Separation and purification, identification and application of antifungal peptide generated by bacillus

Abstract

The invention discloses separation and purification, identification and application of an antifungal peptide generated by bacillus. The invention provides an application of a fermentation product extract of bacillus velezensis YTQ3CCTCC No: M 20211465 in any one of the following aspects: (1) resisting or inhibiting fungi; 2) preparing an antifungal or fungus-inhibiting product; (3) preventing and controlling fruit and vegetable diseases; 4) preparing a fruit and vegetable disease control product; the antifungal peptide prepared from the metabolite generated by fermentation of bacillus velezensis YTQ3 and the purified peptide have a relatively strong inhibition effect on botrytis cinerea, the antifungal peptide playing the bacteriostasis effect mainly comprises Surfactin, Fengycin and Bacilysin, and the antifungal peptide has a relatively good application prospect in the aspect of preventing and treating postharvest diseases of fruits.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control and relates to the separation, purification, identification and application of an antifungal peptide produced by Bacillus. Background Art

[0002] Botrytis cinerea belongs to the genus Botrytis of the phylum Ascomycetes and is a typical necrotic plant pathogen. It has a wide host range and can infect more than 1,400 species of plants and different tissues and organs. It is the second largest plant pathogenic fungus in the world. Gray mold caused by infection with Botrytis cinerea is a global fungal disease that is difficult to prevent and control. It can occur almost throughout the entire growth period of crops, and it develops rapidly and spreads quickly. The economic losses caused to the world each year amount to hundreds of billions of dollars. For a long time, the prevention and control of gray mold has mainly relied on chemical agents such as carbendazim and prochloraz, and large-scale use of drugs inevitably leads to problems such as pesticide residues, pathogen resistance, and environmental pollution. Therefore, it is crucial to develop a more environmentally friendly and effective method to control post-harvest gray mold. Summary of the invention

[0003] The purpose of the present 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] The invention solves the technical problem of how to achieve fruit disease prevention and control.

[0005] In order to solve the above technical problems, the first aspect of the present invention provides an application of a fermentation product extract of Bacillus velez YTQ3 CCTCC No: M 20211465 in any of the following:

[0006] 1) Antifungal or fungistatic;

[0007] 2) Preparation of antifungal or fungistatic products;

[0008] 3) Fruit and vegetable disease prevention and control;

[0009] 4) Preparation of fruit and vegetable disease prevention and control products;

[0010] The fermentation product extract is a product obtained by subjecting the fermentation broth of Bacillus velez YTQ3 CCTCC No: M 20211465 to acid precipitation and alcohol extraction; the fermentation product extract contains surfactin, fengycin and / or bacilysin.

[0011] In the application described above, the acid precipitation and alcohol extraction adopts methanol extraction;

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

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

[0014] In the above, the Fengycin is the Fengycin A; further, the Fengycin A is 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 velez YTQ3 CCTCC No: M 20211465 is obtained by culturing Bacillus velez YTQ3CCTCC No: M 20211465 in a liquid culture medium;

[0018] In the above, the liquid culture medium is LB medium.

[0019] The fermentation liquid is prepared specifically according to the following method:

[0020] (1) Activation of Bacillus Velez YTQ3: Take out the frozen Bacillus Velez CCTCC No: M20211465 strain, dip it with a sterile inoculation loop in the clean bench, and inoculate it on the LB medium by three-zone streak method. Put it in the incubator and culture it in the dark at 28℃ for 24 hours. Pick a single colony and inoculate it on the LB medium, and culture it in the dark at 28℃ for 24 hours to complete the strain activation.

[0021] (2) Preparation of seed solution: A single colony of the activated Bacillus Velez subtilis YTQ3 obtained in step (1) was inoculated into a conical flask containing 20 mL of LB liquid culture medium, and cultured on a shaker at 6×g and 28° C. for 18 h to obtain a seed solution of Bacillus Velez subtilis YTQ3.

