Stenotrophomonas as well as fermentation product and application thereof

Abstract

The invention provides stenotrophomonas sp. As well as a fermentation product and application thereof, and relates to the technical field of biology, the stenotrophomonas sp. D963 is preserved in China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.33555. The stenotrophomonas sp. D963 can be used for directly inhibiting the production of aflatoxin without influencing the growth of aspergillus flavus, and can be used for preparing the fermentation product of aspergillus flavus. The rapid propagation of drug-resistant strains is not easily caused; the compound has a good prevention and control effect on aflatoxin, can effectively inhibit the formation of aflatoxin pollution in crops, and can be used for preparing an aflatoxin pollution inhibitor.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to oligotrophic mononas and fermentation products and applications thereof. Background Art

[0002] Aspergillus flavus is a saprophytic fungus widely distributed in nature. It has strong adaptability and tolerance to the environment. The aflatoxins it produces have caused widespread pollution around the world and have become one of the important threats that require high vigilance in agricultural production. Aspergillus flavus mainly produces aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2). Some strains can also produce aflatoxins such as aflatoxin G and cyclopiazonic acid (CPA). Among them, AFB1 is its main toxin, which is classified as a Class I carcinogen by the International Agency for Research on Cancer (IARC). It can induce liver cancer and is considered to be one of the strongest known chemical carcinogens. During the pre-production, production, and post-production stages of crops, the toxin can contaminate major grain and oil crops such as peanuts, corn, and rice, posing a major threat to food safety and human health.

[0003] Due to the strong chemical stability of aflatoxin, it is less decomposed during agricultural production and processing, and conventional cooking methods are difficult to effectively remove the toxin. Therefore, it is necessary to reduce toxin pollution by controlling the amount of aflatoxin produced in crops. At present, there is a lack of chemical pesticides that can effectively control Aspergillus flavus, and the fungicides commonly used in corn and peanut fields have limited control effects on Aspergillus flavus, and may even stimulate the production of aflatoxin, while also causing the problem of pesticide residue pollution. Degrading toxins through chemical and physical techniques is also a way, while the existing chemical method can degrade thoroughly, but it cannot reach the level of green safety; physical methods can remove toxins quickly, but the degradation is not thorough. In contrast, biological control can effectively remove toxins and ensure food quality, and has strong specificity and biosafety.

[0004] Common biological control methods currently include aflatoxin-degrading bacteria, aflatoxin-free Aspergillus strains, and aflatoxin antagonists. Aflatoxin-degrading bacteria can effectively degrade aflatoxin secreted by Aspergillus flavus, but the toxicity of its degradation or transformation products to humans is unknown and poses potential risks; the introduction of aflatoxin-free Aspergillus strains may bring other biosafety and health risks, including stability issues caused by mutations within the Aspergillus strains, and the production of CPA and other toxins by non-toxin-producing strains. Therefore, it is necessary to find alternative biological control strains and aflatoxin pollution inhibitors that are safe and non-toxic, have high detoxification efficiency, and stable control effects.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] One of the purposes of the present invention is to provide a Stenotrophomonas geniculata D963 to at least solve one of the technical problems existing in the prior art.

[0007] A second object of the present invention is to provide a fermentation product.

[0008] The third object of the present invention is to provide a bacterial agent.

[0009] A fourth object of the present invention is to provide the use of the above-mentioned Stenotrophomonas geniculata D963, the above-mentioned fermentation product or the above-mentioned bacterial agent in preventing and controlling aflatoxin contamination.

[0010] A fifth object of the present invention is to provide the use of the above-mentioned Stenotrophomonas geniculata D963, the above-mentioned fermentation product or the above-mentioned bacterial agent in inhibiting aflatoxin or preparing a product inhibiting aflatoxin.

[0011] A sixth object of the present invention is to provide a preparation for inhibiting aflatoxin.

[0012] The seventh object of the present invention is to provide a method for preventing and controlling aflatoxin contamination.

