Bacillus subtilis complex microbial inoculant capable of simultaneously degrading multiple mycotoxins and application of bacillus subtilis complex microbial inoculant

Abstract

The invention belongs to the technical field of microbial application, and relates to a bacillus subtilis complex microbial inoculant capable of simultaneously degrading multiple mycotoxins and application of the bacillus subtilis complex microbial inoculant, the bacillus subtilis complex microbial inoculant comprises bacillus subtilis HNGD-Mq02 and HNGD-sg5, the preservation number is CCTCC NO: M2025181, and the preservation number is CCTCC NO: M20242743; the complex microbial inoculant has degradation rates of 98.55%, 93.77% and 98.97% for AFB1, AFM1 and OTA in 24 h, has a degradation rate of 95.33% for ZEN in 16 h and a degradation rate of 54.38% for DON in 48 h, has the advantages of simultaneous degradation of a plurality of mycotoxins, short degradation time, high efficiency, high temperature resistance and huge production application potential, provides a new microbial resource for detoxification of mycotoxins, and has wide application prospects. The method can be widely applied to detoxification of fungaltoxin in grains, grains, feed and other processing byproducts.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial application, and relates to a Bacillus subtilis complex bactericide for simultaneously degrading multiple mycotoxins. Background Art

[0002] Mycotoxins are natural toxic secondary metabolites produced by fungi such as Aspergillus ( Aspergillus ), Penicillium ( Penicillium ), and Fusarium ( Fusarium ), and are widely present in various agricultural products worldwide. According to the estimation of the Food and Agriculture Organization of the United Nations, approximately 25% of the feed and agricultural products globally are contaminated with mycotoxins. After consumption, it not only causes a huge economic burden on consumers but also leads to a series of food safety problems.

[0003] Mycotoxins are mainly divided into aflatoxins (AFs), zearalenone (ZEN), ochratoxin (OTA), deoxynivalenol (DON), etc. Aflatoxins are a class of mycotoxins synthesized by various Aspergillus species (including Aspergillus flavus and Aspergillus parasiticus), and are commonly found in crops such as corn, peanuts, rice, and dried fruits. Aflatoxins mainly include aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin M1, and aflatoxin M2, among which aflatoxin B1 and aflatoxin M1 have the strongest toxicity, posing a serious threat to livestock production and human health. The International Agency for Research on Cancer (IARC) of the World Health Organization has classified aflatoxins as Group 1 carcinogens for humans.

[0004] Ochratoxin is a typical polyketide mycotoxin, which is a secondary metabolite produced by certain species of Aspergillus and Penicillium genera. It mainly exists in agricultural products such as grains, fruits, cocoa, and coffee beans, and may further contaminate processed foods and feeds. The most harmful is ochratoxin A (OTA), which is a potent nephrotoxin and also has teratogenic and immunotoxic effects, and is classified as a Group 2B human carcinogen by the International Agency for Research on Cancer (IARC).

[0005] DON and ZEN are mainly produced by Fusarium species such as Fusarium graminearum, contaminating cereal crops such as wheat, corn, and rice. They not only reduce grain yields but also have varying degrees of residues in cereal foods and animal-derived products, causing serious food safety problems. DON and ZEN have reproductive, genetic, cellular, and immunotoxic effects, and these toxins are difficult to be destroyed during the processes of cereal milling, processing, and heat treatment, and can be enriched in the food chain for a long time.

[0006] With the continuous research on mycotoxin biodegradation technology, many microbial strains capable of degrading mycotoxins have been discovered. Patent 202210942045.5 discloses a Bacillus velezensis strain Vel-HNGD-F2, which can effectively degrade vomitoxin and zearalenone, and the degradation effect reaches 70%-80%. Patent 202411541583.9 discloses a Bacillus subtilis IFST-Xd-2, which can efficiently degrade aflatoxin B1 and zearalenone simultaneously. However, when AFB1 and ZEN coexist, the degradation rate of AFB1 by Bacillus subtilis IFST-Xd-2 decreases by 0.41%, and the degradation rate of ZEN decreases by 0.62%. In this context, many physical, chemical, and biological methods have been developed to remove mycotoxins, but most of these methods have limitations such as high cost, complex operation, low degradation efficiency, inability to degrade multiple mycotoxins simultaneously, and activation of secondary metabolites toxic to humans and animals. Biodegradation uses microorganisms and their metabolites to degrade mycotoxins with high specificity and efficiency. The genus Bacillus is considered one of the best biological control agents for mycotoxin production through inhibiting fungal growth and mycotoxin detoxification. It is necessary to further screen broad-spectrum degradation strains, optimize the production process, develop more cost-effective degradation agents, and promote their large-scale application in the feed industry. Summary of the Invention

