Bacillus velezensis DSHBC4 and application thereof

Abstract

The invention discloses bacillus velezensis DSHBC4 and application thereof, and belongs to the technical field of microorganisms. The invention aims to provide the bacillus velezensis capable of degrading vomitoxin, aflatoxin or zearalenone and inhibiting the growth of fusarium graminearum and aspergillus flavus. The invention provides bacillus velezensis DSHBC4, the bacillus velezensis is preserved in the China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO: M20242144, and the preservation time is October 8, 2024. A new solution is provided for biological control of corn crops, and the application potential is wider.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and particularly relates to a Bacillus velezensis DSHBC4 and its application. Background Art

[0002] Fusarium graminearum is the main pathogenic bacterium causing Fusarium head blight of cereals. It not only affects crop yields but also causes mycotoxin contamination of cereals. Deoxynivalenol (DON) is a mycotoxin produced by Fusarium graminearum and is also the mycotoxin with the highest detection rate and exceeding rate in feed. Once humans and animals ingest food contaminated with DON, it will seriously endanger their own health. In recent years, the strategy of detoxification using microorganisms has attracted much attention. Its advantages such as high specificity, mild treatment conditions, good palatability, and environmental friendliness make it an ideal detoxification method.

[0003] Aflatoxin is a secondary metabolite produced by Aspergillus flavus and Aspergillus parasiticus, and has extremely strong toxicity and carcinogenicity, posing a serious threat to human and animal health. The following are the main hazards of aflatoxin: Carcinogenic effect, long-term intake of aflatoxin can significantly increase the risk of liver cancer; Liver damage: Aflatoxin is mainly metabolized in the liver. Long-term exposure can lead to a decline in liver function, presenting symptoms such as hepatitis and cirrhosis, and can even cause liver failure in severe cases; Acute poisoning: Large intake of aflatoxin can cause acute poisoning, manifested as fever, vomiting, ascites, liver failure, etc., with a relatively high fatality rate; Immune system suppression: Aflatoxin can inhibit the function of the immune system, making the human body more susceptible to diseases.

[0004] Zearalenone is a toxic metabolite produced by fungi of the genus Fusarium (such as Fusarium graminearum), and mainly contaminates cereals such as corn, wheat, and rice. Its hazards mainly include the following aspects: Reproductive toxicity: Zearalenone has an estrogen-like effect, which can cause disorders in the reproductive systems of animals (especially females), manifested as elevated estrogen levels, ovarian dysfunction, miscarriage, stillbirth, and teratogenesis. For example, after pigs ingest feed containing ZEN, symptoms such as vulvar swelling and uterine cell enlargement may occur; Immunotoxicity: ZEN and its derivatives can inhibit immune responses, reduce the body's resistance to pathogens, and increase the risk of infection; Liver and kidney damage: ZEN can cause oxidative stress in the body, leading to lipid peroxidation, and thus cause damage to the liver and kidneys; Nervous system effects: High doses of ZEN can cause nervous system excitement, leading to organ hemorrhage, and can even cause animal death in severe cases; Carcinogenicity: Long-term exposure may increase the risk of certain cancers.

[0005] In summary, DON, zearalenone, and aflatoxin widely contaminate feeds and feed raw materials, causing great harm to livestock and poultry farming. Biological detoxification based on microbial degradation is an efficient method for the prevention and control of DON, zearalenone, and aflatoxin. Developing detoxification preparations with high degradation activity for DON, zearalenone, and aflatoxin is an important means to reduce economic losses in the feed and livestock industries and has broad application prospects. Summary of the Invention

[0006] The object of the present invention is to provide a Bacillus velezensis that degrades vomitoxin, aflatoxin, or zearalenone and inhibits the growth of Fusarium graminearum and Aspergillus flavus.

[0007] The present invention provides a Bacillus velezensis DSHBC4, which is preserved in the China Center for Type Culture Collection, with the preservation number of CCTCC NO: M20242144, and the preservation time is October 8, 2024.

[0008] The present invention provides a microbial preparation containing the above-mentioned Bacillus velezensis DSHBC4.

[0009] The present invention provides an application of the above-mentioned Bacillus velezensis DSHBC4 or the above-mentioned microbial preparation in the preparation of a drug for inhibiting Fusarium graminearum or Aspergillus flavus.