[0013] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0014] In a first aspect, the present invention provides a Stenotrophomonas geniculata D963, wherein the Stenotrophomonas geniculata D963 is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration, with a deposit number of CGMCC No.33555.

[0015] Furthermore, the Stenotrophomonas geniculata D963 is derived from rice ears.

[0016] In a second aspect, the present invention provides a fermentation product, and the preparation method comprises: subjecting the above-mentioned Stenotrophomonas geniculata D963 to liquid fermentation, and after the fermentation is completed, taking the fermentation liquid to obtain the fermentation product.

[0017] In a third aspect, the present invention provides a bacterial agent, comprising the above-mentioned Stenotrophomonas geniculata D963 or the above-mentioned fermentation product.

[0018] In a fourth aspect, the present invention provides the use of the above-mentioned Stenotrophomonas geniculata D963, the above-mentioned fermentation product or the above-mentioned bacterial agent in preventing and controlling aflatoxin contamination.

[0019] In a fifth aspect, the present invention provides the use of the above-mentioned Stenotrophomonas geniculata D963, the above-mentioned fermentation product or the above-mentioned bacterial agent in inhibiting aflatoxin or preparing a product inhibiting aflatoxin.

[0020] Furthermore, the aflatoxins include aflatoxin B1 and / or aflatoxin B2.

[0021] In a sixth aspect, the present invention provides a preparation for inhibiting aflatoxin, comprising the above-mentioned Stenotrophomonas geniculata D963, the above-mentioned fermentation product or the above-mentioned bacterial agent.

[0022] Further, it also includes auxiliary agents;

[0023] Preferably, the auxiliary agent includes at least one of a solvent, a cosolvent, an emulsifier, a wetting agent, a dispersant, a tackifier or a stabilizer;

[0024] Preferably, the dosage form of the preparation includes at least one of powder, granule, suspension, oil, fumigant, sustained-release agent, seed coating agent, microcapsule, effervescent tablet or spray.

[0025] In a seventh aspect, the present invention provides a method for preventing and controlling aflatoxin contamination, comprising applying a solution containing the above-mentioned Stenotrophomonas geniculata D963, the above-mentioned fermentation product, the above-mentioned bacterial agent or the above-mentioned preparation to a prevention and control object;

[0026] Preferably, the control targets include crops and / or agricultural products;

[0027] Preferably, applying to crops comprises soaking the seeds of the crops;

[0028] Preferably, the soaking time is at least 10 hours;

[0029] Preferably, the seeds are germinated seeds;

[0030] Preferably, the crops are grain and oil crops;

[0031] Preferably, the grain and oil crops include at least one of peanuts, corn, rice or wheat;

[0032] Preferably, applying to the agricultural product comprises spraying the agricultural product;

[0033] Preferably, the agricultural products are agricultural products in storage period;

[0034] Preferably, the agricultural product comprises at least one of grains, tea leaves or cotton seeds.

[0035] The Stenotrophomonas geniculata D963 provided by the present invention can directly inhibit the production of aflatoxin without affecting the growth of Aspergillus flavus, and is not likely to cause the rapid spread of drug-resistant strains, compared with the antagonistic bacteria that inhibit the growth of Aspergillus flavus; it has a good prevention and control effect on aflatoxin, can effectively inhibit the formation of aflatoxin contamination in crops, and can be used to prepare aflatoxin contamination inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1 The colony morphology of the oligotrophic monocytogenes D963 provided in Example 1 of the present invention;

[0038] Figure 2 A phylogenetic tree of 16S rDNA sequences of the oligotrophomonas D963 and related model strains provided in Example 1 of the present invention;

[0039] Figure 3 Phylogenetic analysis of the gyrB gene sequences of Stenotrophomonas D963 and related model strains provided in Example 1 of the present invention;

[0040] Figure 4 Comparison of the control effects of different biocontrol bacteria on AFB1 and AFB2 pollution provided in Example 2 of the present invention;

