Novel non-ribosomal antifungal peptide maafp1, preparation method therefor and use thereof

Abstract

Provided are a non-ribosomal antifungal peptide MaAFP1, a preparation method therefor and a use thereof. The molecular formula of the antifungal peptide MaAFP1 is C41H74N18O12, wherein an amino acid sequence from the N-terminus to the C-terminus is (D)Arg-(L)Arg(OH)-(D)Orn-(L)Thr-(D)Orn-(L)Arg(OH)-(D)Tyr. Fermentation is performed by using Metarhizium anisopliae CQMa421, a fermentation broth is collected, and separation and purification are performed by means of ion exchange chromatography and high performance liquid chromatography so as to obtain the antifungal peptide MaAFP1. The structure of MaAFP1 is analyzed by comprehensively using an Edman degradation method, mass spectrometry, infrared spectroscopy, nuclear magnetic resonance spectroscopy, and circular dichroism spectroscopy. By performing an in vitro bacteriostatic test, it is found that MaAFP1 has a good inhibitory effect on a variety of animal and plant pathogenic fungi, and has the value of the development of a novel antibacterial drug.

Description

A novel nonribosomal antifungal peptide, MaAFP1, its preparation method and application Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a novel non-ribosomal antifungal peptide MaAFP1, its preparation method, and its applications. Background Technology

[0002] Pathogenic or opportunistic fungi can infect human skin and deep organs, cause plant diseases, and contaminate food and feed, seriously affecting human health and daily life. Individuals with weakened or compromised immune systems, such as those with HIV / AIDS or organ transplants, are highly susceptible to pathogenic fungi and experience extremely high mortality rates. Fungal diseases in crops can lead to reduced yields. Fungal contamination of food and feed can cause spoilage and deterioration; some strains can even produce mycotoxins, harming the health of humans and livestock. Currently, the number of available medical or agricultural antifungal drugs is limited, and their antifungal spectrum is narrow. Long-term use of most antifungal drugs leads to increased drug resistance in pathogens, requiring increasingly higher dosages. Furthermore, many antifungal drugs have strong toxic side effects. Therefore, there is an urgent need for safe, effective, and broad-spectrum new antifungal drugs.

[0003] Insectivorous fungi can produce a large number of natural products with antibacterial, insecticidal, anticancer, and immunomodulatory activities, which act on insects, plants, and other microorganisms to better adapt to environmental changes and occupy habitats. Examples include beauveria bassiana and the terpenoid compound fumosarinone. Whole-genome sequencing and analysis have revealed that several species of Metarhizium anisopliae, such as *Metarhizium anisopliae* and *Metarhizium anisopliae*, possess more than 60 secondary metabolic biosynthetic clusters. However, the fact that only a few bioactive substances have been found in *Metarhizium anisopliae* indicates that the natural product resources of entomopathogenic fungi are abundant and worthy of study, making them an important microbial resource for developing novel drug molecules.

[0004] The *Metarhizium anisopliae* CQMa421 used in this invention has been widely used as a microbial insecticide for agricultural pest control. In its application, it has been found to inhibit the growth of pathogenic fungi on plant leaves. Furthermore, a non-ribosomal antifungal peptide, MaAFP1, was isolated and purified from its fermentation broth. Testing showed that this non-ribosomal peptide exhibited broad-spectrum activity inhibiting fungal germination and growth. Summary of the Invention

[0005] Therefore, one objective of this invention is to provide a novel non-ribosomal antifungal peptide, MaAFP1, with the molecular formula C 41 H 74 N 18 O 12The molecular weight is 1010.575 Da. The amino acid sequence from the N-terminus to the C-terminus is: (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 terms), containing two non-natural amino acids ((L)γ-hydroxyarginine and (D)ornithine) and the non-natural configuration of natural amino acids (D-type arginine and (D)tyrosine), with D-type and L-type amino acids alternating. MaAFP1 is a white powder that gradually turns into colorless, transparent droplets in the form of dewdrops after being exposed to air for several minutes. It is stable in acidic and alkaline solutions, and its activity is not lost after digestion with α-chymotrypsin and proteinase K. There is no significant loss of activity after boiling at 100°C for 30 minutes. It is readily 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 in a sealed, dry, and light-protected environment.

