Microbial anti-mildew storage control method for dried chilies

Abstract

The invention discloses a microbial anti-mildew storage control method for dried chilies, and belongs to the technical field of microorganisms. The bacillus velezensis FP123 is obtained through screening, the bacillus velezensis FP123 is preserved in the China Center for Type Culture Collection on July 31, 2024, and the preservation number of the bacillus velezensis FP123 is CCTCC (China Center for Type Culture Collection) NO: M20241718. The bacillus velezensis FP123 has an obvious antagonistic effect on aspergillus flavus and can be used as an effective dried chili biological mildew-proof preparation. The strain can inhibit aspergillus flavus hypha growth and spore germination by generating volatile compounds such as 2-methylbutyric acid, 3-methylbutyric acid and acetic acid. The method for spraying or fumigating the dried chilies by using the bacillus velezensis FP123 is a novel microbial anti-mildew storage control method, and has extremely high research and application values on the development of novel green antibacterial drugs.

Description

Technical Field

[0001] The invention relates to a microbial mildew-proof storage control method for dried peppers, belonging to the technical field of microorganisms. Background Art

[0002] Dried chili peppers are red chili peppers that have been naturally dried and artificially dehydrated. They are also called dried chili peppers, processed chili peppers, processed chili peppers, etc. Dried chili peppers are an important part of the chili pepper industry. In the process of preparing dried chili peppers, the dried chili peppers are usually put into woven bags and then stacked indoors. Dried chili peppers are easily moldy due to direct contact with air. In addition, factors such as production conditions, packaging materials, and mechanical damage during the production and processing process can lead to mold and excessive microbial content during storage of dried chili peppers. Among them, the main microorganism that causes mold is Aspergillus flavus.

[0003] Aspergillus flavus is a common saprophytic fungus that is widely distributed in the world and often exists in the air and soil. It is a pathogen of many important crops and can also infect crops such as peanuts, corn, and soybeans, inducing crop mildew and disease. At the same time, after Aspergillus flavus infects crops, it will produce a variety of toxic secondary metabolites under certain conditions. Among them, aflatoxin is the most toxic and harmful, and is a hazard to human and animal health and food safety. The problem of excessive aflatoxin has seriously hindered the healthy development of agricultural products. Therefore, controlling the growth of Aspergillus flavus is of great significance to food safety and human health.

[0004] The methods commonly used to inhibit the growth of Aspergillus flavus are physical, chemical and biological control. Physical methods include dehumidification, temperature control, deoxygenation and ultraviolet irradiation, etc., which mainly control environmental conditions according to the growth characteristics of Aspergillus flavus to achieve control effects. Chemical methods mainly use chemical mildew inhibitors to treat agricultural products, the main ingredients of which are chemically synthesized organic acids and their salts or some antioxidants. The use of physical and chemical methods to prevent and control the pollution of Aspergillus flavus and its toxins is not only inefficient and costly, but also causes the loss of food nutrients and the difficulty in removing the residues of toxic compounds, which seriously endangers human health and ecosystem safety.

[0005] Biological control is to achieve control effects by using mutual inhibition between organisms or the production of certain active substances. Studies have shown that Bacillus subtilis, bacteria, lactic acid bacteria, yeast, mold, and actinomycetes can all produce substances that inhibit the growth of Aspergillus flavus. With the continuous development and accumulation of microbial preparations, they have the characteristics of safety, non-toxicity, no residue, and conducive to the sustainable development of the ecological environment, making them gradually become alternatives to traditional antibiotics.

[0006] Therefore, it is of great practical significance to screen and obtain microbial strains that have antagonistic effects on Aspergillus flavus and study their application effects in mildew prevention of dried peppers. Summary of the invention

[0007] In view of the deficiencies of the above-mentioned prior art, the present invention provides a method for controlling the storage of dried peppers to prevent mildew by microorganisms, aiming to solve the technical problem that the use of physical and chemical methods to prevent and control aflatoxin and its toxin pollution is not only inefficient and costly, but also causes the loss of food nutrients and difficulty in removing the residues of toxic compounds, which seriously endangers human health and ecosystem safety.