[0022] (3) Preparation of fermentation broth: The seed liquid of Bacillus Velez subtilis YTQ3 obtained in step (2) was inoculated into a conical flask containing 100 mL of LB liquid culture medium at an inoculum size of 5%, and cultured in a shaking incubator at 6×g and 28°C for 5 days to obtain a fermentation broth.

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

[0024] 1) adjusting the pH of the supernatant of the fermentation broth to 2.0-2.2, allowing the supernatant to stand, and collecting the precipitate;

[0025] 2) resuspending the precipitate with methanol to obtain a suspension, and then adjusting the pH of the suspension to 7.0-7.2 and then extracting to obtain a 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. The conditions for centrifugation to collect the supernatant are specifically centrifugation at 9,600×g for 15 min.

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

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

[0029] The further removing of the liquid in the supernatant is removing methanol and water in the supernatant.

[0030] The removal of methanol and water in the supernatant can be achieved by 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 standing condition is standing at 4° C. for 12-18 hours;

[0034] Or, in step 1), the standing condition is standing at 4° C. for 14 hours;

[0035] And / or, in step 1), the conditions for collecting the precipitate are centrifugation at 6,200×g and 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 is adjusted using a sodium hydroxide aqueous solution, the concentration of the sodium hydroxide aqueous solution is 1-6 mol / L;

[0038] And / or, in step 2), the extraction condition is 20-25° C. for 6-12 h;

[0039] In step 2), the extraction condition is 25°C extraction (extraction (stirring)) for 8h;

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

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

[0042] Or, the fruit and vegetable disease is gray mold;

[0043] Or, the fruit and vegetable disease is gray mold of grape fruit;

[0044] Or, the fruit and vegetable disease control is post-harvest disease control of fruits and vegetables.

[0045] Alternatively, the fruit and vegetable disease control is specifically the control of gray mold of post-harvest grape fruits.

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

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

[0048] In a third aspect, the present invention provides a composition for resisting fungi or fungal diseases, which is the fermentation product extract in the first aspect.

[0049] In a fourth aspect, the present invention provides a product, the active ingredient of which is the fermentation product extract of the first aspect or the composition of the third aspect;

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

[0051] 1) Antifungal or fungistatic;

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

[0053] In the above species, the fungus is Botrytis cinerea;

[0054] Or, the fruit and vegetable disease is gray mold;

[0055] Or, the fruit and vegetable disease is gray mold of grape fruit.

[0056] Or, the fruit and vegetable disease control is post-harvest disease control of fruits and vegetables.

[0057] Alternatively, the fruit and vegetable disease control is specifically the control of gray mold of post-harvest grape fruits.

[0058] The beneficial effects achieved by the present invention are:

[0059] (1) The antifungal peptide produced by the Bacillus Velezii strain of the present invention can effectively inhibit the growth of gray mold hyphae and the germination of gray mold spores, and destroy the morphology of gray mold hyphae. In addition, it can completely inhibit the occurrence of gray mold in grape fruits. Therefore, the antifungal peptide has important practical significance in reducing the occurrence of gray mold in planting. It can be widely used in the prevention and control of gray mold diseases in fruits and vegetables, inhibit the growth and reproduction of gray mold, reduce the probability of fruit and vegetable infection, and extend 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 the antifungal peptide of the present invention is all above 80%.

[0062] (4) The antifungal peptide of the present invention has 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 peptide.

[0063] In summary, the antifungal peptides prepared by the metabolites produced by fermentation of Bacillus Velezii 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 preventing and controlling post-harvest diseases of fruits. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0065] Figure 2 This is a preparative liquid chromatogram for separating antifungal peptides in the present invention.

[0066] Figure 3 The control effect of the antifungal peptide of the present invention on gray mold disease of grape fruit; wherein, (A) is the disease symptom diagram of grape during the entire storage period, (B) is the lesion diameter of grape fruit during storage, and (C) is the incidence rate of grape fruit during storage; different letters represent significant differences; 0: blank control; 2.5: 2.5 mg / mL; 5: 5 mg / mL; 7.5: 7.5 mg / mL.