[0041] Figure 5 Comparison of the antitoxic effects of different relative concentrations of Oligotrophomonas D963 and Aspergillus flavus spores provided in Example 2 of the present invention;

[0042] Figure 6 Comparison of the control effects of the sterile supernatants of different biocontrol bacteria on aflatoxin contamination provided in Example 3 of the present invention;

[0043] Figure 7The stability of the sterile supernatant of Stenotrophomonas D963 provided in Example 3 of the present invention;

[0044] Figure 8 The results are as follows: The fermentation products of Oligotrophomonas D963 obtained by different extraction methods provided in Example 4 of the present invention are compared in terms of the prevention and control effects on aflatoxin contamination. DETAILED DESCRIPTION

[0045] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0046] Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification.

[0047] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] The present invention provides a Stenotrophomonas geniculata D963, which is deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee with a deposit number of CGMCC No. 33555. Stenotrophomonas geniculata D963 CGMCC No. 33555 is hereinafter referred to as Stenotrophomonas geniculata D963.

[0049] Collection Instructions

[0050] Bacterial species name: Stenotrophomonas D963;

[0051] Latin name: Stenotrophomonas geniculate D963;

[0052] Depository: General Microbiology Center, China Microbiological Culture Collection Administration;

[0053] Abbreviation of depository institution: CGMCC;

[0054] Address: No. 3, Yard 1, Beichen Road, Chaoyang District, Beijing;

[0055] Deposit date: February 18, 2025;

[0056] Collection Center Collection Number: CGMCC No.33555.

[0057] This strain was isolated from rice ears. Research has found that compared with antagonistic bacteria that inhibit the growth of Aspergillus flavus, this strain can directly inhibit the production of aflatoxin without affecting the growth of Aspergillus flavus, and is not likely to cause the rapid spread of drug-resistant strains. It has a good prevention and control effect on aflatoxin, can effectively inhibit the formation of aflatoxin contamination in crops, and can be used to prepare aflatoxin contamination inhibitors.

[0058] According to another aspect of the present invention, a fermentation product is provided, and the preparation method comprises: subjecting the above-mentioned oligotrophic mononas D963 to liquid fermentation, and after the fermentation is completed, taking the fermentation liquid to obtain the fermentation product.

[0059] In some specific embodiments, liquid fermentation includes inoculating the pre-cultured oligotrophic mononas D963 bacterial solution into a liquid culture medium for liquid fermentation. In some specific embodiments, pre-culturing includes taking a single colony of oligotrophic mononas D963 and inoculating it into a liquid culture medium for at least 24 hours. In some specific embodiments, the inoculation amount of the pre-cultured oligotrophic mononas D963 bacterial solution inoculated into the liquid culture medium is 0.05% to 0.3%, preferably 0.2%.

[0060] Among them, the culture medium used for culturing Stenotrophomonas D963 can be Sabouraud liquid medium.

[0061] Specifically, a single colony of oligotrophic mononas D963 can be vertically picked with an inoculation needle, and placed in 50 ml of Sabouraud medium for pre-culture. After culturing for 24 hours, 10 ml of bacterial solution is drawn, centrifuged at 8000 rpm for 5 min, and the supernatant is discarded, and then an equal amount of clean water is added for washing, and then centrifuged at 5000 rpm for 5 min after shaking and mixing, and an equal amount of clean water is added again and shaken and mixed, and 100 μL of the mixed bacterial solution is drawn and added to 50 ml of SD liquid medium for culture. After completion, the culture medium containing oligotrophic mononas D963 is placed at 28° C. and shaken for 4 days to obtain the fermentation liquid of oligotrophic mononas D963.

