Methylobacterium torquens capable of efficiently degrading zearalenone and application of methylobacterium torquens
By using methyl trebromosiligo to culture and prepare bacterial agents under specific conditions, the problem of difficult removal of zearalenone was solved, and efficient degradation was achieved, with a degradation rate of 83%, laying the foundation for industrial applications.
Patent Information
- Application Number
- CN202510438034.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-09
- Publication Date
- 2025-07-11
- Estimated Expiration
- 2045-04-09
AI Technical Summary
The prior art is difficult to efficiently remove zearalenone, resulting in animal health risks and economic losses, and the traditional methods do not have significant effect on zearalenone degradation.
Methylobacterium extorquens were used for culture, and bacteria were prepared and zearalenone was degraded under specific conditions. The culture temperature was 29-31℃, the time was 24-48h, and the bacteria content in the bacteria was 1×108cfu/mL-1×109cfu/mL, and synthetic or LB culture medium was used.
Under the conditions of culture in synthetic culture medium, the removal rate of zearalenone can reach 83%, providing the possibility for industrial applications and significantly reducing the content of zearalenone.
Smart Images
Figure CN120290378A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a Methylobacterium torquatum capable of efficiently degrading zearalenone and its application. Background Art
[0002] Mycotoxins are toxic secondary metabolites produced by molds during their growth, mainly including aflatoxins, trichothecenes, zearalenone, ochratoxin A, fumonisins, etc. Raw materials such as grains will be contaminated by molds in the field. If the environmental temperature and humidity are suitable during transportation, processing, and storage, the molds will continue to grow, and the toxin content will increase accordingly. According to the estimation of the Food and Agriculture Organization of the United Nations, 25% of the world's grains are contaminated by mycotoxins every year, and on average, 2% are inedible; coupled with the diseases and deaths caused by animal poisoning due to toxin contamination, it has caused huge economic losses to the food industry and animal husbandry.
[0003] Zearalenone, also known as F-2 toxin, was isolated by Stob et al. from maize contaminated with Gibberella zeae in 1962. It is mainly produced by Fusarium graminearum, Fusarium culmorum, and Fusarium crookwellense. Fusarium is both parasitic and saprophytic, with strong ecological adaptability and wide distribution. Grains such as maize, wheat, barley, and oats are prone to mildew and are easily infected by Fusarium and contaminated by ZEN. Animals absorb ZEN by eating ZEN-contaminated feed. ZEN has estrogen-like effects and can compete with estrogen in the body to bind to the corresponding receptors, activate downstream response elements, trigger estrogen receptor dimerization, and then produce a series of estrogen-like effects, resulting in disorder of estrogen levels in animals and damage to the reproductive systems of animals, especially sows. It is mainly manifested as decreased appetite, immunosuppression, and slow growth. Continuous poisoning will cause reproductive disorders in female livestock, leading to infertility, miscarriage, and stillbirth. ZEN also has strong carcinogenicity and can affect human edible meat products and dairy products through animal ingestion, posing a serious threat to human health. Summary of the Invention
[0004] In order to solve the above technical problems, the present invention provides a Methylobacterium torquatum capable of efficiently degrading zearalenone and its application. The Methylobacterium torquatum obtained in the present invention Methylobacterium extorquens can efficiently degrade zearalenone. Methylobacterium torquatum was deposited in the China General Microbiological Culture Collection Center (CGMCC) on January 2, 2024. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 29497, and the microbial taxonomic name is Methylobacterium torquatum Methylobacterium extorquens .
[0005] The present invention provides a Methylobacterium torulosum capable of efficiently degrading zearalenone. The Methylobacterium torulosum was deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 2, 2024. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 29497, and the microbial taxonomic name is Methylobacterium torulosum Methylobacterium extorquens .
[0006] The present invention also provides a bacterial agent containing the Methylobacterium torulosum
[0007] Furthermore, the preparation method of the bacterial agent includes: culturing the Methylobacterium torulosum in a culture medium
[0008] Furthermore, the temperature of the culturing is 29°C - 31°C, and the culturing time is 24h - 48h
[0009] Furthermore, after the culturing is completed, each milliliter of the bacterial agent contains 1×10 8 cfu / mL - 1×10 9 cfu / mL of Methylobacterium torulosum
[0010] Furthermore, the culture medium includes one of a synthetic culture medium and an LB culture medium
[0011] The present invention also provides the application of the Methylobacterium torulosum in degrading zearalenone
[0012] The present invention also provides the application of the bacterial agent containing the Methylobacterium torulosum in degrading zearalenone
[0013] The embodiments of the present invention have the following technical effects The Methylobacterium torulosum isolated and screened in the present invention is cultured in a synthetic culture medium. Under the optimal culture conditions, the removal rate of zearalenone can reach 83%. Moreover, it is found that high concentrations of zearalenone have no obvious inhibition on the growth and degradation of Methylobacterium torulosum, providing a possibility for the industrial application of strains for the biological treatment of zearalenone Description of the Drawings
[0014] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings
[0015] Figure 1 It is the test result provided by the experimental group of the present invention Detailed Embodiments
[0016] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without any creative efforts fall within the scope protected by the present invention.