[0008] The first technical solution provided by the present invention is a strain of Bacillus velezensis FP123, which was deposited in the China Center for Type Culture Collection on July 31, 2024, and the strain collection number is: CCTCC NO: M20241718.

[0009] The second technical solution provided by the present invention is a microbial preparation containing the Bacillus Velez FP123 described in the first technical solution.

[0010] In certain embodiments, the concentration of the Bacillus Velez FP123 in the microbial preparation is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0011] Furthermore, the concentration of the Bacillus Velez FP123 in the microbial preparation is at least 1×10 8 CFU / mL or 1×10 8 CFU / g.

[0012] The third technical solution provided by the present invention is a biological control agent, which contains the Bacillus Velez FP123 described in the first technical solution or the microbial preparation described in the second technical solution.

[0013] In certain embodiments, the amount of the Bacillus Velez FP123 added to the biological control agent is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0014] Furthermore, the amount of the Velez subtilis FP123 added to the biological control agent is at least 1×10 8 CFU / mL or 1×10 8 CFU / g.

[0015] The fourth technical solution provided by the present invention is the use of the Bacillus Velez FP123 described in the first technical solution, the microbial preparation described in the second technical solution, or the biological control agent described in the third technical solution to prevent and control Aspergillus flavus in dried peppers.

[0016] In certain embodiments, the control of Aspergillus flavus refers to the use of 2-methylbutyric acid, 3-methylbutyric acid and / or acetic acid produced by Bacillus Velezii FP123 to inhibit the growth of Aspergillus flavus hyphae and spore germination.

[0017] The fifth technical solution provided by the present invention is a method for preventing and controlling Aspergillus flavus, which uses the Bacillus Velez FP123 described in the first technical solution, the microbial preparation described in the second technical solution, or the biological control agent described in the third technical solution to spray or fumigate dried peppers.

[0018] In certain embodiments, the control of Aspergillus flavus refers to the production of 2-methylbutyric acid, 3-methylbutyric acid and / or acetic acid to inhibit the growth of Aspergillus flavus hyphae and spore germination.

[0019] In certain embodiments, the concentration of the Bacillus Velez FP123 sprayed on the dried pepper is at least 1×10 8 CFU / mL.

[0020] The sixth technical solution provided by the present invention is a method for increasing the anti-aflatoxin substances in dried peppers, wherein the method comprises spraying or fumigating the dried peppers with the Bacillus Velez FP123 described in the first technical solution, the microbial preparation described in the second technical solution, or the biological control agent described in the third technical solution, wherein the anti-aflatoxin substances include 2-methylbutyric acid, 3-methylbutyric acid and acetic acid.

[0021] In certain embodiments, the concentration of the sprayed Bacillus Velez FP123 is at least 1×10 8 CFU / mL.

[0022] The technical effects of the present invention are as follows:

[0023] The present invention obtains a strain of Bacillus velezensis FP123 through screening, which has been deposited in the China Center for Type Culture Collection on July 31, 2024, and the strain deposit number is: CCTCC NO: M20241718. Bacillus velezensis FP123 has a significant antagonistic effect on Aspergillus flavus and can be used as an effective biological mildew-proof preparation for dried peppers. The strain can inhibit the growth of Aspergillus flavus hyphae and spore germination by producing volatile compounds such as 2-methylbutyric acid, 3-methylbutyric acid, and acetic acid. Spraying or fumigating dried peppers with Bacillus velezensis FP123 is a new microbial mildew-proof storage control method, which has extremely high research and application value for the development of new green antibacterial drugs.

[0024] Biomaterial Deposit

[0025] Bacillus velezensis FP123, with the taxonomic name Bacillus velezensis, was deposited in the China Center for Type Culture Collection on July 31, 2024. The deposit address is Wuhan University, Wuhan, China. The strain deposit number is: CCTCCNO:M20241718. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is to initially screen the inhibitory effects of some strains on the standard strain of Aspergillus flavus.