[0062] In some specific embodiments, after taking the fermentation broth, the fermentation product is also extracted. In some specific embodiments, the extraction method includes extraction with an extractant or extraction with a macroporous resin adsorption method (MR); preferably, the extractant is selected from at least one of methanol, ethyl acetate (EA), dichloromethane (DCM) or petroleum ether (PET). The polarity of these three organic solvents increases successively, and the polarity range is wide, which can ensure that the active secondary metabolites in the fermentation broth of Oligotrophomonas D963 are extracted. At the same time, the secondary metabolites of Oligotrophomonas D963 can also be extracted quickly and efficiently by macroporous resin adsorption method (MR), and the fermentation products of Oligotrophomonas D963 can be obtained based on the MR extraction method.

[0063] According to another aspect of the present invention, a bacterial agent is also provided, comprising the above-mentioned Stenotrophomonas D963 or the above-mentioned fermentation product.

[0064] Since the oligotrophic mononas D963 and its fermentation products have a good inhibitory effect on aflatoxin, the oligotrophic mononas D963, its fermentation products, or a bacterial agent containing oligotrophic mononas D963 or its fermentation products can be used as an effective ingredient to prevent and control aflatoxin contamination, or inhibit aflatoxin, or prepare aflatoxin-inhibiting products.

[0065] According to another aspect of the present invention, there is also provided the use of the above-mentioned Oligotrophomonas D963, the above-mentioned fermentation product or the above-mentioned bacterial agent in preventing and controlling aflatoxin contamination.

[0066] According to another aspect of the present invention, there is also provided the use of the above-mentioned Oligotrophomonas D963, the above-mentioned fermentation product or the above-mentioned bacterial agent in inhibiting aflatoxin or preparing a product inhibiting aflatoxin.

[0067] In some specific embodiments, the aflatoxins include aflatoxin B1 (AFB1 toxin) and / or aflatoxin B2 (AFB2 toxin).

[0068] According to another aspect of the present invention, there is also provided a preparation for inhibiting aflatoxin, comprising the above-mentioned Stenotrophomonas D963, the above-mentioned fermentation product or the above-mentioned bacterial agent.

[0069] In some specific embodiments, an auxiliary agent is also included;

[0070] In some specific embodiments, the auxiliary agent includes at least one of a solvent, a cosolvent, an emulsifier, a wetting agent, a dispersant, a sticker or a stabilizer;

[0071] In some specific embodiments, the dosage form of the preparation includes at least one of powder, granule, suspension, oil, fumigant, sustained-release agent, seed coating agent, microcapsule, effervescent tablet or spray.

[0072] According to another aspect of the present invention, a method for preventing and controlling aflatoxin contamination is also provided, comprising applying a solution containing the above-mentioned Oligotrophomonas D963, the above-mentioned fermentation product, the above-mentioned bacterial agent or the above-mentioned preparation to the prevention and control object; the content of aflatoxin in crops can be effectively inhibited.

[0073] In some specific embodiments, the control targets include crops and / or agricultural products.

[0074] The crops are grain and oil crops; preferably, the grain and oil crops include at least one of peanuts, corn, rice or wheat. The agricultural products are agricultural products in storage period; preferably, the agricultural products include at least one of grains, tea or cotton seeds.

[0075] In some specific embodiments, applying to crops comprises soaking the seeds of the crops.

[0076] In some specific embodiments, the seed soaking treatment lasts for at least 10 hours.

[0077] Specifically, the soaking time can be, but is not limited to, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 16 hours or any other time of more than 10 hours. In order to achieve a better prevention and control effect, in some specific embodiments, the seeds are germinated seeds.

[0078] In some specific embodiments, applying to the produce comprises spraying the produce.

[0079] In order to achieve a better prevention and control effect of aflatoxin contamination, in some specific embodiments, the concentration of Oligotrophomonas D963 in the liquid sprayed on the agricultural products is 10 3 ~10 7 CFU / mL; or, the concentration of the fermentation product of Oligotrophomonas D963 in the liquid sprayed on agricultural products is 200-400 μg / mL.

