Achromobacter xylosoxidans ZJS2-1 and its application

Abstract

The present invention discloses a strain of Achromobacter xylosoxidans ZJS2‑1 and its application, and the Achromobacter xylosoxidans ZJS2‑1 has been preserved in China Center for Type Culture Collection on March 6, 2024, with a deposit number of CCTCCNO: M2024396, and a deposit address of Wuhan University, Wuhan, China. A strain of Achromobacter xylosoxidans ZJS2‑1 provided by the present invention affects its growth and development by destroying the cell wall and cell membrane of Aspergillus flavus, and has a very good Aspergillus flavus inhibition effect, and the inhibition rate can reach up to 100%; In the Aspergillus flavus and aflatoxin control research conducted on peanuts and corn, experiments have shown that the active substances in the bacterial cell fermentation broth of the present invention can inhibit the spore production of Aspergillus flavus by 90%, and the inhibition rate of toxin production can reach 100%; The present invention constructs a rich microbial resource library for biological control of Aspergillus flavus or aflatoxin pollution.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Achromobacter xylosoxidans ZJS2-1 and an application thereof. Background Art

[0002] Aspergillus flavus is a common soil saprophytic fungus. Aflatoxin B1 (AFB1), a secondary metabolite produced by Aspergillus flavus, is one of the most toxic and carcinogenic natural compounds discovered so far. Peanuts and corn are both important oil crops and cash crops in my country, accounting for a considerable part of the world's total output. Aspergillus flavus can infect many important crops, such as peanuts, corn, cotton, etc., and can contaminate crops before and after harvest, causing huge economic losses to agricultural production around the world.

[0003] Current studies have shown that the growth and toxin synthesis of Aspergillus flavus are affected by many environmental factors, such as temperature and light. There are two main strategies for the detoxification of mycotoxins: (1) preventing mold contamination and growth; (2) detoxification of contaminated products. Mycotoxin contamination can be prevented by storing crops before or after harvest. However, toxin contamination is inevitable, and the detoxification pathways of contaminated food after harvest have always been the focus of in-depth research by scientists. Commonly used detoxification methods include: physical methods, chemical methods, and biological methods. In the field of environmental science, the use of microorganisms, especially microorganisms with probiotic effects, to reduce AFB1 is regarded as an environmentally friendly and efficient method. This biological detoxification strategy is not only low-cost, safe and harmless, but also its action process is irreversible, which fully reflects the concept of green technology.

[0004] According to the World Health Organization (WHO) and the Food and Agriculture Organization, the appropriate application of biocontrol bacteria has a positive impact on agricultural production and food. If the biocontrol bacteria themselves are non-toxic and harmless, and even have many health-promoting effects on the host, then the strain will become a more suitable additive for food and feed. Different types of biocontrol bacteria inhibit the growth or toxin production of Aspergillus flavus in different ways. Some can kill or inhibit Aspergillus flavus, such as the non-toxin-producing Aspergillus oryzae in CN109170509A and Lactobacillus paracasei in CN116640700A; some can convert AFB1 into other non-toxic or low-toxic secondary products or isomers, such as Bacillus subtilis in CN110878265B and lactic acid bacteria in CN106261474B.

[0005] In general, there are currently the following major problems in the prevention and control of aflatoxin or aflatoxin-producing microorganisms: (1) The strains that have the ability to inhibit the growth and toxin production of A. flavus are relatively limited; (2) The strains currently reported have certain limitations in inhibiting the growth and toxin production of A. flavus.

[0006] The discovery and application of new strains or new characteristics of strains are of great value, but there are currently no reports on the biological control of Aspergillus flavus by Achromobacterium. Summary of the invention

[0007] The object of the present invention is to provide a strain of Achromobacter xylose oxidans ZJS2-1 and its application, wherein the strain can effectively inhibit the growth of Aspergillus flavus and / or the production of aflatoxin.

[0008] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is:

[0009] A strain of Achromobacter spp. ZJS2-1, which was deposited in the China Center for Type Culture Collection on March 6, 2024, with a deposit number of CCTCCNO: M2024396, and a deposit address of Wuhan University, Wuhan, China.

[0010] In order to achieve the above-mentioned object of the invention, the present invention also provides the following technical solutions:

[0011] The application of the Achromobacter xylose oxidans ZJS2-1 in inhibiting the growth of Aspergillus flavus and / or the production of aflatoxin.