[0027] Figure 2 The morphological characteristics of the strains; A: morphological characteristics of Bacillus Velez FP123 on PDA medium, B: morphological characteristics of Bacillus Velez FP123 under a microscope.

[0028] Figure 3 This is a growth curve of Bacillus velez FP123 in different culture media; PDA: potato dextrose agar medium, ZT: sucrose medium, PTT: glucose medium, LB: LB medium, YPD: yeast extract peptone dextrose medium.

[0029] Figure 4 This is the inhibitory effect of Bacillus Velez FP123 strain on Aspergillus flavus for 2-7 days; A: the effect of the strain on the growth of Aspergillus flavus, B: the effect of the strain on the colony diameter of Aspergillus flavus, C: the effect of the strain on the hyphae growth of Aspergillus flavus, D: the effect of the strain on the number of Aspergillus flavus spores.

[0030] Figure 5 This is the inhibition of Aspergillus flavus by different fermentation components of Bacillus Velez FP123 in different culture media.

[0031] Figure 6 The colony morphology is shown in Table 2.

[0032] Figure 7 This is the effect of 2-methylbutyric acid and 3-methylbutyric acid on the germination of Aspergillus flavus spores.

[0033] Figure 8 This is a picture of Bacillus Velez FP123 controlling Aspergillus flavus in dried peppers.

[0034] Fig. 9 A picture showing the volatile substances produced by Bacillus Velez FP123 in controlling Aspergillus flavus in dried peppers. DETAILED DESCRIPTION

[0035] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0036] The culture medium involved in the following embodiments is:

[0037] 1. Potato dextrose agar (PDA) medium: 10 g / L peptone, 5 g / L yeast extract powder, 20 g / L glucose, 15 g / L agar;

[0038] 2. Potato dextrose liquid (PDB) medium: 10 g / L peptone, 5 g / L yeast extract powder, 20 g / L glucose;

[0039] 3. Sucrose (ZT) medium: 10 g / L sucrose, 10 g / L peptone, 10 g / L sodium chloride, 1 g / L KH2PO4, 0.5 g / LMg2S04·7H2O, 15 g / L agar;

[0040] 4. Glucose (PTT) medium: 10 g / L glucose, 10 g / L peptone, 10 g / L sodium chloride, 1 g / L KH2PO4, 0.5 g / L Mg2S04·7H2O, 15 g / L agar;

[0041] 5. LB medium: 10g / L peptone, 10g / L sodium chloride, 5g / L yeast powder, 15g / L agar;

[0042] 6. Yeast extract peptone dextrose (YPD) medium: 10 g / L peptone, 5 g / L yeast extract powder, 20 g / L glucose, 15 g / L agar.

[0043] Example 1: Screening and identification of Bacillus Velezii FP123

[0044] 1. Preparation of Aspergillus flavus spore suspension

[0045] The standard strain of Aspergillus flavus used in the experiment was purchased from the China General Microbiological Culture Collection Administration Center with the collection number of CGMCC 3.4408. Aspergillus flavus was inoculated on PDA medium and cultured in a 28°C incubator for 7 days. 10 mL of sterile saline was used to scrape the Aspergillus flavus spores with a sterile glass rod and filtered through four layers of gauze to obtain 10 7 The spore suspension of 1000 cells / mL is ready for use.

[0046] 2. Screening of Bacillus Velez FP123

[0047] Wash the skin of fresh peppers, moldy dried peppers, kiwi fruit and pickled peppers with 10 mL of sterile water, collect the liquid and culture it on LB medium at 28°C for 24 h for gradient dilution (10 -1 -10 -6 ) was spread, and the single colonies cultured were purified and numbered. The strains were inoculated into PDB medium in the order of numbering for overnight culture. Five 10 mm paper slips were pasted on the PDA medium using the five-point paper slip method, and the middle paper slip was inoculated with 20 μL of Aspergillus flavus spore suspension (10 7 / mL), four paper pieces on the cross line 25mm away from the center were inoculated with 20μL of overnight culture solution of the numbered strain and cultured at 37℃ for 5 days. The results of the antagonism experiment are shown in Figure 1 , and screened out the strain FP123 with antagonistic effect against Aspergillus flavus.