[0080] Specifically, the concentration of the oligotrophic mononas D963 in the sprayed liquid may be, but is not limited to, 10 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL, 10 6 CFU / mL or 10 7 CFU / mL, or 10 3 ~10 7The concentration of the fermentation product of Oligotrophomonas in the sprayed liquid may be, but is not limited to, 200 μg / mL, 240 μg / mL, 280 μg / mL, 300 μg / mL, 340 μg / mL, 380 μg / mL or 400 μg / mL, or may be any concentration value between 200 and 400 μg / mL.

[0081] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0082] Sabouraud medium: 10.0 g of peptone and 40.0 g of glucose, dissolved in distilled water and fixed to 1 L, sterilized at 121°C for 20 min. Add 2% agar when preparing solid Sabouraud medium.

[0083] Aspergillus flavus CGMCC No.3.4408.

[0084] The biocontrol bacteria (Aspergillus cvjetkovicii, Trichoderma virens PS1-7, Bacillus subtilis, B. velezensis, B. licheniformis, Stenotrophomonas geniculate D963, Pseudomonas mosselii S3-F1, P. mosselii S3-F2, P. hibiscicola, Sphingomonas melonis and Streptomyces N201) are from the biocontrol bacteria library constructed by the Molecular Ecology of Plant-Microbe Interactions Laboratory, Institute of Pesticides and Environmental Toxicology, Zhejiang University.

[0085] Example 1

[0086] 1. Isolation and purification of strain D963: Rice ear samples were collected in August 2021, rinsed with sterile water, and then ground the ears with sterile water in a sterilized mortar. 2 ml of the grinding solution was taken and diluted 2 times, 5 times and 10 times respectively; 100 μL of the diluted solution was spread on LB medium and cultured at 28°C for 3 days. Multiple colonies grew on the plate. According to the color and morphology, single colonies were screened after 3 times of streaking purification.

[0087] 2. Identification of strain D963:

[0088] (a) Morphological characteristics: Figure 1 As shown, the single colony of strain D963 on Sabouraud medium is light yellow, round, smooth, convex, and opaque.

[0089] (ii) 16S rRNA gene analysis: Species identification was performed using the 16S rRNA gene sequence. The primers used for PCR amplification were 27F / 1492R, and a 1465bp gene sequence was obtained (the nucleotide sequence is shown in SEQ ID NO: 1). Based on the standard strain sequence in the NCBI database, the homology was analyzed by sequence alignment, and a phylogenetic tree was constructed using MEGA11 software to further determine the species affiliation of each isolated strain. The phylogenetic analysis was calculated using the maximum likelihood method (ML) and the GTR model, and the reliability of the phylogenetic relationship was evaluated in combination with the Bootstrap support rate. The constructed phylogenetic tree is shown in Figure 1. Figure 2 shown.

[0090] (III) GyrB gene analysis: The gyrB gene sequence was used for species identification. The primers used for PCR amplification were 5'-ATGAGCGACGAACAGAACACC-3' (SEQ ID NO: 3) and 5'-TCAGATATCCAGGTTTGCGACC-3' (SEQ ID NO: 4), and a 2366bp gene sequence was obtained (the nucleotide sequence is shown in SEQ ID NO: 2). Based on the standard strain sequence in the NCBI database, its homology was analyzed by sequence alignment, and a phylogenetic tree was constructed using MEGA11 software to further determine the species affiliation of each isolated strain. The phylogenetic analysis was calculated using the maximum likelihood method (ML) and the GTR model, and the reliability of the phylogenetic relationship was evaluated in combination with the Bootstrap support rate. The constructed phylogenetic tree is shown in Figure 2. Figure 3 shown.

[0091] Based on morphological and gene sequence characteristics, strain D963 was identified as Stenotrophomonas geniculata.

[0092] Example 2 Control effect of strain D963 on aflatoxin production

[0093] 1. Construction of a co-culture system of biocontrol bacteria and Aspergillus flavus: Take out the spore suspension of Aspergillus flavus 3.4408 from the refrigerator and activate it. When the mycelium on the plate grows to 6 cm, use a hole puncher to punch out a 5 mm bacterial cake and inoculate it into 50 mL of Sabouraud medium (SD). At the same time, inoculate 50 μL of the activated biocontrol bacteria solution.

