Wickerhamomyces anomalus capable of producing volatile compounds and application of Wickerhamomyces anomalus in prevention and treatment of aspergillus flavus

Abstract

The invention discloses Wickerhamomyces anomalus capable of producing volatile compounds and application of the Wickerhamomyces anomalus in prevention and treatment of aspergillus flavus, and belongs to the technical field of microorganisms. According to the invention, a strain of Wickhamomyces anomalus FP246 is obtained through screening, the strain of Wickhamomyces anomalus FP246 is preserved in the China Center for Type Culture Collection on July 31, 2024, and the preservation number of the strain is CCTCC (China Center for Type Culture Collection) NO: M 20241719. The Vickerhamomyces anomalus FP246 has an obvious antagonistic effect on aspergillus flavus, and can be used as an effective biological mildew-proof preparation for the dried chilies. 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 the dried chilies by utilizing the Vickerhamomyces anomalus FP246 is a novel biological control method, and has extremely high research and application values on the development of novel green bacteriostatic drugs.

Description

Technical Field

[0001] The invention relates to an abnormal Wickham yeast producing volatile compounds and application thereof in preventing and controlling Aspergillus flavus, belonging to the technical field of microorganisms. Background Art

[0002] 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.

[0003] 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.

[0004] 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.

[0005] 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. Summary of the invention

[0006] In view of the deficiencies of the above-mentioned prior art, the present invention provides an abnormal Wickham yeast producing volatile compounds and its application in preventing and controlling Aspergillus flavus, 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.

[0007] The first technical solution provided by the present invention is a strain of abnormal Wickham yeast (Wickerhamomycesanomalus) FP246, which was deposited in the China Center for Type Culture Collection on July 31, 2024, and the strain collection number is: CCTCC NO: M 20241719.

[0008] The second technical solution provided by the present invention is a microbial preparation containing the abnormal Wickham yeast FP246 described in the first technical solution.

[0009] In certain embodiments, the concentration of the abnormal Wickham yeast FP246 in the microbial preparation is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0010] Furthermore, the concentration of the abnormal Wickham yeast FP246 in the microbial preparation is at least 1×10 8 CFU / mL or 1×10 8 CFU / g.

[0011] The third technical solution provided by the present invention is a biological control agent, which contains the abnormal Wickham yeast FP246 described in the first technical solution or the microbial preparation described in the second technical solution.

[0012] In certain embodiments, the amount of the abnormal Wickham yeast FP246 added to the biological control agent is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0013] Furthermore, the abnormal Wickham yeast FP246 is added to the biological control agent in an amount of at least 1×10 8 CFU / mL or 1×10 8 CFU / g.

[0014] The fourth technical solution provided by the present invention is the use of the abnormal Wickham yeast FP246 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.

[0015] In certain embodiments, the control of Aspergillus flavus refers to using 2-methylbutyric acid, 3-methylbutyric acid and / or acetic acid produced by abnormal Wickham yeast FP246 to inhibit the growth of Aspergillus flavus hyphae and spore germination.

[0016] The fifth technical solution provided by the present invention is a method for controlling aflatoxin in dried peppers, wherein the method is to spray the abnormal Wickham yeast FP246 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 on the surface of the dried peppers, or to fumigate the dried peppers using the abnormal Wickham yeast FP246 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.

[0017] 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.

[0018] In certain embodiments, the concentration of the abnormal Wickham yeast FP246 sprayed on the dried pepper is at least 1×10 8 CFU / mL.

[0019] 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 dried peppers with the abnormal Wickham yeast FP246 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.

[0020] In certain embodiments, the concentration of the abnormal Wickham yeast FP246 sprayed on the dried pepper is at least 1×10 8 CFU / mL.