[0007] The present invention provides a Bacillus subtilis HNGD-Mq02 that can simultaneously degrade AFB1, AFM1, OTA, ZEN, and DON, and a Bacillus subtilis HNGD-sg5 that can simultaneously degrade AFB1, ZEN, and DON. The compounding of the two agents significantly improves the efficiency of simultaneously degrading multiple mycotoxins, solving the deficiencies in the prior art.

[0008] The technical solution of the present invention is realized as follows: The Bacillus subtilis ( Bacillus subtilis )HNGD-Mq02 in this application was isolated from farm-made wheat koji paste, and its taxonomic name is Bacillus subtilis , and it was deposited at the China Center for Type Culture Collection on January 17, 2025. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2025181; the Bacillus subtilis ( Bacillus subtilis )HNGD-sg5 was isolated from sweet garlic, and its taxonomic name is Bacillus subtilis , and it was deposited at the China Center for Type Culture Collection on December 6, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M20242743.

[0009] Based on this, the present application provides a Bacillus subtilis complex bactericide for simultaneously degrading multiple mycotoxins, which comprises Bacillus subtilis HNGD-Mq02 and Bacillus subtilis HNGD-sg5.

[0010] Preferably, the above-mentioned Bacillus subtilis complex bactericide specifically comprises the fermentation broth or fermentation supernatant of Bacillus subtilis HNGD-Mq02 and Bacillus subtilis HNGD-sg5.

[0011] Preferably, the main active substance for degradation in the above fermentation broth or fermentation supernatant is extracellular enzyme.

[0012] Preferably, the volume ratio of the fermentation broth of Bacillus subtilis HNGD-Mq02 and Bacillus subtilis HNGD-sg5 is 1:1, and the volume ratio of the fermentation supernatant is 1:1.

[0013] Furthermore, the preparation method of the above-mentioned Bacillus subtilis complex bactericide: inoculate the seed solutions of Bacillus subtilis HNGD-Mq02 and Bacillus subtilis HNGD-sg5 into the LB medium with an initial pH of 7 at an inoculation amount of 0.2%, and place it in a shaker at 37 °C and 150 - 200 r / min for 24 - 48 h.

[0014] In the second aspect, the above-mentioned Bacillus subtilis complex bactericide is used for simultaneously degrading multiple mycotoxins.

[0015] Preferably, the above-mentioned mycotoxins are one or more of aflatoxin B1, aflatoxin M1, ochratoxin A, zearalenone, and deoxynivalenol.

[0016] In the third aspect, a method for simultaneously degrading multiple mycotoxins, the steps are: mixing the above-mentioned Bacillus subtilis complex bactericide with the sample to be treated.

[0017] Preferably, the use concentration of the above-mentioned Bacillus subtilis complex bactericide is 1×10 6 -1×10 7 CFU / mL.

[0018] In the fourth aspect, the above-mentioned Bacillus subtilis complex bactericide is used as a feed additive.

[0019] Preferably, in the above application, the Bacillus subtilis complex bactericide is used for biological detoxification of feed.

[0020] The Bacillus subtilis complex bactericide can efficiently degrade aflatoxin B1, aflatoxin M1, ochratoxin A, zearalenone, and / or deoxynivalenol, and can be widely used for mycotoxin detoxification in grains, food, feeds, etc. and their processing by-products.