[0067] Figure 4The effect of the antifungal peptides of the present invention on the hyphae growth of Botrytis cinerea; wherein, (A) is the morphology of Botrytis cinerea colonies after being treated with antifungal peptides at different concentrations for 1, 2, 3, 4 and 5 days, (B) is the diameter of Botrytis cinerea colonies after being treated with antifungal peptides at different concentrations for 1, 2, 3, 4 and 5 days, (C) is the inhibition rate of Botrytis cinerea after being treated with antifungal peptides at different concentrations for 1, 2, 3, 4 and 5 days; 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 is the determination of the minimum inhibitory concentration (MIC) of the antifungal peptides of the present invention against Botrytis cinerea.

[0069] Figure 6 The effect of the antifungal peptides of the present invention on the germination of Botrytis cinerea spores; wherein, (A) is a graph of Botrytis cinerea spore germination after being treated with antifungal peptides at different concentrations for 2, 4, 6 and 8 hours, (B) is a graph of Botrytis cinerea spore germination after being treated with antifungal peptides at different concentrations for 2, 4, 6 and 8 hours, (C) is a graph of the length of the spore tube after being treated with antifungal peptides at different concentrations for 2, 4, 6 and 8 hours, 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 The scanning electron microscope images (SEM) of Botrytis cinerea treated with different concentrations of the antifungal peptides (0, 1, 3 and 5 mg / mL) of the present invention; wherein, CK: blank control.

[0071] Figure 8 The antibacterial activity of the antifungal peptides of the present invention after being treated with different pH (A), temperature (B), ultraviolet irradiation (C), metal ions (D), organic solvents (E), and surfactants (F). DETAILED DESCRIPTION

[0072] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0073] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0074] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0075] Bacillus velezensis CCTCC No: M 20211465, Latin name Bacillus velezensis, was deposited in China Center for Type Culture Collection (CCTCC) on November 22, 2021, with the deposit address at No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Center for Type Culture Collection, Wuhan University, with the deposit number CCTCC No: M20211465. It has been authorized by the Chinese invention patent with the authorization announcement number CN 117264847 B published on April 26, 2024.

[0076] The gray mold (Botrytis cinerea B05.10) in the following examples was provided by Professor Tian Shiping of the Institute of Botany, Chinese Academy of Sciences; it is recorded in the following literature: 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 extracts of fermentation products of Bacillus velez YTQ3

[0078] 1. Preparation of fermentation products of Bacillus velez YTQ3

[0079] 1. Activation of Bacillus velez YTQ3

[0080] Take out the frozen Bacillus Velez YTQ3CCTCC No: M 20211465 strain from the -80℃ refrigerator, dip it with a sterile inoculation loop in the clean bench, and inoculate it on the LB medium by the three-zone streak method. Put it in the incubator and culture it in the dark at 28℃ for 24h. Pick a single colony and inoculate it on the LB medium, and culture it in the dark at 28℃ for 24h to complete the strain activation. The activated Bacillus Velez is placed in a 4℃ refrigerator for storage.

[0081] 2. Preparation of seed solution

[0082] The activated single colony of Bacillus Velez subtilis YTQ3 obtained in step 1 was inoculated into a conical flask containing 20 mL of LB liquid culture medium, and cultured on a shaker at 6×g and 28° C. for 18 h to obtain a seed solution of Bacillus Velez subtilis YTQ3.

[0083] 3. Preparation of fermentation broth

[0084] The seed liquid of Bacillus Velez YTQ3 obtained in step 2 was inoculated into a conical flask containing 100 mL of LB liquid culture medium at an inoculation rate of 5%, and cultured in a shaking incubator at 6×g and 28° C. for 5 days to obtain a fermentation liquid.