[0017] In a first aspect, in some embodiments of the present invention, a Methyloversatilis torques capable of efficiently degrading zearalenone is provided. The Methyloversatilis torques was deposited at the China General Microbiological Culture Collection Center (CGMCC) on January 2, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 29497, and the microbial taxonomic name is Methyloversatilis torques. Methylobacterium extorquens .
[0018] In a second aspect, in some embodiments of the present invention, a bacterial agent containing the Methyloversatilis torques is also provided.
[0019] In some embodiments, the preparation method of the bacterial agent includes: culturing the Methyloversatilis torques in a culture medium.
[0020] In some embodiments, the temperature of the culture is 29°C - 31°C, and the culture time is 24h - 48h.
[0021] In some embodiments, after the culture is completed, each milliliter of the bacterial agent contains 1×10 8 cfu / mL - 1×10 9 cfu / mL of Methyloversatilis torques.
[0022] In some embodiments, the culture medium includes one of a synthetic culture medium and an LB culture medium.
[0023] In a third aspect, in some embodiments of the present invention, the application of the Methyloversatilis torques in degrading zearalenone is also provided.
[0024] In a fourth aspect, in some embodiments of the present invention, the application of the bacterial agent containing the Methyloversatilis torques in degrading zearalenone is also provided.
[0025] The following is a detailed elaboration in combination with examples and comparative examples: Example 1: (1)Strains were screened in Xinjiang Uygur Autonomous Region, China. The colonies were round with a diameter of 1-2 mm, having an opaque, pink color, a raised center, a bright and smooth surface, and were moist and viscous. The screened Methylobacterium thiooxidans was sequenced. Total DNA of the bacterial solution was extracted and used as a template for bacterial solution PCR. PCR amplification was performed using 27F and 1492R amplification primers. The PCR amplification reaction system (10 μL) was as follows: cDNA (1 μL), 10× Buffer (1 μL), 2.5 mM dNTP (0.8 μL), 10 μM forward and reverse amplification primers each (0.5 μL), rTaq enzyme (0.05 μL), ddH2O (6.15 μL); The 27F amplification primer was as shown in SEQ ID NO.2, and SEQ ID NO.2 was AGAGTTTGATCCTGGCTCAG.
[0026] The 1492R amplification primer was as shown in SEQ ID NO.3, and SEQ ID NO.3 was GGTTACCTTGTTACGACTT.
[0027] PCR amplification reaction conditions: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 51°C for 30 s, extension at 72°C for 1 min, 30 cycles, and extension at 72°C for 10 min; After the entire amplification reaction was completed, the reaction product was detected by 1% agarose gel electrophoresis. The target band was cut back under a gel imaging system, and DNA recovery and purification were carried out according to the instructions of the Sangon SanPrep column kit. The recovered product was ligated with the PMD18-T cloning vector, and the ligation was carried out overnight at 16°C to obtain the PMD18-T recombinant vector of the target strain gene sequence. The ligation system was as follows: recovered product 4.5 μL, PMD18-T 0.5 μL; All of the ligation system product was transferred into the prepared Escherichia coli competent DH5α, incubated in an ice bath for 30 min, heat-shocked at 42°C for 30 s, and then placed on ice. 650 μL of LB medium preheated to 37°C was added, and the mixture was shaken at 37°C for 1 h. Then, it was centrifuged at 5000 rpm for 3 min, most of the supernatant was discarded, and the remaining approximately 150 μL of supernatant and the precipitate were pipetted and mixed evenly, and then spread on an LB plate containing 100 mg / mL sodium ampicillin (Amp). After incubating upright at 37°C for 1 h, it was then incubated upside down for 12 - 16 h. Well-grown single colonies on the plate were picked, cultured at 37°C for 6 h, and then screened for positive clones using the amplification primers 27F and 1492R. The correctly ligated bacterial solution was sent to Sangon Biotech Co., Ltd., Nanjing Branch for sequencing. Finally, the cDNA sequence of the target strain was subjected to Blast alignment to determine the strain species.
[0028] The 16S rDNA sequence of the obtained strain is shown in SEQ ID NO.1.
[0029]
[0030] (2) Cultivate the Methylobacterium torquens of the present invention to prepare a synthetic medium (10 mL of methanol, 3 g of (NH4)2HPO4, 3 g of Na2HPO4, 1.5 g of KH2PO4, 2 g of MgSO4·7H2O, 0.03 g of CaCl2·2H2O, 0.005 g of MnSO4·H2O, 0.005 g of ZnSO4·7H2O, 0.005 g of CuSO4·5H2O, and disperse the above components in 1 L of water), adjust the pH value of the synthetic medium to 7.2; autoclave the obtained synthetic medium at 121 °C for 20 minutes. Inoculate the Methylobacterium torquens strain into the synthetic medium and culture it in a shaker flask at 30 °C and 170 rpm for 24 - 28 hours to obtain the activated Methylobacterium torquens strain.
