Methylibium versatilis capable of efficiently degrading zearalenone and application thereof

Abstract

The application relates to the field of microbiological technology, and discloses a methylbacterium with high-efficiency zearalenone degradation and application thereof, the methylbacterium has been preserved in the China General Microbiological Culture Collection Center on January 2, 2024, the address is No. 1, Xiliujia, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC No. 29497, and the microbial classification name is methylbacterium. Methylobacterium extorquens The methylbacterium obtained by the application can efficiently degrade zearalenone.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, and in particular to a methylbacterium that can efficiently degrade zearalenone and application thereof. BACKGROUND

[0002] Mycotoxins are toxic secondary metabolites produced by molds during growth, mainly including aflatoxins, trichothecenes, zearalenone, ochratoxin A and fumonisin, etc. Raw materials such as grains can be contaminated by molds in the field, and if the environmental temperature and humidity are suitable during transportation, processing and storage, molds will continue to grow and the toxin content will also increase.

[0003] Zearalenone, also known as F-2 toxin, was isolated from corn contaminated by Gibberella zeae in 1962. It is mainly produced by Fusarium graminearum, Fusarium culmorum and Fusarium graminearum. Fusarium is both parasitic and saprophytic, has strong ecological adaptability and is widely distributed. Cereals such as corn, wheat, barley and oats are susceptible to mold infection and ZEN contamination. Animals absorb ZEN by eating ZEN-contaminated feed. ZEN has estrogen-like effects, can compete with estrogen to bind to the corresponding receptor in the body, activate the downstream response element, trigger estrogen receptor dimerization, and then produce a series of estrogen-like effects, causing estrogen levels in the body to be disordered, and damaging the reproductive system of animals, especially sows. The main manifestations are decreased appetite, immune suppression and slow growth. Persistent poisoning can cause reproductive disorders in sows, leading to infertility, abortion and stillbirth. ZEN also has strong carcinogenicity and can affect human edible meat and dairy products through animal intake, posing a serious threat to human health. SUMMARY

[0004] To solve the above technical problems, the present application provides a methylbacterium that can efficiently degrade zearalenone and application thereof. The methylbacterium obtained by the present application Methylobacterium extorquens can efficiently degrade zearalenone. The methylbacterium has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on January 2, 2024, the address of which is No. 1, Yihuangyuan 3rd, Beichen West Road, Chaoyang District, Beijing, China, the preservation number of which is CGMCC No. 29497, and the microbial classification name of which is methylbacterium Methylobacterium extorquens .

[0005] The application provides a methylibium tortuosum capable of efficiently degrading zearalenone, the methylibium tortuosum has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on January 2, 2024, the address of preservation is the Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC No. 29497, and the microbial classification and naming is methylibium tortuosum Methylobacterium extorquens .

[0006] The application further provides a microbial agent containing the methylibium tortuosum.

[0007] Further, the preparation method of the microbial agent comprises the following steps: culturing the methylibium tortuosum in a culture medium.

[0008] Further, the temperature of the culture is 29-31 DEG C, and the culture time is 24-48 hours.

[0009] Further, after the culture, the methylibium tortuosum is 1x10 8 cfu / mL-1x10 9 cfu / mL in the microbial agent per milliliter.

[0010] Further, the culture medium comprises one of a synthetic culture medium and an LB culture medium.

[0011] The application further provides application of the methylibium tortuosum in degradation of zearalenone.

[0012] The application further provides application of the microbial agent containing the methylibium tortuosum in degradation of zearalenone.

[0013] The application has the following technical effects:

[0014] The methylibium tortuosum separated and screened by the application is cultured in a synthetic culture medium, under optimal culture conditions, the removal rate of zearalenone can reach 83%, and it is found that high-concentration zearalenone has no obvious inhibition on the growth and degradation of the methylibium tortuosum, which provides a possibility for industrial application of the strain to zearalenone biological treatment. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the specific embodiments or the prior art, the following will briefly introduce the drawings needed to be used in the specific embodiments or the prior art description. Obviously, the drawings in the following description are some embodiments of the application, and those skilled in the art can also obtain other drawings according to these drawings without any creative effort.

[0016] Figure 1 are the test results provided by the experimental group of the application. DETAILED DESCRIPTION

[0017] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0018] In a first aspect, some embodiments of the present application provide a methylotrophic bacterium capable of efficiently degrading zearalenone, wherein the methylotrophic bacterium has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on January 2, 2024, the address of the preservation is No. 1, Yitian West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 29497, and the microbial classification name is Methylotrophicus torquens. Methylobacterium extorquens .