[0010] Further limited, the concentration of Bacillus velezensis DSHBC4 is 8.661×10 9 CFU / mL.

[0011] The present invention provides an application of the above-mentioned Bacillus velezensis DSHBC4 or the above-mentioned microbial preparation in degrading vomitoxin, aflatoxin, or zearalenone.

[0012] Further limited, the concentration of Bacillus velezensis DSHBC4 is 8.661×10 9 CFU / mL.

[0013] The present invention provides a method for inhibiting Fusarium graminearum, which is to add the above-mentioned Bacillus velezensis DSHBC4 or the above-mentioned microbial preparation to a culture medium containing Fusarium graminearum mycelial cakes and react for 7 days.

[0014] Further limited, the Fusarium graminearum is made into mycelial cakes with a diameter of 3 mm, and Bacillus velezensis is inoculated 2.5 cm away from the mycelial cakes.

[0015] The present invention provides a method for degrading vomitoxin, which is characterized in that the above-mentioned Bacillus velezensis DSHBC4 or the above-mentioned microbial preparation is added to a reaction solution containing vomitoxin, and the reaction is carried out for 48 hours.

[0016] Further limited, the concentration of vomitoxin is 5 μg / mL.

[0017] Beneficial effects: The present invention collects corn straw from the experimental field of Northeast Agricultural University in Harbin, Heilongjiang Province, and uses the plate coating method to isolate and purify the samples. After mixing 5 g of corn straw with 25 mL of sterile water and shaking well, it is diluted and coated on an LB solid medium for cultivation, and finally the strain is obtained through purification. Through morphological, physiological and biochemical, and molecular biological identifications, the obtained strain is confirmed to be Bacillus velezensis DSHBC4, which has significant effects in inhibiting Fusarium graminearum, degrading DON, and promoting the detoxification of corn. This strain can not only effectively inhibit the growth of Fusarium graminearum, with an inhibition rate of more than 70%, but also has a degradation rate of more than 80% for DON, and can reach 95.35% after optimization. This provides a new solution for the biological control of corn crops and has more extensive application potential.

[0018]

Biological preservation information

[0019] Figure 1 It is the colony morphology diagram of Bacillus velezensis DSHBC4;

[0020] Figure 2 It is the scanning electron microscope image of Bacillus velezensis DSHBC4;

[0021] Figure 3 It is the phylogenetic tree of Bacillus velezensis DSHBC4 based on 16S rDNA sequence;

[0022] Figure 4 It is the effect diagram of the destruction of the hyphae of Fusarium graminearum by Bacillus velezensis DSHBC4;

[0023] Figures 5 - 7 It is the high performance liquid chromatography diagram of the degradation effect of Bacillus velezensis DSHBC4 on DON (cultured for 24 hours);

[0024] Figure 8Effect of fermentation temperature on the degradation of DON by the fermentation broth of Bacillus velezensis DSHBC4;

[0025] Figure 9 Effect of fermentation time on the degradation of DON by the fermentation broth of Bacillus velezensis DSHBC4;

[0026] Figure 10 Effect of initial fermentation pH on the degradation of DON by the fermentation broth of Bacillus velezensis DSHBC4;

[0027] Figure 11 Effect of bacterial concentration on the degradation of DON by the fermentation broth of Bacillus velezensis DSHBC4;

[0028] Figure 12 Semi - normal probability effect plot of factor standardized effects;

[0029] Figure 13 Pareto chart of factor standardized effects;

[0030] Figure 14 Response surface interaction diagrams of factors affecting the degradation rate of DON by Bacillus velezensis DSHBC4 (A is the 2D diagram of the interaction between bacterial concentration and initial fermentation pH; B is the 3D diagram of the interaction between bacterial concentration and initial fermentation pH; C is the 2D diagram of the interaction between bacterial concentration and fermentation time; D is the 3D diagram of the interaction between bacterial concentration and fermentation time; E is the 2D diagram of the interaction between initial fermentation pH and fermentation time; F is the 3D diagram of the interaction between initial fermentation pH and fermentation time);

[0031] Figure 15 Degradation rate of DON in corn by Bacillus velezensis DSHBC4;