[0048] 3. Identification of Bacillus Velezii FP123 strain

[0049] The genome of strain FP123 was extracted using a kit, and the selected strains were identified according to the national standard GB T 33682-2017 "General Methods for Identification of Microorganisms by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry", and classified according to Bergey's Manual of Bacteriology. The classification of the strains was identified using 16s rDNA primer PCR, and Bacillus velezensis FP123 was obtained. The appearance and microscopic morphology of the colony are as follows Figure 2 The strain is milky white, round, smooth, slightly convex, 1.0-1.5mm, and has a viscous texture; Gram staining is positive, and the bacterial cells are rod-shaped.

[0050] 4. Optimal culture medium selection

[0051] 1 mL of Bacillus Velez FP123 culture liquid was inoculated into PDB, LB, PTT and ZT medium respectively and cultured at 28°C and 180 rpm for 24 h. The growth curve of Bacillus Velez FP123 was measured using a microbial growth curve analyzer. 150 μL of different culture media were added to a 96-well plate, and 2% of the cultured fermentation liquid was inoculated. Different culture media were used as controls, and the optical density at a wavelength of 600 nm was measured every 1 hour. The growth curve of Bacillus Velez FP123 is shown in Figure 1. Figure 3 As shown, PDB medium was most suitable for the growth of Bacillus velez FP123. Therefore, PDA and PDB medium were used in subsequent experiments for antagonism experiments.

[0052] 5. Inhibition of Bacillus Velez FP123 on Aspergillus flavus

[0053] (1) Radial hyphae growth inhibition

[0054] Using the five-point paper disc method, five 10 mm paper discs were pasted on the PDA medium, and 20 μL of the prepared aflatoxin spore suspension (10 7 / mL), four paper pieces on the cross line 25mm away from the center were inoculated with 20μL of fermentation broth of Bacillus Velez FP123, spore suspension and water were inoculated as controls, and cultured at 37℃ for 7d. The radial growth of each hyphae colony was monitored with a vernier caliper every day. The percentage of inhibition of the growth of Aspergillus flavus hyphae by the bacteria in the treatment group from 2nd to 7th day was calculated according to the formula. The calculation formula for the inhibition of fungal radial hyphae growth is: (R1×R2 / R1)×100%, where R1 is the radial hyphae growth diameter of the control group, and R2 is the radial hyphae growth diameter of the strain treatment.

[0055] (2) Conidia inhibition rate

[0056] The spore germination test was performed using the five-point paper disc method. Five 10 mm paper discs were pasted on the PDA medium, and 20 μL of the prepared aflatoxin spore suspension (10 7 The four paper pieces on the cross line 25 mm from the center were inoculated with 20 μL of fermentation liquid of Bacillus Velez FP123, and the spore suspension and water were inoculated as controls. The plates were cultured at 37°C for 7 days. The plates with the entire growing colony were washed twice with 10 mL of normal saline, and gently scraped with a glass rod. After filtering, 10 μL of the suspension was counted on a hemocytometer, and the number of conidia per mL was calculated to determine the number of conidia per cm 2Conidia concentration of hyphae. The inhibition percentage of the fungus in the treatment group on the 3rd to 7th day on the growth of Aspergillus flavus hyphae was calculated according to the formula. The calculation formula for the fungal conidia inhibition rate is (Z1×Z2 / Z1)×100%, where Z1 is the conidia concentration of the control group and Z2 is the conidia concentration of the strain treatment.

