Isolation, purification, identification and application of an anti-fungal peptide produced by bacillus

Abstract

The application discloses separation and purification, identification and application of an anti-fungal peptide produced by bacillus. The application provides application of a fermentation product extract of bacillus velezensis YTQ3 CCTCC No: M 20211465 in any one of the following aspects: 1) anti-fungal or fungal inhibition; 2) preparation of an anti-fungal or fungal inhibition product; 3) fruit and vegetable disease prevention; 4) preparation of a fruit and vegetable disease prevention product; the anti-fungal peptide prepared from metabolites produced by fermentation of bacillus velezensis YTQ3 has a strong inhibitory effect on botrytis cinerea, and the anti-fungal peptide playing an inhibitory role is mainly Surfactin, Fengycin and Bacilysin; the anti-fungal peptide has a good application prospect in prevention of postharvest diseases of fruits.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology and relates to the isolation, purification, identification and application of an antifungal peptide produced by Bacillus. Background Technology

[0002] Botrytis cinerea, belonging to the genus Botrytis in the phylum Ascomycota, is a typical necrotic plant pathogen. It has a wide host range, infecting over 1400 plant species and various tissues and organs, making it the world's second most prevalent plant pathogenic fungus. Gray mold, caused by Botrytis cinerea, is a global fungal disease that is difficult to control. It can occur almost throughout the entire crop growth cycle, with rapid onset and spread. It causes economic losses of hundreds of billions of dollars annually worldwide. For a long time, gray mold control has relied mainly on chemical agents such as carbendazim and iprodione. However, large-scale application of these agents inevitably leads to pesticide residues, pathogen resistance, and environmental pollution. Therefore, developing a more environmentally friendly and effective method to control postharvest gray mold is crucial. Summary of the Invention

[0003] The purpose of this invention is to address public concerns about food safety, develop consumer-acceptable and environmentally friendly methods to reduce the use of chemical fungicides in controlling postharvest diseases, and provide an antifungal peptide produced by microbial metabolism that can effectively prevent and control postharvest diseases.

[0004] This invention addresses the technical problem of how to prevent and control fruit diseases.

[0005] To address the aforementioned technical problems, the first aspect of this invention provides the application of a fermentation product extract of Bacillus belyssus YTQ3 CCTCC No: M 20211465 in any of the following:

[0006] 1) Antifungal or antifungal;

[0007] 2) Prepare antifungal or antifungal products;

[0008] 3) Prevention and control of fruit and vegetable diseases;

[0009] 4) Prepare products for the prevention and control of fruit and vegetable diseases;

[0010] The fermentation product extract is obtained by acid precipitation and alcohol extraction of Bacillus baileyi YTQ3 CCTCC No: M 20211465 fermentation broth; the fermentation product extract contains Surfactin, Fengycin and / or Bacilysin.

[0011] In the above-described application, the acid precipitation and alcohol extraction is performed using methanol extraction;

[0012] Alternatively, the mass ratio of Surfactin, Fengycin, and Bacilysin in the fermentation product extract is 6-10:1-5:1-5;

[0013] Alternatively, the mass ratio of Surfactin, Fengycin, and Bacilysin in the fermentation product extract is 102.7:18.3:14.8.

[0014] In the above text, Fengycin refers to Fengycin A; further, Fengycin A refers to C. 15 -C 17 Fengycin A;

[0015] The Surfactin is C 14 -C 16 Surfactin.

[0016] In the applications described above,

[0017] The fermentation broth of Bacillus belye YTQ3 CCTCC No: M 20211465 was obtained by culturing Bacillus belye YTQ3 CCTCC No: M 20211465 in liquid culture medium.

[0018] The liquid culture medium mentioned above is LB medium.

[0019] The above fermentation broth was prepared according to the following method:

[0020] (1) Activation of Bacillus belyss YTQ3: Take out the frozen Bacillus belyss strain CCTCC No: M20211465, dip it into the LB medium using a sterile inoculation loop in a clean bench, and inoculate it onto the LB medium using the three-zone streak method. Incubate at 28°C in the dark for 24 hours. Pick a single colony and inoculate it onto LB medium, and incubate at 28°C in the dark for 24 hours to complete the activation of the strain.

[0021] (2) Preparation of seed culture: The activated Bacillus berleis YTQ3 obtained in step (1) was inoculated into an Erlenmeyer flask containing 20 mL of LB liquid medium and cultured on a shaker at 6×g and 28℃ for 18 h to obtain the seed culture of Bacillus berleis YTQ3.

[0022] (3) Preparation of fermentation broth: The seed culture of Bacillus berreatus YTQ3 obtained in step (2) was inoculated into a conical flask containing 100 mL of LB liquid medium at an inoculation rate of 5%, and placed on a shaker and cultured at 6×g and 28℃ for 5 days to obtain the fermentation broth.

[0023] The fermentation product extract described above was prepared according to method 1, which includes the following steps:

[0024] 1) Adjust the pH of the supernatant of the fermentation broth to 2.0-2.2, let it stand, and collect the precipitate;

[0025] 2) The precipitate was resuspended in methanol to obtain a suspension, and the pH of the suspension was adjusted to 7.0-7.2 before extraction to obtain the fermentation product extract.