[0021] The technical effects of the present invention are as follows:

[0022] The present invention obtained a strain of abnormal Wickerham yeast (Wickerhamomyces anomalus) FP246 through screening, which was deposited in the China Center for Type Culture Collection on July 31, 2024, and the strain collection number is: CCTCC NO: M20241719. Abnormal Wickerham yeast FP246 has a significant antagonistic effect on Aspergillus flavus and can be used as an effective biological mildew prevention 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 dried peppers with abnormal Wickerham yeast FP246 is a new biological control method, which has extremely high research and application value for the development of new green antibacterial drugs.

[0023] Biomaterial Deposit

[0024] Abnormal Wickham yeast FP246, with the taxonomic name Wickerhamomyces anomalus, 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: CCTCC NO:M 20241719. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is to initially screen the inhibitory effect of some strains on the standard strain of Aspergillus flavus.

[0026] Figure 2 The morphological characteristics of the strains; A: morphological characteristics of strains FP246 and 232 on PDA medium, B: morphological characteristics of strains FP246 and 232 under a microscope.

[0027] Figure 3 This is a growth curve of abnormal Wickham yeast FP246 in different culture media; PDA: potato dextrose agar medium, ZT: sucrose medium, PTT: glucose medium, LB: LB medium, YPD: yeast extract peptone dextrose medium.

[0028] Figure 4 This is the inhibitory effect of strain FP246 on Aspergillus flavus for 2-7 days; A: The inhibitory effect of the two strains on Aspergillus flavus for 2-7 days, B: The inhibition of the diameter of Aspergillus flavus colonies by the two strains for 2-7 days, C: The inhibition of Aspergillus flavus hyphae growth by the two strains, D: The inhibition of the number of Aspergillus flavus spores by the two strains.

[0029] Figure 5 This is the inhibition of Aspergillus flavus by different fermentation components of strain FP246 in different culture media.

[0030] Figure 6 The colony morphology is shown in Table 2.

[0031] Figure 7 This is the effect of 3-methylbutyric acid and 2-methylbutyric acid on the germination of Aspergillus flavus spores.

[0032] Figure 8 This is a picture of the abnormal Wickham yeast FP246 controlling Aspergillus flavus in dried peppers.

[0033] Fig. 9 A picture showing the volatile compounds produced by the anomalous Wickham yeast FP246 against Aspergillus flavus in dried pepper. DETAILED DESCRIPTION

[0034] 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.

[0035] The culture medium involved in the following embodiments is:

[0036] 1. Potato dextrose agar (PDA) medium: 10 g / L peptone, 5 g / L yeast extract powder, 20 g / L glucose, 15 g / L agar;

[0037] 2. Potato dextrose liquid (PDB) medium: 10 g / L peptone, 5 g / L yeast extract powder, 20 g / L glucose;

[0038] 3. Sucrose (ZT) medium: 10g / L sucrose, 10g / L peptone, 10g / L sodium chloride, 1g / L KH 2 PO 4 , 0.5g / LMg 2 S0 4 7H 2 O, 15 g / L agar;

[0039] 4. Glucose (PTT) medium: 10g / L glucose, 10g / L peptone, 10g / L sodium chloride, 1g / L KH 2 PO, 0.5 g / L Mg 2 S0 4 7H 2 O, 15 g / L agar;

[0040] 5. LB medium: 10g / L peptone, 10g / L sodium chloride, 5g / L yeast powder, 15g / L agar;

[0041] 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.

[0042] Example 1: Screening and identification of antagonistic bacteria to Aspergillus flavus

[0043] 1. Preparation of Aspergillus flavus spore suspension

[0044] 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.

[0045] 2. Screening of antagonistic strains

[0046] Wash the fresh dried pepper, moldy dried pepper, kiwi fruit and pickled pepper skin with 10 mL of sterile water, collect the liquid and culture it on MRS and YPD medium at 28℃ 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 uL of Aspergillus flavus spore suspension (10 7 / mL), four paper pieces on the cross line 25mm away from the center were inoculated with 20uL of overnight culture solution of the numbered strain and cultured at 37℃ for 5 days. The results of the antagonism experiment are as follows Figure 1 , and screened out strains FP246 and 232 with antagonistic effects on Aspergillus flavus.