[0021] The present invention has the following beneficial effects: 1. In this application, Bacillus subtilis HNGD-Mq02 and Bacillus subtilis HNGD-sg5 with the ability to degrade multiple toxins are isolated. Among them, strain HNGD-Mq02 can efficiently degrade AFB1, AFM1, OTA, ZEN, and DON within 72 h, and the degradation rates are 95.5%, 93.1%, 98.3%, 59.15%, and 42.58% respectively; the degradation rates of strain HNGD-sg5 for AFB1, ZEN, and DON within 72 h are 97.27%, 93.81%, and 49.74% respectively. The degradation abilities of strain HNGD-Mq02 and strain HNGD-sg5 are relatively excellent, and they have good application potential in treating feeds contaminated with multiple mycotoxins.

[0022] 2. Active substance localization shows that the supernatant of strain HNGD-Mq02 has a good degradation effect, and there is no significant difference from the fermentation broth, indicating that the degradation active substance belongs to extracellular metabolites; the fermentation supernatant of strain HNGD-sg5 has the highest degradation rate for ZEN, and the degradation rates for AFB1 and DON are also significantly higher than those of the cell suspension and intracellular extract, indicating that the degradation active substance is in the fermentation supernatant and belongs to extracellular metabolites. Qualitative analysis of the active substance found that the substance that plays a major role in the degradation of AFB1 by strain HNGD-Mq02 is extracellular protein; the substance that plays a major degradation effect in strain HNGD-sg5 is extracellular enzyme, and the degradation rate of AFB1 under heat treatment is 68.90% ± 2.36%, indicating that the degradation active substance of AFB1 has certain high-temperature resistance characteristics.

[0023] 3. The Bacillus subtilis composite bactericide can achieve efficient degradation of AFB1 (98.55%), AFM1 (93.77%), and OTA (98.97%) within 24 h. Among them, the degradation rates of AFB1 and OTA are 3.05% and 0.67% higher than those of the single strain treatment for 72 h respectively, and the treatment time is shortened by 67%; the degradation rate of ZEN within 16 h is as high as 95.33%, which is 61.6% higher than the 72 h degradation efficiency (59.15%) of the single strain HNGD-Mq02, and the degradation rate per unit time is increased by 8.1 times. This indicates that there is a synergistic effect between strain HNGD-Mq02 and strain HNGD-sg5; this composite bactericide can simultaneously degrade multiple mycotoxins in a short time, has a high degradation efficiency and high-temperature resistance, and has great production application potential. It provides a new microbial resource for mycotoxin detoxification and can be widely used for mycotoxin detoxification in grains, foods, feeds, and their processing by-products. Description of the Drawings

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0025] Figure 1 For the morphological identification of two strains, where A is Bacillus subtilis The strain morphology diagram of HNGD-Mq02; B is Bacillus subtilis The Gram staining diagram of HNGD-Mq02; C is Bacillus subtilis The electron microscopy diagram of HNGD-Mq02; D is Bacillus subtilis The strain morphology diagram of HNGD-sg5; E is Bacillus subtilis The Gram staining diagram of HNGD-sg5; F is Bacillus subtilis The electron microscopy diagram of HNGD-sg5.

[0026] Figure 2 For the phylogenetic tree constructed based on the 16S rDNA gene sequence, where A is Bacillus subtilis The phylogenetic tree of HNGD-Mq02 strain; B is Bacillus subtilis The phylogenetic tree of HNGD-sg5 strain.

[0027] Figure 3 For Bacillus subtilis The high performance liquid chromatography diagrams of HNGD-Mq02 strain before and after degrading toxins, where A is AFB1 toxin; B is AFM1 toxin; C is OTA toxin; D is ZEN toxin; E is DON toxin.

[0028] Figure 4 For Bacillus subtilis The high performance liquid chromatography diagrams of HNGD-sg5 strain before and after degrading toxins, where A is AFB1 toxin; B is ZEN toxin; C is DON toxin.

[0029] Figure 5 For Bacillus subtilis The active substance localization diagram of HNGD-Mq02 strain degrading the mycotoxin AFB1.

[0030] Figure 6 For Bacillus subtilis The active substance localization diagram of HNGD-sg5 strain degrading mycotoxins, where A is AFB1 toxin; B is ZEN toxin; C is DON toxin.