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

[0007] Preferably, the separation and purification method includes centrifuging, ion exchange chromatography, and high performance liquid chromatography (HPLC) of the fermentation broth, followed by stepwise separation and purification at the ultraviolet absorption peak of 280 nm.

[0008] A second objective of this invention is to provide an antibacterial agent containing the aforementioned antifungal peptide MaAFP1.

[0009] A third objective of this invention is to provide a method for preparing the above-mentioned antifungal peptide MaAFP1, comprising the following steps:

[0010] 1) Fermentation broth preparation: Inoculate a suspension of Metarhizium anisopliae CQMa421 spores into 1 / 4 SDAY liquid medium at pH 6.5 to a final concentration of 5 × 10⁻⁶. 6 Fermentation broth was obtained by incubating 1 spore / mL at 28℃ and 200rpm for 36h; the preservation number of the Metarhizium anisopliae CQMa421 was CGMCC NO.4609; Alternaria alternata was used as the indicator strain for fermentation; note that the liquid volume should be 20%.

[0011] 2) Preparation of crude antifungal peptide: Centrifuge the fermentation broth obtained in step 1) and collect the supernatant; add citrate-sodium phosphate buffer (pH 5.6) to the supernatant to a concentration of 20 mM, incubate in a boiling water bath for 30 minutes, then transfer to 4°C and let stand overnight. Filter the solution sequentially using filter paper and a 0.22 μm filter membrane to obtain the filtrate; purify the filtrate using ion exchange chromatography, the purification method including connecting an SP Sepharose resin column to… Using Prime Plus, the detector wavelength was set to 280 nm. The resin was equilibrated with 20 mM citrate-sodium phosphate buffer at pH 5.6. The filtrate was circulated and loaded onto the resin at a flow rate of 0.5 mL / min. After loading, the resin was washed with equilibration buffer until the UV absorbance remained constant. Then, 20 mM citrate-sodium phosphate buffer containing 1 M sodium chloride at pH 5.6 was used as the eluent. The parameters were adjusted to mix the eluent and equilibration buffers, increasing the eluent percentage from 0% to 100% within 60 column volumes. The fraction eluted for 139.1 min was collected. The crude antimicrobial peptide was obtained by freeze-drying.

[0012] 3) Preparation of pure antifungal peptide: The crude antifungal peptide obtained in step 2) was separated and purified by high performance liquid chromatography. The separation and purification method included: using ultrapure water and methanol containing 0.1% TFA as the mobile phase, and a C18 reversed-phase column as the stationary phase; setting the program as follows: 0 min methanol 15%, 5 min methanol 17%, 45 min methanol 27%, 50 min methanol 100%; each injection was 500 μL, the flow rate was 2 mL / min; the detection wavelength was 280 nm; the fraction with a retention time of 29.4 min was collected; and the antifungal peptide MaAFP1 was obtained by freeze drying.

[0013] The fourth objective of this invention is to provide the application of the above-mentioned antifungal peptide MaAFP1 in the preparation of antifungal products.

[0014] Preferably, the product includes pharmaceuticals, cosmetics, toiletries, food packaging, antibacterial coatings, or pesticides.

[0015] Preferably, the fungi in the antifungal agents 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 Magnaphorthe oryzae.