[0012] Furthermore, the application process is: adding one of the bacteria of Achromobacter xylosoxidans ZJS2-1, the fermentation liquid of Achromobacter xylosoxidans ZJS2-1, and the active crude extract of Achromobacter xylosoxidans ZJS2-1 to the object to be treated to inhibit the growth of Aspergillus flavus and / or the production of aflatoxin in the object to be treated; the object to be treated includes food, feed and agricultural products.

[0013] Furthermore, the method for preparing the fermentation broth of Achromobacter xylose oxidans ZJS2-1 is as follows: inoculating the strain of Achromobacter xylose oxidans ZJS2-1 into LB liquid culture medium and culturing and enriching the bacteria at 37° C. for 3 days with shaking, and the obtained culture broth is the fermentation broth.

[0014] Furthermore, the preparation method of the active crude extract of Colorless Bacillus xylose oxidizing ZJS2-1 is: inoculate Colorless Bacillus xylose oxidizing ZJS2-1 into LB liquid culture medium and culture at 37°C for 3 days, extract the culture solution with an equal volume of ethyl acetate, and obtain the active crude extract of Colorless Bacillus xylose oxidizing ZJS2-1 after rotary evaporation.

[0015] Preferably, the agricultural product is peanuts or corn.

[0016] The present invention also provides a microecological preparation for inhibiting the growth of Aspergillus flavus and / or the production of aflatoxin, which is prepared by using the above-mentioned Achromobacter xyloseoxidans ZJS2-1.

[0017] Preferably, the preparation is in the form of a liquid, a spray powder, a dry wettable powder, or a dry wettable granule.

[0018] The research process of the present invention is as follows:

[0019] (1) The method for isolating and screening Achromobacter xylose oxidizing ZJS2-1 of the present invention is to separate the sample on LB solid culture medium by using the dilution spreading method; the samples for separation and screening are derived from peanut endodermis and rhizosphere soil.

[0020] (2) The identification method of Achromobacter is a combination of morphology, molecular biology and other methods. In terms of morphological identification, the colony or cell morphology of Achromobacter xylosoxidans ZJS2-1 was observed using ordinary optical microscope, fluorescence microscope and scanning electron microscope; in terms of molecular biological identification, the 16S DNA characteristic sequence was extracted and the evolutionary tree was constructed by comparing the database to identify the strain at the genus level.

[0021] (3) The method for the strain to inhibit the growth of Aspergillus flavus was to evaluate the strain's inhibition of the growth of Aspergillus flavus by combining the plate confrontation method and the microplate method. For the confrontation plate method, the colorless bacillus was streaked on the upper, left, and right 1 / 4 of the plate, and 1 μL of Aspergillus flavus was inoculated on the upper, lower, left, and right 1 / 4 of the plate, respectively, and cultured at 37°C for 3 days; for the microplate method, the bold substance gradient was added to the PDA culture medium, and 1 mL of PDA was added to each well of the 12-well plate. After the culture medium solidified, 1 μL of Aspergillus flavus was inoculated in the middle of the plate, and cultured at 37°C for 3 days.

[0022] (4) The inhibition of aflatoxin production by the strain was achieved by adding A. flavus and Achromobacter or gradient Achromobacter active crude extracts to PDB liquid culture medium and culturing at 28°C for 3 days. The residual toxins in 500 μL of fermentation broth were extracted with 700 μL of dichloromethane, and the aflatoxin content was detected by silica gel plate chromatography.

[0023] (5) The application experiment of the strain in inhibiting the production of aflatoxin by Aspergillus flavus was conducted on peanuts and corn. Peanut and corn seeds were sterilized with 1% sodium hypochlorite and washed. A 2 mm hole was cut in the embryo and placed in sterile water containing crude extract of Achromobacterium for 4 h. Aflatoxin spores were removed and inoculated to simulate aflatoxin infection. After 7 days of culture, the content of aflatoxin spores and toxins was detected.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The strain Achromobacter xylosoxidans ZJS2-1 provided by the present invention affects the growth and development of Aspergillus flavus by destroying the cell wall and cell membrane of Aspergillus flavus, and has a very good Aspergillus flavus inhibition effect, with an inhibition rate of up to 100%;

[0026] (2) In the research on the prevention and control of Aspergillus flavus and aflatoxin on peanuts and corn, the present invention has experimentally proved that the active substances in the fermentation liquid of the bacterial strains of the present invention can inhibit the spore production of Aspergillus flavus by 90%, and the inhibition rate of toxin production can reach 100%;