[0026] The supernatant of the fermentation broth described above is prepared as follows: the fermentation broth is centrifuged to collect the supernatant, which is the supernatant of the fermentation broth. Specifically, the centrifugation conditions for collecting the supernatant are centrifugation at 9,600 × g for 15 min.

[0027] In the above-described application, the extraction includes the following steps: adjusting the pH of the suspension to 7.0-7.2 and then extracting, collecting the supernatant of the extract, and then removing the liquid from the supernatant to obtain the fermentation product extract.

[0028] In the above text, the supernatant of the collected extract is collected by centrifugation;

[0029] The further removal of liquid from the supernatant involves removing methanol and water from the supernatant.

[0030] The removal of methanol and water from the supernatant can be achieved through evaporation and drying.

[0031] In the applications described above,

[0032] In step 1), the pH value is adjusted using a hydrochloric acid aqueous solution, and the concentration of the hydrochloric acid aqueous solution is 1-6 mol / L;

[0033] And / or, in step 1), the settling conditions are settling at 4°C for 12-18 hours;

[0034] Alternatively, in step 1), the settling conditions are settling at 4°C for 14 hours;

[0035] And / or, in step 1), the conditions for collecting the precipitate are 6,200 × g, centrifuged at 4°C for 20-25 min;

[0036] And / or, in step 2), the amount of methanol added is 0.1-1 times the volume of the supernatant of the fermentation broth;

[0037] And / or, in step 2), the pH adjustment is performed using an aqueous sodium hydroxide solution, the concentration of which is 1-6 mol / L;

[0038] And / or, in step 2), the extraction conditions are 20-25℃ for 6-12 hours;

[0039] In step 2), the extraction conditions are: extraction (extraction (stirring)) at 25°C for 8 hours;

[0040] And / or, in step 2), the conditions for collecting the supernatant of the extract are centrifugation at 4°C and 1,500×g for 10-15 min.

[0041] In the above application, the fungus is Botrytis cinerea;

[0042] Alternatively, the disease affecting the fruits and vegetables may be gray mold;

[0043] Alternatively, the fruit and vegetable disease mentioned is gray mold of grapes;

[0044] Alternatively, the aforementioned fruit and vegetable disease control refers to post-harvest disease control.

[0045] Alternatively, the aforementioned prevention and control of fruit and vegetable diseases specifically refers to the prevention and control of gray mold disease in grape fruits after harvest.

[0046] In the applications described above, the product is a microbial agent, a drug, a composition, or other biological product.

[0047] In a second aspect, the present invention provides a method for preparing the fermentation product extract described in the first aspect, comprising method 1 described in the first aspect.

[0048] Thirdly, the present invention provides an antifungal or antifungal disease composition, which is the fermentation product extract described in the first aspect.

[0049] Fourthly, the present invention provides a product whose active ingredient is the fermentation product extract described in the first aspect or the composition described in the third aspect;

[0050] The product has any of the following functions:

[0051] 1) Antifungal or antifungal;

[0052] 2) Prevention and control of fruit and vegetable diseases.

[0053] The fungus mentioned above is Botrytis cinerea;

[0054] Alternatively, the disease affecting the fruits and vegetables may be gray mold;

[0055] Alternatively, the fruit and vegetable disease mentioned is gray mold of grapes.

[0056] Alternatively, the aforementioned fruit and vegetable disease control refers to post-harvest disease control.

[0057] Alternatively, the aforementioned prevention and control of fruit and vegetable diseases specifically refers to the prevention and control of gray mold disease in grape fruits after harvest.

[0058] The beneficial effects achieved by this invention are:

[0059] (1) The antifungal peptide produced by the *Bacillus belye* strain of the present invention can effectively inhibit the growth of *Botrytis cinerea* mycelium and the germination of *Botrytis cinerea* spores, and damage the morphology of *Botrytis cinerea* mycelium. Furthermore, it can completely inhibit the occurrence of gray mold in grape berries. Therefore, this antifungal peptide has significant practical implications for reducing the occurrence of gray mold in cultivation. It can be widely used in the prevention and control of gray mold in fruits and vegetables, inhibiting the growth and reproduction of *Botrytis cinerea*, reducing the probability of fruit and vegetable infection, and extending the shelf life of fruits and vegetables.

[0060] (2) The antifungal peptides of the present invention mainly include Fengycin, Surfactin and Bacilysin.

[0061] (3) The purity of the five substances obtained after purification of the antifungal peptide of the present invention is all above 80%.

[0062] (4) The antifungal peptides of the present invention have good stability and can maintain high biological activity under acidic, alkaline, high temperature and ultraviolet irradiation conditions. Metal salts, organic solvents and surfactants have no effect on the activity of the antifungal peptides.

[0063] In summary, the antifungal peptides prepared by the fermentation of Bacillus vesiculosus YTQ3 and the purified peptides have a strong inhibitory effect on Botrytis cinerea. The antifungal peptides that exert the antibacterial effect are mainly Surfactin, Fengycin and Bacilysin. These antifungal peptides have good application prospects in the prevention and control of postharvest diseases of fruits. Attached Figure Description

[0064] Figure 1 This is the Fourier transform infrared (FTIR) spectrum of the antifungal peptide in this invention.

[0065] Figure 2 This is a liquid chromatogram of the preparation of antifungal peptides isolated in this invention.