[0047] 3. Strain identification

[0048] The bacterial cells were collected and the genome of the strains was extracted. The screened 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 Wickerhamomyces anomalus FP246 and Saccharomyces cerevisiae 232 were obtained. Figure 2 As shown, the colonies of strain FP246 are round, white, with radially raised surfaces, sticky, 2-4 mm, oval, Candida-type hyphae, and asexual reproduction is budding. The colonies of strain 232 are round, white, flat, with neat edges, and the reproduction method is budding.

[0049] 4. Optimal culture medium selection

[0050] 1 mL of abnormal Wickham yeast FP246 and Saccharomyces cerevisiae 232 bacterial liquid were inoculated into PDB, LB, PTT and ZT medium at 28°C and 180 rpm for 24 h to cultivate the seed liquid. Abnormal Wickham yeast FP246 uses a 48-well plate, 800uL of culture medium is added to each well, and 2% seed fermentation liquid is accessed. The corresponding culture medium is used as a control, and the microbial growth curve analyzer is used for measurement: the optical density at a wavelength of 600nm is measured every 2 hours. The growth curve of abnormal Wickham yeast FP246 is shown in the figure Figure 3 Combined with the growth curve, the abnormal Wickham yeast FP246 grew best under the conditions of PDB medium, so PDA and PDB medium were used in subsequent experiments.

[0051] 5. Inhibition of Abnormal Wickham Yeast FP246 and Saccharomyces cerevisiae 232 on Aspergillus flavus

[0052] (1) Mycelium growth detection

[0053] 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 20uL of fermentation broth of abnormal Wickham yeast FP246 and brewer's yeast 232, inoculated with spore suspension and water as controls, and cultured at 37℃ for 7 days. 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 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.

[0054] (2) Spore germination detection

[0055] Using the five-point paper disc method, five 10 mm paper discs were pasted on the PDA medium, and 20 uL of the prepared aflatoxin spore suspension (10 7 20uL of fermentation broth of abnormal Wickham yeast FP246 and cerevisiae 232 were inoculated on four paper pieces on the cross line 25mm away from the center, and spore suspension and water were inoculated as controls. The plates were cultured at 37℃ for 7d, and the entire plate with growing colonies was washed twice with 10mL of normal saline, gently scraped with a glass rod, filtered, and conidia were counted on a hemocytometer after 10μL of the suspension, and the number of conidia per mL was calculated to determine the number of conidia per cm 2 Conidia 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.

[0056] (3) Conidia germination and viability

[0057] 40 μL of antagonistic bacteria (abnormal Wickham yeast FP246 or Saccharomyces cerevisiae 232) suspension (10 μL) was inoculated into 2 mL of sterilized buffered peptone water (10 g / L peptone, 5 g / L sodium chloride, 3.6 g / L anhydrous disodium hydrogen phosphate, 1.5 g / L potassium dihydrogen phosphate) 9 / mL) and 40μL of Aspergillus flavus spore suspension (10 6 / mL), and cultured at 28℃ for 24h. The control group was sterile buffered peptone water without bacteria. 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, and the experiment was repeated three times. After the conidia were counted, 100μL of each replicate was inoculated and spread on PDA medium, cultured at 28℃ for 5d, the survival rate of conidia was determined, and the colony forming units (CFU) were counted. The experiment was repeated three times.

[0058] like Figure 4 As shown, FP246 and 232 began to inhibit the growth of Aspergillus flavus from the 4th day, and the inhibitory effect of FP246>232. On the 7th day, the inhibition rates of FP246 and 232 on the growth of Aspergillus flavus hyphae were 52% and 44%, and the inhibition rates on the number of Aspergillus flavus spores were 86.30% and 79.63%, respectively.

[0059] As shown in Table 1, the conidia of Aspergillus flavus did not germinate after inoculation with abnormal Wickham yeast FP246 and cerevisiae 232. According to the inhibition of the mycelial growth and spore germination of Aspergillus flavus and the survival number of the two strains, the strain with the better effect was screened out as abnormal Wickham yeast FP246.