[0016] The antifungal peptide MaAFP1 of this invention is a novel non-ribosomal antifungal heptapeptide that inhibits the growth of various pathogenic fungi of animals and plants, obtained by isolating and purifying the cell-free supernatant fermentation broth of Metarhizium anisopliae CQMa421. The molecular formula of MaAFP1 is C 41 H 74 N 18 O 12The amino acid sequence from the N-terminus to the C-terminus is: (D)Arg-(L)Arg(OH)-(D)Orn-(L)Thr-(D)Orn-(L)Arg(OH)-(D)Tyr. Shake-flask fermentation was performed using *Metarhizium anisopliae*, and the fermentation broth was collected. The antifungal peptide MaAFP1 was obtained by separation and purification using ion exchange chromatography and high-performance liquid chromatography. The structure of MaAFP1 was determined by a combination of methods including the Edman degradation method, mass spectrometry, infrared spectroscopy, nuclear magnetic resonance spectroscopy, and circular dichroism spectroscopy. In vitro antibacterial tests revealed that MaAFP1 exhibits good inhibitory effects against various animal and plant pathogenic fungi, particularly against spore germination and hyphal growth of animal pathogenic fungi such as *Aspergillus flavus*, *Aspergillus fumigatus*, *Aspergillus nidulans*, *Aspergillus niger*, *Mucor racemosus*, *Trichophyton mentagrophytes*, *Fusarium verticillioides*, *Candida albicans*, and *Cryptococcus neoformans*. It also showed strong inhibitory effects on *Alternaria alternata*, *Alternaria solani*, *Alternaria tenuissima*, *Botrytis cinerea*, *Botryosphaeria dothidea*, *Colletotrichum fioriniae*, *Fusarium fujikuroi*, *Fusarium graminearum*, *Fusarium oxysporum*, *Pestalotiopsis microspora*, *Magnaporthe oryzae*, and *Sclerotinia*. It exhibits strong inhibitory effects on the spore germination and hyphal growth of plant pathogenic fungi such as *Sclerotiorum*; it does not inhibit yeasts such as *Pichia pastoris* and *Saccharomyces cerevisiae*, nor bacteria such as *Escherichia coli*, *Erwinia carotovora*, *Pseudomonas syringae*, and *Bacillus subtilis*; and it shows no hemolytic activity against mouse and human blood cells in vitro. The antifungal peptide MaAFP1 of this invention exhibits broad-spectrum inhibitory activity against fungal growth, demonstrating significant advantages and possessing value for the development of novel antifungal drugs with broad application prospects. Attached Figure Description

[0017] Figure 1 shows the UV absorption peaks during the isolation and purification process of the antifungal peptide MaAFP1, where A is the UV absorption peak of ion exchange chromatography and B is the UV absorption peak of high performance liquid chromatography.

[0018] Figure 2 shows the mass spectrometry, infrared spectroscopy, and Edman degradation spectroscopy of MaAFP1. In the figure, A is the determination of the purity of purified MaAFP1 by analytical liquid chromatography, B is the determination of the molecular weight of MaAFP1 by mass spectrometry, C is the determination of the functional groups of MaAFP1 by infrared spectroscopy, and D is the determination of the peptide sequence of MaAFP1 by Edman degradation method.

[0019] Figure 3 shows the two-dimensional nuclear magnetic resonance (NMR) measurement. 1 H, 1 H-COSY spectrum;

[0020] Figure 4 shows the two-dimensional nuclear magnetic resonance (NMR) measurement. 1 H, 13 C-HSQC spectra;

[0021] Figure 5 shows the two-dimensional nuclear magnetic resonance (NMR) measurement. 1 H, 13 C-HMBC spectrum;

[0022] Figure 6 shows the amino acid configuration determined by circular dichroism spectroscopy.

[0023] Figure 7 shows the secondary mass spectrum of MaAFP1. Detailed Implementation

[0024] The present invention will be described in detail below with reference to embodiments. These embodiments are for illustrative purposes only 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. Any modifications and variations made without departing from the spirit of the present invention should be included within the scope of the present invention. Unless otherwise specified, the reagents or materials mentioned in the embodiments are commercially available.

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

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

[0027] The strain is Metarhizium anisopliae var. anisopliae CQMa421, which has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with the accession number CGMCC NO.4609. This strain has been granted a patent and is used to control the rice leaf roller, a rice pest (patent number: CN201110105462.6).

[0028] Method: Inoculate sterile 1 / 4 SDAY liquid medium (1% glucose, 0.5% yeast extract, 0.25% peptone, pH 6.5) to a final concentration of 5 × 10⁻⁶. 6 A spore suspension of *Metarhizium anisopliae* CQMa421 (spores / mL) was prepared at 20% capacity and incubated at 28°C and 200 rpm for 36 hours to obtain the fermentation broth. Note: *Alternaria alternata* was used as the indicator bacterium.