[0019] In a second aspect, some embodiments of the present application further provide a microbial agent containing the methylotrophic bacterium.

[0020] In some embodiments, the preparation method of the microbial agent comprises: culturing the methylotrophic bacterium in a culture medium.

[0021] In some embodiments, the temperature of the culture is 29-31℃, and the culture time is 24-48h.

[0022] In some embodiments, after the culture, the microbial agent contains 1×10 8 cfu / mL-1×10 9 cfu / mL of the methylotrophic bacterium per milliliter.

[0023] In some embodiments, the culture medium comprises one of a synthetic culture medium and an LB culture medium.

[0024] In a third aspect, some embodiments of the present application further provide the application of the methylotrophic bacterium in degrading zearalenone.

[0025] In a fourth aspect, some embodiments of the present application further provide the application of the microbial agent containing the methylotrophic bacterium in degrading zearalenone.

[0026] The embodiments and comparative examples will be described in detail as follows:

[0027] Embodiment 1:

[0028] (1) In Xinjiang Uygur Autonomous Region of China, the strain obtained by screening is 1-2 mm round edge, opaque, pink, raised in the middle, surface bright and smooth, wet and sticky. The methylbacterium obtained by screening is sequenced, the total DNA of the bacterial liquid is extracted, and used as the PCR template of the bacterial liquid. 27F and 1492R amplification primers are used for PCR amplification;

[0029] The PCR amplification reaction system (10 μL) is as follows:

[0030] cDNA (1 μl), 10x Buffer (1 μL), 2.5 mM dNTP (0.8 μL), 10 μM forward and reverse amplification primers (0.5 μL each), rTaq enzyme (0.05 μL), ddH2O (6.15 μL);

[0031] The 27F amplification primer is shown in SEQ ID NO. 2, and SEQ ID NO. 2 is AGAGTTTGATCCTGGCTCAG.

[0032] The 1492R amplification primer is shown in SEQ ID NO. 3, and SEQ ID NO. 3 is GGTTACCTTGTTACGACTT.

[0033] The PCR amplification reaction conditions are as follows: 94°C pre-denaturation for 5 min, 94°C unwinding for 30 s, 51°C annealing for 30 s, 72°C extension for 1 min, 30 cycles, 72°C extension for 10 min;

[0034] After the whole amplification reaction is completed, the reaction product is detected by 1% agarose gel electrophoresis, the target band is cut back under the gel imaging system, and the DNA is recovered and purified according to the SanPrep column kit instructions;

[0035] The recovered product is connected with PMD18-T cloning vector, and the connection is carried out at 16°C overnight to obtain the PMD18-T recombinant vector of the target strain gene sequence, and the connection system is as follows: recovered product 4.5 μL, PMD18-T 0.5 μL;

[0036] The product of the connection system is transferred into the prepared E. coli competent DH5α, and the ice bath is 30 min, the heat shock is 42°C for 30 s, and it is placed on ice;

[0037] 650 μL of LB medium preheated to 37°C is added, and the culture is shaken at 37°C for 1 h, then centrifuged at 5000 rpm for 3 min, most of the supernatant is discarded, and the remaining about 150 μL of supernatant is mixed by blowing and coating on the LB plate containing 100 mg / mL ampicillin sodium (Amp), and then incubated at 37°C for 1 h, and then incubated for 12-16 h;

[0038] The single colony with good growth on the streak plate was picked and cultured at 37°C for 6 h, and then positive clones were screened using amplification primers 27F and 1492R. The correct ligation liquid was sent to Nanjing Branch of Shengong Bioengineering Co., Ltd. for sequencing. Finally, the cDNA sequence of the target strain was subjected to Blast comparison to determine the species of the strain.

[0039] The 16S rDNA sequence of the obtained strain is shown as SEQ ID NO. 1.

[0040]

[0041] (2) The methyl-rod-shaped bacteria of the present application is cultured to prepare a synthetic culture medium (methanol 10 mL, (NH4)2HPO4(3 g), Na2HPO4(3 g), KH2PO4(1.5 g), MgSO4·7H2O (2 g), CaCl2·2H2O (0.03 g), MnSO4·H2O (0.005 g), ZnSO4·7H2O (0.005 g), CuSO4·5H2O (0.005 g), which are dispersed in 1 L of water), and the pH value of the synthetic culture medium is adjusted to 7.2; the obtained synthetic culture medium is autoclaved at 121°C for 20 minutes. The methyl-rod-shaped bacteria strain is inoculated into the synthetic culture medium, and is cultured at a temperature of 30°C and 170 rpm for 24-28 hours to obtain the activated methyl-rod-shaped bacteria strain.