[0027] (3) The present invention constructs a rich microbial resource library for biological control of Aspergillus flavus or aflatoxin contamination. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The separation effect diagram of Achromobacter in the present invention in LB solid medium, (A) phylogenetic tree of Achromobacter xylosoxidans ZJS2-1 strain; (B) streaking diagram; (C) single colony diagram; (D) bacterial morphology image taken by fluorescence microscope after Gram staining; (E) bacterial morphology image under high-power scanning electron microscope;

[0029] Figure 2 The effect diagram of the xylose-oxidizing Achromobacterium ZJS2-1 in the present invention on inhibiting the growth of Aspergillus flavus, (A) is the effect diagram of ZJS2-1 inhibiting the growth of Aspergillus flavus on solid culture medium; the streaked Achromobacterium flavus grows on PDA culture medium, the unstreaked group serves as a blank group, and the Escherichia coli group serves as a negative control; (B) ZJS2-1 and Aspergillus flavus are mixed and cultured in a ratio of 1:1 to verify the effect diagram of inhibiting hyphae production;

[0030] Figure 3 The effect diagram of the xylose-oxidizing Achromobacter ZJS2-1 in the present invention on inhibiting the production of aflatoxin by Aspergillus flavus. Different proportions of mixed Achromobacter and Aflatoxin cells were added to PDB for cultivation to inhibit the production of aflatoxin by Aspergillus flavus; (A) is a thin chromatogram of AFB1 in PDB, and (B) is a statistical analysis of the relative production of AFB1 in Figure A;

[0031] Figure 4 The figures are the inhibitory effect of the crude extract of Achromobacter xylose oxidans ZJS2-1 on the growth and spore formation of Aspergillus flavus in the present invention; (A) is the minimum inhibitory concentration of the Aspergillus flavus strain treated with the crude extract of ZJS2-1 measured on PDA, (B) is the statistical graph of the growth diameter of the Aspergillus flavus colony, and (C) is the statistical graph of the spore formation of Aspergillus flavus;

[0032] Figure 5 The figures are the effects of different concentrations of the crude extract of Achromobacter xylose oxidans ZJS2-1 in the present invention on inhibiting the production of aflatoxin by Aspergillus flavus; (A) is the thin chromatography analysis of AFB1 in PDB, and (B) is the statistical analysis of the relative production of AFB1 in Figure A;

[0033] Figure 6The inhibitory effect of the crude extract of Achromobacter xylosoxidans ZJS2-1 on Aspergillus flavus infection in the present invention is shown in Figure 1. (A) Schematic diagram of the infection of seeds soaked with crude extract of Achromobacter xylosoxidans ZJS2-1 at different concentrations by Aspergillus flavus; (B) Statistical analysis diagram of the number of spores produced on seeds; (C) Schematic diagram of the residual AFB1 on seeds after infection; (D) Statistical analysis diagram of AFB1 content;

[0034] Figure 7 Schematic diagram showing that the treatment with the crude extract of Achromobacter xylosoxidans ZJS2-1 in the present invention will damage the cell wall and membrane integrity of Aspergillus flavus; (A) fluorescence microscopy detection of the fluorescence coating of Aspergillus flavus hyphae after Congo red staining; (B) flow cytometric analysis of Congo red staining of Aspergillus flavus hyphae; (C) quantitative analysis of the Congo red fluorescence ratio with or without crude extract treatment; (D) fluorescence microscopy detection of the fluorescence of Aspergillus flavus hyphae after MM 4-64 staining; (E) flow cytometric analysis of MM4-64 staining of Aspergillus flavus hyphae; (F) quantitative analysis of the MM 4-64 fluorescence ratio with or without crude extract treatment; (G) scanning electron microscopic images of Aspergillus flavus spores and hyphae after crude extract treatment; (H) absorbance comparison of the culture supernatant at 280 nm after crude extract treatment;

[0035] Figure 8 This is a schematic diagram of fluorescence microscopy detection of the effect of the crude extract of Achromobacter xylosoxidans ZJS2-1 on DNA and nuclear damage of Aspergillus flavus in the present invention. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with specific embodiments.

[0037] The experimental methods in the following examples are conventional methods unless otherwise specified; the materials and reagents used are obtained from commercial sources unless otherwise specified.