[0032] Figure 16 Effect of Bacillus velezensis DSHBC4 on the mycelial growth of Fusarium graminearum in corn (the lowercase letters a, b, c, d, e, f, g, h, i, and j in the figure are respectively: Fusarium graminearum + C4 fermentation broth (6d), Fusarium graminearum (6d), Fusarium graminearum + C4 fermentation broth (12d), Fusarium graminearum (12d), Fusarium graminearum + C4 fermentation broth (18d), Fusarium graminearum (18d), Fusarium graminearum + C4 fermentation broth (24d), Fusarium graminearum (24d), Fusarium graminearum + C4 fermentation broth (30d), Fusarium graminearum (30d));

[0033] Figure 17 The bacteriostatic rate of Aspergillus flavus is the result diagram of confrontation culture. Detailed implementation methods

[0034] Example 1. Isolation of Bacillus velezensis DSHBC4

[0035] Corn straw was collected from the experimental field of Northeast Agricultural University in Harbin, Heilongjiang Province. After removing gravel and sieving, the straw was packed into sterile plastic bags and stored at -20 °C for later use. Under sterile conditions, 5 g of corn straw was weighed and dissolved in a conical flask containing 25 mL of sterile water. After shaking on a shaker at 180 rpm for 20 min and then standing still, it was diluted and spread on LB solid medium. After culturing at 37 °C for 3 - 5 d, different colonies grown on the plate were picked, the colony morphology was observed, and after subculturing more than three times, the strains were placed in LB liquid medium for further purification. Fifteen strains were obtained, and the fermentation broth was prepared by shaking culture at 37 °C and 180 rpm for 12 h for later use.

[0036] Example 2. Morphological, molecular biological and physiological and biochemical identification of strain DSHBC4

[0037] Morphological identification of the screened strain DSHBC4: The cells of DSHBC4 were short rod-shaped under a 400-fold optical microscope and were motile; the colonies formed on LB medium were round, with neat edges, folds in the middle, light yellow and opaque ( Figure 1 ), and were Gram-positive. Scanning electron microscope observation showed that the cells were short rod-shaped ( Figure 2 ).

[0038] Molecular biological identification of DSHBC4: A purified single colony was inoculated from the LB plate medium into 25 mL of LB medium and cultured on a shaker at 37 °C for 24 h, and the genomic DNA of DSHBC4 was extracted. After amplifying 16s rDNA with specific primers, sequencing was performed.

[0039] DNA sequences of the sequencing primers:

[0040] 16S rDNA (forward primer): 5'-CAGAGTTTGATCCTGGCT-3' (SEQ ID NO.1);

[0041] 16S rDNA (reverse primer): 5'-AGGAGGTGATCCAGCCGCA-3' (SEQ ID NO.2);

[0042] In the Genbank database, the 16s rDNA sequence of strain DSHBC4 was retrieved by BLAST, and it was found that it had a high similarity with strains within the genus Bacillus velezensis. To further determine the genetic characteristics of this strain, a phylogenetic tree was constructed using MEGA5.0 software ( Figure 3 ).

[0043] The physiological and biochemical results of strain DSHBC4 showed that casein hydrolysis, catalase, oxidase, nitrate reduction, starch hydrolysis, semi-solid agar, amygdalin, and L-arginine dihydrolase tests were all positive, while indole, methyl red, V-P, gelatin liquefaction, xylose-gelatin, peptone water, urease, lysine decarboxylase, Simmons citrate, and ornithine decarboxylase tests were negative. This strain was able to produce acid using arabinose, xylose, glucose, maltose, mannitol, sucrose, mannose, and sorbitol, but not using melibiose, lactose, and rhamnose, and was tolerant to 3% NaCl.

[0044] Table 1 Physiological and biochemical tests of strain DSHBC4

[0045]

[0046] For the identification of the taxonomic status of the strain involved in the present invention, the classic microbiological classification manuals were referred to: for example, the content of George M. Garrity's "Bergey’s Mannual of Systematic Bacteriology" Vol. Ⅷ, 1974 edition. According to its morphological characteristics, physiological and biochemical characteristics, and the results of 16S rDNA gene sequence alignment, through polyphasic classification and identification, the strain isolated in the present invention is a Bacillus velezensis. The applicant named the isolated strain Bacillus velezensis DSHBC4, Bacillus velezensis DSHBC4, and deposited this strain with the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China on October 8, 2024. The deposit number is CCTCC NO: M20242144.

