include: 1) Preparation of fermentation broth: A spore suspension of Metarhizium anisopliae CQMa421 was inoculated into a 1 / 4SDAY liquid medium with a pH of 6.5 to a final concentration of 5×10 6 spores / ml, and cultured at 28°C and 200rpm for 36h to obtain a fermentation liquid; the preservation number of the green muscardine fungus CQMa421 is: CGMCC NO.4609; 2) Preparation of crude antimicrobial peptides: centrifuge the fermentation broth obtained in step 1) and collect the supernatant of the fermentation broth; add pH 5.6 citric acid-sodium phosphate buffer to the supernatant to a concentration of 20 mM, incubate in a boiling water bath for 30 minutes, transfer to 4°C and stand overnight, and then filter using filter paper and a Φ=0.22 μm filter membrane in turn to obtain a filtrate; The filtrate is separated and purified by ion exchange chromatography, and the separation and purification method comprises: connecting a chromatography column of SP Sepharose resin to prime plus, set the detector wavelength to 280nm, use 20mM, pH5.6 citric acid-sodium phosphate buffer to balance the resin, circulate the filtrate at a flow rate of 0.5mL / min, and continue to wash the resin with the balance buffer until the ultraviolet absorbance value remains unchanged after the loading is completed, and then use 20mM, pH5.6 citric acid-sodium phosphate buffer containing 1M sodium chloride as the eluent, adjust the parameters of the mixed eluent and the balance solution so that the proportion of the eluent increases from 0% to 100% within 60 column bed volumes, and collect the component with an elution time of 139.1min; obtain the crude antimicrobial peptide by freeze drying; 3) Preparation of pure antimicrobial peptide: The crude antimicrobial peptide obtained in step 2) was separated and purified using high performance liquid chromatography, and the separation and purification method included: using ultrapure water and methanol containing 0.1% TFA as mobile phases and a C18 reverse phase chromatography column as a stationary phase; setting the program to 15% methanol at 0 min, 17% methanol at 5 min, 27% methanol at 45 min, and 100% methanol at 50 min, injecting 500 μL each time, with a flow rate of 2 mL / min; the detection wavelength was 280 nm; the component with a retention time of 29.4 min was collected; and freeze-drying was performed to obtain the pure antifungal peptide MaAFP1.

6. Use of the antifungal peptide MaAFP1 according to claim 1 in the preparation of antifungal products.

7. The use according to claim 6, characterized in that The products include medicines, cosmetics, toiletries, food packaging, antimicrobial coatings or pesticides.

8. The use according to claim 7, characterized in that The fungi in the antifungal agent include Aspergillus spp., Fusarium spp., Mucor racemosus, Candida albicans, Cryptococcus neoformans, Alternaria spp., Trichophyton mentagrophytes, Sclerotinia sclerotiorum, Botrytis cinerea, Botryosphaeria dothidea, Colletotrichum fioriniae, Pestalotiopsis microspora and Magnaporthe oryzae.

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