[0031] Figure 7 For Bacillus subtilis The degradation rate of AFB1 by different treatment methods of HNGD-Mq02 strain.

[0032] Figure 8 For Bacillus subtilis Degradation rates of mycotoxins by different treatment methods of HNGD-sg5 strain, where A is the degradation rate of AFB1 toxin; B is the degradation rate of ZEN toxin; C is the degradation rate of DON toxin.

[0033] Figure 9 For the strain Bacillus subtilis HNGD-Mq02 and Bacillus subtilis Degradation of mycotoxins by the composite bacterium agent of HNGD-sg5. Specific implementation manners

[0034] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0035] The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0036] The Bacillus subtilis ( Bacillus subtilis ) HNGD-Mq02 used in this application was isolated from farm wheat koji sauce in Henan Province in September 2023, and its classification name is Bacillus subtilis , and was deposited in the China Center for Type Culture Collection on January 17, 2025. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2025181; Bacillus subtilis ( Bacillus subtilis ) HNGD-sg5 was isolated from sugar garlic in Henan Province in July 2024, and its classification name is Bacillus subtilis , and was deposited in the China Center for Type Culture Collection on December 6, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M20242743.

[0037] Example 1: Screening and identification of strains Weigh 10 g of samples from each type of sample taken and place them in 90 mL of sterile physiological saline, shake at a constant temperature of 150 r / min for 12 h, and dilute to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10-8 Absorb 200 μL of each gradient dilution and spread it on the primary screening medium plate with coumarin as the sole carbon source. Place it in an incubator at 37°C and incubate it upside down for 3 - 7 days to observe the growth status of the strains. Select the strains with good growth, subculture them multiple times on the primary screening medium to obtain single colonies, and store the colonies in a -80°C refrigerator with 40% glycerol.

[0038] Example 2: Functional verification of degradation of AFB1, AFM1, OTA, ZEN, and DON Activate the single colonies obtained by primary screening and purification in LB liquid medium. Take 975 μL of the strain fermentation broth and 25 μL of the AFB1 standard (100 μg / mL) and place them in a 10 mL sterilized centrifuge tube to make the final concentration of AFB1 2.5 μg / mL. Incubate it in a shaking incubator at 37°C and 150 r / min for 72 h, and detect the AFB1 content using a high-performance liquid chromatograph. The detection methods for the contents of AFM1, ZEN, OTA, and DON toxins are the same, with their final concentrations being 1.5 μg / mL, 5 μg / mL, 5 μg / mL, and 5 μg / mL respectively. Use sterile fermentation medium plus toxins as blank controls. After the strain and the toxin standard are co-incubated, add three volumes of dichloromethane (use ethyl acetate for DON and add 1 M hydrochloric acid for OTA) to the system and vortex and shake for extraction. Take out and discard the aqueous layer, and place the remaining dichloromethane layer in a nitrogen blower to blow dry until dry. Dissolve the residue with 0.5 mL of mobile phase, vortex and shake for 60 s, and filter it through a 0.22 μm organic phase filter membrane. Use HPLC to detect the toxin content.

[0039] Detection method for AFB1 and AFM1: Chromatographic column: C18 column (150 mm × 4.6 mm, 4 μm); Mobile phase: water: methanol: acetonitrile (60:20:20, v / v / v, 35°C); Detection wavelength: 365 nm; Flow rate: 1.0 mL / min; Injection volume: 20 μL.

[0040] Detection method for OTA: Chromatographic column: C18 column (150 mm × 4.6 mm, 4 μm); Mobile phase: acetonitrile: water: acetic acid (99:99:2, v / v / v); Fluorescence detection, excitation and emission wavelengths are 330 nm and 460 nm respectively. Flow rate: 1.0 mL / min; Injection volume: 20 μL.

[0041] Detection method for ZEN: Chromatographic column C18 (4.6 × 250 mm, 5 μm); Mobile phase: (water: methanol = 20:80); Flow rate: 1.0 mL / min; Column temperature: 35°C; Injection volume: 20 μL; UV detection wavelength: 236 nm.