[0047] Example 3. Antagonistic ability of Bacillus velezensis DSHBC4 against Fusarium graminearum and the effect of Bacillus velezensis on the mycelium of Fusarium graminearum

[0048] The antibacterial activity of Fusarium graminearum was determined by the plate confrontation method. The pathogenic bacteria cultured on the PDA plate for 14 days were punched into bacterial cakes with a diameter of 3 mm and inoculated in the center of the prepared PDA medium. Then, filter paper was used to inoculate the fermentation broth, supernatant, and cell suspension of Bacillus velezensis DSHBC4 at a distance of 2.5 cm from the bacterial cake. Four points were inoculated on each PDA plate, and sterile water was used as a control. It was cultured in an incubator at 28°C for 7 days. The colony diameters of Fusarium graminearum in the control group and the treatment group were measured respectively. The results are shown in Table 2. At the same time, the medium growing at the edge of the pathogenic bacteria strain was cut and fixed with FAA for 2 days, and the morphological characteristics of the mycelium in the control group and the treatment group were observed by scanning electron microscopy. The antibacterial rate = [(colony diameter of the control group - colony diameter of the experimental group / colony diameter of the control group)] × 100%.

[0049] Table 2 Antagonistic ability of Bacillus velezensis DSHBC4 against Fusarium graminearum

[0050]

[0051] Observation of the effect of Bacillus velezensis DSHBC4 on the mycelial growth of Fusarium graminearum under scanning electron microscope Figure 4 Figure shows the effect of Bacillus velezensis DSHBC4 on the destruction of Fusarium graminearum hyphae; in the control group, the hyphae were long, straight, and uniform in thickness; the hyphae treated with the DSHBC4 fermentation broth were uneven in thickness, rough on the surface, curved and disordered, severely shrunk, and there was ablation in some parts of the hyphae; it shows that the DSHBC4 fermentation broth has an obvious inhibitory effect on the hyphae of Fusarium graminearum

[0052] Example 4. Determination of the ability of Bacillus velezensis DSHBC4 to degrade DON

[0053] The DON standard was prepared with chromatographic methanol into standard solutions with concentrations of 5 μg / mL, 10 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL. The peak areas of DON at different concentrations were detected by high-performance liquid chromatography. With the concentration as the abscissa and the peak area as the ordinate, a standard curve was plotted. The linear regression equation was y = 6134.2x - 37273, R 2 = 0.9999

[0054] ( Figure 5 )

[0055] The strain was activated, and the DSHBC4 fermentation broth was inoculated into the LB liquid medium with a DON concentration of 5 μg / mL and cultured at a constant temperature of 37°C. The LB liquid medium containing 5 μg / mL DON without inoculation was used as the blank control. The content of DON at 0 h and 24 h was detected by high-performance liquid chromatography. The results are as Figure 6 (control group), Figure 7 (treatment group) as shown in Table 3

[0056] Table 3 Ability of Bacillus velezensis DSHBC4 to degrade DON

[0057]

[0058] The results showed that after 24 h of treatment with Bacillus velezensis DSHBC4 on DON, the degradation rate was 80.65%, indicating that it has a high and good ability to degrade DON

[0059] Example 5. Effect of single fermentation factors on the degradation of DON by Bacillus velezensis DSHBC4

[0060] Taking the fermentation temperature, fermentation time, initial fermentation pH, and bacterial concentration as screening targets, the degradation rate of DON by the fermentation broth of Bacillus velezensis DSHBC4 under different conditions was determined, and the fermentation conditions of Bacillus velezensis DSHBC4 affecting the degradation rate of DON were screened. The specific fermentation scheme is shown in Table 4.

[0061] Table 4 Screening test scheme for factors affecting the degradation rate of DON by the fermentation process of Bacillus velezensis DSHBC4

[0062]