[0038] Embodiment 1

[0039] Isolation and identification of Achromobacter

[0040] 1. Isolation and acquisition of Achromobacter

[0041] The Achromobacter xylose oxidizing ZJS2-1 of the present invention is obtained by screening from peanut planting soil, and the obtaining process is as follows:

[0042] (1) Separation of peanut endophytes and peanut rhizosphere microorganisms: Sodium hypochlorite was used to kill the bacteria on the surface of the sampled peanuts. After the surface was sterilized, other bacteria attached to the surface were removed. The rhizosphere soil was directly ground to obtain the supernatant without sterilization. The shells were peeled and placed in a mortar. 10 mL of sterile water was added per gram of sample, and the peanut shells and soil were ground to obtain the supernatant to form a homogenate. The upper homogenate was aspirated and diluted to 10 by 10-fold volume dilution method. -3 , 10 -4 , 10 -5 ; Take 10 -5 100 μL of the dilution solution was coated on the LB plate at the concentration of 1 μg / mL, and each sample was repeated 3 times; the coated plate was re-sealed, inverted and placed in a constant temperature incubator at 28°C for 48 hours. Figure 1 As shown in B, after the colonies grow, use an inoculation loop to pick out a single colony with a transparent circle or typical morphological characteristics of achromobacterium.

[0043] (2) Purification of strains: Use a pipette tip to pick up the above single colony and streak the bacteria on an experimental plate. Inoculate the bacteria on an LB plate and culture at 37°C. Subculture the bacteria three times.

[0044] (3) Preservation of strains: The purified Achromobacter (named ZJS2-1) was cultured in LB liquid overnight, the culture was inoculated into 50% glycerol cryovials at a ratio of 1:1, and stored in a -80°C refrigerator.

[0045] 2. Identification of strains

[0046] The obtained Achromobacter ZJS2-1 was identified by morphology and molecular biology:

[0047] (1) Morphological identification

[0048] The purified strain ZJS2-1 was streaked onto LB agar medium and cultured at 37°C for 3 days, and then normal and macro photography were performed with a Canon camera to observe the morphological characteristics of the colonies. Achromobacter was inoculated into LB liquid medium and cultured at 37°C overnight. The bacterial precipitate was collected by centrifugation, washed with sterile PBS, and then stained with Gram staining method. The bacterial color was observed under an optical microscope and a fluorescence microscope, respectively. The above-mentioned bacteria were resuspended in 2.5% glutaraldehyde solution, placed at 4°C overnight to fix the cells, and washed with sterile PBS for 2-3 times after centrifugation. The cells were gradient dehydrated with different concentrations of alcohol (30%, 50%, 70%, 90%, 100%), mixed evenly and each dehydration time was 20 minutes, and then the alcohol containing the bacteria was dripped onto the clean surface of the conductive silicon wafer, and dried in an environment not higher than 40°C. The conductivity of the bacteria was enhanced by gold spraying with a gold spraying instrument, and the gold spraying time was 90 seconds. Then, the cell morphology of Achromobacter xylosus was observed with a scanning electron microscope (SEM), and the cell length and width were counted. The results are as follows Figure 1 As shown in C, the colonies formed by this bacterium on LB culture are about 1 cm in diameter, with round shape, neat edges, hemispherical protrusions, milky yellow color, creamy texture, and opaque. Under the microscope, the cells are red ( Figure 1 D), presumed to be Gram-negative bacteria, with a long rod-shaped morphology ( Figure 1 E), the surface is not smooth and has wrinkles, the cell length is 2-3 μm, and the cell width is 0.5-0.7 μm. Morphologically, this bacterium has the characteristics of colorless bacillus.

[0049] (2) Molecular biological identification of 16S rDNA

[0050] The purified strain ZJS2-1 was inoculated into LB liquid culture and cultured overnight at 37°C until it became obviously turbid. The bacterial solution was used directly as a PCR template. PCR amplification system (20 μL): 2 μL 10×Hifi Buffer I, 0.48 μL bacterial solution, 0.4 μL forward primer, 0.4 μL reverse primer, 2 μL dNTPs, 0.2 μL Hifi enzyme, 14.52 μL ddH2O. Universal primers for bacterial amplification 27F (SEQ ID NO.1): 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R (SEQ ID NO.2): 5'-AAGGAGGTGATCCAGCCGCA-3'; PCR amplification conditions: 95°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1.5 min, 28 cycles; 72°C extension for another 7 min. The PCR amplification product was stored at 4°C and sent to GeneWeiZhi Biotechnology Co., Ltd. for sequencing. After removing the heterogeneous peaks and homologous splicing, the obtained nucleotide sequence is shown in SEQ ID NO.3, with a length of 2006 bp. BLAST similarity comparison was performed in GenBank, and the strain phylogenetic tree is shown in Figure 1 As shown in A, it was found that the ZJS2-1 strain provided by the present invention has a similarity of 100% with Achromobacter xylosoxidans. Combined with other identification methods, it was shown that the strain was Achromobacter xylosoxidans.