[0066] Figure 3 The antifungal peptides in this invention are shown to control gray mold disease in grapes. Among them, (A) is a diagram of disease symptoms of grapes during the entire storage period, (B) is the diameter of lesions in grapes during storage, and (C) is the incidence rate of grapes during storage. Different letters indicate significant differences. 0: blank control; 2.5: 2.5 mg / mL; 5: 5 mg / mL; 7.5: 7.5 mg / mL.

[0067] Figure 4This invention illustrates the effect of antifungal peptides on the mycelial growth of *Botrytis cinerea*. (A) shows the colony morphology of *Botrytis cinerea* after 1, 2, 3, 4, and 5 days of cultivation following treatment with different concentrations of antifungal peptides; (B) shows the colony diameter of *Botrytis cinerea* after 1, 2, 3, 4, and 5 days of cultivation following treatment with different concentrations of antifungal peptides; (C) shows the inhibition rate of *Botrytis cinerea* after 1, 2, 3, 4, and 5 days of cultivation following treatment with different concentrations of antifungal peptides. Different letters indicate significant differences: 0: blank control; 0.5: 0.5 mg / mL; 1: 1 mg / mL; 1.5: 1.5 mg / mL; 2: 2 mg / mL.

[0068] Figure 5 This invention relates to the determination of the minimum inhibitory concentration (MIC) of the antifungal peptide against Botrytis cinerea.

[0069] Figure 6 This invention illustrates the effect of antifungal peptides on the germination of *Botrytis cinerea* spores. (A) shows the germination rate of *Botrytis cinerea* spores after 2, 4, 6, and 8 hours of culture following treatment with different concentrations of antifungal peptides; (B) shows the germination rate of *Botrytis cinerea* spores after 2, 4, 6, and 8 hours of culture following treatment with different concentrations of antifungal peptides; (C) shows the spore tube length after 2, 4, 6, and 8 hours of culture following treatment with different concentrations of antifungal peptides. Different letters indicate significant differences: 0: blank control; 0.25: 0.25 mg / mL; 0.5: 0.5 mg / mL; 0.75: 0.75 mg / mL.

[0070] Figure 7 Scanning electron microscope (SEM) images of Botrytis cinerea after treatment with different concentrations of antifungal peptides (0, 1, 3 and 5 mg / mL) in this invention; where CK: blank control.

[0071] Figure 8 The antifungal peptides in this invention exhibit antibacterial activity after treatment with different pH (A), temperature (B), ultraviolet irradiation (C), metal ions (D), organic solvents (E), and surfactants (F). Detailed Implementation

[0072] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0073] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0074] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0075] Bacillus velezensis, CCTCC No: M 20211465, was deposited on November 22, 2021, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China. The accession number is CCTCC No: M20211465. It has been granted a Chinese invention patent with publication number CN 117264847 B, published on April 26, 2024.

[0076] The gray mold (Botrytis cinerea B05.10) used in the following examples was provided by Professor Tian Shiping of the Institute of Botany, Chinese Academy of Sciences; it is described in the following reference: He, C., Zhang, ZQ, Li, BQ, Xu, Y., Tian, ​​SP, 2019. Effect of natamycin on Botrytis cinerea and Penicillium expansum—Postharvest pathogens of grape berries and jujube fruit. Postharvest Biol. Tec. 151, 134-141. https: / / orcid.org / 10.1016 / j.postharvbio.2019.02.009.

[0077] Example 1: Preparation and identification of Bacillus belyssus YTQ3 fermentation product extract

[0078] I. Preparation of fermentation products of Bacillus belyssus YTQ3

[0079] 1. Activation of Bacillus belyssus YTQ3

[0080] Remove the frozen Bacillus belye strain YTQ3CCTCC No: M 20211465 from the -80℃ freezer. Using a sterile inoculation loop, transfer the strain to LB agar using the three-zone streak method. Incubate at 28℃ in the dark for 24 hours. Pick a single colony and inoculate it onto LB agar, then incubate at 28℃ in the dark for another 24 hours to complete the activation of the strain. Store the activated Bacillus belye strain at 4℃ for later use.

[0081] 2. Preparation of seed solution

[0082] The activated Bacillus berleis YTQ3 obtained in step 1 was inoculated into an Erlenmeyer flask containing 20 mL of LB liquid medium and cultured on a shaker at 6×g and 28℃ for 18 h to obtain the seed culture of Bacillus berleis YTQ3.

[0083] 3. Preparation of fermentation broth

[0084] The seed culture of Bacillus berreatus YTQ3 obtained in step 2 was inoculated at a rate of 5% into an Erlenmeyer flask containing 100 mL of LB liquid medium and placed on a shaker at 6 × g and 28 °C for 5 days to obtain the fermentation broth.

