CN114231432A

CN114231432A - Bacillus belgii capable of degrading vomitoxin and zearalenone simultaneously and application of bacillus belgii

Info

Publication number: CN114231432A
Application number: CN202111172665.7A
Authority: CN
Inventors: 谢岩黎; 曹荣耀; 马卫宾; 孙淑敏; 李倩; 杨玉辉
Original assignee: Henan University of Technology
Current assignee: Henan University of Technology
Priority date: 2021-10-08
Filing date: 2021-10-08
Publication date: 2022-03-25
Also published as: CN115895934A; CN115895934B

Abstract

The bacillus beilesensis Vel-HNGD-F2 strain is preserved in China general microbiological culture collection center with the preservation number of CGMCCNO: 23365. the strain provided by the invention can effectively degrade vomitoxin and zearalenone, the degradation effect reaches 70% -80%, and the strain has a good high-temperature resistance effect and can be well applied to processing of food or feed.

Description

Bacillus belgii capable of degrading vomitoxin and zearalenone simultaneously and application of bacillus belgii

Technical Field

The invention relates to the technical field of microbiology and plant disease biocontrol, in particular to bacillus beijerinckii for simultaneously degrading vomitoxin and zearalenone and application thereof.

Background

Vomitoxin is also called deoxynivalenol toxin (DON), is a type B trichothecene toxin, is mainly a secondary metabolite produced by Fusarium graminearum (Fusarium graminearum) and Fusarium flavum (Fusarium culmorum), and is widely present in polluted grains, feeds and foods. The vomitoxin has higher cytotoxicity and immunosuppressive property, enters human bodies and animal bodies to influence digestive systems to cause symptoms such as vomit and the like, reduces the immunity of the organisms, and causes serious harm to the health of the human bodies and the animals.

At present, the vomitoxin is removed by mainly adopting a physical method, a chemical method and a biological method at home and abroad. Physical and chemical methods have the disadvantages of non-ideal detoxification effect, introduction of other harmful substances and the like. Compared with the traditional physical and chemical methods, the biological method can convert the vomitoxin into nontoxic or low-toxic substances without causing the nutrient loss of agricultural products, so that the method is a green and safe detoxification method and is considered as the best method for removing the vomitoxin.

Zearalenone (ZEN) is a steroid mycotoxin with estrogen toxicity effect generated by fungi such as zearalenone and fusarium graminearum, is widely present in grains and poses great threat to human beings and animals, and after the human beings or the animals eat foods polluted by the zearalenone, cytotoxicity, reproductive toxicity, immunotoxicity and reproductive toxicity appear, thus posing great threat to the human beings and the animals.

Zearalenone can also be detoxified by physical, chemical and biological methods, and the use of biodegradation has become the mainstream since physical and chemical methods are difficult to use in life, biodegradation has minimal impact on the ecological environment and product quality, and zearalenone in food and feed is reduced or eliminated mildly and effectively.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a method for simultaneously degrading vomitoxin and zearalenone and application thereof.

The invention is realized by the following technical scheme: the bacillus beilesensis Vel-HNGD-F2 strain is preserved in China general microbiological culture collection center with the preservation number of CGMCCNO: 23365.

preferably, the strain can degrade vomitoxin and zearalenone simultaneously generated by fusarium mycotoxin.

Preferably, the fermentation liquor of the Bacillus belgii Vel-HNGD-F2 strain degrades vomitoxin when the thallus concentration is 10⁸-10⁹The degradation rate is 70-80% when CFU/mL.

Preferably, the Bacillus belgii Vel-HNGD-F2 strain has a zearalenone degradation effect, and the zearalenone degradation rate is 70% -80%.

Preferably, the Bacillus belgii Vel-HNGD-F2 has a significant degradation effect on vomitoxin and zearalenone as an extracellular enzyme.

Preferably, the Bacillus belgii Vel-HNGD-F2 strain is used in food or feed processing to degrade the biologically toxic secondary metabolite emetic toxin and zearalenone produced by Fusarium fungi.