[0085] 2. Preparation of extracts from fermentation products of Bacillus velez YTQ3

[0086] 1. Extraction by acid precipitation and alcohol extraction

[0087] The fermentation liquid obtained in step 1 was centrifuged at a speed of 9,600×g for 15 minutes, the supernatant of the fermentation liquid was collected, and then 1 mol / L hydrochloric acid aqueous solution was added to the supernatant of the fermentation liquid until the pH was 2.0; after standing at 4°C for 14 hours, it was centrifuged at 6,200×g and 4°C for 25 minutes to collect the precipitate. Methanol was added to the obtained precipitate, and the amount of methanol added was 0.1 times the volume of the supernatant of the fermentation liquid to obtain a resuspension, and then the resuspension was adjusted to pH 7.0 with 1 mol / L sodium hydroxide aqueous solution, and extracted (stirred) at room temperature (25°C) for 8 hours to obtain an extract. The extract was centrifuged at 4°C and 1,500×g for 10 minutes, the supernatant of the extract was taken, and the methanol was removed by a rotary evaporator (45°C, speed of 200rpm), and vacuum freeze drying was adopted (conventional freeze drying can be achieved, generally freeze drying for 48 hours, temperature is -55°C), and the obtained dry product is the YTQ3 fermentation product extract.

[0088] After testing, the yield of YTQ3 fermentation product extract in the supernatant of every 1L of fermentation broth was 712mg / L.

[0089] 2. Identification

[0090] 1) Liquid chromatography-mass spectrometry

[0091] The YTQ3 fermentation product extract obtained in 1 was measured by ultra-high performance liquid chromatography-electrospray ionization mass spectrometry (UHPLC-ESI-MS) (Thermo Fisher Scientific, USA). The test conditions are as follows: ACQUITY HSS T3 (2.1×100 mm, 1.8 μm) (Waters, Milford, MA, USA) chromatographic column, flow rate of 0.3 mL / min, column temperature of 40°C, injection volume of 2 μL. Positive ion mode, mobile phase of 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 ion source (ESI), spray voltage of 3.50 kV, sheath gas 40 arb, auxiliary gas 10 arb. Capillary temperature 325°C, scanning range m / z 100-1500.

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

[0093] The compound obtained by structural identification is indeed the target compound.

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

[0095] C15-C17 refers to the different lengths of fatty acid chains, with one more carbon atom in sequence;

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

[0097] C15-C17 refers to the different lengths of fatty acid chains, with one more carbon atom in sequence;

[0098] The structural formula of Bacilysin is as follows:

[0099]

[0100] The quantitative results of secondary mass spectrometry showed that Surfactin was the most abundant, accounting for 54.7% of the total amount of the fermentation product extract.

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

[0102]

[0103] 2) FTIR spectroscopy analysis

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

[0105] Take 1 mg of the YTQ3 fermentation product extract powder obtained in 1 above, grind it into fine powder in an agate mortar, mix it evenly with dry potassium bromide powder (about 100 mg, particle size 200 mesh), put it into a mold, and press it into tablets on a tablet press. Put the prepared sample into the sample holder and insert it into a fixed position in the instrument sample chamber. In the transmission mode, at 400 and 4000 cm- 1 4cm- 1 Scan the sample at high resolution 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 There is a broad band between -OH and NH, indicating the stretching of -OH and NH. The absorption peak is shown at 1653.01 cm- 1 There is an intermediate bond or peptide bond at 1237.67 cm-1, which is generated by the amide I band formed by CO stretching vibration. 1 The peak at 1404.11 cm- 1 The other major peak observed at corresponds to carbon-hydrogen bond bending vibrations, which are usually seen in compounds with aliphatic chains.

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

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

[0109] (1) Liquid chromatography separation and purification

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

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

[0112] (2) Antibacterial test

[0113] The pure powders of the 12 peaks obtained in (1) above were re-dissolved in sterile water and their antibacterial effects on Botrytis cinerea were determined. A sterile 96-well plate was used for the experiment, and 180 μL of a Botrytis cinerea spore suspension (1×10 6 CFU mL -1 ), and 20 μL of pure solution with a final concentration of 0.5 mg / mL (solvent is water, A1). Sterile water replaces the pure solution as a negative control (A2). After all the additions are completed, the 96-well plate is sealed with a sealing film and placed in a 22°C incubator for 48 hours, and the OD is measured using a spectrophotometer. 600 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 to 5 had significant antibacterial activity against Botrytis cinerea (Table 2), while the substances corresponding to other peaks had no antibacterial effect against Botrytis cinerea.