[0085] II. Preparation of Bacillus vesiculosus YTQ3 fermentation product extract

[0086] 1. Extraction using acid precipitation and alcohol extraction method

[0087] The fermentation broth obtained in step one was centrifuged at 9,600 × g for 15 min, and the supernatant was collected. Then, 1 mol / L hydrochloric acid aqueous solution was added to the supernatant until the pH reached 2.0. After standing at 4°C for 14 h, it was centrifuged at 6,200 × g at 4°C for 25 min, and the precipitate was collected. Methanol was added to the precipitate, with the amount of methanol added being 0.1 times the volume of the supernatant of the fermentation broth, to obtain a resuspension. The pH of the resuspension was then adjusted to 7.0 with 1 mol / L sodium hydroxide aqueous solution, and extracted (stirred) at room temperature (25°C) for 8 h to obtain the extract. The extract was centrifuged at 1,500 × g for 10 min at 4°C, and the supernatant was collected. Methanol was removed using a rotary evaporator (45°C, 200 rpm), and the product was then freeze-dried under vacuum (any conventional freeze dryer can achieve this, typically freeze-drying for 48 h at -55°C). The resulting dried product was the YTQ3 fermentation product extract.

[0088] The test results showed that the yield of YTQ3 fermentation product extract in the supernatant of each 1L of fermentation broth was 712mg / L.

[0089] 2. Identification

[0090] 1) Liquid Chromatography-Mass Spectrometry

[0091] The YTQ3 fermentation product extract obtained in step 1 above was analyzed using ultra-high performance liquid chromatography-electrospray ionization mass spectrometry (UHPLC-ESI-MS) (Thermo Fisher Scientific, USA). The test conditions were as follows: ACQUITY... HSS T3 (2.1×100mm, 1.8μm) column (Waters, Milford, MA, USA), flow rate 0.3 mL / min, column temperature 40℃, injection volume 2 μL. Positive ion mode, mobile phase 0.1% formic acid acetonitrile (B) and 0.1% formic acid water (A), gradient elution program: 0–1 min, 10% B; 1–5 min, 10%–98% B; 5–6.5 min, 98% B; 6.5–6.6 min, 98%–10% B; 6.6–8 min, 10% B. Mass spectrometry conditions: electrospray ionization (ESI), spray voltage 3.50 kV, sheath gas 40 alb, auxiliary gas 10 alb. Capillary temperature 325℃, scan range m / z 100–1500.

[0092] The results are shown in Table 1. The fermentation product extract produced by strain YTQ3 is mainly C. 15 -C 17 Fengycin A、C 14 -C 16 Surfactin and Bacilysin.

[0093] The compound obtained through structural identification was indeed the target compound.

[0094] C 15 -C 17 The general structural formula of Fengycin A is as follows: Equation 1:

[0095] C15-C17 refers to the different lengths of the fatty acid chains, with each chain having one more carbon atom than the last.

[0096] C 14 -C 16 The general structural formula of Surfactin is as follows: Equation 2:

[0097] C15-C17 refers to the different lengths of the fatty acid chains, with each chain having one more carbon atom than the last.

[0098] The structural formula for Bacilysin is shown in Equation 3 below:

[0099]

[0100] Quantitative results from secondary mass spectrometry showed that Surfactin was the most abundant component, accounting for 54.7% of the total content of the fermentation product extract.

[0101] Table 1 shows the composition of antifungal peptides in strain YTQ3.

[0102]

[0103] 2) FTIR spectral analysis

[0104] The YTQ3 fermentation product extract obtained in step 1 above was subjected to FTIR spectroscopy analysis using a Nicolet iS10 (Thermo, USA) to determine the characteristic components of the antifungal peptides.

[0105] Take 1 mg of the YTQ3 fermentation product extract powder obtained in step 1 above, grind it into a fine powder in an agate mortar, and mix it evenly with dry potassium bromide powder (approximately 100 mg, particle size 200 mesh). Place the mixture into a mold and compress it into tablets using a tablet press. Place the prepared sample into the sample holder and insert it into the fixed position in the instrument's sample chamber. In transmission mode, at 400 and 4000 cm⁻¹... 1 Between 4cm 1 High-resolution scanning is used to obtain the sample spectrum.

[0106] The results are as follows Figure 1 As shown, the FTIR spectrum of the YTQ3 fermentation product extract is in the range of 3000-3600 cm⁻¹. 1 There is a relatively broad band in between, indicating the stretching of -OH and NH. The absorption peak is located at 1653.01 cm⁻¹. 1 There is an intermediate bond or peptide bond at this point, which is generated by the amide I band formed by the CO stretching vibration. 1237.67cm- 1 The peak value at 1404.11 cm⁻¹ indicates that the amide III band generated by the NH bond in the deformation mode is combined with the CN stretching mode. 1 Other major peaks observed correspond to carbon-hydrogen bond bending vibrations, which are commonly found in compounds with aliphatic chains.

[0107] Therefore, YTQ3 fermentation product extract contains antimicrobial peptides, especially lipopeptides.

[0108] 3) Purification and identification of YTQ3 fermentation product extract

[0109] (1) Separation and purification by liquid chromatography

[0110] Weigh 1g of the YTQ3 fermentation product extract powder obtained in step 1 above, dissolve it in 20mL of chromatographic grade methanol, vortex mix, and filter through a 0.22μm pinhole organic filter membrane. Separate the fermentation product extract using preparative liquid chromatography (HPLC). chromatographic column: Agilent PrepHT XDB-C. 18(21.2×250mm 7-Micron), mobile phase A was an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B was an acetonitrile solution containing 0.1% trifluoroacetic acid. Flow rate: 20 mL / min, wavelength: 210 nm, injection volume: 500 μL. Elution conditions: 0 min, 80% A; 0–6 min, 50% A; 6–20 min, 28% A; 20–20.5 min, 80% A; 20.5–25 min, 80% A.

