Bacillus velezensis and application thereof
By providing a Bacillus Veles that can efficiently degrade zearalenone, the problem of insufficient degradation ability of the toxin in the prior art is solved, and effective control of mycotoxin pollution and agricultural safety improvement are achieved.
Patent Information
- Application Number
- CN202510079172.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-17
- Publication Date
- 2025-05-09
AI Technical Summary
The degradation capacity of zearalenone (ZEN) in the prior art is insufficient, making it difficult to effectively prevent and control mycotoxin pollution, affecting the safety of food and animal husbandry.
A strain of Bacillus velezensis was provided, with the storage number CGMCC No. 27321, which significantly inhibits the growth of Fusarium and efficiently degrades zearalenone in a short period of time.
Bacillus Veles significantly improved the inhibitory effect on pathogens and achieved a 100% ZEN degradation rate in the experiment, effectively reducing mycotoxin pollution, improving food utilization and agricultural security.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of microorganisms, and in particular to Bacillus Velez Bacillus velezensis ) and its applications. Background Art
[0002] Zearalenone (ZEN), also known as F-2 toxin, has the chemical name 6-(10-hydroxy-6-oxy-undecenyl)-β-razolactone, which is mainly produced by Fusarium graminearum ( Fusarium graminearum )、Fusarium trilineatum( Fusarium tricinctum )、Yellow Fusarium( Fusarium culmorum ) and other fungi, which widely contaminate corn, wheat and other grains and their by-products. ZEN, as an analog of estrogen, mainly acts on the reproductive system of animals, causing estrogen syndrome in livestock and poultry, and impairing reproductive function; and ZEN will enter the metabolites of animals (such as tissues, milk and eggs), migrate and accumulate along the food chain, and thus pose a serious threat to human health. Therefore, mycotoxin pollution not only causes huge economic losses to the food and animal husbandry industries, but also seriously threatens the health of humans and animals.
[0003] The production of ZEN is affected by many factors. Toxigenic fungi are widely distributed in nature. Food crops are good hosts for fungi. They may be infected by toxin-producing fungi and contaminated by mycotoxins during their planting, drying, storage, and processing. At present, the main methods for controlling the harm of fungi and their metabolites are biological control. Biological control mainly refers to the use of microorganisms to antagonize fungi, inhibit the growth and reproduction of fungi, and degrade their metabolites. This method has the advantages of strong specificity, mild action conditions, environmental friendliness, and little effect on the sensory properties and palatability of raw materials, and has become a research hotspot. Bacillus has strong stress resistance and storage resistance, and can produce a variety of enzymes and antibacterial compounds after fermentation. It is widely used in agriculture, feed and fermentation industries.
[0004] The degradation strains reported so far have a low degradation rate and weak degradation ability for ZEN, and there is still a certain gap from actual production application. Summary of the invention
[0005] The purpose of the present invention is to overcome the problems existing in the prior art and provide a strain of Bacillus Velezii and its application. The Bacillus Velezii can inhibit the growth of pathogenic bacteria and has excellent degradation ability for the toxin zearalenone produced by the pathogenic bacteria.
[0006] In order to achieve the above object, the present invention provides a strain of Bacillus Velezii ( Bacillus velezensis ), the deposit number of the Bacillus Velez is CGMCC No.27321.
[0007] The second aspect of the present invention provides a bacterial agent, which contains the Bacillus Velezii as described above; preferably, the bacterial agent is a liquid bacterial agent.
[0008] The third aspect of the present invention provides the use of the aforementioned Bacillus Velezii or the aforementioned bacterial agent in inhibiting the growth of pathogenic bacteria.
[0009] The fourth aspect of the present invention provides the use of the aforementioned Bacillus Velezii or the aforementioned bacterial agent in degrading zearalenone.
[0010] The fifth aspect of the present invention provides the use of the aforementioned Bacillus Velez subtilis or the aforementioned bacterial agent in detoxification of grains or feeds.