[0063] The DON content was measured at 20 - 60 °C, and the degradation rate of DON by the fermentation broth of Bacillus velezensis DSHBC4 fermented at different temperatures was calculated. The results are shown in Figure 8 . When the fermentation temperature was between 25 - 35 °C, the degradation rate of the fermentation broth for DON increased rapidly with the increase in temperature; when the temperature reached 37 °C, it was the optimal fermentation temperature for degrading DON, and the degradation rate was 82.21%. From Figure 9 , it can be seen that the optimal fermentation time for the fermentation broth to degrade DON was 48 h. During the fermentation time of 24 - 48 h, the degradation rate of DON increased with the extension of time; during the fermentation of 48 - 72 h, the degradation rate of DON decreased with the increase in time; at 48 h, the degradation rate of DON reached the highest value, up to 86.61%. From Figure 10 The results show that when the initial fermentation pH was in the range of 5 - 11, the degradation rate of DON by the DSHBC4 fermentation broth was above 80%; when pH < 7, the degradation rate of DON increased with the increase in pH; when pH > 7, the degradation rate of DON decreased with the increase in pH. When the initial fermentation pH was 7, the highest degradation rate reached 90.06%. Figure 11 It was shown that when the bacterial concentration was in the range of 8.18×10 4 -39.76×10 8 , the degradation rate of DON was above 60%, and the degradation rate of DON increased with the increase in bacterial concentration; when the bacterial concentration was 39.76×10 8 , the degradation rate of DON reached the highest, which was 81.34%. It can be seen from this that the fermentation temperature, fermentation time, initial fermentation pH, and bacterial concentration of Bacillus velezensis DSHBC4 all affected the degradation rate of DON by the fermentation broth.

[0064] Example 6. Optimization of the fermentation process for Bacillus velezensis DSHBC4 to degrade DON

[0065] Design-Expert software was used to design the Plackett-Burman experiment, creating an experiment with the number of trials N = 12. Four factors, namely A (fermentation temperature), B (fermentation time), C (initial pH of fermentation), and D (bacterial concentration), were investigated. Another dummy factor was set to investigate the experimental error. The effect of each factor on the DON content was investigated with the DON content as the response value (see Table 5).

[0066] Table 5 Factor levels and coded values of Plackett-Burman design

[0067]

[0068] Based on the single-factor experiment, an experimental design with the number of trials N = 12 was selected, with the DON degradation rate as the response value. The experimental design and results are shown in Table 6. The experimental data were analyzed by variance analysis. The Plackett-Burman test results were used to identify significant effects by the Lenth method, and the half-normal probability effect plot and Pareto chart of the standardized effects of the factors were obtained. From Figure 12 it can be seen that the standardized effect points of factors D and C are far from the fitting line, so they are significant influencing factors (P < 0.05), that is, the significant factors affecting the DON degradation rate are the bacterial concentration and the initial pH of fermentation, and the standardized effect points of other factors are relatively small. The Pareto chart (see Figure 13 ) further determined the magnitude and importance of the effects, and factor D exceeded the t-value and was a significant factor. As can be seen from Table 7, the P-value of the model is 0.0001 < 0.05, indicating a significant difference, so the model is reliable, that is, the model fits well in the entire regression region under study; the multiple correlation coefficient R2 = 0.9508, indicating a good correlation; the adjusted determination coefficient R2adj = 0.8553, indicating that 85.53% of the variability of the experimental data can be explained by this regression model; usually, the lower the coefficient of variation (CV), the higher the credibility and precision of the experiment. The CV value is equal to 4.46%, indicating that the Plackett-Burman experiment has good credibility and precision; the precision is the ratio of the effective signal to the noise (Adeq Precisior), and a value greater than 4.0 is considered reasonable. The precision of this experiment reached 14.5340. By performing multiple regression fitting on the data, the regression equation was obtained: Y = 77.73 + 1.42A + 2.22B + 3.16C + 10.89D.

[0069] Table 6 Plackett-Burman experimental design and results

[0070]

[0071]

[0072] Table 7 Factors, levels and significance levels of Plackett-Burman test

[0073]

[0074] The steepest ascent test uses the gradient direction of the response value change as the climbing direction and determines the change step size according to the magnitude of the significant factor effect values, which can quickly and economically approach the maximum response region. After the Plackett-Burman test, three most significant factors, namely fermentation time, initial fermentation pH, and bacterial concentration, were selected for the steepest ascent test.

[0075] From the regression equation, it can be seen that the partial regression coefficient of factor D is 10.89, indicating that the effect of factor D on the DON degradation rate is a positive effect, that is, as factor D increases, the DON degradation rate shows an upward trend; factors A, B, and C are positive effects. When conducting response surface tests, examining more than 3 factors will significantly increase the number of tests (20 treatments for 3 factors and 31 treatments for 4 factors). At the same time, considering that the increase in factor B, that is, temperature, will lead to a significant increase in actual production costs, further optimization of this factor is discarded. Considering factors such as cost and workload, the experimental design and results are shown in Table 8. It can be seen from Table 8 that the DON degradation rate of treatment 5 is the highest, so treatment 5 is used as the center point of the response surface test. The bacterial concentration is fixed at 9.888×10 9 CFU / mL, the initial fermentation pH is 7, and the fermentation time is 48 h.