[0111] The results of liquid chromatography showed that... Figure 2 As shown, a total of 12 peaks were detected. The substances corresponding to these 12 peaks were collected, the organic solvent was removed by rotary evaporation, and the pure powder was obtained by freeze drying.

[0112] (2) Antibacterial test determination

[0113] The pure powder containing the 12 peaks obtained in (1) above was reconstituted with sterile water, and its antibacterial effect against Botrytis cinerea was determined. The experiment was conducted using sterile 96-well plates. 180 μL of a Botrytis cinerea spore suspension (1 × 10⁻⁶) resuspended in potato dextrose broth (PDB) was added to each well. 6 CFU mL -1 ), and 20 μL of a pure solution with a final concentration of 0.5 mg / mL (solvent: water, A1). Sterile water was used as a negative control instead of the pure solution (A2). After all the contents were added, the 96-well plate was sealed with sealing film and incubated at 22°C for 48 h. The OD was then measured using a spectrophotometer. 600 Value. The inhibition rate was calculated according to formula (1). Each treatment group was repeated three times.

[0114] Inhibition rate (%) = (A2-A1) / A2×100 (1)

[0115] As a result, the substances corresponding to peaks 1-5 showed significant antibacterial activity against Botrytis cinerea (Table 2). The substances corresponding to other peaks had no antibacterial effect against Botrytis cinerea.

[0116] (3) Mass spectrometry identification

[0117] The pure solutions corresponding to peaks 1-5, which showed antibacterial activity as detected in (2) above, were analyzed by UHPLC-ESI-MS under the following conditions: ACQUITY HSS T3 (2.1×100mm, 1.8μm) column (Waters, Milford, MA, USA), flow rate 0.3 mL / min, column temperature 40℃, injection volume 2 μL. Positive ion mode, mobile phase 0.1% formic acid acetonitrile (B) and 0.1% formic acid water (A), gradient elution program: 0–1 min, 10% B; 1–5 min, 10%–98% B; 5–6.5 min, 98% B; 6.5–6.6 min, 98%–10% B; 6.6–8 min, 10% B. Mass spectrometry conditions: electrospray ionization (ESI), spray voltage 3.50 kV, sheath gas 40 alb, auxiliary gas 10 alb. Capillary temperature 325℃, scan range m / z 100–1500.

[0118] UHPLC-ESI-MS results showed that the compounds obtained by structural identification were indeed the target compounds. Substances 1-5 were C15-Surfactin, C15-Fengycin A, Bacilysin, Bacilysin, and C17-Fengycin A, respectively, with a purity of over 80% (Table 2).

[0119] Therefore, the fermentation product extract of strain YTQ3 contains a variety of antifungal peptides, with the yields from highest to lowest being Surfactin, Fengycin, and Bacilysin.

[0120] Table 2 shows the antifungal activity and yield of the purified antifungal peptides.

[0121]

[0122] Example 2: Determination of the control effect of Bacillus vesiculosus YTQ3 fermentation product extract on gray mold in fruits.

[0123] Different amounts of the YTQ3 fermentation product extract obtained in Example 1 were dissolved in water to obtain solutions of different concentrations, which were denoted as antifungal peptide solutions.

[0124] Botrytis cinerea B05.10 spore suspension was prepared as follows: Pre-stored Botrytis cinerea B05.10 was removed from a -80℃ freezer and incubated at 25℃ for approximately 20 minutes. It was then inoculated onto potato dextrose agar (PDA) medium and incubated at 25℃ for approximately 7 days, until the Botrytis cinerea completely covered the agar plate. One or two plates confluent with Botrytis cinerea were taken, and an appropriate amount of sterile distilled water was added. Under sterile conditions, the spores were collected and filtered through four layers of gauze to remove mycelia. The spores were counted using a hemocytometer, and the concentration of the spore suspension was adjusted to the desired level with sterile distilled water to obtain the Botrytis cinerea spore suspension.

[0125] Grapes were harvested from local orchards at their commercial ripening stage. Uninfected or undamaged grapes of uniform size, color, shape, and weight were selected. The fruit surface was disinfected with a 2% (v / v) sodium hypochlorite solution for 2 minutes, rinsed with distilled water, and air-dried at room temperature before use. Holes (3 mm deep, 3 mm wide) were punched near the equator on the fruit using a sterile punch. First, 10 μL of antifungal peptide solutions of different concentrations (2.5, 5, and 7.5 mg / mL) were inoculated. After incubation at room temperature for 2 hours, 10 μL of a Botrytis cinerea spore suspension (1×10⁻⁶) was inoculated. 5 CFU / mL). Fruits inoculated solely with a suspension of *Botrytis cinerea* spores served as a control group (denoted as 0 mg / mL antifungal peptide concentration in the figure). Three replicates were prepared for each treatment (15 fruits per group, one wound per fruit). All fruits were stored at 25°C and 90% RH. Observations were made daily after the diameter of the control group reached a measurable size. The incidence rate (DI) for each infected fruit / vegetable was calculated: DI(%) = (number of rotten wounds / total number of wounds) × 100%. Lesion diameter (LD) was assessed by measuring the average diameter of the damaged area.