[0031] (3) Test the obtained Methylobacterium torquens strain for the degradation of zearalenone: The control group is 14.85 mL of liquid LB medium supplemented with 150 μL of ZEN with a mass concentration of 1 μg / μL (dissolve and disperse ZEN in acetonitrile); the experimental group is 14.80 mL of liquid LB medium supplemented with 150 μL of ZEN with a mass concentration of 1 μg / μL, and 50 μL of Methylobacterium torquens bacterial liquid is added respectively; culture at 30 °C and 170 rpm for 48 h; sample at 0 h, 6 h, 12 h, 24 h, 36 h, and 48 h respectively, and take three samples for each group. After separating the experimental group and the control group, keep fermenting at 30 °C for 2 days, and then analyze the zearalenone content in the fermentation broth to calculate the degradation rate. Detect the zearalenone content by high performance liquid chromatography. Note: When detecting by high performance liquid chromatography, there will be an error with the theoretical concentration of zearalenone in the solution. Therefore, the zearalenone content measured by high performance liquid chromatography at 0 h is used as the initial content of zearalenone.
[0032] The formula for calculating the degradation rate of zearalenone is as follows: (Initial zearalenone content - Degraded zearalenone content / Initial zearalenone content) × 100%.
[0033] Table 1 Test conditions of high performance liquid chromatography
[0034] Results and analysis: Table 2 Degradation rates of zearalenone in the experimental group and the control group
[0035] Use high performance liquid chromatography to test the zearalenone content in the experimental group and the control group at 0 h of fermentation. The test results are shown in Table 1 and Figure 1As shown in the figure. It can be found that as time goes by, the content of zearalenone in the fermentation broth gradually decreases, indicating that the degradation rate gradually increases with the prolongation of time. It approaches the maximum on the second day, reaching 83%. When the time continues to extend, its degradation rate does not increase further. Therefore, the fermentation time of the experiment is 2 days. After 2 days of fermentation, the content of zearalenone is only 1.55 μg / mL, and the degradation rate reaches 83%. While the content of ZEN in the control is still greater than 9 μg / mL without change, indicating that the Methylobacterium thiooxydans provided by the present invention can effectively degrade ZEN. There have been many reports that many microorganisms have a certain degradation effect on ZEN. Risa et al. (see Current Microbiology, Volume 75 (2018), pages 907 - 917 by Risa, A., Krifaton, C., etc. (Risa, A., Krifaton, C. et al., Curr Microbiol, 2018, 75, 907 - 917)) found 1 strain that can degrade ZEN from 42 strains of Rhodococcus. Yu et al. (see Microbiological Research, Volume 167, Issue 3 (2012), pages 121 - 126 by Yuanshan Yu, Hui Wu, etc. (Yuanshan Yu, Hui Wu, et al., Microbiological Research, 2012, 3(167):121 - 126)) isolated 1 strain of Acinetobacter from the soil. After treating it with extracellular enzymes in the culture medium for 12 h, a decrease in ZEN and estrogen - like metabolites could be found. Tan (see Mycotoxin Research, Volume 30 (2014), pages 191 - 196 by Tan, H., Hu, Y., etc. (Tan, H., Hu, Y. et al., Mycotoxin Res, 2014, 30, 191 - 196)) isolated 2 strains of Pseudomonas from the soil. After culturing in a medium containing 2 μg / mL ZEN for 72 h, they could degrade 68% and 51% of ZEN respectively. Compared with other existing strains that degrade zearalenone, the Methylobacterium thiooxydans strain obtained in the present invention also has a good degradation efficiency.
[0036] Finally, it should be noted that the above - mentioned embodiments are only used to illustrate the technical solutions of the present invention, not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present invention.
Claims
1. Rhodococcus toruloides capable of efficiently degrading zearalenone, characterized in that, The said Methylobacterium torquens was deposited at the China General Microbiological Culture Collection Center on January 2, 2024. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 29497, and the microbial taxonomic name is Methylobacterium torquens Methylobacterium extorquens .
2. A bacterial agent, characterized in that, The microbial agent includes the microbial agent of Methylobacterium extorquens described in claim 1.
3. The microbial agent according to claim 2, wherein The preparation method of the microbial agent includes: culturing Methylobacterium extorquens in a culture medium.
4. The microbial agent according to claim 2, wherein The temperature of the culturing is 29°C - 31°C, and the culturing time is 24h - 48h.
5. The microbial agent according to claim 2, characterized in that, After the cultivation, each milliliter of the bacterial agent contains 1×10 8 cfu / mL - 1×10 9 cfu / mL of *Methylobacterium thiocyanatum*.
6. Use of Methylobacterium extorquens described in claim 1 in degrading zearalenone.
7. Use of the microbial agent described in any one of claims 2 - 5 in degrading zearalenone.
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