[0057] (3) Conidia germination and viability

[0058] The experiment used an 8 mL test tube, filled with 2 mL of sterile peptone water. At the same time, 40 μL of bacterial suspension (10 9 / mL) and 40μL of Aspergillus flavus spore suspension (10 6 / mL) and incubated at 28℃ for 24h. The control group was a test tube without bacteria, which was replaced by sterile peptone water. After 24h, 200 conidia were counted in each replicate and the proportion of germinated conidia was determined. Conidia were considered to be germinated when the length of the tooth tube doubled. The experiment was repeated three times.

[0059] After conidia were counted, 100 μL of each replicate was inoculated on the surface of a PDA plate and cultured at 28°C for 5 days. The survival rate of conidia was determined and the colony forming units were counted. The experiment was repeated three times.

[0060] (4) Figure 4 As shown in Table 1, the FP123 strain began to inhibit the growth of Aspergillus flavus spores and hyphae on the 3rd day. The FP123 strain had a 61% inhibition rate on the growth of Aspergillus flavus hyphae on the 7th day, and a 98.04% inhibition rate on the number of Aspergillus flavus spore germination. As shown in Table 1, after inoculation with Bacillus Velezii FP123, the number of Aspergillus flavus conidia germination was significantly reduced, and the survival rate of Aspergillus flavus was low.

[0061] Table 1 Effects of Bacillus Velez FP123 on conidia and viability of Aspergillus flavus

[0062] Spore germination number Survival count (log CFU / mL) CK 90.33 1.96 Bacillus Velez FP123 23.67 1.37

[0063] Example 2: Inhibition of Aspergillus flavus by different fermentation ingredients

[0064] 1. Preparation of fermentation broth and bacterial suspension of Bacillus Velez FP123

[0065] Inoculate Bacillus Velez FP123 on PDA medium and culture at 28°C for 24h; take a full loop of the strain on the above PDA solid medium and inoculate it in PDB medium, and culture it at 28°C and 180rpm for 24h; take 2mL of the bacterial liquid after culturing for 24h and inoculate it in 100mL of PDB medium, and culture it at 28°C and 180rpm for 48h to prepare fermentation liquid; centrifuge the fermentation liquid after culturing for 48h, centrifuge it at 5000×g for 15min at 4°C, then recover the fermentation supernatant and bacteria respectively, wash the bacteria after centrifugation, wash the bacterial precipitate 3 times with sterile peptone water, and then reset it in peptone water to make a bacterial suspension, and adjust the bacterial suspension to 10 under a microscope using a hemocytometer and sterile peptone water. 7 Pieces / mL.

[0066] 2. Inhibition of Aspergillus flavus by different fermentation ingredients

[0067] (1) Effects of volatile substances on Aspergillus flavus

[0068] Inverted plate method: two culture media, the upper layer is inverted PDA for culturing the standard strain of Aspergillus flavus (sticking 10mm paper, adding 20μL of Aspergillus flavus spore suspension), and the lower layer is used with the corresponding five culture media (PDA, YPD, LB, glucose (PTT), sucrose medium (ZT)), and 100μL of the fermentation liquid of Bacillus Velez FP123 is applied respectively, and the blank culture medium without culturing bacteria in the lower layer is used as a control, and each group is repeated 3 times. The edges of the two plates are wrapped together with sealing film to prevent air leakage. After all culture dishes were cultured at 28°C for 6 days, the effect of volatile substances produced by the strain on Aspergillus flavus was evaluated by comparing the mycelial growth diameter of the standard strain of Aspergillus flavus with and without bacterial bodies.

[0069] (2) Effects of bacteria on Aspergillus flavus

[0070] Using the five-point paper disc method, five 10 mm paper discs were pasted on five culture media, and 20 μL of the prepared aflatoxin spore suspension (10 7 / mL), inoculate 20μL of antagonistic strain suspension on four paper pieces on the cross line 25mm away from the center, and culture at 37℃ for 5d. Wash the entire plate with growing colonies twice with 10mL of normal saline, gently scrape with a glass rod, filter and count conidia in 10μL of suspension on a hemocytometer, and calculate the inhibition rate of antagonistic strains on Aspergillus flavus spores according to the fungal conidia inhibition rate formula.