[0029] 2. Ion exchange chromatography for separation of active substances

[0030] The fermentation broth was centrifuged at 10,000 rpm for 10 min, and the supernatant was collected. Citrate-sodium phosphate buffer (pH 5.6) was added to the supernatant to a concentration of 20 mM, and the mixture was incubated in a boiling water bath for 30 min. The supernatant was then transferred to 4°C and allowed to stand overnight. The mixture was then filtered sequentially using filter paper and a filter membrane (Φ = 0.22 μm) to obtain the filtrate. The supernatant, i.e., the filtrate, was purified using cation exchange resin (SP Sepharose). A chromatography column containing 5 mL of SP Sepharose resin was connected to... Prime Plus was used to equilibrate the resin using 20 mM citrate-sodium phosphate buffer (pH 5.6). One liter of culture medium was circulated and loaded at a flow rate of 0.5 mL / min. After loading, the resin was washed with the equilibration buffer until the UV absorbance remained constant. 20 mM citrate-sodium phosphate buffer (containing 1 M sodium chloride) at pH 5.6 was used as the eluent. The parameters were adjusted to mix the eluent and equilibration buffer, increasing the eluent percentage from 0% to 100% over 60 column volumes. 2 mL of eluent was collected per tube until the UV detection curve returned to baseline. The UV absorption peak during the separation process is shown in Figure 1A, where the arrow indicates the MaAFP1 activity peak, with an elution time of 139.1 min. All collected solutions below this peak were combined and freeze-dried to obtain the crude antimicrobial peptide.

[0031] 3. High-performance liquid chromatography (HPLC) separation of target substance MaAFP1

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

[0033] Experimental Example 2: Structural Identification of MaAFP1

[0034] 1. Determination of the precise molecular weight of MaAFP1 using mass spectrometry

[0035] Analytical liquid chromatography (HPLC) confirmed that the purity of MaAFP1 obtained through the above separation and purification reached 99% (Figure 2A). The molecular weight of MaAFP1 was determined using an orbital trap mass spectrometer (Q Exactive Plus). As shown in Figure 2B, four main peaks were observed in cation mode, with m / z values ​​of 253.651, 337.866, 506.296, and 1011.583. Calculations showed that these peaks corresponded to identical molecules with charges of 4, 3, 2, and 1, respectively, indicating that MaAFP1 possesses at least four primary amino groups. Subtracting the hydrogen ion mass from 1011.583 yielded the precise molecular weight of MaAFP1, 1010.575 Da.

[0036] 2. Sequence resolution using infrared spectroscopy and Edman degradation method

[0037] The major functional groups of MaAFP1 were determined using infrared spectroscopy. One mg of MaAFP1 powder was mixed with potassium bromide, compressed into a tablet, and then dried under an infrared lamp. The detection results are shown in Figure 2C, with values ​​at 617.72, 839.38, 1384.24, 1636.57, 2850.80, 2919.26, and 3446.01 cm⁻¹. -1 It exhibits absorption peaks at 3446.01 and 1636.57 cm⁻¹. -1 The strongest absorption peak is located at 3446.01 cm⁻¹, which is speculated to be the secondary amine group and carbonyl group in the amide bond, respectively. Furthermore, based on the peak area, it is estimated to be... -1 It may also contain primary amino groups.

[0038] The MaAFP1 sequence was further determined from the N-terminus using the Edman degradation method. 2 mg of MaAFP1 was dissolved in water, spotted onto a glass fiber plate, and immobilized. The plate was then immersed in a 12% trimethylamine solution, and 100 μL of phenyl isothiocyanate was added. Nitrogen gas was purged to replace the air, and the mixture was incubated at 40 °C for 90 min to convert the peptide into phenylaminothioformyl peptide. 200 mL of 100% trifluoroacetic acid was added, and the mixture was kept at 40 °C for 15 min to cleave the N-terminal residues into the form of 2-phenylaminothiazolinone. The trifluoroacetic acid concentration was diluted to 25% to convert it into a stable phenylhydantoin N-urea derivative (PTH-AA). PTH-AA was then fed into a high-performance liquid chromatography (HPLC) system and detected using a Wako-Pack WS-PTH Column II (4.6 × 250 mm). The results, as shown in Figure 2D, indicate that MaAFP1 is a symmetrical peptide with the sequence Arg-X-Trp-Thr-Trp-X-Tyr. X showed no signal during detection, suggesting it is a non-natural amino acid.