[0076] Table 8 Design and results of the steepest ascent test

[0077]

[0078] In this experiment, a 3-factor and 3-level experiment was carried out using Design-expert software based on the steepest ascent test, with the DON concentration as the response value to determine the optimal fermentation conditions (see Table 9).

[0079] Table 9 Variables and levels in Box-Behnken test

[0080]

[0081] According to the steepest ascent test, a Box-Behnken test was carried out with treatment 5 as the center point. Three factors, namely bacterial concentration, pH, and fermentation time, were selected as independent variables. A 3-factor and 3-level experiment was established according to the Box-Behnken design, with the DON degradation rate as the response value. The experimental design and results are shown in Table 10.

[0082] By performing quadratic multiple regression fitting on the data, a quadratic polynomial equation was obtained:

[0083] Y = 95.65 + 4.30A + 0.3863B + 0.7C - 0.0425AB + 0.725AC + 0.7BC - 6.7A 2 - 3.32B 2 - 3.89C 2 The multiple correlation coefficient of the equation is R 2 = 0.9790, indicating that the model fits the actual situation well. The experimental results can be analyzed through the equation. The results of the response surface test variance analysis are shown in Table 11. It can be seen from Table 11 that the model is extremely significant (P < 0.05) while the lack-of-fit term is not significant (P > 0.05). The correlation coefficient R of the model 2 = 0.9790, and the adjusted coefficient R 2 adj = 0.8495, indicating that the model has a good fitting degree. The model can explain 84.95% of the change in the response value. This model can be used to analyze and predict the DON degradation rate. It can be seen from the results in Table 12 that the first-order terms of factors A, B, and C have a significant impact on the DON degradation rate; the interaction terms AB, AC, and BC are not significant; the second-order terms A2, B2, and C2 have a significant impact on the DON degradation rate. This shows that the relationship between each factor and the response value is not a simple linear relationship. The influence degree of each factor is in turn: A > B > C, that is, the concentration of bacteria > the initial pH of fermentation > the fermentation time. The regression model is used to predict the DON degradation rate under each fermentation condition, and the response surface analysis diagram is drawn by Design-expert. The results are shown in Figure 14 . It can be clearly seen from the response surface 3D diagram that the range of the experiments conducted includes the area where the maximum value is located.

[0084] Table 10 Box - Behnken experimental design and its results

[0085]

[0086] Table 11 Model suitability analysis

[0087]

[0088] Table 12 Variance analysis of the Box - Behnken experimental design quadratic model

[0089]

[0090] At a bacteria concentration of 8.661×10 9Under the conditions of CFU / mL, initial fermentation pH 6.5, and fermentation time 49 h, a verification test was carried out. The reaction time with DON was 48 h, the initial concentration of DON was 5 μg / mL, and the DON degradation rate could reach 95.35%. The test value was very close to the theoretical predicted value. It can be seen that this model can better predict the actual fermentation situation, thus proving the feasibility of optimizing the process of degrading DON by Bacillus velezensis DSHBC4 using response surface methodology.

[0091] Example 7. Application of Bacillus velezensis DSHBC4 in antagonizing molds and detoxification ability during corn storage

[0092] Fusarium graminearum was inoculated on a PDA plate and activated at 28 °C for 14 d, and then the strain was made into a spore suspension of 1.07×10 3 CFU / mL with sterile water.

[0093] Bacillus velezensis DSHBC4 was streaked and cultured on an LB plate medium, and after culturing at 37 °C for 24 h, it was transferred to an LB culture solution and cultured at 37 °C and 180 rpm for 24 h. It was propagated in an LB liquid medium for 48 h at an inoculation amount of 3%. 5 mL of Fusarium graminearum spore solution was mixed with 15 mL of Bacillus velezensis DSHBC4 bacterial solution and 30 mL of sterile water, vortexed and then added to 100 g of corn, and placed in a constant temperature anaerobic fermentation at 37 °C. A liquid containing only 5 mL of Fusarium graminearum spore suspension and 45 mL of sterile water was used as a blank control. Samples were taken at 6, 12, 18, 24, and 30 d respectively to detect the content of DON (shown in Table 13 and Figure 15 ), and the mycelial growth status was observed (shown in Table 14 and Figure 16 ).