[0071] (3) Figure 5 As shown, the volatile substances produced by FP123 can inhibit the growth of Aspergillus flavus spores. The effects of volatile compounds on FP123 in different culture media are: PDA>LB>ZT>PTT>YPD.

[0072] The inhibition rate of the bacteria on Aspergillus flavus spores: Under the conditions of PTT, ZT, and LB, the standard strain of Aspergillus flavus can only produce a small amount of spores. When the FP123 strain is used as the inhibitory strain, the hyphae growth of the standard strain of Aspergillus flavus is reduced under the conditions of these three culture media (the hyphae inhibition rate is calculated based on the colony diameter), and no spores are produced. However, under the conditions of PDA culture medium, the standard strain of Aspergillus flavus produces a large number of spores and hyphae. When FP123 is used as the inhibitory strain, the hyphae and spores are inhibited.

[0073] Therefore, the optimal culture medium for FP123 is PDA, which can inhibit the growth of Aspergillus flavus to the greatest extent under appropriate culture medium conditions.

[0074] Example 3: Inhibition of Aspergillus flavus by specific volatile compounds

[0075] 1. Headspace solid phase microextraction (HS-SPME) of volatile organic compounds in antagonistic bacteria

[0076] The Bacillus Velez FP123 bacterial suspension in Example 2 was added into 20 mL SPME tubes containing 5 mL PDB and YPD liquid culture media, respectively, and the concentration of the bacterial suspension was adjusted to 1×10 6 CFU / mL. The SPME tubes were tightly closed with polytetrafluoroethylene (PTFE) caps and cultured on a rotary shaker at 28°C and 180 rpm for 3, 5, and 7 days. SPME tubes containing 5 mL of PDB and YPD liquid culture medium were used as controls. After equilibration at 40°C for 40 min, the SPME fiber was inserted into the vial and VOCs were extracted at 40°C for 30 min. The syringe depth was 40 mm. The analytes were desorbed in the GC injector at 270°C in a non-split mode for 10 min.

[0077] 2. Gas chromatography-mass spectrometry analysis of volatile organic compounds

[0078] VOCs were analyzed using a gas chromatograph (Themofisher Trace-1300) and a quadruple mass spectrometer (Themofisher Trace-ISO) in electron ionization (EI) mode (70 eV).

[0079] A TR-5MS capillary column (30 m × 0.25 mm, 0.25 um film thickness, Thermo Scientific) and a TriPlusRSH autosampler were used to separate different volatile organic compounds.

[0080] The GC oven temperature was set to an initial temperature of 40°C for 3 min, then increased to 180°C at 10°C / min, and then increased to 270°C at 40°C / min and maintained for 4 min. The temperature of the ion source and mass spectrometer transmission line were both set to 250°C. The mass spectra were scanned in the range of m / z 35 to 500 amu. VOCs were identified by comparing mass spectra, direct matching (Sl) and reverse matching (RSl) with VOCs in the National Institute of Standards and Technology (NIST) library.

[0081] The volatile compounds obtained after matching are phenylethanol, 2-methylbutyric acid, 3-methylbutyric acid, 2.3-butanediol, acetic acid, 3-methyl-1-butanol, isoamyl acetate, 2-methyl-1-propanol, ethyl 2-methylbutyrate, n-propanol, isobutyl acetate, propyl acetate and ethyl acetate, and the corresponding peak areas are 393456185, 31462090, 86628973, 156323542, 16450622, 924461354, 53962597, 58745042, 28453811, 19068209, 4990338, 3800431 and 295578086, respectively. According to relevant literature reports, the antibacterial effect of ester compounds is not obvious. Therefore, phenylethanol, 2-methylbutyric acid, 3-methylbutyric acid, acetic acid, 2,3-butanediol, 3-methyl-1-butanol, and 2-methyl-1-propanol were selected to evaluate the inhibitory effect on enzymes. The details are as follows: (1) Evaluation of the in vitro inhibitory effect of single identified VOCs on fungal pathogens