[0039] 3. Two-dimensional nuclear magnetic resonance spectroscopy analysis of the MaAFP1 sequence

[0040] Combination 1 H, 1 H-COSY, 1 H, 13 C-HSQC and 1 H, 13 The side chain structure of residues was determined by two-dimensional nuclear magnetic resonance spectroscopy, including C-HMBC. 50 mg of MaAFP1 powder was dissolved in 600 μL of deuterated dimethyl sulfoxide. The solution was transferred to an NMR tube, and the structures were analyzed using a 600 MHz NMR spectrometer. 1 H, 1 H-COSY, 1 H, 13 C-HSQC and 1 H, 13 C-HMBC. Then, 50 μL of heavy water was added to the NMR tube, the mixture was shaken to mix, and measurements were taken again using a 600 MHz NMR spectrometer. 1 H, 1 H-COSY was used to determine the chemical shift of active hydrogen. Figures 3, 4, and 5 show the determination of... 1 H, 1 H-COSY, 1 H, 13 C-HSQC and 1 H, 13The C-HMBC spectrum shows that MaAFP1 contains arginine (Arg), ornithine (Orn), threonine (Thr), tyrosine (Tyr), and γ-hydroxyarginine (Arg(OH)), but lacks tryptophan (Trp). Because the Orn side chain has a primary amino group at its terminal, it can react with phenyl isothiocyanate in the Edman degradation method, thus increasing its retention time in liquid chromatography, and was therefore misidentified as Trp.

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

[0042] Weigh 1 mg of MaAFP1 into a hard glass tube and add 1 mL of 6M hydrochloric acid. Purge with nitrogen and seal the tube. Place the glass tube in an oil bath and heat at 110°C for 12 hours. Remove the hydrolysate and adjust the pH to 8.0 with sodium hydroxide. Add 50 mM potassium borate buffer (pH 8.5) and 2 molar amounts of 2,4-dinitrofluorobenzene (DNFB), and incubate at 60°C for 40 minutes. Add 50 μL of 1M hydrochloric acid to acidify the reaction solution to terminate the reaction. Simultaneously, label the purchased L and D types of arginine, ornithine, tyrosine, and threonine using the above labeling method. Separate the labeled amino acid standards (DNB-AA) using a semi-preparative high-performance liquid chromatography (HPLC) system, and then separate the labeled hydrolysate; determine the amino acid type corresponding to each retention time in the hydrolysate by the retention time. Measure the spectra of the standard amino acids and each amino acid in the hydrolysate sample in the 200-400 nm range using circular dichroism spectroscopy (CDS). By comparing the spectra of the sample and the standard (Figure 6), it was found that Tyr, Orn and Arg are D-type, Thr is L-type; there is no standard for Arg(OH), but after comparing it with the spectrum of arginine, it was determined to be L-type.

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

[0044] One microgram of MaAFP1 was dissolved in ultrapure water and analyzed using a Q Exactive Plus orbital trap mass spectrometer. The precursor ion with m / z 1011.58371 was selected and fragmented by applying a 20 eV voltage. Ion peaks in the secondary mass spectrum (Figure 7) were identified, confirming the sequence of MaAFP1 as: (D)Arg-(L)Arg(OH)-(D)Orn-(L)Thr-(D)Orn-(L)Arg(OH)-(D)Tyr.

[0045] Example 3: Determination of half-maximal inhibitory concentration (HMC) and minimum inhibitory concentration (MIC) of MaAFP1 against pathogens

[0046] Different concentrations of MaAFP1 were prepared using ultrapure water and mixed with molten PDA medium to prepare plates. Pathogen spores were suspended and spread onto the plates, then incubated at 28°C. Germination rates were measured at 1-hour intervals. Separately, a drop of the cell suspension was added to the plates and incubated at 28°C for 3 days, and colony diameters were measured. A concentration-diameter / germination rate curve was fitted, and the minimum inhibitory concentration (MIC) for inhibiting the germination of half of the fungal spores was calculated based on 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 concentrations (MICs) for common plant pathogenic fungi are... 50 As shown in Table 2, the antifungal peptide MaAFP1 exhibited strong inhibitory effects on spore germination and hyphal growth of animal pathogenic fungi such as Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Mucor racemosus, Trichophyton mentagrophytes, Fusarium verticillioides, Candida albicans, and Cryptococcus neoformans; and on plant pathogenic fungi such as Alternaria alternata, Alternaria solani, Alternaria tenuissima, Botrytis cinerea, Botryosphaeria dothidea, Colletotrichum fioroniae, Fusarium fujikuroi, Fusarium graminearum, Fusarium oxysporum, Pestalotiopsis microspora, Magnaporthe oryzae, and Sclerotinia. MaAFP1 exhibits strong inhibitory effects on spore germination and hyphal growth of *Sclerotiorum*; however, it does not inhibit yeasts such as *Pichia pastoris* and *Saccharomyces cerevisiae*, nor bacteria such as *Escherichia coli*, *Erwinia carotovora*, *Pseudomonas syringae*, and *Bacillus subtilis*. Hemolysis experiments also showed that MaAFP1 has no hemolytic effect on mouse and human erythrocytes. Therefore, the antifungal peptide MaAFP1 possesses broad-spectrum antifungal activity against filamentous pathogenic fungi and shows promising application prospects.