[0116] (3) Mass spectrometry identification

[0117] The pure solution corresponding to the peaks 1-5 detected as having antibacterial activity in (2) above was subjected to mass spectrometry analysis using UHPLC-ESI-MS. The test conditions were as follows: ACQUITY HSS T3 (2.1×100 mm, 1.8 μm) (Waters, Milford, MA, USA) chromatographic column, flow rate of 0.3 mL / min, column temperature of 40°C, injection volume of 2 μL. Positive ion mode, mobile phase of 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 ion source (ESI), spray voltage of 3.50 kV, sheath gas 40 arb, auxiliary gas 10 arb. Capillary temperature 325°C, scanning range m / z 100-1500.

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

[0119] Therefore, the fermentation product extract of strain YTQ3 contained a variety of antifungal peptides, and the production was ranked from high to low as Surfactin, Fengycin and Bacilysin.

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

[0121]

[0122] Example 2: Determination of the control effect of the extract of Bacillus Velezii YTQ3 fermentation product on fruit gray mold

[0123] Different amounts of the YTQ3 fermentation product extract obtained in Example 1 (2) were dissolved in water to obtain solutions of different concentrations, which were recorded as antifungal peptide solutions.

[0124] The gray mold spore suspension is prepared as follows: the gray mold (Botrytis cinerea B05.10) stored in advance is taken out from a -80°C refrigerator, placed in a 25°C constant temperature incubator for about 20 minutes, and then inoculated on a potato dextrose agar medium (PDA), and cultured at 25°C for about 7 days until the gray mold grows all over the plate. Take 1-2 plates full of gray mold, add an appropriate amount of sterile distilled water thereto, collect the spores of gray mold under sterile conditions, and filter through four layers of gauze to remove the hyphae. Count with a hemocytometer, adjust the spore suspension to the desired concentration with sterile distilled water, and obtain a gray mold spore suspension.

[0125] Grape berries were harvested from local orchards at their commercial maturity. Uninfected or uninjured berries of uniform size, color, shape, and weight were selected, and the fruit surface was disinfected with a 2% (v / v) sodium hypochlorite aqueous solution for 2 min, washed with distilled water, and air-dried at room temperature before use. A hole (3 mm deep and 3 mm wide) was punched near the equator of the fruit with a sterile puncher. First, 10 μL of antifungal peptide solution of different concentrations (2.5, 5, and 7.5 mg / mL) was inoculated, and after standing at room temperature for 2 h, 10 μL of Botrytis cinerea spore suspension (1×10 5 CFU / mL). Fruits inoculated with a suspension of gray mold spores alone were used as the control group (represented as an antifungal peptide concentration of 0 mg / mL in the figure). Three replicates were prepared for each treatment (15 fruits per group, 1 wound per fruit). All fruits were stored at 25°C and 90% RH. After the diameter of the control group reached a measurable size, observations were made every day. The incidence rate (DI) of each infected fruit and vegetable was calculated: DI (%) = (number of rotten wounds / total number of wounds) × 100%. The lesion diameter (LD) was evaluated by measuring the average diameter of the damaged area.

[0126] The results are as follows Figure 3 As shown in the results, different concentrations of antifungal peptide solutions had significant effects on the lesion diameter and incidence of gray mold in grape berries. During the entire storage period, the lesion diameter of the antifungal peptide treatment group was significantly lower than that of the control group (P<0.05) ( Figure 3 (B)); The fruits in the control group began to develop disease on the second day, and the incidence rate reached 100% on the third day, while the group treated with 2.5 mg / mL antifungal peptide began to develop disease on the fourth day, with an incidence rate of only 6.8%; on the sixth day of storage, the group treated with 5 mg / mL antifungal peptide began to develop disease slowly ( Figure 3 (C)); On the 7th day of storage, the diameter of the lesions in the control group reached 16.7 mm, while the diameters of the lesions 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)). However, treatment with 7.5 mg / mL antifungal peptides completely inhibited the occurrence of gray mold in grape berries.