[0126] The results are as follows Figure 3 As shown, different concentrations of antifungal peptide solutions significantly affected the diameter and incidence of gray mold lesions on grape berries. Throughout the storage period, the diameter of lesions in the antifungal peptide-treated group was significantly lower than that in the control group (P<0.05). Figure 3 (B)); The control group's fruit began to show symptoms on the 2nd day, and the incidence rate reached 100% on the 3rd day, while the 2.5 mg / mL antifungal peptide treatment group began to show symptoms on the 4th day, with an incidence rate of only 6.8%; by the 6th day of storage, the 5 mg / mL antifungal peptide treatment group only began to slowly show symptoms. Figure 3 (C)); On the 7th day of storage, the lesion diameter in the control group reached 16.7 mm, while the lesion diameters in the 2.5 mg / mL and 5 mg / mL treatment groups were 10 mm and 2.6 mm, respectively. Figure 3 (A) and Figure 3 (B)). Treatment with 7.5 mg / mL antifungal peptides can completely inhibit the occurrence of gray mold on grape fruits.

[0127] Therefore, it can be seen that the YTQ3 fermentation product extract can prevent the occurrence of gray mold in grapes.

[0128] Example 3: Determination of the inhibitory effect of Bacillus belye YTQ3 fermentation product extract on Botrytis cinerea growth.

[0129] Different amounts of the YTQ3 fermentation product extract obtained in Example 1 were dissolved in water to obtain solutions of different concentrations, which were denoted as antifungal peptide solutions.

[0130] 1. Determination of the effect of antifungal peptide solution on the mycelial growth of Botrytis cinerea

[0131] Botrytiscinerea mycelium was prepared as follows: Botrytiscinerea was cultured on PDA medium for 7 days and then punched with a sterile punch (9 mm in diameter) to obtain Botrytiscinerea mycelium.

[0132] Experimental group: 1.5 mL of antifungal peptide solutions with final concentrations of 0.5, 1, 1.5, and 2 mg / mL were mixed with 15 mL of PDA medium to prepare PDA medium containing antifungal peptides. Botrytis cinerea mycelial cakes cultured for 7 days were broken into mycelial cakes using a sterile 9 mm diameter punch and placed in the center of the PDA medium containing antifungal peptides, with the mycelial side facing down. The cakes were incubated at 22℃. The diameter of the colonies was measured daily, and the inhibition rate was calculated.

[0133] Control group: The only difference from the experimental group was that no antifungal peptide solution was added.

[0134] Colony diameter was measured using the cross-sectional method and inhibition rate was calculated using formula (2). Each treatment was repeated in triplicate, and the entire experiment was repeated twice.

[0135]

[0136] The results are as follows Figure 4 As shown, the inhibitory effect on the mycelial growth of *Botrytis cinerea* gradually increased with increasing concentration of the antifungal peptide solution. After 3 days of culture, the mycelium in the control group grew rapidly, with a colony diameter reaching approximately 83.5 mm. Figure 4 (A) and (B)), at this point, the 0.5 mg / mL antifungal peptide solution already showed an inhibition rate of 73.6% on the mycelial growth of Botrytis cinerea. Figure 4 (C) showed a significant inhibitory effect. After 5 days of culture, the inhibition rate of Botrytis cinerea mycelial growth in the 2 mg / mL antifungal peptide solution treatment group remained at 82.4%, and the colony diameter did not grow at all during the entire culture period.

[0137] This demonstrates that the extract of YTQ3 fermentation products has a significant inhibitory effect on the mycelial growth of Botrytis cinerea.

[0138] 2. Determination of the minimum inhibitory concentration (MIC) of antifungal peptides

[0139] The experiment was conducted using sterile 96-well plates. 180 μL of a *Botrytis cinerea* spore suspension (1 × 10⁻⁶) resuspended in PDB medium was added to each well. 6 CFU mL -1Add 20 μL of antifungal peptide solutions with final concentrations of 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL to a 96-well plate. Sterile water (0 mg / mL) serves as a negative control instead of the antifungal peptide solution. After adding all the antifungal peptide solution, seal the 96-well plate with sealing film and incubate at 22°C for 48 hours. Then, measure the OD using a spectrophotometer. 600 Value. Repeated three times for each treatment group.

[0140] The results are as follows Figure 5 As shown, the inhibitory effect on Botrytis cinerea gradually increases with increasing concentration of the antifungal peptide solution. The minimum inhibitory concentration (MIC) of the antifungal peptide solution against Botrytis cinerea is 0.1 mg / mL.

[0141] 3. Determination of the effect of antifungal peptide solution on the survival rate of Botrytis cinerea spores

[0142] 500 μL of antifungal peptide solutions with final concentrations of 0, 0.25, 0.5, and 0.75 mg / mL were mixed with 5 mL of PDA medium to obtain PDA medium containing antifungal peptides. 100 μL of a 1×10⁻⁶ mg / mL solution was then added. 6 The *Botrytis cinerea* spore suspension prepared in Example 2 (CFU / mL) was uniformly spread on PDA medium containing antifungal peptide solution and placed in a 22°C incubator. Microscopic observation was performed at 2, 4, 6, and 8 hours of culture, with at least 200 spores observed each time, along with the germ tube diameter. Spore germination was considered complete when the germ tube diameter was equal to or greater than the spore diameter. Germination rate was calculated according to formula (3). Each treatment was repeated in triplicate, with the experiment conducted twice.