[0060] Table 1 Effects of abnormal Wickham yeast FP246 and Saccharomyces cerevisiae 232 on conidia and viability of Aspergillus flavus

[0061] Spore germination number Survival count (log CFU / mL) CK 90.33 1.96 Abnormal Wickham yeast FP246 0 0 Saccharomyces cerevisiae 232 0 0

[0062] Example 2: Inhibition of Aspergillus flavus by different fermentation components of abnormal Wickham yeast FP246

[0063] 1. Inhibitory effect of volatile substances on Aspergillus flavus

[0064] Using the inverted plate method, the upper medium was PDA for culturing the standard strain of Aspergillus flavus (10 mm paper was pasted and 20 uL of Aspergillus flavus spore suspension was added), and the lower medium was the corresponding 5 culture media (PDA, YPD, LB, PTT and ZT culture media), and the abnormal Wickham yeast FP246 (OD 600 =1) 100uL of fermentation liquid was applied, and a blank medium without antagonistic bacteria was used as a control. Each group was repeated 3 times. The edges of the two plates were wrapped together with sealing film to prevent air leakage. After all culture plates were cultured at 28°C for 6 days, the effect of volatile substances produced by the strains on Aspergillus flavus was evaluated by comparing the spore inhibition rate of Aspergillus flavus standard strains. Figure 5As shown, the volatile substances produced by abnormal Wickham yeast FP246 in five culture media can inhibit the growth of Aspergillus flavus spores, and the inhibition intensity of volatile compounds (VOCs) in different culture media is YPD>PDA>LB>PTT>ZT.

[0065] 2. Inhibitory effect of fermentation supernatant on Aspergillus flavus

[0066] Abnormal Wickham yeast FP246 was inoculated into PDB medium and cultured at 28°C and 180rpm for 24h; the seed liquid was added to 100mL PDB medium at a 2% inoculation amount and cultured at 28°C and 180rpm for 48h to prepare fermentation liquid; the fermentation liquid after 48h of culture was centrifuged and centrifuged at 5000×g for 15min at 4°C to collect the fermentation supernatant. Using the five-point paper disc method, five 10mm paper discs were pasted on five culture media, and 20uL of the prepared aflatoxin spore suspension (10 7 / mL), and 20uL of antagonistic bacteria fermentation supernatant was inoculated on four paper pieces on the cross line 25mm away from the center and cultured at 37℃ for 6d. The inhibitory effect of abnormal Wickham yeast FP246 fermentation supernatant on Aspergillus flavus was not obvious.

[0067] 3. Inhibitory effect of bacteria on Aspergillus flavus

[0068] The abnormal Wickham yeast FP246 was inoculated into PDB medium and cultured at 28°C and 180 rpm for 24 h; the seed liquid was added to 100 mL of PDB medium at a 2% inoculum and cultured at 28°C and 180 rpm for 48 h to prepare the fermentation liquid; the fermentation liquid after 48 h of culture was centrifuged at 5000×g for 15 min at 4°C to collect the bacteria. The bacterial precipitate was washed 3 times with sterile buffered peptone water, reset in buffered peptone water, and the bacterial suspension was adjusted to 10 7 Using the 5-point paper disc method, 5 10 mm paper discs were pasted on 5 culture media, and 20 uL of the prepared aflatoxin spore suspension (10 7 / mL), four paper pieces on the cross line 25mm away from the center were inoculated with 20uL of bacterial suspension and cultured at 37℃ for 6d. In YPD and PDA medium, the standard strain of Aspergillus flavus produced a large number of spores and hyphae. When the abnormal Wickham yeast FP246 was used as the inhibitory strain, the hyphae and spores of Aspergillus flavus were inhibited.

[0069] Therefore, the optimal culture medium for the abnormal Wickham yeast FP246 strain is YPD, which can inhibit the growth of Aspergillus flavus to the greatest extent under appropriate culture medium conditions.