Preferably, the Bacillus belgii Vel-HNGD-F2 has high temperature resistance, and can survive the bacterial cell number up to 10 at 85 ℃ for 20min⁸-10⁹CFU/mL。

The preparation method of the Bacillus belgii Vel-HNGD-F2 full culture solution culture comprises the following steps:

the strain frozen and stored in-80 ℃ B.beilai Vel-HNGD-F2 is streaked on a fermentation medium for culturing for 48 hours, a single colony is selected and inoculated into a conical flask filled with 50mL of fermentation liquid medium, and the conical flask is shake-cultured for 48 hours at 37 ℃.

On the basis of the scheme, the preparation method of the Bacillus belgii Vel-HNGD-F2 bacterial suspension comprises the following steps:

and (2) centrifuging 20mL of a culture of the Vel-HNGD-F2 full culture solution of the Bacillus belgii for 10min at 10000r/min, separating to obtain thalli and a supernatant, washing the prepared thalli with a phosphate buffer solution, centrifuging, adding the phosphate buffer solution to make up to 20mL, and suspending the thalli to obtain a Vel-HNGD-F2 strain suspension.

On the basis of the scheme, the preparation method of the extracellular metabolite of the Bacillus belgii Vel-HNGD-F2 comprises the following steps:

and (3) centrifuging 20mL of the Bacillus belgii Vel-HNGD-F2 whole culture solution culture at 10000r/min for 10min, and separating to obtain thalli and a supernatant, wherein the supernatant is the extracellular metabolite of the Bacillus belgii Vel-HNGD-F2.

On the basis of the scheme, the preparation method of the crude extract of the Bacillus belgii Vel-HNGD-F2 comprises the following steps:

after the suspension of the Vel-HNGD-F2 strain of the Bacillus belgii is subjected to low-temperature ultrasonic crushing, the liquid obtained by centrifugation at 10000r/min for 10min is filtered by a filter membrane of 0.22 mu m to prepare the crude extract of the Vel-HNGD-F2 strain.

The strain provided by the invention can effectively degrade vomitoxin and zearalenone, the degradation effect reaches 70% -80%, and the strain has a good high-temperature resistance effect and can be well applied to processing of food or feed.

Drawings

FIG. 1 is a high performance liquid chromatogram of the Vel-HNGD-F2 strain before and after degradation of vomitoxin;

FIG. 2 is a morphogram of the Vel-HNGD-F2 strain;

FIG. 3 is a high performance liquid chromatogram of Vel-HNGD-F2 strain before and after degradation of zearalenone.

Detailed Description

In the description of the present invention, it should also be noted that, unless otherwise explicitly specified or limited, the terms "disposed," "mounted," "connected," and "connected" are to be construed broadly and may, for example, be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 screening and identification of Bacillus beilesiensis Vel-HNGD-F2

1. Taking a sample from pig intestinal microorganisms, weighing 10g of the sample, adding the sample into 90ml of physiological saline, shaking at a constant temperature of 150r/min for 2h, sucking 0.2ml of suspension into a sterilized fermentation medium, and shaking in a shaking table at 150r/min and at 37 ℃ for 48 h. Diluting the fermentation medium cultured for 48h to 10^-1、10^-2、10^-3、10^-4、10^-5、10^-6Then 200 mul of diluted fermentation culture solution with different dilutions was respectively sucked and spread on primary screening culture medium containing 20 mug/ml DON, and inverted cultured in 37 ℃ incubator for 48 h. Observing the growth condition of the strain, selecting a larger single bacterial colony, carrying out streak purification in a primary screening culture medium, obtaining the single bacterial colony after 3 generations of purification, selecting the bacterial colony in a liquid fermentation culture medium, putting the bacterial colony into a shaking table at 37 ℃ for 48h at 150rpm, sucking 500 mu l of bacterial liquid into a 1.5ml centrifugal tube, adding glycerol (50 percent), and storing the purified bacterial liquid in a refrigerator at-80 ℃. Numbered Vel-HNGD-F2.