[0011] The Bacillus Velezii provided by the present invention has an obvious inhibitory effect on pathogens, especially Fusarium. In addition, the strain can efficiently degrade zearalenone produced by pathogens in a short time. Furthermore, using the strain for detoxification of grains or feed is of great significance for improving food utilization and ensuring safe production in animal husbandry and agriculture.
[0012] Biological Deposit Bacillus Velezii of the present invention Bacillus velezensis , numbered Z1, and was deposited on May 12, 2023 at the General Microbiology Center of China Microorganism Culture Collection Committee (address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101) (the abbreviation of the depository unit is CGMCC), and the deposit number is CGMCC No. 27321. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 The strain morphology of Bacillus Velez Z1 of the present invention under an optical microscope; Figure 2 The cell morphology of Bacillus Velez Z1 of the present invention under a transmission electron microscope; Figure 3 is a phylogenetic tree of Bacillus Velezii Z1 of the present invention; Figure 4 is a liquid chromatogram of the degradation of zearalenone in Example 3; Figure 5 This is a plate confrontation diagram of the antagonism of Bacillus Velezii Z1 against Fusarium oxysporum of the present invention; Figure 6 It is a plate confrontation diagram of the antagonism of Bacillus Velez subtilis Z1 of the present invention against Fusarium graminearum. DETAILED DESCRIPTION
[0014] The endpoints and any values of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and the individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.
[0015] The first aspect of the present invention provides a strain of Bacillus Velezii ( Bacillus velezensis ), the deposit number of the Bacillus Velez is CGMCC No. 27321. In the present invention, it is numbered as Z1.
[0016] According to the present invention, the sequence of 16S rDNA of Bacillus Velezii is shown as SEQ ID NO:1.
[0017] SEQ ID NO: 1:
[0018] The Velez subtilis bacteria of the present invention is milky white on the surface of the LB culture medium, has uneven edges, is moist and convex on the surface, and has a round single colony.
[0019] The Velez bacteria described in the present invention is a Gram-positive bacterium. When observed under a Leica optical microscope, the strain is rod-shaped with spores growing at the tip (e.g. Figure 1 Observation of Bacillus Velez Z1 using transmission electron microscopy showed that the strain had flagella and was a peritrichous fungus (as shown in Figure 2 as shown).
[0020] The Bacillus Velezii described in the present invention is compared by using BLAST to construct a phylogenetic tree, and it is found that the Bacillus Velezii has a close homology relationship with Bacillus subtilis and Bacillus amyloliquefaciens.
[0021] A second aspect of the present invention provides a bacterial agent, wherein the bacterial agent contains the Bacillus Velezii as described above.
[0022] Preferably, the bacterial agent is a liquid bacterial agent.
[0023] In the present invention, the method for preparing the bacterial agent may include: culturing the aforementioned Bacillus Velez subtilis in a liquid culture medium.
[0024] Preferably, the method for preparing the bacterial agent may further include: resuspending the cultured Bacillus Velez subtilis in a buffer solution.
[0025] According to the present invention, preferably, the number of viable bacteria of Bacillus Velezii in the bacterial agent is 1×10 8 -1×10 10 CFU / mL.
[0026] The third aspect of the present invention provides the use of the aforementioned Bacillus Velezii or the aforementioned bacterial agent in inhibiting the growth of pathogenic bacteria.
[0027] In the present invention, the Bacillus Velezii has an inhibitory effect on pathogens that produce zearalenone, and the pathogens are mainly Fusarium, including but not limited to Fusarium graminearum, Fusarium moniliforme, Fusarium trilineatum, Fusarium equisetum and Fusarium rubrum.
[0028] According to the present invention, preferably, the pathogenic bacteria include Fusarium oxysporum and Fusarium graminearum.
[0029] The fourth aspect of the present invention provides the use of the aforementioned Bacillus Velezii or the aforementioned bacterial agent in degrading zearalenone.