[0070] Example 3: Inhibition of VOCs of abnormal Wickham yeast FP246 on Aspergillus flavus

[0071] 1. Headspace solid phase microextraction (HS-SPME) of VOCs from abnormal Wickham yeast FP246

[0072] The abnormal Wickham yeast FP246 (1×10 6 CFU / mL) was inoculated into a 20mL SPME tube containing 5mL YPD liquid medium and cultured on a rotary shaker at 28°C and 180rpm for 5d. The SPME tube containing 5mL YPD liquid medium was used as a control. After equilibration at 40°C for 40min, the SPME fiber was inserted into the vial and VOCs were extracted at 40°C for 30min. The syringe depth was 40mm. The analytes were desorbed in a GC injector at 270°C in a non-split mode for 10 minutes.

[0073] 2. Gas chromatography-mass spectrometry analysis of VOCs

[0074] VOCs were analyzed by gas chromatograph (Themofisher Trace-1300) and quadruple mass spectrometer (Themofisher Trace-ISO) in electron ionization (EI) mode (70 eV). A TR-5MS capillary column (30 m×0.25 mm, 0.25 um film thickness, ThermoScientific) and a TriPlus RSH autosampler were used to separate different volatile organic compounds. 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 was set to 250°C. Mass spectra were obtained by scanning in the range of m / z 35-500 amu. VOCs were identified by comparing mass spectra, direct matching, and reverse matching with VOCs in the National Institute of Standards and Technology (NIST) library.

[0075] The volatile compounds obtained after matching are phenylethanol, 2-methylbutyric acid, 3-methylbutyric acid, acetic acid, 3-methyl-1-butanol, isoamyl acetate, 2-methyl-1-propanol, ethyl 2-methylbutyrate, n-propanol, isobutyl acetate, propyl acetate, ethyl acetate, butyl acetate and phenylethyl acetate, and the corresponding peak areas are 1404174189, 54396886, 178018763, 8644511, 1649229262, 207058111, 762396719, 107058111, 9330215, 721382799, 188404918, 991127260, 20256219, 7168432, respectively. According to relevant literature reports, the antibacterial effect of ester compounds is not obvious. Therefore, the inhibitory effects of phenylethanol, 2-methylbutyric acid, 3-methylbutyric acid, acetic acid, 3-methyl-1-butanol, and 2-methyl-1-propanol on Aspergillus flavus were evaluated.

[0076] (1) Evaluation of the in vitro inhibitory effect of VOCs on fungal pathogens

[0077] A 10 mm paper was pasted in the center of the PDA medium and inoculated with 20 μL of Aspergillus flavus spore suspension. Six pure standards (phenylethanol, 2-methylbutyric acid, 3-methylbutyric acid, acetic acid, 3-methyl-1-butanol, 2-methyl-1-propanol) were diluted with sterile water to 4 concentration gradients (1000, 100, 10, 1 μL / mL). 20 μL of the pure standard 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 hyphae colony was measured with a vernier caliper, and the entire plate of the growing colony was washed twice with 10 mL of saline, gently scraped with a glass rod, and 10 μL of the suspension was filtered and counted for conidia on a blood cell counting plate. The inhibition rate of different volatile compounds on the growth of Aspergillus flavus hyphae was calculated according to the formula.

[0078] 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, with an inhibition rate of 100% at the highest concentration. 3-Methylbutyric acid and phenylethanol have the second-best 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.

[0079] Table 2 Effects of selected pure compounds on the growth of Aspergillus flavus after 5 days of cultivation at 28°C.

[0080]

[0081] 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.

[0082] (2) Determination of minimum inhibitory concentration (MIC) value

[0083] 1 mL of Aspergillus flavus spore suspension (2%, 10 5 PDB medium containing 7 compounds (μL / mL) was used as a control, and phenylethanol, 2-methylbutyric acid, 3-methylbutyric acid, acetic acid, 3-methyl-1-butanol and 2-methyl-1-propanol were added to make the final concentrations of the 7 compounds in the PDB medium 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4 μL / mL, respectively. The above culture medium was cultured at 28°C for 24 hours, and 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 each time, and a total of three measurements were made to confirm the MIC value of each compound.