[0042] (3) The obtained methyl-rod-shaped bacteria strain is tested for degradation of zearalenone: the control group is 14.85 mL of liquid LB medium added with 150 μL of ZEN with a mass concentration of 1 μg / μL (ZEN is dissolved and dispersed in acetonitrile); the experimental group is 14.80 mL of liquid LB medium added with 150 μL of ZEN with a mass concentration of 1 μg / μL, and 50 μL of methyl-rod-shaped bacteria liquid is inoculated respectively; the culture is carried out at a temperature of 30°C and 170 rpm for 48 hours; sampling is carried out at 0h, 6h, 12h, 24h, 36h and 48h, and each group is sampled three times. After the experimental group and the control group are separated, fermentation is carried out at 30°C for 2 days, and then the zearalenone content in the fermentation liquor is analyzed to calculate the degradation rate. The zearalenone content is detected by high performance liquid chromatography. Note: when the detection is carried out by high performance liquid chromatography, there is an error in the theoretical concentration of zearalenone in the solution, therefore, the zearalenone content detected by high performance liquid chromatography at 0h is taken as the initial content of zearalenone.

[0043] The calculation formula of the degradation rate of zearalenone is as follows: (initial zearalenone content-degraded zearalenone content / initial zearalenone content) × 100%.

[0044] Table 1 Test conditions of high performance liquid chromatography

[0045]

[0046] Results and analysis:

[0047] Table 2 Degradation rates of zearalenone in the experimental group and the control group

[0048]

[0049] The contents of zearalenone in the fermentation 0h of the experimental group and the control group were tested by high performance liquid method, and the test results are shown in Table 1 and Figure 1 It can be found that the content of zearalenone in the fermentation liquid gradually decreases with the extension of time, indicating that the degradation rate gradually increases with the extension of time, and approaches the maximum of 83% on the second day. When the time continues to extend, the degradation rate does not further improve, so the experiment takes 2 days as the fermentation time. The content of zearalenone after 2 days of fermentation is only 1.55 μg / mL, and the degradation rate reaches 83%. The content of ZEN in the control is still greater than 9 μg / mL, which does not change, indicating that the provided Bacillus vallitutus can effectively degrade ZEN. It has been reported that many microorganisms have a certain degradation effect on ZEN. Risa et al. (see Risa, A., Krifaton, C. et al., Curr Microbiol, 2018, 75, 907-917) found a strain that can degrade ZEN from 42 strains of Rhodococcus. Yu et al. (see Yuanshan Yu, Hui Wu, et al., Microbiological Research, 2012, 3(167): 121-126) isolated a strain of Acinetobacter from soil, which can find the reduction of ZEN and estrogen-like metabolites after 12h of treatment in the culture medium with extracellular enzymes. Tan (see Tan, H., Hu, Y. et al., Mycotoxin Res, 2014, 30, 191-196) isolated 2 strains of Pseudomonas from soil, which can degrade 68% and 51% of ZEN respectively after 72h of culture in the medium containing 2 μg / mL ZEN. Compared with other strains that degrade zearalenone, the Bacillus vallitutus strain obtained in the present application also has a good degradation efficiency.

[0050] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or make equivalent replacement for part 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 application.

Claims

1. A type of *Methylobacterium taurenzopes* that degrades zearalenone ( Methylobacterium extorquens ), characterized in that, The *Tylomycinobacter tauren* was deposited on January 2, 2024, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 29497 and microbiological classification name *Tylomycinobacter tauren*.

2. A microbial agent, characterized in that, The bacterial agent includes *Methylobacterium tauren* as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The preparation method of the bacterial agent includes: culturing *Methylobacterium tauren* in a culture medium.

4. The microbial agent according to claim 3, characterized in that, The culture temperature is 29℃-31℃, and the culture time is 24h-48h.

5. The microbial agent according to claim 3, characterized in that, After the culture is completed, the bacterial agent contains 1×10 8 cfu / mL -1×10 9 CFU / mL of *Tytosylmethyl*.

6. The use of *Typhatidylcholine* as described in claim 1 in the degradation of zearalenone.

7. The use of the microbial agent according to any one of claims 2-5 in the degradation of zearalenone.

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