[0042] DON detection method: C18 (4.6×250 mm, 5 μm); column temperature 35°C; sample loading volume 20 μL; mobile phase: (water: methanol = 70:30); UV detection wavelength 218 nm; flow rate 1 mL / min.

[0043] The degradation rate calculation formula is as follows: D=(C-S) / C×100%.

[0044] Where: C is the toxin peak area of the blank control group; S is the toxin peak area after experimental degradation; D is the degradation rate of the toxin (%).

[0045] As Figure 3 shown, the degradation rates of strain HNGD-Mq02 for AFB1, AFM1, OTA, ZEN, and DON are 95.5%, 93.1%, 98.3%, 59.15%, and 42.58% respectively.

[0046] As Figure 4 can be seen, strain HNGD-sg5 has a degradation efficiency of 97.27% for AFB1, 93.81% for ZEN, and 49.74% for DON.

[0047] Compared with most strains that can only degrade single mycotoxins at present, strains HNGD-Mq02 and HNGD-sg5 have excellent degradation capabilities and have good application potential in dealing with feeds contaminated with multiple mycotoxins.

[0048] Example 3: Morphological identification of the strain The strain was streaked on LB solid medium and cultured at 37°C for 18 h. Observe the morphology, color, etc. of the single colonies, and then perform Gram staining and observe the strain using a scanning electron microscope.

[0049] As Figure 1 shown in A and D, Bacillus subtilis the single colonies of HNGD-Mq02 and HNGD-sg5 are large and flat, with an irregular morphology with serrated edges. The colony surface is rough, white, and opaque, and the texture is dry and easy to pick up.

[0050] Figure 1 B and E are the states of strains Bacillus subtilis HNGD-Mq02 and HNGD-sg5 after staining under an optical microscope. It can be seen that the strain is blue and the cells are short rod-shaped, indicating that the strain is a Gram-positive bacterium.

[0051] As Figure 1 shown in C and F, Bacillus subtilis the scanning electron microscope images of HNGD-Mq02 and HNGD-sg5 show that the strain has a short rod structure of 2 - 3.5 μm.

[0052] Example 4: Molecular Biological Identification of Strains Extract the DNA of the strain using the Ezup Column Bacterial Genomic DNA Extraction Kit. Use primers 27F (5’-AGTTTGATCMTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’) to amplify 16S rDNA with the genomic DNA as the template. The reaction system is: 15 μL of ddH2O, 12 μL of taq enzyme, 1 μL of primer 27F, 1 μL of primer 1492R, and 1 μL of the bacterial liquid template. The amplification program is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 55°C for 30 s; extension at 72°C for 10 min. After the amplification is completed, electrophoresis is carried out at 110 V and 100 mA for 40 min to observe the results and judge whether the PCR amplification is successful. The amplified product is purified and sequenced by Shanghai Sangon Biotech Co., Ltd. The sequencing results are compared and analyzed in the NCBI database, and a phylogenetic tree is constructed using the MEGA 8.0 software.

[0053] The phylogenetic tree is as Figure 2 shown. The similarity of both strains is the highest with the genus Bacillus subtilis. Based on the comprehensive morphological characteristics, physiological and biochemical characteristics, and the results of 16S rDNA sequencing and homology analysis, both strains belong to Bacillus subtilis ( Bacillus ), and are named Bacillus subtilis HNGD-Mq02 and Bacillus subtilis HNGD-sg5 respectively, and have been deposited in the China Center for Type Culture Collection with the deposit numbers of CCTCC NO: M2025181 and CCTCC NO: M20242743 respectively.

[0054] Example 5: Localization of the Active Substance for Degrading Mycotoxins by Strains To explore the localization of the degradation active substances of strains HNGD-Mq02 and HNGD-sg5, strains HINGD-Mq02 and HNGD-sg5 were activated and fermented. 10 mL of the fermentation broth of each strain was centrifuged at 12,000 r / min for 15 - 20 min at 4°C, and the supernatant was collected for standby. The precipitate was washed with phosphate buffered saline (PBS) and redissolved to the same volume as the supernatant to form a cell suspension, which was reserved for later use. The obtained cell suspension was further subjected to cell disruption, and the resulting milky suspension was centrifuged for 15 - 20 min. Then, the supernatant obtained after centrifugation was filtered through a 0.22 μm filter membrane to obtain the intracellular extract of the strain. The fermentation broth, supernatant, cell suspension, and intracellular extract of the strain were respectively mixed with the toxin standard and incubated together to test the toxin degradation effect of different components of strains HNGD-Mq02 and HNGD-sg5. The blank control group was LB medium containing PBS, and there were three parallels in each group.