[0051] Embodiment 2

[0052] Inhibition of Aspergillus flavus growth by strains

[0053] 1. Confrontation plate method

[0054] The growth inhibition of Achromobacter xylose oxidans ZJS2-1 on Aspergillus flavus was detected by confrontation plate method:

[0055] (1) Collection of fresh spores of Aspergillus flavus

[0056] 2 μL of wild-type Aspergillus flavus spores were inoculated on PDA solid medium and cultured in the dark at 37°C for 7 days; the spores were washed with 0.1% Tween 80 spore washing solution, the mycelium was filtered with four layers of lens paper, and the conidia were collected in a 10 mL centrifuge tube. The spores were diluted and counted, and the spore solution concentration was uniformly diluted to 10 7 Pieces / mL.

[0057] (2) Cultivation of Achromobacterium

[0058] Inoculate 10 μL of Achromobacter xylosoxidans ZJS2-1 from the glycerol tube and culture in 1 mL of LB liquid at 37°C and 200 r / min overnight until the growth logarithmic phase. The concentration is about 5×10 8 CFU / mL(OD 600 value between 0.5-0.7), and then diluted to about 10 7 CFU / mL.

[0059] (3) Plate culture

[0060] Use the tip of a pipette to dip the bacterial solution and draw a line on the top, bottom, left and right of the PDA plate. Inoculate 2 μL of Aspergillus flavus spore solution in the center of the PDA. Use the E. coli line as a negative control and the unstreaked area as a blank control. After culturing at 37℃ for 3 days, compare the E. coli and unstreaked areas. Figure 2 A: The growth of Aspergillus flavus in the marked area was obviously inhibited.

[0061] 2. Co-culture Method

[0062] The co-culture method was used to detect the inhibition of Achromobacter xylose oxidans ZJS2-1 on the growth of Aspergillus flavus:

[0063] (1) Preparation of Achromobacter cells

[0064] Prepare according to the preparation method of colorless bacteria in step (2) of the confrontation plate method, and then dilute the bacteria to about 10 6 CFU / mL.

[0065] (2) Preparation of Aspergillus flavus spores

[0066] The spores of Aspergillus flavus were prepared according to step (1) of the confrontation plate method, and the spore solution was uniformly diluted to a concentration of 10 6 Pieces / mL.

[0067] (3) Co-culture of strains and inhibitory effects

[0068] The liquids prepared in step (1) and step (2) were mixed in a ratio of 1:1, then inoculated into PDB medium and cultured at 37°C and 200 r / min for 3 days. Only the Aspergillus flavus spore liquid prepared in step (2) was inoculated as the control group. Figure 2 As shown in B, Achromobacter xylose oxidizing ZJS2-1 strongly inhibited the production of Aspergillus flavus hyphae, with the inhibition rate reaching up to 100%.

[0069] Embodiment 3

[0070] Inhibition of aflatoxin B1 by strains

[0071] To test the inhibition of Achromobacter xylosoxidans ZJS2-1 on aflatoxin B1, follow the steps below:

[0072] (1) Activation of Achromobacter

[0073] The preparation method is the same as step (1) of the co-culture method in Example 2.

[0074] (2) Preparation of Aspergillus flavus spores

[0075] The preparation method is the same as step (2) of the co-cultivation method in Example 2.

[0076] (3) Inhibition of aflatoxin production by strains

[0077] Achromobacter cells were mixed with Aspergillus flavus spores at a ratio of 1:1, 1:10 and 1:100, respectively, inoculated into PDB, and cultured at 37°C and 200 r / min for 3 days. Then 500 μL of the co-cultured fermentation liquid was aspirated and placed in a 1.5 mL centrifuge tube with 700 μL of dichloromethane, shaken for 30 seconds, centrifuged at 9000 r / min for 5 minutes, and 400 μL of the bottom dichloromethane was carefully aspirated into a new 1.5 mL centrifuge tube. After drying, 15 μL of dichloromethane was used to repeatedly blow and wash the bottom and wall of the tube to fully dissolve AFB1. After the dichloromethane evaporated to less than 10 μL, all the liquid was aspirated and spotted on a silica gel plate, and the toxin was separated in a developing agent (chloroform: acetone = 9:1).

[0078] The results are as follows Figure 3 As described above, the figure shows that the inhibition efficiency of Achromobacter xylosoxidans ZJS2-1 on aflatoxin B1 is 99%, 48% and 0% respectively.

[0079] Embodiment 4

[0080] Inhibition of the growth and toxin production of Aspergillus flavus by active substances from strains

[0081] (1) Preparation of Aspergillus flavus spores

[0082] The preparation method is the same as step (1) of the co-culture method in Example 2.