[0030] According to the present invention, preferably, the amount of the Bacillus Velezii in terms of viable bacteria per mg of zearalenone is 3×10 8 -1×10 11 CFU.
[0031] More preferably, the amount of the Bacillus Velezii in terms of viable bacteria per mg of zearalenone is 3×10 8 -1×10 10 CFU.
[0032] The fifth aspect of the present invention provides the use of the aforementioned Bacillus Velez subtilis or the aforementioned bacterial agent in detoxification of grains or feeds.
[0033] In the present invention, the dosage of the Bacillus Velezii or the bacterial agent can be determined according to the content of zearalenone in feed or grains.
[0034] According to the present invention, the application comprises: inoculating Bacillus Velez or its bacterial agent into grains or feed, and culturing at 30-37° C. for 24-72 hours.
[0035] The present invention will be described in detail below by way of examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention, and are not intended to limit the present invention.
[0036] Unless otherwise specified, the reagents and materials used in the following examples were purchased from regular chemical reagent suppliers and were of analytical grade.
[0037] In the following examples, zearalenone is represented by the abbreviation ZEN.
[0038] The culture medium formula used in the following examples is as follows: Trace element solution: 1.5g FeCl2•4H2O, 0.19g CoCl2•6H2O, 0.1g MnCl2•4H2O, 0.07gZnCl2, 0.062g H3BO3, 0.036g Na2MoO4•2H2O, 0.024g NiCl2•6H2O, 0.017g CuCl2•2H2O, 0.01gAlK(SO4)2•12H2O, 1 L distilled water.
[0039] MM medium: 1.6 g Na2HPO4, 1 g KH2PO4, 0.5 g MgSO4•7H2O, 0.5 g NaNO3, 0.5 g (NH4)2SO4, 0.025 g CaCl2•7H2O, 1 L distilled water, 2 mL trace element solution, 10 mg or 20 mg ZEN.
[0040] LB solid medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 15 g agar, 1 L distilled water.
[0041] PDA solid medium: 300 g potatoes, 20 g glucose, 15 g agar, 1 L distilled water.
[0042] ZEN content detection method: (1) Take 0.8 mL of the test solution, add 10 times the volume of PBS solution and mix well. Load the entire solution onto the zearalenone immunoaffinity column and control the flow rate at 1-3 mL / min. Elute the immunoaffinity column with 10 mL of deionized water at the same flow rate.
[0043] (2) Elute the immunoaffinity column twice with 1 mL of methanol solution and collect the eluate; blow dry with nitrogen at 55°C, add 1 mL of 50 vol% acetonitrile to dissolve the precipitate and filter through a 0.2 μm PTFE filter membrane; collect the liquid in a 2 mL injection vial to obtain the sample solution, which is then tested by HPLC.
[0044] HPLC detection conditions are: Mobile phase: Phase A is acetonitrile: water = 1:1 Phase B is methanol; isocratic elution, 8% B; Chromatographic column: C18 column: 150 mm × 4.6 mm, 5 μm; Mobile phase flow rate: 1.0 mL / min; Detector: fluorescence detector, Em=440 nm Ex=274 nm; Column temperature: 40℃; injection volume: 20 μL.
[0045] ZEN degradation rate (%) = (remaining ZEN content in the control group - remaining ZEN content in the experimental group) / remaining ZEN content in the control group × 100%.
[0046] Example 1 This example is used to illustrate the enrichment, screening and identification of Bacillus Velezii Z1 of the present invention.