[0094] Table 13 Effects of Bacillus velezensis DSHBC4 on detoxification of corn

[0095]

[0096] It was proved by experiments that the detoxification efficiency of Bacillus velezensis DSHBC4 was as high as over 71.87% within 24 d, showing excellent detoxification effect.

[0097] Table 14 Effects of Bacillus velezensis DSHBC4 on the growth of Fusarium

[0098]

[0099] It has been experimentally proven that under the condition of adding the same concentration of Fusarium graminearum spore solution, within 6d, 12d, 18d, 24d and 30d, as maize was continuously cultured over time, it was found that the spore germination rate of the control group with only sterile water added increased continuously due to the infection rate over time. The maize treated with the DSHBC4 fermentation broth showed an inhibitory ability against the germination of foreign spores in this experimental group, and had different degrees of antagonistic effects on the subsequent growth of Fusarium graminearum hyphae. The longer the time, the more obvious the effect of the DSHBC4 bacteria (as Figure 16 shown).

[0100] The bacteriostatic rate of the cell suspension of Bacillus velezensis DSHBC4 against Aspergillus flavus was 69.74% when the inoculation amount was 50 μL and the time was 7d; at 48h, when the inoculation amount of the DSHBC4 cell suspension was 2%, the degradation rate of ZEN with a concentration of 0.625 μg / mL was 63.01%; at 36h, the highest degradation rate of the supernatant of DSHBC4 with an inoculation amount of 2% against AFB1 (aflatoxin) with a concentration of 0.04 μg / mL was 96.57%; when treated for 24d, the degradation rates of DSHBC4 against ZEN (zearalenone) with a concentration of 45.18 μg / mL and AFB1 with a concentration of 0.002142 μg / mL in maize were 99% and 83.53% respectively.

[0101] The bacteriostatic rate against Aspergillus flavus was determined by confrontation culture, and the Aspergillus flavus in the middle was directly inoculated with a fungal cake.( Figure 17 In

[0102] As mentioned above, the above are only the preferred specific embodiments of the present invention, and the protection scope of the present invention is not limited thereto. Any simple changes or equivalent replacements of the technical solutions that can be obtained by those skilled in the art within the technical scope disclosed by the present invention all fall within the protection scope of the present invention.

Claims

1. A Bacillus velezensis DSHBC4, characterized in that The Bacillus Velezii is deposited in the China Center for Type Culture Collection, with the deposit number being CCTCC NO: M20242144 and the deposit date being October 8, 2024.

2. A microbial preparation containing the Bacillus Velezii DSHBC4 according to claim 1.

3. Use of the Bacillus Velezii DSHBC4 according to claim 1 or the microbial preparation according to claim 2 in the preparation of a drug for inhibiting Fusarium graminearum or Aspergillus flavus.

4. The use according to claim 3, characterized in that: The concentration of Bacillus velez DSHBC4 was 8.661×10 9 CFU / mL.

5. Use of the Bacillus Velezii DSHBC4 according to claim 1 or the microbial preparation according to claim 2 in degrading vomitoxin, aflatoxin or zearalenone.

6. The use according to claim 4, characterized in that: The concentration of Bacillus velez DSHBC4 was 8.661×10 9 CFU / mL.

7. A method for inhibiting Fusarium graminearum, characterized in that: The Bacillus Velez subtilis DSHBC4 described in claim 1 or the microbial preparation described in claim 2 is added to a culture medium containing a Fusarium graminearum cake and reacted for 7 days.

8. The method according to claim 7, characterized in that The Fusarium graminearum was beaten into a 3 mm cake and inoculated with Bacillus Velezii 2.5 cm from the cake.

9. A method for degrading vomitoxin, characterized in that: The Bacillus Velez subtilis DSHBC4 described in claim 1 or the microbial preparation described in claim 2 is added to the reaction solution containing vomitoxin and reacted for 48 hours.

10. The method according to claim 9, characterized in that The concentration of DON was 5ug / mL.

Images