[0127] It can be seen that the YTQ3 fermentation product extract can prevent the occurrence of gray mold in grape berries.

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

[0129] Different amounts of the YTQ3 fermentation product extract obtained in Example 1 (2) were dissolved in water to obtain solutions of different concentrations, which were recorded as antifungal peptide solutions.

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

[0131] The gray mold cake was prepared as follows: the gray mold (Botrytis cinerea) cultured on the PDA medium for 7 days was taken out, and holes (diameter 9 mm) were punched with a sterile puncher to obtain the gray mold cake.

[0132] Experimental group: 1.5 mL of antifungal peptide solution with final concentrations of 0.5, 1, 1.5 and 2 mg / mL was mixed with 15 mL of PDA medium to prepare PDA medium containing antifungal peptide. The 7-day-old Botrytis cinerea cake was punched into a cake with a sterile 9 mm diameter puncher and placed in the center of the PDA medium containing antifungal peptide with the hyphae side facing down. The cake was cultured in a constant temperature incubator at 22°C, and the diameter of the colony was measured every day and its inhibition rate was calculated.

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

[0134] The colony diameter was measured by the cross method and the inhibition rate was calculated according to formula (2). Each treatment was repeated 3 times and the whole experiment was repeated 2 times.

[0135]

[0136] The results are as follows Figure 4 As shown in the figure, as the concentration of the antifungal peptide solution increases, the inhibitory effect on the growth of Botrytis cinerea gradually increases. After 3 days of cultivation, the mycelium of the control group grew rapidly, and the colony diameter reached about 83.5 mm ( Figure 4 (A) and (B)), at this time, the inhibition rate of 0.5 mg / mL antifungal peptide solution on the mycelium growth of Botrytis cinerea has reached 73.6% ( Figure 4 (C)), showing a significant inhibitory effect. After 5 days of culture, the inhibition rate of the 2 mg / mL antifungal peptide solution treatment group on the mycelium growth of Botrytis cinerea was still maintained at 82.4%, and the colony diameter did not grow substantially during the entire culture period.

[0137] It can be seen that the extract of YTQ3 fermentation product has a significant inhibitory effect on the growth of Botrytis cinerea mycelium.

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

[0139] A sterile 96-well plate was used for the experiment, and 180 μL of a suspension of Botrytis cinerea spores (1×10 6 CFU mL -1), and 20 μL of antifungal peptide solution with a final concentration 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 was used instead of antifungal peptide solution as a negative control (0 mg / mL). After all the additions were completed, the 96-well plate was sealed with a sealing film and placed in a 22°C incubator for 48 hours, and the OD was measured using a spectrophotometer. 600 Each treatment group was repeated three times.

[0140] The results are as follows Figure 5 As shown, with the increase of the concentration of the antifungal peptide solution, the inhibitory effect on Botrytis cinerea gradually increased. The minimum inhibitory concentration (MIC) of the antifungal peptide solution on Botrytis cinerea was 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 1×10 6 CFU / mL The gray mold spore suspension prepared in Example 2 was evenly coated on a PDA medium containing an antifungal peptide solution and placed in a constant temperature incubator at 22°C. At the 2nd, 4th, 6th and 8th hour of culture, observation was performed under a microscope, and the germination of at least 200 spores and the diameter of the germ tube were observed each time. When the length of the germ tube diameter was equal to or greater than the spore diameter, the spores were considered to have germinated. The germination rate was calculated according to formula (3). Three replicates were set for each treatment in the experiment, and the test was repeated 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 the 4th hour of culture, the spore germination rate of the control group was 88%, while the spore germination rate of the group treated with 0.25 mg / mL antifungal peptide solution was significantly reduced to 51% ( Figure 6 (B)). At this time, the spores in the group treated with 0.5 mg / mL antifungal peptide solution had just begun to germinate, with a germination rate of 12%. As the concentration of the antifungal peptide solution increased, the inhibitory effect on the survival of Botrytis cinerea spores increased significantly. At the 8th hour of culture, the diameter of the spore tubes in the control group (0 mg / mL antifungal peptide solution) reached 95 μm ( Figure 6 (C)), the spore germination rate was 100%, while the spore germination rate of the group treated with 0.75 mg / mL antifungal peptide solution was almost 0%, indicating that the 0.75 mg / mL antifungal peptide solution can completely inhibit the germination of Botrytis cinerea spores.