[0083] (2) Preparation of Achromobacter fermentation broth

[0084] The preparation method is the same as step (2) of the co-culture method in Example 2. After activation, the mixture is added to a fermenter containing LB liquid culture medium and cultured at 37° C. for 3 days with aeration.

[0085] (3) Extraction and separation of active substances in fermentation broth

[0086] Pour ethyl acetate into the fermentation liquid at a volume ratio of 1:1, and ventilate and tumble extract for 6 hours. Stop the gas, and release the waste fermentation liquid from the lower port after stratification. Ventilate again to resuspend the fermentation liquid that has not been released, and stop the gas to release the remaining fermentation liquid after stratification. Then release the upper layer of ethyl acetate and add it to the rotary evaporator for rotary evaporation. After completion, the active substance extracted from 1L of fermentation liquid is dissolved in 1mL DMSO.

[0087] (4) Inhibition of the growth of Aspergillus flavus by active substances of Achromobacter

[0088] The active substance was added to the PDA plate at a final concentration of 10 μL / mL, 20 μL / mL, 30 μL / mL, 40 μL / mL, 50 μL / mL, 60 μL / mL, and 70 μL / mL, and 1 μL of the spore solution in (1) was spotted on the plate and cultured at 37°C for 3 days. Figure 4 The active substances of Achromobacter can effectively inhibit the diameter and spore production of Aspergillus flavus, with the minimum inhibitory concentration (MIC) of 30μL / mL and the complete inhibitory concentration of 60μL / mL.

[0089] (5) Inhibition of aflatoxin production by active substances of Achromobacter

[0090] The active substances with final concentrations of MIC and 2MIC were added to PDB, and Aspergillus flavus was cultured at 28°C and 200 r / min for 4 days. The fermentation broth was aspirated to extract AFB1, and the same method as step (3) of Example 3 was used. Figure 5 It can be seen that the active substances of Achromobacter can effectively inhibit the production of aflatoxin by Aspergillus flavus, with an inhibition rate of 100%.

[0091] Embodiment 5

[0092] Application of strains to inhibit aflatoxin production in corn and peanut seeds

[0093] In this example, Achromobacter xylosoxidans ZJS2-1 was used to prevent and control aflatoxin contamination in corn and peanut seeds, and the following steps were followed:

[0094] (1) Preparation of Aspergillus flavus spores

[0095] The preparation method is the same as step (2) of the co-culture method in Example 2, and the spore concentration is adjusted to 10 6 Pieces / mL.

[0096] (2) Preparation of Achromobacter active substances

[0097] The preparation method is the same as step (2) in Example 4.

[0098] (3) Sterilization of corn and peanut seeds

[0099] Peel the corn and peanut kernels, keep them intact, and make a small cut at the root of the seeds. Place the seeds in a 1% sodium hypochlorite solution and shake them on a shaker for 10 minutes. Wash them twice with sterile water, then wash them with 70% ethanol for 2 minutes, and then wash them twice with sterile water.

[0100] (4) Cultivation of Aspergillus flavus on corn and peanut seeds

[0101] Pour out the seeds and dry them with filter paper. Take an appropriate amount of seeds and place them evenly in a 50mL tube. Add 30mL of sterile water or active substance solution with a final concentration of MIC / 2MIC, place at 37℃, and shake at 120r / min for 30min. Place two filter papers in each plate, moisten with 2mL of sterile water or active substance solution of corresponding concentration, take 6 seeds and place them in the plate, inoculate each seed with 10μL of the Aspergillus flavus spore solution prepared in step (1), and culture at 29℃ for 5d.

[0102] (5) Extraction of spores from corn and peanut seeds

[0103] Transfer the corn kernels in the plate to a 50mL centrifuge tube, add 10mL of eluent to the tube, and shake to mix. Pipette 10μL of eluent into a blood cell counting plate, count the number of spores under a microscope, and calculate the spore concentration. Figure 6 As shown, the Achromobacter xylose oxidans ZJS2-1 provided by the present invention is used to inhibit the spore production of Aspergillus flavus on peanut and corn seeds, and the inhibition rate of the active substance on the spore production of Aspergillus flavus is higher than 90%.

[0104] (6) Extraction of toxins from corn and peanut seeds

[0105] Add 2 mL of dichloromethane to the tube in step (5), shake and mix, and after complete separation, pipette 400 μL of the dichloromethane extract at the bottom into a new 1.5 mL centrifuge tube, and follow the same steps as step (5) of Example 4. Figure 6 As shown, the Achromobacter xylose oxidans ZJS2-1 provided by the present invention is used to inhibit the spore production of Aspergillus flavus on peanut and corn seeds, and the inhibition rate of the active substance on the toxin production of Aspergillus flavus is 100%.