[0047] (1) Take 10 g of plant root soil from a corn field in Beijing and add it to a 250 mL conical flask. Add 100 mL of physiological saline. Oscillate at 180 rpm for 30 min at 37°C, then place in a water bath at 85°C for 10 min and let stand for 30-60 s. Take 1 mL of the upper bacterial liquid and add it to MM medium containing 10 mg / L ZEN. Incubate at 37°C at 200 rpm for 7 days. Take another 1 mL of the enriched bacterial liquid and add it to MM medium containing 20 mg / L ZEN. Incubate at 37°C at 200 rpm for further 14 days. (2) Dilute the enriched culture solution to 10 -5 , 10 -6 , 10 -7 times, spread on LB solid plates, and culture at 37°C for 24 h; select plates with moderate colony growth density, pick strains with inconsistent colony morphology on the plates and inoculate them into LB liquid culture medium, culture at 37°C and 200 rpm for 16 h, and then transfer them to MM culture medium containing 10 mg / L ZEN at an inoculum of 1 vol%, and culture at 37°C and 200 rpm for 12 h to obtain candidate ZEN-degrading strains; screen all candidate ZEN-degrading strains, detect their degradation efficiency of ZEN, and screen out the strain with the highest degradation efficiency, named Z1, and store strain Z1 in glycerol tubes at -80°C for later use.
[0048] (3) After strain Z1 was activated by inoculation on LB solid medium using the plate streak method, it was cultured at 37°C for 24 h and the morphological characteristics of the colonies were observed. The colonies of strain Z1 were milky white, with uneven edges, moist and raised surfaces, and single colonies were round.
[0049] After Gram staining, the morphology was observed using a Leica optical microscope. Figure 1 Strain Z1 is a Gram-positive bacterium with a rod shape and spore tip growth.
[0050] Using transmission electron microscopy, we observed Figure 2 As shown, the strain has flagella and belongs to the Perichritic fungus.
[0051] The 16S rDNA gene fragment was amplified using universal primers 27F / 1492R, and the amplified product was sequenced to obtain the 16S rDNA sequence of strain Z1 as shown in SEQ ID NO: 1. The obtained sequence SEQ ID NO: 1 was compared using BLAST, and a phylogenetic tree was constructed. The results are shown in Figure 3 shown.
[0052] The strain Z1 was identified as Bacillus velezinsis ( Bacillus velezensis ), the strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration with the deposit number CGMCC No. 27321.
[0053] Example 2 This example is used to illustrate the inhibitory effect of the Bacillus Velezii Z1 of the present invention on pathogenic bacteria.
[0054] (1) Inoculate Bacillus velezensis Z1 into LB solid culture medium and incubate at 37°C for 24 h. (2) Inoculate Fusarium oxysporum and Fusarium graminearum in the center of the PDA solid culture medium, and inoculate Bacillus Velez Z1 3 cm from the center. Incubate in a 30°C constant temperature incubator for 7 days, and observe the growth of mycelium every other day until the mycelium fills the plate. (3) Use a vernier caliper to measure the radius of the inhibition zone.
[0055] The experiment was repeated three times and the average value was taken. The average radius of the inhibition zone of Bacillus Velez Z1 against Fusarium oxysporum was 4.35 mm, and the average radius of the inhibition zone against Fusarium graminearum was 6.50 mm. The plate confrontation effect of Bacillus Velez Z1 against Fusarium oxysporum and Fusarium graminearum was as follows: Figure 5 and Figure 6 shown.
[0056] Example 3 This example is used to illustrate the degradation effect of Bacillus Velezii Z1 of the present invention on ZEN.
[0057] (1) Bacillus Velezii Z1 was inoculated into LB liquid culture and cultured at 37°C and 220 rpm for 24 h to obtain a bacterial agent with a live bacterial concentration of 3×10 8 CFU / mL; (2) Dissolve 10 mg of ZEN standard in 10 mL of 50 vol% acetonitrile to obtain a 1000 mg / L ZEN solution; (3) Take 1 mL of the bacterial agent prepared in step (1) and place it in a 2 mL centrifuge tube. Add 10 µL of the ZEN solution prepared in step (2) to the tube. After thorough oscillation and mixing, incubate at 37°C and 160 rpm for 8 h, centrifuge at 12,000 rpm for 10 min, and collect the supernatant to obtain the experimental group solution. (4) Take 1 mL of LB liquid culture medium and place it in a 2 mL centrifuge tube. Add 10 µL of the ZEN solution prepared in step (2) to the tube. After thorough vortexing and mixing, incubate at 37°C and 160 rpm for 8 h. Centrifuge at 12,000 rpm for 10 min and collect the supernatant to obtain the control group solution. (5) Liquid chromatography was used to test the ZEN content in the experimental group solution and the control group solution. The test results are as follows: Figure 4 shown.