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

[0146] Example 4: Scanning electron microscopy observation of morphological changes in Botrytis cinerea mycelium after antifungal peptide treatment

[0147] Different amounts of the YTQ3 fermentation product extract obtained in Example 1 (2) were dissolved in water to obtain solutions of different concentrations, which were recorded as antifungal peptide solutions.

[0148] Scanning electron microscopy was used to observe the morphological changes of Botrytis cinerea hyphae after treatment with antifungal peptide solution.

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

[0150] Weigh 0.1g mycelium (wet weight) into a centrifuge tube and add antifungal peptide solutions with final concentrations of 1, 3 and 5mg / mL. Incubate in a 22℃ constant temperature incubator for 12h. PBS replaces antifungal peptide as the control group (CK). Collect mycelium by centrifugation at 1,500×g, add 4% (v / v) glutaraldehyde and fix overnight at 4℃ (about 12h), then dehydrate with 100μL ethanol gradient (30, 50, 70, 90, 100%), each treatment for 10min. After natural air drying, the sample was mounted on the stage and sprayed with gold, and loaded into the scanning electron microscope for imaging.

[0151] The results are as follows Figure 7 As shown, the mycelial surface of the control group was normal and smooth (CK), but after being treated with antifungal peptides, the mycelial surface was damaged to varying degrees. When the mycelial surface was treated with 1 mg / mL of antifungal peptides, the mycelial surface shrank and was no longer smooth, while after the mycelial surface was treated with high concentrations of antifungal peptides (5 mg / mL), the mycelial contents were severely lost, the entire mycelial surface collapsed, and the mycelial surface 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 (2) were dissolved in water to obtain solutions of different concentrations, which were recorded as antifungal peptide solutions.

[0154] 1. Thermal stability

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

[0156] 2. Acid and alkali stability

[0157] The antifungal peptide solution (concentration of 1 mg / mL, solvent is water) was adjusted to pH 2, 4, 6, 8 and 10 respectively with 4 mol / L concentrated hydrochloric acid aqueous solution and 4 mol / L sodium hydroxide aqueous solution, and after standing for 1 hour, the pH was adjusted back to 7.0. The antibacterial effect on Botrytis cinerea was determined using a 96-well plate, and the method was referred to the above 1.

[0158] 3. Anti-ultraviolet stability

[0159] The antifungal peptide solution (concentration of 1 mg / mL, solvent is water) was placed at 20 cm under an ultraviolet lamp (UV-C, 30W) and irradiated for 10 min, 20 min, 30 min, 45 min and 60 min respectively. The antibacterial effect on Botrytis cinerea was determined using a 96-well plate, according to the method mentioned above 1.

[0160] 4. Metal ion stability

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

[0162] 5. Organic solvent stability

[0163] The antifungal peptide solution (concentration of 1 mg / mL, solvent is water) was mixed with methanol, ethanol, isopropanol, chloroform and acetone solutions at a ratio of 1:1 (v / v). After standing for 1 hour, the antibacterial effect on Botrytis cinerea was determined using a 96-well plate, according to the method mentioned above 1.

[0164] 6. Surfactant stability

[0165] The antifungal peptide solution (concentration of 1 mg / mL, solvent is 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 hour, the antibacterial effect on Botrytis cinerea was determined using a 96-well plate, according to the method mentioned above 1.