[0143]

[0144] The results are as follows Figure 6 As shown, the antifungal peptide solution has a strong inhibitory effect on the spores of Botrytis cinerea. Figure 6 (A)). At 4 hours of culture, the spore germination rate in the control group was 88%, while the spore germination rate in the group treated with 0.25 mg / mL antifungal peptide solution was significantly reduced to 51%. Figure 6 (B)). At this point, the spores in the 0.5 mg / mL antifungal peptide solution treatment group had just begun to germinate, with a germination rate of 12%. With increasing concentration of the antifungal peptide solution, the inhibitory effect on the survival of *Botrytis cinerea* spores significantly increased. At 8 hours of culture, the spore tube diameter in the control group (0 mg / mL antifungal peptide solution) reached 95 μm. Figure 6 (C) showed a spore germination rate of 100%, while the spore germination rate in the 0.75 mg / mL antifungal peptide solution treatment group was almost 0%. This indicates that the 0.75 mg / mL antifungal peptide solution can completely inhibit the germination of Botrytis cinerea spores.

[0145] Therefore, the TQ3 fermentation product extract can inhibit the germination of Botrytis cinerea spores.

[0146] Example 4: Morphological changes of Botrytis cinerea mycelium after treatment with antifungal peptides observed by scanning electron microscopy.

[0147] Different amounts of the YTQ3 fermentation product extract obtained in Example 1 were dissolved in water to obtain solutions of different concentrations, which were denoted as antifungal peptide solutions.

[0148] The morphological changes of Botrytis cinerea hyphae after treatment with antifungal peptide solution were observed using scanning electron microscopy.

[0149] 100 μL of the solution prepared in Example 2 with a concentration of 1×10⁻⁶ was inoculated into 25 mL of PDB medium. 6 CFU mL -1 The botrytis cinerea spore suspension was cultured at 22°C with shaking for 48 hours. The mycelia were collected, washed three times with PBS, and resuspended.

[0150] Weigh 0.1 g of mycelium (wet weight) into a centrifuge tube and add antifungal peptide solutions to final concentrations of 1, 3, and 5 mg / mL. Incubate at 22 °C for 12 h. Use PBS instead of antifungal peptides as a control (CK). Collect mycelium by centrifugation at 1,500 × g, add 4% (v / v) glutaraldehyde and fix overnight at 4 °C (approximately 12 h). Then dehydrate with a gradient of 100 μL ethanol (30, 50, 70, 90, 100%) for 10 min each time. After air drying, mount the samples on a stage, sputter-coated with gold, and mount for scanning electron microscopy (SEM) imaging.

[0151] The results are as follows Figure 7 As shown, the mycelial surface in the control group was normal and smooth (CK), but the mycelia were damaged to varying degrees after treatment with antifungal peptides. When the mycelia were treated with 1 mg / mL of antifungal peptides, the mycelia shrank and the surface was no longer smooth. After treatment with high concentrations of antifungal peptides (5 mg / mL), the mycelial contents were severely lost, the entire mycelium collapsed, and it shrank and deformed severely.

[0152] Example 5: Stability Study of Antifungal Peptides

[0153] Different amounts of the YTQ3 fermentation product extract obtained in Example 1 were dissolved in water to obtain solutions of different concentrations, which were denoted as antifungal peptide solutions.

[0154] 1. Thermal stability

[0155] The antifungal peptide solution (concentration 1 mg / mL, solvent: water) was treated in water baths at 4℃, 28℃, 37℃, 65℃, and 90℃ for 1 h. The control was not subjected to heat treatment. The treated solutions were then rapidly cooled to room temperature on ice. The antifungal effect against *Botrytis cinerea* was determined using a 96-well plate. The specific method was as follows: 180 μL of *Botrytis cinerea* spore suspension (1 × 10⁻⁶) resuspended in PDB medium was added to each well. 6 CFU mL -1 20 μL of antifungal peptide solution treated under different conditions was added to a 96-well plate (A1). PDB was used as a negative control instead of the antifungal peptide solution (A2). After all the contents were added, the 96-well plate was sealed with sealing film and incubated at 22°C for 48 h. The OD was then measured using a spectrophotometer. 600 Value. The inhibition rate was calculated according to formula (1). Each treatment group was repeated three times.

[0156] 2. Acid-base stability

[0157] The antifungal peptide solution (concentration 1 mg / mL, solvent: water) was adjusted to pH 2, 4, 6, 8, and 10 respectively using 4 mol / L concentrated hydrochloric acid aqueous solution and 4 mol / L sodium hydroxide aqueous solution. After standing for 1 h, the pH was adjusted back to 7.0. The antifungal effect against Botrytis cinerea was determined using a 96-well plate, following the method described in section 1 above.

[0158] 3. UV resistance stability

[0159] The antifungal peptide solution (concentration of 1 mg / mL, solvent of water) was placed 20 cm under a UV lamp (UV-C, 30W) for 10 min, 20 min, 30 min, 45 min and 60 min respectively. The antifungal effect against Botrytis cinerea was determined by using a 96-well plate, and the method was the same as described in 1 above.