[0106] Embodiment 6

[0107] Destruction of the cell wall and cell membrane of Aspergillus flavus by the strain

[0108] The experiment to detect the effect of Achromobacter xylosoxidans ZJS2-1 on the cell wall and cell membrane of Aspergillus flavus was carried out according to the following steps:

[0109] (1) Preparation of Aspergillus flavus spores

[0110] The preparation method is the same as step (2) of the co-culture method in Example 2, and the spore concentration is adjusted to 106 Pieces / mL.

[0111] (2) Preparation of Achromobacter active substances

[0112] The preparation method is the same as step (2) in Example 4.

[0113] (3) Fluorescence microscopy to detect cell wall and cell membrane integrity

[0114] 100 μL of spore solution (containing 10 6 spores) were mixed into 900 μL PDB medium, and then the mixture was incubated at 37°C for 12 hours with shaking at 200 rpm. After incubation, the samples were rinsed twice with distilled water; then, the cells were collected and treated with two dyes, 0.025% Congo Red or 1.5 μM compound SynaptoRedTM C2 (called MM 4-64), and shaken at 28°C in the dark for 30 minutes; finally, the samples were examined under a fluorescence microscope. The results are shown in Figure 7 A, 7B and 7C, the higher the concentration of the active substance provided by the present invention, the more obvious the red fluorescence of Aspergillus flavus is.

[0115] (4) Flow cytometry to detect cell wall and cell membrane integrity

[0116] The treatment method was the same as step (3) in Example 6, but the culture system was different, and PDB was replaced with PBS. After the treatment, the sample was placed in a flow cytometer for detection. The results are shown in FIG. Figure 7 D, 7E and 7F, the higher the concentration of the active substance provided by the present invention, the higher the proportion of cell wall-positive cells increased from 29.2% to 46.7%, and the higher the proportion of cell membrane-positive cells increased from 1.8% to 44.2%.

[0117] (5) Detection of intracellular substance release

[0118] 100 μL of spore solution (containing 10 6 spores) were mixed into 900 μL PDB medium, and the mixture was then incubated at 37°C for 12 hours with shaking at 200 rpm. The Aspergillus flavus culture was collected and fixed by soaking it in a 2.5% glutaraldehyde solution for two hours. Excess glutaraldehyde was removed by rapid centrifugation for five minutes. The sample was dehydrated in a series of ethanol baths of increasing strength (30%, 50%, 70%, 90%, 100%), each lasting 20 minutes. The sample was centrifuged again at 4°C, 12000rpm for 5 minutes, and the ethanol was discarded. Filter paper absorbed the remaining moisture. The sample was freeze-dried for 12 hours, and detailed images of the fungal structure were taken using a scanning electron microscope. The results are shown in Figure 7 G, application of the active substance provided by the present invention to treat Aspergillus flavus results in dehydration, depression and aggravated surface damage of Aspergillus flavus mycelium and spores.

[0119] (6) Flow cytometry to detect cell wall and cell membrane integrity

[0120] 2 mL of spore solution (containing 10 6 spores) in 20 mL PDB and incubated at 28 ° C with continuous shaking at 120 rpm for 48 hours. After incubation, the culture medium was centrifuged, the cells were washed twice with PBS, and they were suspended in PBS with different active substance concentrations (0.5, 1 and 2 MIC). Anhydrous ethanol was used as a negative control. The treated cultures and control cultures were cultured again with shaking at 28 ° C and samples were taken at 0, 1, 2, 4, 8, 16 and 32 hours. After incubation, we centrifuged the cells at 8000 rpm for 5 minutes at 28 ° C and measured the absorbance of the supernatant at 280 nm using a microplate reader and compared it with a blank sample without Aspergillus flavus. The results are shown in Figure 7 H. When the active substance provided by the present invention is used to treat Aspergillus flavus, the contents of the Aspergillus flavus cells leak out, and the absorbance of the culture system increases significantly after the treatment.

[0121] Embodiment 7

[0122] Application of strains in inducing apoptosis of Aspergillus flavus cells

[0123] (1) Preparation of Aspergillus flavus spores

[0124] The preparation method is the same as step (2) of the co-culture method in Example 2, and the spore concentration is adjusted to 10 6 Pieces / mL.