[0166] The results are as follows Figure 8 As shown, the antifungal peptide solution has good stability against adverse conditions. In an environment with a pH of 2-10, the antifungal peptide solution has little effect on the antibacterial activity against Botrytis cinerea ( Figure 8 (A)). The antifungal peptide solution can still maintain a 95% inhibition rate at high temperature of 65-90°C ( Figure 8 (B)). UV irradiation for 60 min had no effect on the antibacterial activity of the antifungal peptide solution ( Figure 8 (C)). In addition, metal salts, organic solvents and surfactants had no effect on the activity of antifungal peptide solutions ( Figure 8 (D)- Figure 8 (F)).

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

[0168] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Application of the fermentation product extract of Bacillus velez YTQ3 CCTCC No: M 20211465 in any of the following: 1) Antifungal or fungistatic; 2) Preparation of antifungal or fungistatic products; 3) Fruit and vegetable disease prevention and control; 4) Preparation of fruit and vegetable disease prevention and control products; The fermentation product extract is a product obtained by subjecting the fermentation broth of Bacillus velez YTQ3 CCTCC No: M 20211465 to acid precipitation and alcohol extraction; the fermentation product extract contains surfactin, fengycin and / or bacilysin.

2. The use according to claim 1, characterized in that: The acid precipitation and alcohol extraction adopts methanol extraction; Or, the mass ratio of Surfactin, Fengycin and Bacilysin in the fermentation product extract is 6-10:1-5:1-5; Or, the mass ratio of Surfactin, Fengycin and Bacilysin in the fermentation product extract is 102.7:18.3:14.

8.

3. The use according to claim 1 or 2, characterized in that: The fermentation broth of Bacillus velez YTQ3 CCTCC No: M 20211465 is obtained by culturing Bacillus velez YTQ3CCTCC No: M 20211465 in a liquid culture medium; The fermentation product extract is prepared according to method 1, which comprises the following steps: 1) adjusting the pH of the supernatant of the fermentation broth to 2.0-2.2, allowing the supernatant to stand, and collecting the precipitate; 2) resuspending the precipitate with methanol to obtain a suspension, and then adjusting the pH of the suspension to 7.0-7.2 and then extracting to obtain a fermentation product extract.

4. The use according to claim 3, characterized in that: The extraction comprises the following steps: adjusting the pH of the suspension to 7.0-7.2, extracting, collecting the supernatant of the extract, and removing the liquid in the supernatant to obtain a fermentation product extract.

5. The use according to claim 3 or 4, 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 1), the standing condition is standing at 4° C. for 12-18 hours; And / or, in step 1), the conditions for collecting the precipitate are centrifugation at 6,200×g and 4° C. for 20-25 min; And / or, in step 2), the amount of methanol added is 0.1-1 times the volume of the supernatant of the fermentation broth; And / or, in step 2), the pH is adjusted using a sodium hydroxide aqueous solution, the concentration of the sodium hydroxide aqueous solution is 1-6 mol / L; And / or, in step 2), the extraction condition is 20-25° C. for 6-12 h; And / or, in step 2), the supernatant of the extract is collected by centrifugation at 4°C and 1,500×g for 10-15 min.

6. The use according to any one of claims 1 to 5, characterized in that: The fungus is Botrytis cinerea; Or, the fruit and vegetable disease is gray mold; Or, the fruit and vegetable disease is gray mold of grape fruit.

7. The use according to any one of claims 1 to 6, characterized in that: The product is a bacterial agent, a medicine, a composition or other biological product.

8. A method for preparing the fermentation product extract according to any one of claims 1 to 6, characterized in that: The method comprises the method 1 described in any one of claims 3-7.

9. A composition for resisting fungi or fungal diseases, characterized in that: The composition is the fermentation product extract according to any one of claims 1-7.

10. A product, the active ingredient of which is the fermentation product extract according to any one of claims 1 to 7 or the composition according to claim 9; The product has any of the following functions: 1) Antifungal or fungistatic; 2) Prevention and control of fruit and vegetable diseases.

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