[0094] Among them, the biocontrol bacteria specifically selected Aspergillus cvjetkovicii, Trichoderma virensPS1-7, Bacillus subtilis, B.velezensis, B.licheniformis, Stenotrophomonasgeniculate D963, Pseudomonas mosselii S3-F1, P.mosselii S3-F2, P.hibiscicola, Sphingomonas melonis and Streptomyces N201, as well as a mixed agent (mix) of T.virens PS1-7, Stenotrophomonasgeniculate D963 and P.hibiscicola as biocontrol bacteria. At the same time, a control group was set up, using metabolites instead of biocontrol bacteria, and 100ppm of 2,4-di-tert-butylphenol (DTBP) was selected as the metabolite. After inoculation, it was placed at 28°C and 150rpm for four days.

[0095] 2. Detection of aflatoxin production by thin layer chromatography: Take out the culture solution of the co-culture system of biocontrol bacteria and Aspergillus flavus constructed in step 1, and centrifuge it at 12000rpm for 10min, take the supernatant and filter out the mycelium with filter cloth. Extract the supernatant with ethyl acetate, and spot it on a silica gel plate with a capillary tube with a pore size of 3 microns. At the same time, prepare 25μg / mL of aflatoxin AFB1, AFB2, AFG1 and 3μg / mL AFG2 standard solutions. Then use a developing agent of chloroform: acetone = 9:1 to run to 0.5cm from the upper edge of the silica gel plate. After the silica gel plate is blown dry, observe the toxin production under a 365nm ultraviolet lamp. Screen out biocontrol bacteria with dim or even no fluorescence of aflatoxin AFB1, that is, biocontrol bacteria that inhibit the toxin production effect. The results are as follows Figure 4 As shown, A is the standard solution and B is different co-culture systems.

[0096] from Figure 4 The results showed that after 48h, 72h and 96h of co-cultivation, compared with Aspergillus flavus (CK), the inhibitory effects of T. virens PS1-7, P. hibiscicola, Stenotrophomonas geniculate D963 and the mixed bacterial agent (mix) were relatively obvious, among which Stenotrophomonas geniculate D963 had the best toxin inhibition effect, and P. mosselii S3-F2 had a certain inhibitory effect at 48h and 72h.

[0097] 3. Effect of relative concentrations of Aspergillus flavus spores (Af) and strain D963 (Sg) on ​​the inhibitory ability in co-culture system: 100 μL 5×10 6 spores / mL of spore suspension was added to 10 mL of Sabouraud medium, and then 10 μL of 5×10 6 CFU / mL, 5×10 7 CFU / mL, 5×10 8 CFU / mL, 5×10 9 CFU / mL of D963 bacterial solution was co-cultured with Aspergillus flavus spores. Treatments with Af:Sg = 10:1, Af:Sg = 1:1, Af:Sg = 1:10 and Af:Sg = 1:100 were obtained respectively. Figure 5 As shown, A shows the growth of the co-culture system under different treatments, and B shows the thin layer chromatography detection.

[0098] from Figure 5 The results show that when the ratio of the number of Aspergillus flavus 3.4408 spores to the number of strain D963 colonies is less than 1:1, the inhibitory ability against aflatoxin is significantly improved.

[0099] Example 3: Effect of sterile supernatant on the prevention and control of Aspergillus flavus

[0100] In this example, the fermentation supernatant of Trichoderma virens PS1-7, P.hibiscicola, Stenotrophomonas geniculate D963, and a mixed bacterial agent (mix) of Trichoderma virens PS1-7, P.hibiscicola, Stenotrophomonas geniculate D963 and P.mosselii S3-F2, which have better toxin inhibition effects in Example 2, was used to culture Aspergillus flavus, and the inhibitory effects of different fermentation products on Aspergillus flavus were compared.