[0160] 4. Stability of metal ions

[0161] The antifungal peptide solution (concentration of 1 mg / mL, solvent of water) was mixed with aqueous solutions of MgSO4, CuSO4, ZnSO4, CaCl2 and FeSO4 at a concentration of 1 mg / mL at a ratio of 1:1 (v / v). After standing for 1 h, the antifungal effect against Botrytis cinerea was determined using a 96-well plate, following the method described in section 1 above.

[0162] 5. Organic solvent stability

[0163] The antifungal peptide solution (concentration of 1 mg / mL, solvent of water) was mixed with methanol, ethanol, isopropanol, chloroform and acetone solutions at a ratio of 1:1 (v / v). After standing for 1 h, the antifungal effect against Botrytis cinerea was determined using a 96-well plate, following the method described in section 1 above.

[0164] 6. Surfactant stability

[0165] The antifungal peptide solution (concentration of 1 mg / mL, solvent of water) was mixed with sodium tridecyl sulfate, urea, Tween 20 and Triton X-100 at a ratio of 1:1 (v / v). After standing for 1 h, the antifungal effect against Botrytis cinerea was determined using a 96-well plate, following the method described in section 1 above.

[0166] The results are as follows Figure 8 As shown, the antifungal peptide solution exhibits good stress stability. Under conditions of pH 2-10, the antifungal peptide solution has virtually no effect on its antifungal activity against *Botrytis cinerea*. Figure 8 (A) The antifungal peptide solution maintained a 95% inhibition rate even under high temperature conditions of 65-90℃. Figure 8 (B) UV irradiation for 60 min had no effect on the antifungal peptide solution's antibacterial activity. Figure 8 (C)). Furthermore, metal salts, organic solvents, and surfactants had no effect on the activity of the antifungal peptide solution. Figure 8 (D)- Figure 8 (F)).

[0167] This indicates that the YTQ3 fermentation product extract has good stability and broad application prospects.

[0168] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Application of the fermentation product extract of Bacillus belyssus YTQ3 CCTCC No: M20211465 in any of the following: 1) Antifungal or antifungal agents not intended for disease diagnosis or treatment; 2) Prepare antifungal or antifungal products; 3) Prevention and control of fruit and vegetable diseases; 4) Prepare products for the prevention and control of fruit and vegetable diseases; The fungus is Botrytis cinerea; The disease affecting the fruits and vegetables is gray mold. The fermentation product extract is a product obtained by acid precipitation and alcohol extraction of Bacillus belicei YTQ3 CCTCC No: M20211465 fermentation broth; the fermentation product extract contains Surfactin, Fengycin and Bacilysin. Surfactin is C15 Surfactin; Fengycin is C15 Fengycin A and C17 Fengycin A; The fermentation product extract was prepared according to method 1, which includes the following steps: 1) Adjust the pH of the supernatant of the fermentation broth to 2.0-2.2 and let it stand, then collect the precipitate; the standing conditions are 4℃ for 12-18 h; the precipitate collection conditions are 6200×g, centrifuged at 4℃ for 20-25 min. 2) Resuspend the precipitate in methanol to obtain a suspension. Adjust the pH of the suspension to 7.0-7.2 and extract. Collect the supernatant of the extract and remove the liquid from the supernatant to obtain the fermentation product extract. The amount of methanol added is 0.1-1 times the volume of the supernatant of the fermentation broth. The extraction conditions are 20-25℃ for 6-12 h. The supernatant of the extract is collected by centrifugation at 4℃ and 1500×g for 10-15 min. The mass ratio of Surfactin, Fengycin, and Bacilysin in the fermentation product extract is 6-10:1-5:1-5.

2. The application according to claim 1, characterized in that: The mass ratio of Surfactin, Fengycin, and Bacilysin in the fermentation product extract was 102.7:18.3:14.

8.

3. The application according to claim 1 or 2, characterized in that: The fermentation broth of Bacillus belye YTQ3 CCTCC No: M20211465 was obtained by culturing Bacillus belye YTQ3 CCTCC No: M20211465 in liquid culture medium.

4. The application according to claim 1 or 2, characterized in that: In step 1), the pH value is adjusted using a hydrochloric acid aqueous solution, and the concentration of the hydrochloric acid aqueous solution is 1-6 mol / L; And / or, in step 2), the pH adjustment is performed using an aqueous solution of sodium hydroxide, the concentration of which is 1-6 mol / L.

5. Use according to claim 1 or 2, characterized in that: The gray mold mentioned refers to gray mold on grape fruits.

6. The application according to claim 1 or 2, characterized in that: The product is a microbial agent.

7. The application according to claim 1 or 2, characterized in that: The product is a medicine.

8. The application according to claim 1 or 2, characterized in that: The product is a composition.

9. A method for preparing the fermentation product extract according to any one of claims 1-4, characterized in that: The method includes method 1 as described in any one of claims 1-5.

10. A composition that is antifungal or antifungal disease, characterized in that: The composition is the fermentation product extract of any one of claims 1-8.

11. A product wherein the active ingredient is the fermentation product extract of any one of claims 1-8 or the composition of claim 10; The product has any of the following functions: 1) Antifungal or antifungal; 2) Prevention and control of fruit and vegetable diseases.

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