[0084] After MIC value determination, 2-methylbutyric acid and 3-methylbutyric acid had the lowest MIC values ​​against Aspergillus flavus, which were 1.4μL / mL and 1.2μL / mL respectively. Figure 7 This is the effect of 3-methylbutyric acid and 2-methylbutyric acid on the germination of Aspergillus flavus spores. With the increase of compound concentration, the spore germination rate decreases and the inhibition rate increases.

[0085] Example 4 Application of abnormal Wickham yeast FP246 in controlling Aspergillus flavus in dried peppers

[0086] After selecting intact and uniform-sized dried peppers and sterilizing them with ultraviolet light for 30 minutes, 10 8 1 mL of abnormal Wickham yeast FP246 with a CFU / mL was sprayed on the surface of the dried pepper, and then 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 without spraying the abnormal Wickham yeast FP246 suspension were used as the control. Each experiment was repeated 3 times, with 6 dried peppers in each repeat. Figure 8 As shown, the mildew rate of dried peppers was significantly reduced after spraying with No. 246 bacteria.

[0087] The fumigation method was used to evaluate the VOC in vivo antibacterial effect of abnormal Wickham yeast FP246. The abnormal Wickham yeast FP246 fumigated dried peppers using the inverted plate method, and 100 μL of abnormal Wickham yeast FP246 bacterial solution was added to the upper YPD medium and cultured for 24 hours. The dried peppers with complete and uniform size were selected for ultraviolet irradiation for 30 minutes and sprayed with 1 mL of Aspergillus flavus spore suspension (10 7 / mL), placed in the lower culture dish, cultured at 28℃, and observed the mildew of the dried pepper during storage. The dried pepper without inoculation of abnormal Wickham yeast FP246 in the upper YPD medium was used as a control. Each experiment was repeated 3 times, and 6 dried peppers were treated in each repeat. The dried peppers that were fumigated with FP246 strain and then sprayed with Aspergillus flavus spores did not mildew.

[0088] Example 5 Application of VOCs in the Control of Aflatoxin in Dried Peppers

[0089] 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 the dried pepper during storage. The dried pepper in the upper YPD medium without VOC was used as a control. Each experiment was repeated 3 times, and each repeat treated 6 dried peppers. The results are as follows Fig. 9 As shown, when the addition amount of 3-methylbutyric acid and 2-methylbutyric acid was 10 μL, the dried pepper did not mold. When the addition amount of acetic acid and phenylethanol was 20 μL and 40 μL, respectively, the dried pepper did not mold.

[0090] 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 Wickerhamomyces anomalus FP246, 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:M 20241719.

2. A microbial preparation containing the abnormal Wickham yeast FP246 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The concentration of the abnormal Wickham yeast FP246 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 abnormal Wickham yeast FP246 according to claim 1 or the microbial preparation according to claim 2 or 3.

5. Use of the abnormal Wickham yeast FP246 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 abnormal Wickham yeast FP246 to inhibit the growth of Aspergillus flavus hyphae and spore germination.

7. A method for preventing and controlling Aspergillus flavus in dried peppers, characterized in that: The method comprises spraying the abnormal Wickham yeast FP246 of claim 1, the microbial preparation of claim 2 or 3, or the biological control agent of claim 4 on the surface of dried peppers, or fumigating dried peppers with the abnormal Wickham yeast FP246 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 method for preventing and controlling Aspergillus flavus refers to using 2-methylbutyric acid, 3-methylbutyric acid and / or acetic acid produced by abnormal Wickham yeast FP246 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 abnormal Wickham yeast FP246 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 abnormal Wickham yeast FP246 according to claim 1, the microbial preparation according to claim 2 or 3, or the biological control agent according to claim 4, and the anti-aflatoxin substances include 2-methylbutyric acid, 3-methylbutyric acid and acetic acid.

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