[0055] It can be Figure 5 seen that the degradation rates of the fermentation broth, supernatant, cell suspension, and intracellular extract of strain HNGD-Mq02 for the toxin were 89.66%, 87.72%, 23.87%, and 16.03% respectively. Among them, the intracellular extract had almost no degradation effect on AFB1; the cell suspension had a certain degradation effect on AFB1, but the effect was not good, indicating that strain HNGD-Mq02 had a certain adsorption effect on the mycotoxin, but the effect was not obvious; there was no significant difference in the degradation effect between the supernatant of the strain and the fermentation broth, and the degradation effect was good, indicating that the degradation active substance was an extracellular metabolite of the cell.

[0056] It can be Figure 6 seen from Fig. A that there were significant differences in the degradation ability of different components of strain HNGD-sg5 for ZEN. Among them, the fermentation supernatant had the highest degradation rate for ZEN, which was 78.01% ± 1.32%, significantly higher than that of the intracellular extract (11.28% ± 0.79%), indicating that the active substance for degrading ZEN mainly existed extracellularly; the degradation rate of the cell suspension for ZEN was 58.49% ± 0.95%, and the degradation rate of the inactivated cells for ZEN was 44.41% ± 1.60%, both higher than that of the intracellular extract degradation rate, inferring that strain HNGD-sg5 had a certain cell adsorption effect. The degradation rates of the fermentation supernatant of strain HNGD-sg5 for AFB1 and DON were significantly higher than those of the cell suspension and intracellular extract. As shown in Figure 6 Figs. B and C, the degradation rates were 96.71% ± 0.31% and 35.15% ± 1.53% respectively, indicating that the degradation active substance mainly existed in the fermentation supernatant and was an extracellular metabolite.

[0057] Example 6: Qualitative analysis of the active substance in the supernatant of the strain for degrading mycotoxin The fermentation supernatants of strains HNGD-Mq02 and HNGD-sg5 were divided into four components to study the effects of proteinase K (PK), sodium dodecyl sulfate (SDS), PK+SDS, and heat treatment (boiling for 20 min) on toxin degradation. Subsequently, each component was incubated with true toxins at different final concentrations at 37°C, with sterile PBS as a control. After 72 h of reaction, the residual toxin content was detected by the method described above.

[0058] It can be seen from Figure 7 that after treatment with PK, SDS, and PK+SDS, the degradation effect of the supernatant of strain HNGD-Mq02 was significantly weakened, indicating that proteinase K and SDS inhibited the protein substances in the supernatant, and further speculated that the substance mainly responsible for the degradation effect on AFB1 was extracellular protein.

[0059] It can be seen from Figure 8 that after treating the fermentation supernatant of strain HNGD-sg5, it was found that the toxin degradation rates of proteinase K, SDS, and heat treatment were significantly reduced, indicating that proteinase K and SDS inhibited the protein, and further speculated that the substance mainly responsible for the degradation effect was extracellular enzyme. In addition, the AFB1 degradation rate under heat treatment was 68.90% ± 2.36%, indicating that the AFB1-degrading active substance had certain high-temperature resistance characteristics.

[0060] Example 7: Degradation of mycotoxins by composite strains The fermentation broths of strains HNGD-Mq02 (1×10 6 -1×10 7 CFU / mL) and HNGD-sg5 (1×10 6 -1×10 7 CFU / mL) were mixed evenly at a ratio of 1:1. The concentration of the mixed bacterial solution was about 1×10 6 -1×10 7 CFU / mL. According to the method in Example 2, it was mixed with the toxin so that the final concentrations of AFB1, AFM1, OTA, ZEN, and DON were 2.5 μg / mL, 1.5 μg / mL, 5 μg / mL, 5 μg / mL, and 5 μg / mL respectively, and the content of the toxin was detected by HPLC during different incubation times in a shaking incubator.