[0125] (2) Preparation of Achromobacter active substances

[0126] The preparation method is the same as step (2) in Example 4.

[0127] (3) Detection of cell apoptosis fluorescence using fluorescence microscopy

[0128] TUNEL fluorescent probe was used to evaluate the apoptosis of cells treated with active substances. 6 Aspergillus flavus spores were incubated with active substances (0, 1 / 2MIC, MIC) at 37°C for 12 hours. The cells were fixed with 4% formaldehyde for 30 minutes, washed twice with PBS, and permeabilized with 0.2% Triton X-100. After washing both groups with PBS, staining was performed using TdT enzyme (terminal deoxynucleotidyl transferase) and TUNEL (transferase-mediated dUTP nick end labeling assay) reaction buffer at 37°C in the dark for 1.5 hours with gentle shaking every 15 minutes. After staining, the cells were washed and then incubated with DAPI (4',6-diamidino-2-phenylindole) staining solution at room temperature in the dark for 30 minutes. Finally, the cells were suspended in PBS and examined under a fluorescence microscope. The results are shown in Figure 8 When the active substance provided by the present invention was used to treat Aspergillus flavus, the green fluorescence intensity indicating DNA breakage in the cells treated with increasing concentrations was significantly stronger than that in the control group.

[0129] In summary, the strain of Achromobacter xylosoxidans ZJS2-1 provided by the present invention induces cell apoptosis and affects its growth and development by destroying the cell wall and cell membrane of Aspergillus flavus, and has a very good Aspergillus flavus inhibition effect, with an inhibition rate of up to 100%; in the research on the prevention and control of Aspergillus flavus and aflatoxin on peanuts and corn, experiments have proved that the active substances in the cell fermentation liquid of the strain of the present invention can inhibit the spore production of Aspergillus flavus by 90%, and the inhibition rate of toxin production can reach 100%.

Claims

1. A strain of Achromobacter xylosoxidans ( Achromobacter spp. )ZJS2-1, characterized in that The xylose-oxidizing Achromobacter ZJS2-1 was deposited in the China Center for Type Culture Collection on March 6, 2024, with the deposit number CCTCCNO:M 2024396, and the deposit address is Wuhan University, Wuhan, China.

2. Use of the Achromobacter xylosoxidans ZJS2-1 according to claim 1 in inhibiting the growth of Aspergillus flavus and / or the production of aflatoxin.

3. The use of Achromobacter xylosoxidans ZJS2-1 according to claim 2 in inhibiting the growth of Aspergillus flavus and / or the production of aflatoxin, characterized in that: The application process is: adding the bacterial body of Achromobacter xylosoxidans ZJS2-1 or the supernatant of the fermentation liquid of Achromobacter xylosoxidans ZJS2-1 or the active crude extract of Achromobacter xylosoxidans ZJS2-1 to the object to be treated, so as to inhibit the growth of Aspergillus flavus and / or the production of aflatoxin in the object to be treated; the object to be treated includes food, feed and agricultural products.

4. The use of Achromobacter xylosoxidans ZJS2-1 according to claim 3 in inhibiting the growth of Aspergillus flavus and / or the production of aflatoxin, characterized in that: The method for preparing the fermentation liquid of Achromobacter xylose oxidans ZJS2-1 is as follows: inoculating the strain of Achromobacter xylose oxidans ZJS2-1 into LB liquid culture medium and culturing at 37° C. for 3 days to enrich the bacteria, and the obtained culture liquid is the fermentation liquid.

5. The use of Achromobacter xylosoxidans ZJS2-1 according to claim 3 in inhibiting the growth of Aspergillus flavus and / or the production of aflatoxin, characterized in that: The preparation method of the active crude extract of Achromobacter xylose oxidans ZJS2-1 is as follows: Achromobacter xylose oxidans ZJS2-1 is inoculated into LB liquid culture medium and cultured at 37° C. for 3 days, the culture solution is extracted with an equal volume of ethyl acetate, and the active crude extract of Achromobacter xylose oxidans ZJS2-1 is obtained after rotary evaporation.

6. The use of Achromobacter xylosoxidans ZJS2-1 according to claim 3 in inhibiting the growth of Aspergillus flavus and / or the production of aflatoxin, characterized in that: The agricultural product is peanuts or corn.

7. A microecological preparation for inhibiting the growth of Aspergillus flavus and / or the production of aflatoxin prepared by using the Achromobacter xylosoxidans ZJS2-1 described in claim 1.

8. The preparation according to claim 7, characterized in that The preparation is in the form of liquid, spray powder, dry wettable powder, or dry wettable granules.

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