[0082] A 10 mm paper was pasted in the center of the PDA medium and inoculated with 20 μL of Aspergillus flavus spore suspension. Seven portions of pure standard products were diluted with sterile water into four concentration gradients (1000, 100, 10, 1 μL / mL). 20 μL of pure standard product was dropped onto a 10 mm filter paper, pasted on the inner cover of the culture dish, sealed in time, and incubated upside down at 28°C for 5 days. Three replicate sample units were used for each dose, and the same volume of sterile water was used as a control. The radial growth of each mycelial colony was measured with a vernier caliper, and the inhibition radial growth rate of the mycelial colony was calculated.

[0083] The results are shown in Table 2. 2-methylbutyric acid and acetic acid have the strongest inhibitory effect on the growth of Aspergillus flavus mycelium, and the inhibition rate is 100% at the highest concentration. 3-methylbutyric acid and phenylethanol have the second strongest inhibitory effect on Aspergillus flavus, with inhibition rates of 89.12% and 79.82%, respectively. The other three compounds have an inhibition rate of less than 50% on the growth of Aspergillus flavus mycelium, among which 3-methyl-1-butanol and 2-methyl-1-propanol mycelium turned white after treatment, and 2,3-butanediol had no antibacterial effect on Aspergillus flavus.

[0084] Table 2 Effects of selected pure compounds on the growth of Aspergillus flavus after 5 days of cultivation at 28°C.

[0085]

[0086] Note: Each value is the mean ± standard error of three replicates. The values ​​in the column behind different letters are significantly different at the P < 0.05 level by Duncan's multiple range test. The images correspond to the colonies of Aspergillus flavus grown on PDA for 5 days at a treatment concentration of 1000 μL / mL.

[0087] (2) Determination of minimum inhibitory concentration (MIC) value

[0088] Different amounts of standard compounds were added into 1 mL of a suspension containing Aspergillus flavus spores (2%, 10 5 / mL) in PDB medium, and the final concentrations were 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 μL / mL, respectively. The control group was a parallel treatment without compound, and all samples were cultured at 28°C for 24h. The spore germination rate after incubation was recorded using an optical microscope. When the germination length exceeded half of the maximum size of the spore, the spore was considered to have entered the germination stage. At least 200 spores were measured for each counting calculation, and a total of three measurements were made. The minimum concentration of the standard compound that did not allow Aspergillus flavus spores to germinate was considered to be the minimum inhibitory concentration (MIC).

[0089] After MIC value determination, it was found that 3-methylbutyric acid had the lowest MIC value, which was 1.2 μL / mL. Figure 6 The effect of 3-methylbutyric acid and 2-methylbutyric acid on the germination of Aspergillus flavus spores. As the concentration of 3-methylbutyric acid increases, the spore germination rate decreases and the inhibition rate increases. When the concentration of 3-methylbutyric acid is 1.2μL / mL and above, the spore germination inhibition rate reaches 100%. Secondly, the MIC value of 2-methylbutyric acid is 1.4μL / mL.

[0090] Example 4 Application of Bacillus Velez FP123 in the method for controlling the mildew-proof storage of dried peppers

[0091] After selecting intact and uniform-sized dried peppers and sterilizing them with ultraviolet light for 30 minutes, 10 8 1 mL of Bacillus Velezii FP123 with a CFU / mL was sprayed on the surface of the dried pepper, and then sprayed with 1 mL of Aspergillus flavus spore suspension (10 7 / mL), placed in a petri dish at 28℃, and observed the mildew of the dried peppers during storage. The dried peppers that were not sprayed with the suspension of Bacillus Velez FP123 were used as the control. Each experiment was repeated 3 times, with 6 dried peppers in each repeat. Figure 8 As shown, the dried peppers did not become moldy after being sprayed with No. 123 bacteria, and the storage mold rate of the dried peppers was reduced by 100%.