[0101] 1. Preparation of sterile supernatant: Use an inoculation needle to pick up a single colony vertically, place it in 50ml Sabouraud medium for pre-culture, and after culturing for 24 hours, take 10mL of bacterial solution, centrifuge at 8000rpm for 5min, discard the supernatant, add an equal amount of clean water to wash, shake and mix, centrifuge at 5000rpm for 5min, add an equal amount of clean water again and shake and mix, take 100μL of the mixed bacterial solution and add it to 50mL SD liquid medium for culture. After completion, place the culture medium containing oligotrophic monocytogenes at 28℃ for constant temperature and shaking culture for 4 days to obtain the fermentation liquid. After centrifugation at 12000rpm for 10min, take the supernatant, and filter through a 0.22μm microporous filter membrane to obtain a sterile supernatant containing the fermentation product.

[0102] 2. Comparison of the inhibitory effect on Aspergillus flavus

[0103] The four fermentation supernatants prepared in step 1 were used to culture Aspergillus flavus 3.4408 for four days to obtain a co-cultured bacterial solution, which was treated and detected by the thin layer chromatography method in Example 2. The results are as follows: Figure 6 As shown, wherein A: Aspergillus flavus 3.4408 bacterial solution, T: Trichodermavirens PS1-7 bacterial solution, Ph: P.hibiscicola bacterial solution, Sg: Stenotrophomonas geniculate D963 bacterial solution, M: bacterial solution of co-culture of Trichoderma virens PS1-7, P.hibiscicola, and Stenotrophomonas geniculate D963, Tm: sterile supernatant of co-culture of A and T, Pm: sterile supernatant of co-culture of A and Ph, Sm: sterile supernatant of co-culture of A and Sg, Mm: sterile supernatant of co-culture of A and M.

[0104] from Figure 6 The results show that for Trichoderma virens PS1-7, A+T has the effect of inhibiting aflatoxin, but A+Tm is ineffective, indicating that the inhibitory effect of the bacteria on aflatoxin is not derived from Tm; for P.hibiscicola, the effects of different treatments on inhibiting aflatoxin are poor; for Stenotrophomonasgeniculate D963, compared with A, the bands of A+Sg, Sm and A+Sm are weaker, indicating that Stenotrophomonasgeniculate D963 bacterial solution and its sterile supernatant containing fermentation products can inhibit the production of aflatoxin; for the co-cultured bacterial solution, A+M, Mm, and A+Mm all achieved a high inhibitory effect, indicating that the bacterial solution or sterile supernatant of strain D963, or the co-cultured bacterial solution or sterile supernatant of mixed strains containing strain D963 can inhibit the production of aflatoxin by Aspergillus flavus.

[0105] 2. Stability test

[0106] The fermentation supernatant of strain D963 obtained in step 1 was sterilized at 121°C and then cultured with Aspergillus flavus 3.4408 for four days to obtain a co-cultured bacterial solution, which was treated and detected for aflatoxin production using the thin layer chromatography method in Example 2. The results are shown in FIG. Figure 7As shown in the figure, compared with Af, the band after sterilization still showed a weakening, which shows that the activity after sterilization was slightly reduced compared with that before sterilization, but it still had a certain inhibitory effect and had a certain chemical stability. Therefore, strain D963 and its fermentation products have application prospects in the preparation of aflatoxin pollution inhibitors.

[0107] Example 4 Control effect of strain D963 fermentation product on Aspergillus flavus

[0108] In this example, the fermentation supernatant of strain D963 prepared by the method in Example 3 was selected, extracted with ethyl acetate (EA), dried, concentrated, and dissolved in 200 μL of DMSO solution to obtain the fermentation product of strain D963 (Sg metabolite), and added to 50 mL of liquid SD culture medium to obtain a culture medium containing Sg metabolites extracted by EA. At the same time, 50 mL of the fermentation supernatant of strain D963 prepared by the method in Example 3 was taken as a culture medium containing unextracted Sg metabolites. Aspergillus flavus 3.4408 was inoculated into SD culture medium containing unextracted Sg metabolites and SD culture medium containing Sg metabolites extracted by EA, respectively, and the co-cultured bacterial liquids on the 4th day and the 6th day were taken, respectively, and the production of aflatoxin in the co-cultured bacterial liquid was treated and detected by the thin layer chromatography method in Example 2.