2. Extraction of DNA from bacteria using the Ezup column bacterial genomic DNA extraction kit 16S rDNA was amplified using genomic DNA as a template using primers 7F (5'-CAGAGTTTGATCCTGGCT-3') and 1540R (5'-AGGAGGTGATCCAGCCGCA-3'). The reaction system is; 2.5. mu.l of 10 XBuffer (with Mg2+), 0.5. mu.l of F (10uM), 0.5. mu.l of R (10uM), 25. mu.l of dd H2O 25, 0.5. mu.l of Template (20-50 ng/. mu.l of genomic DNA), 1. mu.l of dNTPs (2.5 mM each), 0.2. mu.l of enzyme. The amplification procedure is as follows; (1) pre-denaturation at 94 ℃ for 4 min; (2) denaturation at 94 ℃ for 45 s; (3) annealing at 55 ℃ for 45 s; (4) extending for 1min at 72 ℃; (6) circulating the steps (2) to (4) for 30 times; (6) repairing and extending for 10min at 72 ℃. And (3) carrying out electrophoresis by using 1% agarose, taking 5 mu l of each PCR product and DNA marker to a sample application hole, carrying out electrophoresis at 150V for 20min and 100mA, observing a band under an ultraviolet lamp after the electrophoresis is finished, and judging whether the PCR amplification is successful or not. The amplified products were purified and sequenced by Shanghai Biotech GmbH, and the sequencing results were compared in NCBI database.

3. The morphological characteristics and physiological and biochemical characteristics of the strain are identified, and the result is as follows:

morphological characteristics: the single colony of the strain on a solid fermentation culture medium is convex, light yellow, opaque, round in surface, dry in surface and irregular in edge.

The physiological and biochemical characteristics are shown in Table 1(+ positive, -negative).

TABLE 1 physiological and biochemical experiment results table

The results of comprehensive morphological characteristics, physiological and biochemical characteristics, 16S rDNA sequencing and homology analysis are Bacillus belgii, named as Vel-HNGD-F2, which is preserved in the China general microbiological culture Collection center, with the preservation date of 2021, 9 and 6 days, and the addresses are as follows: western road No. 1, north chen, chaoyang district, beijing, ministry of sciences, china, institute of microbiology, zip code 100101; the preservation number of the preservation unit Henan university of industry is CGMCC NO: 23365.

Example 2 degradation of vomitoxin by Bacillus belgii

1. Culture of Bacillus belgii Vel-HNGD-F2 for degrading emetic toxin

The culture medium of the bacillus belgii for degrading vomitoxin is a fermentation culture medium: 3g of beef extract, 10g of peptone, 6g of glucose, 5g of NaCl and distilled water to 1000ml, wherein the pH value is 7.2-7.4.

Taking out the frozen Bacillus belgii from a refrigerator at the temperature of-80 ℃, cooling, selecting a bacterial liquid, streaking the bacterial liquid onto a solid fermentation culture medium, culturing for 48 hours in a constant-temperature incubator at the temperature of 37 ℃, selecting and culturing a larger single bacterial colony into a liquid fermentation culture medium, and putting the liquid fermentation culture medium into a shaking table at the temperature of 37 ℃ at the speed of 150rpm for 48 hours.

2. Degradation of vomitoxin by Bacillus belgii Vel-HNGD-F2

Putting 950 mu L of Bacillus belgii bacterial liquid into a 1.5mL centrifuge tube, adding 50 mu L of 100ppm vomitoxin standard substance (the final concentration is 5ppm), replacing the bacterial liquid with a sterile fermentation medium for a control group, putting the control group into a shaking table at 37 ℃ for 48 hours at 150rpm, using an immunoaffinity column for vomitoxin, enabling the supernatant to pass through the immunoaffinity column for 1-2 seconds, washing the immunoaffinity column with 10mL of distilled water, enabling 3mL of methanol to pass through the immunoaffinity column for 1-2 seconds, collecting methanol solution, drying by using a nitrogen blower to collect methanol solution, re-dissolving by using a mobile phase, and collecting the experimental group solution and the blank group solution.