[0092] The fumigation of dried peppers with Bacillus Velez FP123 was carried out by the inverted plate method. 100 μL of Bacillus Velez FP123 bacterial solution was added to the upper YPD medium and cultured for 24 h. The dried peppers with complete and uniform size were selected for ultraviolet irradiation for 30 min and sprayed with 1 mL of Aspergillus flavus spore suspension (10 7 / mL), placed in the lower culture dish, cultured at 28 ° C, and observed the mildew during the storage of peppers. The upper PDA medium was used as a control without inoculation of Bacillus Velez FP123. Each experiment was repeated 3 times, and each repeat treated 6 peppers. The storage mildew rate of the peppers fumigated with FP123 strain was reduced by 50%, and Aspergillus flavus did not produce spores at the mildewed site.

[0093] Example 5 Application of VOCs in the Control of Aflatoxin in Dried Peppers

[0094] The inverted plate method was used to add filter paper in the center of the culture dish lid, and a 10 mm paper sheet was attached to the upper culture medium. Different volumes of single VOC (10, 20, 40 μL) were added. The dried peppers with complete and uniform size were selected for ultraviolet irradiation for 30 min, and 1 mL of Aspergillus flavus spore suspension (10 7 / mL), placed in the lower culture dish, cultured at 28℃, and observed the mildew of peppers during storage. The upper culture medium without VOC was used as the control, and each experiment was repeated 3 times, with 6 peppers in each repeat. Fig. 9 As shown, when the addition amount of 3-methylbutyric acid and 2-methylbutyric acid is 10 μL, the dried pepper will not mold. The addition amount of acetic acid and phenylethanol is 20 μL and 40 μL, respectively, for the dried pepper to prevent mold.

[0095] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Bacillus velezensis FP123, characterized in that: It was deposited in the China Center for Type Culture Collection on July 31, 2024, and the strain accession number is: CCTCC NO:M20241718.

2. A microbial preparation containing the Bacillus Velez FP123 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The concentration of the Bacillus Velez FP123 in the microbial preparation is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

4. A biological control agent, characterized in that The biological control agent contains the Bacillus Velez FP123 of claim 1 or the microbial preparation of claim 2 or 3.

5. Use of the Bacillus Velez FP123 according to claim 1, the microbial preparation according to claim 2 or 3, or the biological control agent according to claim 4 in controlling Aspergillus flavus in dried peppers.

6. The use according to claim 5, characterized in that: The method for preventing and controlling Aspergillus flavus refers to using 2-methylbutyric acid, 3-methylbutyric acid and / or acetic acid produced by Bacillus Velezii FP123 to inhibit the growth of Aspergillus flavus hyphae and spore germination.

7. A method for preventing and controlling Aspergillus flavus, characterized in that: The method comprises spraying or fumigating dried peppers with the Bacillus Velez FP123 of claim 1 , the microbial preparation of claim 2 or 3 , or the biological control agent of claim 4 .

8. The method according to claim 7, characterized in that The control of Aspergillus flavus refers to the production of 2-methylbutyric acid, 3-methylbutyric acid and / or acetic acid to inhibit the growth of Aspergillus flavus hyphae and spore germination.

9. The method according to claim 7, characterized in that: The concentration of the Velez Bacillus FP123 sprayed on the dried pepper is at least 1×10 8 CFU / mL.

10. A method for increasing the anti-aflatoxin substances in dried peppers, characterized in that: The method comprises spraying or fumigating dried peppers with the Bacillus Velez FP123 described in claim 1, the microbial preparation described in claim 2 or 3, or the biological control agent described in claim 4, and the anti-aflatoxin substances include 2-methylbutyric acid, 3-methylbutyric acid and acetic acid.

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