[0109] The results are as follows Figure 8 As shown, A: Aspergillus flavus 3.4408 and medium without Sg metabolites, Sm: A and medium containing Sg metabolites. From the three repeated groups 1, 2, and 3, it can be seen that the fermentation product of strain D963 extracted with ethyl acetate has a certain inhibitory effect on aflatoxin, but the unextracted sterile supernatant has the best inhibitory effect on aflatoxin.

[0110] In summary, Oligotrophomonas and its fermentation products can be used for the prevention and control of aflatoxin contamination in crops, and can be mainly used to prepare aflatoxin contamination inhibitors for crops such as peanuts and corn. The preparation of the embodiment of the present invention is applied to grain storage to prevent and control aflatoxin contamination.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Stenotrophomonas geniculata D963, characterized in that: The Stenotrophomonas geniculata D963 is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number: CGMCC No.33555.

2. The Stenotrophomonas geniculata D963 according to claim 1, characterized in that The Stenotrophomonas geniculata D963 is derived from rice ears.

3. A fermentation product, characterized in that The preparation method comprises: subjecting the Stenotrophomonas geniculata D963 described in claim 1 or 2 to liquid fermentation, and taking the fermentation liquid after the fermentation is completed to obtain a fermentation product.

4. A bacterial agent, characterized in that The invention comprises the Stenotrophomonas geniculata D963 of claim 1 or 2 or the fermentation product of claim 3.

5. Use of the Stenotrophomonas geniculata D963 described in claim 1 or 2, the fermentation product described in claim 3 or the bacterial agent described in claim 4 in preventing and controlling aflatoxin contamination.

6. Use of the Stenotrophomonas geniculata D963 according to claim 1 or 2, the fermentation product according to claim 3 or the bacterial agent according to claim 4 in inhibiting aflatoxin or preparing a product that inhibits aflatoxin.

7. The use according to claim 5 or 6, characterized in that: The aflatoxins include aflatoxin B1 and / or aflatoxin B2.

8. A preparation for inhibiting aflatoxin, characterized in that: It includes the Stenotrophomonas geniculata D963 described in claim 1 or 2, the fermentation product described in claim 3, or the bacterial agent described in claim 4.

9. The preparation according to claim 8, characterized in that It also includes adjuvants; Preferably, the auxiliary agent includes at least one of a solvent, a cosolvent, an emulsifier, a wetting agent, a dispersant, a tackifier or a stabilizer; Preferably, the dosage form of the preparation includes at least one of powder, granule, suspension, oil, fumigant, sustained-release agent, seed coating agent, microcapsule, effervescent tablet or spray.

10. A method for preventing and controlling aflatoxin contamination, characterized in that: The method comprises applying a solution containing Stenotrophomonas geniculata D963 according to claim 1 or 2, the fermentation product according to claim 3, the bacterial agent according to claim 4 or the preparation according to claim 8 or 9 to a control target; Preferably, the control targets include crops and / or agricultural products; Preferably, applying to crops comprises soaking the seeds of the crops; Preferably, the soaking time is at least 10 hours; Preferably, the seeds are germinated seeds; Preferably, the crops are grain and oil crops; Preferably, the grain and oil crops include at least one of peanuts, corn, rice or wheat; Preferably, applying to the agricultural product comprises spraying the agricultural product; Preferably, the agricultural products are agricultural products in storage period; Preferably, the agricultural product comprises at least one of grains, tea leaves or cotton seeds.

Images