3. Analysis of ability of Bacillus beleisi Vel-HNGD-F2 to degrade emetic toxin

The collected experimental and blank solutions were filtered through a 0.22 μm filter and then assayed for the remaining vomitoxin content in the medium using HPLC. The following conditions for detecting vomitoxin by HPLC are adopted, and the chromatographic column comprises: c18 column (250 mm. times.4.6 mm, 5 μm); mobile phase: methanol-water (30:70, v/v); the detection wavelength is 218 nm; column temperature: 35 ℃; flow rate: 1.0 mL/min; sample introduction amount: 20 μ L.

Vomitoxin degradation rate (A)₀-A₁)÷A₀×100％

In the formula: vomitoxin content of A0 control group; a1 is the content of emetic toxin in the experimental group.

The result is shown in figure 1, and the result shows that the Bacillus belgii Vel-HNGD-F2 has good degradation effect on vomitoxin at 27 ℃, and the degradation rate is 76.7%.

Example 3 degradation of zearalenone by Bacillus belgii Vel-HNGD-F2

Placing 950 μ L of Bacillus beilisi bacterial liquid in 1.5mL centrifuge tube, adding 50 μ L of 100ppm zearalenone standard (final concentration is 5ppm), replacing the bacterial liquid with sterile fermentation medium for control group, incubating at 37 deg.C for 48 hr, extracting with dichloromethane, collecting organic phase, extracting toxin, blowing dry organic phase with nitrogen blower, re-dissolving with mobile phase, filtering with 0.22 μm filter, and detecting with HPLC.

The detection conditions for detecting zearalenone by HPLC are as follows: a chromatographic column: c18 column (250 mm. times.4.6 mm, 4 μm); mobile phase: methanol-water (80:20, v/v); the detection wavelength is 236 nm; column temperature: 30 ℃; flow rate: 1.0 mL/min; sample introduction amount: 20 μ L.

Zearalenone degradation rate (B)₀-B₁)÷B₀×100％

In the formula: b0 control zearalenone content; b1 is the zearalenone content of the experimental group.

The results are shown in FIG. 3, which shows that Bacillus belgii Vel-HNGD-F2 has good effect of degrading zearalenone at 37 ℃, and the degradation rate is 70.2%.

Example 4 Bacillus belgii Vel-HNGD-F2 high temperature resistance assay

1. Diluting Bacillus beilis Vel-HNGD-F2 bacterial liquid by 1 × 10 by adopting a common plate counting method⁶、1×10⁷、1×10⁸Coating on solid fermentation culture medium at 85 deg.C for 20min, recording total number of heated bacterial colonies, and calculating viable count to 10⁸-10⁹CFU/mL. The results are shown in Table 2.

N ∑ C/(M1+0.1 × M2)5 d. In the formula, N is the total number of viable bacteria in the fermentation liquor, Sigma C is the sum of the bacterial colonies of the plate (containing the bacterial colonies in the proper range), M1 is the number of the plate with the first dilution (low dilution factor), M2 is the number of the plate with the second dilution (high dilution factor), and d is the dilution factor (first dilution factor).

TABLE 2 viable count results after different batches of heating

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments or portions thereof without departing from the spirit and scope of the invention.

AGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTATGGAGCCA

Claims

1. A Bacillus beleisi for simultaneously degrading vomitoxin and zearalenone, which is characterized in that: the Bacillus belgii is Bacillus belgii (Bacillus velezensis) Vel-HNGD-F2, and is preserved in China general microbiological culture preservation management center with the preservation number: CGMCCNO: 23365.

2. the use of Bacillus belgii for the simultaneous degradation of emetic toxin and zearalenone according to claim 1 wherein the strain Vel-HNGD-F2 is effective in degrading emetic toxin for the degradation of emetic toxin.

3. The use of the strain of Bacillus belgii for the simultaneous degradation of emetic toxin and zearalenone according to claim 1, wherein the strain Vel-HNGD-F2 is effective for the degradation of zearalenone.

4. A biocontrol agent comprising the Bacillus belgii strain, spore or fermentation product of claim 1.

5. The use of bacillus beilesensis Vel-HNGD-F2 according to claim 1 in microbial feed, feed additives.

6. The use of bacillus beilesiensis Vel-HNGD-F2 according to claim 1 for microbial fertilizer, plant disease control.