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

[0049] Table 2 Minimum inhibitory concentration of MaAFP1 against plant pathogenic fungi

[0050] The conventional techniques and solutions not described in detail in the above embodiments are all well known in the art, and therefore will not be elaborated upon here. The above embodiments and / or experimental examples describe the preferred embodiments of the present invention in detail. However, 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, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within 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 the N-terminus to the C-terminus is: (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.

2. The antifungal peptide MaAFP1 as described in claim 1, characterized in that, The antifungal peptide MaAFP1 was isolated and purified from the fermentation broth of Metarhizium anisopliae strain CQMa421, which is deposited at the China General Microbiological Culture Collection Center with the accession number CGMCC NO.4609.

3. The antifungal peptide MaAFP1 as described in claim 2, characterized in that, The separation and purification method includes centrifuging, ion exchange chromatography, and high performance liquid chromatography of the fermentation broth, followed by stepwise separation and purification at the 280 nm ultraviolet absorption peak.

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

5. A method for preparing the antifungal peptide MaAFP1 according to claim 1, characterized in that, Includes the following steps: 1) Fermentation broth preparation: Inoculate a suspension of Metarhizium anisopliae CQMa421 spores into 1 / 4 SDAY liquid medium at pH 6.5 to a final concentration of 5 × 10⁻⁶. 6 The fermentation broth was obtained by culturing 1 spore / mL at 28℃ and 200rpm for 36h; the preservation number of the Metarhizium anisopliae CQMa421 is: CGMCC NO.4609; 2) Preparation of crude antimicrobial peptide: Centrifuge the fermentation broth obtained in step 1) and collect the supernatant of the fermentation broth; add citrate-sodium phosphate buffer (pH 5.6) to the supernatant to a concentration of 20 mM and incubate in a boiling water bath for 30 minutes, then transfer to 4°C and let stand overnight. Filter the solution using filter paper and a filter membrane with a diameter of 0.22 μm to obtain the filtrate. The filtrate was separated and purified using ion exchange chromatography, the purification method comprising: connecting an SP Sepharose resin chromatography column to... Using Prime Plus, the detector wavelength was set to 280 nm. The resin was equilibrated with 20 mM citrate-sodium phosphate buffer at pH 5.

6. The filtrate was circulated and loaded onto the resin at a flow rate of 0.5 mL / min. After loading, the resin was washed with equilibration buffer until the UV absorbance remained constant. Then, 20 mM citrate-sodium phosphate buffer containing 1 M sodium chloride at pH 5.6 was used as the eluent. The parameters were adjusted to mix the eluent and equilibration buffers, increasing the eluent percentage from 0% to 100% within 60 column volumes. The fraction eluted for 139.1 min was collected. The crude antimicrobial peptide was obtained by freeze-drying. 3) Preparation of pure antimicrobial peptide: The crude antimicrobial peptide obtained in step 2) was separated and purified by high performance liquid chromatography. The separation and purification method included: using ultrapure water and methanol containing 0.1% TFA as the mobile phase, and a C18 reversed-phase column as the stationary phase; setting the program as follows: 0 min methanol 15%, 5 min methanol 17%, 45 min methanol 27%, 50 min methanol 100%, with 500 μL injected each time at a flow rate of 2 mL / min; the detection wavelength was 280 nm; the fraction with a retention time of 29.4 min was collected; and the antifungal peptide MaAFP1 was obtained by freeze drying.

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

7. The application as described in claim 6, characterized in that, The products include pharmaceuticals, cosmetics, personal care products, food packaging, antibacterial coatings, or pesticides.

8. The application as described in claim 7, characterized in that, The fungi mentioned in the antifungal list 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 Magnaphorthe oryzae.

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