[0061] It can be seen from Figure 9It can be seen that the results of this study show that the composite microbial agent (HNGD-Mq02 + HNGD-sg5) has significantly better degradation efficiency and reaction rate for various mycotoxins than single strains. Under the same treatment conditions, the degradation rates of AFB1, AFM1, OTA, ZEN and DON by the fermentation broth of the single strain HNGD-Mq02 after 72 h of reaction were 95.5%, 93.1%, 98.3%, 59.15% and 42.58% respectively, while the degradation rates of AFB1, ZEN and DON by the HNGD-sg5 strain within 72 h were 97.27%, 93.81% and 49.74% respectively. In contrast, the composite microbial agent achieved efficient degradation of AFB1 (98.55%), AFM1 (93.77%) and OTA (98.97%) within 24 h. Among them, the degradation rates of AFB1 and OTA were 3.05% and 0.67% higher than those of the single strain after 72 h of treatment, respectively, and the treatment time was shortened by 67%. For the ZEN toxin, the degradation rate of the composite microbial agent reached 95.33% within 16 h, which was 61.6% higher than the 72 h degradation efficiency (59.15%) of the single strain HNGD-Mq02, and the degradation rate per unit time increased by 8.1 times. This indicates that there is a synergistic effect between the strains HNGD-Mq02 and HNGD-sg5, and the overall degradation ability may be improved through the complementarity of metabolic pathways. Although the degradation effect of the composite strain on DON is relatively weak (the degradation rate after 48 h is 54.38%), its high degradation ability for AFB1, AFM1, OTA and ZEN provides potential possibilities for its application in the food and feed industries.

[0062] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Bacillus subtilis composite agent for simultaneously degrading multiple mycotoxins, characterized in that: Contains Bacillus subtilis HNGD-Mq02 and Bacillus subtilis HNGD-sg5; the classification name of Bacillus subtilis HNGD-Mq02 is Bacillus subtilis , deposited in China Center for Type Culture Collection on January 17, 2025, with the deposit address at Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2025181; the classification name of Bacillus subtilis HNGD-sg5 is Bacillus subtilis , deposited in the China Center for Type Culture Collection on December 6, 2024, with the deposit address at Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M20242743.

2. The Bacillus subtilis composite agent for simultaneously degrading multiple mycotoxins according to claim 1, characterized in that: Specifically comprises the fermentation broth or fermentation supernatant of Bacillus subtilis HNGD-Mq02 and Bacillus subtilis HNGD-sg5.

3. The Bacillus subtilis composite agent for simultaneously degrading multiple mycotoxins according to claim 2, characterized in that: The degradation effect in the fermentation broth or fermentation supernatant is exerted by the extracellular enzyme.

4. The Bacillus subtilis composite agent for simultaneously degrading multiple mycotoxins according to claim 3, characterized in that: The volume ratio of the fermentation broth of Bacillus subtilis HNGD-Mq02 and Bacillus subtilis HNGD-sg5 is 1:1, and the volume ratio of the fermentation supernatant is 1:

1.

5. Use of the Bacillus subtilis composite agent according to any one of claims 1 to 4 in simultaneously degrading multiple mycotoxins.

6. The use according to claim 5, characterized in that: The fungal toxins are one or more of aflatoxin B1, aflatoxin M1, ochratoxin A, zearalenone and deoxynivalenol.

7. A method for simultaneously degrading multiple mycotoxins, characterized in that: The method comprises the following steps: mixing the Bacillus subtilis composite bacterial agent described in any one of claims 1 to 4 with a sample to be processed.

8. The method for simultaneously degrading multiple mycotoxins according to claim 7, characterized in that: The concentration of the Bacillus subtilis composite agent is 1×10 6 -1×10 7 CFU / mL.

9. Use of the Bacillus subtilis composite agent according to any one of claims 1 to 4 in feed additives.

10. The use according to claim 9, characterized in that: The Bacillus subtilis composite bacterial agent is used for biological detoxification of feed.

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