[0058] Bacillus Velez Z1 had an excellent degradation effect on ZEN, with a degradation rate of 100%.
[0059] Example 4 This example is used to illustrate the detoxification effect of Bacillus Velezii Z1 of the present invention on feed.
[0060] (1) Inoculate Bacillus velezensis Z1 into LB liquid culture and culture at 37°C and 220 rpm for 24 h to prepare seed solution; (2) The cultured seed solution was transferred to LB medium containing 10 g / L sucrose at an inoculum rate of 2 vol%, and fermented at 30 °C and 220 rpm for 72 h; (3) Take the fermented bacterial liquid, centrifuge it at 6500 rpm for 20 min, and collect the bacterial cells; (4) Suspend the cells in pH 8.0 Tris-HCl buffer to a concentration of 1 × 10 9 CFU / mL; (5) 5 mL of the above-mentioned inoculum was inoculated into 30 g of corn germ meal (from a corn oil factory) with a solid content of 50% and 40%, respectively. The initial ZEN concentrations in the corn germ meal were 1596 μg / kg and 1510 μg / kg, respectively. The mixture was stirred evenly and placed in a 30°C incubator for static culture. (6) After fermentation for 24 h and 48 h, 10 g of sample was weighed and added with 100 mL of 84 vol% acetonitrile. After ultrasonic treatment for 1 h, the sample was extracted and the supernatant was collected by centrifugation. The remaining ZEN content in the sample was determined. The results are shown in Table 1.
[0061] Table 1
[0062] Bacillus Velez Z1 can effectively degrade ZEN in corn germ meal, with a removal rate of more than 80%, achieving effective detoxification of corn germ meal feed.
[0063] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.
Claims
1. A strain of Bacillus velez Bacillus velezensis ), characterized in that, The deposit number of the Bacillus Velez is CGMCC No.27321.
2. The Bacillus Velezii according to claim 1, wherein The sequence of 16S rDNA of the Bacillus velez is shown in SEQ ID NO:
1.
3. A bacterial agent, wherein: The bacterial agent contains the Bacillus Velezii according to claim 1 or 2, and the bacterial agent is a liquid bacterial agent.
4. The bacterial agent according to claim 3, wherein The number of viable bacteria of Bacillus Velez in the bacterial agent is 1×10 8 -1×10 10 CFU / mL.
5. Use of the Bacillus Velezii described in claim 1 or 2 or the bacterial agent described in claim 3 or 4 in inhibiting the growth of pathogenic bacteria.
6. The use according to claim 5, wherein: The pathogenic bacteria include Fusarium oxysporum and Fusarium graminearum.
7. Use of the Bacillus Velezii described in claim 1 or 2 or the bacterial agent described in claim 3 or 4 in degrading zearalenone.
8. The use according to claim 7, wherein: The amount of the Velez subtilis in terms of viable bacteria is 3×10 8 -1×10 11 CFU.
9. Use of the Bacillus Velezii described in claim 1 or 2 or the bacterial agent described in claim 3 or 4 in detoxification of grains or feed.
10. The use according to claim 9, wherein: The application comprises: inoculating the Bacillus Velez described in claim 1 or 2 or the bacterial agent described in claim 3 or 4 into grains or feed, and culturing at 30-37° C. for 24-72 hours.