CN113862201B

CN113862201B - Microbacterium for degrading aflatoxin B1 and application thereof

Info

Publication number: CN113862201B
Application number: CN202111333406.8A
Authority: CN
Inventors: 徐嘉良; 闫怡; 任清; 陈海燕; 张心悦
Original assignee: Beijing Technology and Business University
Current assignee: Beijing Technology and Business University
Priority date: 2021-11-11
Filing date: 2021-11-11
Publication date: 2023-04-25
Anticipated expiration: 2041-11-11
Also published as: CN113862201A

Abstract

The invention discloses microbacterium for degrading aflatoxin B1 and application thereof. The strain is a micro-bacillus Microbacterium proteolyticum B, and the collection number of the micro-bacillus is GDMCC NO:62022. the microbacterium Microbacterium proteolyticum B of the invention has remarkable effect of degrading aflatoxin B1, and has the advantages of low preparation cost, simple process and convenient mass production.

Description

Microbacterium for degrading aflatoxin B1 and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to a microbacterium for degrading aflatoxin B1 and application thereof.

Background

Aspergillus flavus (Aspergillus flauvs) is a common pathogen for many important crops and animals that are common worldwide. The secondary metabolite aflatoxin B1 (AFB 1) produced by aspergillus flavus is one of the natural compounds currently found to be the most toxic and carcinogenic. Aspergillus flavus can infect many important crops, such as peanut, corn, cotton, etc., and can contaminate both pre-harvest and post-harvest crops, causing significant economic losses to agricultural production around the world. According to the statistics of grain and agriculture organizations of the united nations, huge economic losses are caused for agriculture. AFB1 is detected in a plurality of sampled processed products such as soy sauce, aquatic feed and the like, because aflatoxin B1 has relatively stable physicochemical properties, the AFB1 is difficult to degrade, once the polluted feed is eaten by livestock, the AFB1 is subjected to hydroxylation metabolism in an animal body to form a derivative AFM1 which is basically similar to toxicity and carcinogenicity of the AFB1, a part of the derivative of the AFB1 can be discharged along with urine and milk, and a large part of the derivative can be found in dairy products and meat products.

In summary, aflatoxin B1 brings different degrees of harm to agricultural production and human health, and research and development of a microbial agent for degrading aflatoxin B1 is needed.

Disclosure of Invention

For this reason, the invention provides microbacterium for degrading aflatoxin B1 and application thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

in one aspect, the present invention provides a strain, which is a microbacterium Microbacterium proteolyticum B, deposited under the accession number GDMCC NO:62022.

in another aspect, the invention provides a microbial inoculum, the active ingredient of which is the strain and/or the metabolite of the strain.

The invention also provides application of the strain or the microbial inoculum in the following (A1), the following (A2) or the following (A3): (A1) degrading aflatoxin B1; (A2) preparing a product for degrading aflatoxin B1; (A3) preparing a product for detoxification of food.

In one embodiment of the present invention, the 16S rDNA sequence of the strain in the micro-bacillus Microbacterium proteolyticum B is shown in SEQ ID No: 1.

In one embodiment of the present invention, the degradation rate of aflatoxin B1 by the strain in microbacterium Microbacterium proteolyticum B is 87%.

In one embodiment of the invention, the strain in the microbacterium Microbacterium proteolyticum B is selected and cultured by using coumarin selective medium.

In one embodiment of the invention, the preparation method of the stigmadin selection medium comprises the following steps: weighing KH ₂ PO ₄ 0.25g，NH ₄ NO ₃ 1g，MgSO ₄ 0.001g，FeSO ₄ 0.001g,20g agar, and adding 500ml deionized water, and sterilizing.

Strain preservation description: the microbacterium Microbacterium proteolyticum B of the present invention was deposited at the collection of microorganism strains, the Guangdong province, at 2021, 11, 01, accession number: building 5, guangzhou city, first middle road 100, college No. 59, with accession number GDMCC NO:62022.

the invention has the following advantages:

the test proves that: the microbacterium Microbacterium proteolyticum B of the invention has remarkable effect of degrading aflatoxin B1, and has the advantages of low preparation cost, simple process and convenient mass production.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.

The structures, proportions, sizes, etc. shown in the present specification are shown only for the purposes of illustration and description, and are not intended to limit the scope of the invention, which is defined by the claims, so that any structural modifications, changes in proportions, or adjustments of sizes, which do not affect the efficacy or the achievement of the present invention, should fall within the ambit of the technical disclosure.

FIG. 1 is a photograph showing the morphology of a colony of Microbacterium Microbacterium proteolyticum B provided by the invention 1;

FIG. 2 is a phylogenetic tree of strain B204 constructed based on 16S provided by the invention;

FIG. 3 is a graph showing the effect of temperature provided by the present invention on degradation of AFB1 by Microbacterium Microbacterium proteolyticum B;

FIG. 4 is a graph showing the effect of pH on degradation of AFB1 by Microbacterium Microbacterium proteolyticum B provided by the present invention;

FIG. 5 is a graph showing the effect of inoculum size on the degradation of AFB1 by Microbacterium Microbacterium proteolyticum B in accordance with the invention;

FIG. 6 is a graph showing the effect of rotational speed on degradation of AFB1 by Microbacterium Microbacterium proteolyticum B in accordance with the present invention;

FIG. 7 is a bar graph showing the detoxification effect of micro-bacilli Microbacterium proteolyticum B204 of the present invention on various samples.

Detailed Description

Other advantages and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, which, by way of illustration, is to be read in connection with certain specific embodiments, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1 isolation and identification of Microbacterium Microbacterium proteolyticum B204

1. Primary screening media configuration

Coumarin selection medium: weighing KH ₂ PO ₄ 0.25g，NH ₄ NO ₃ 1g，MgSO ₄ 0.001g，FeSO ₄ Adding 500ml deionized water into 0.001g and 20g agar, placing into a high-pressure steam sterilizing pot, setting the program at 121 ℃ for 20min, placing into an ultra-clean bench to be opened after sterilization is finished, adding weighed coumarin into a primary screening culture medium, and gently shaking until the coumarin is completely dissolved. Simultaneously preparing LB liquid culture medium and LB solid culture medium required by experiments as activating and purifying culture medium.

AFB1 stock: 1mg AFB1 standard (C ₁₇ H ₁₂ O ₆ The purity is more than or equal to 98 percent) is dissolved in chromatographic grade acetonitrile, the volumetric flask is fixed to volume to 10mL, the final concentration is 100mg/L, the solution is filtered by a 0.22 mu m filter membrane, and the solution is transferred into a reagent bottle for sealing and then stored at the temperature of minus 20 ℃ in a dark place.

2. Screening of degradation AFB1 Strain

And (3) primary screening of strains: weighing 1g of cow dung, soil, wine medicine, wine mash, distiller's yeast, glutinous rice and oat sample in a sterile environment, adding into a sterile centrifuge tube, adding 10mL of sterile water, swirling the sample for 3min, and mixing uniformly to obtain 10 ^-1 Diluting the solution, and sequentially diluting to 10 according to experimental operation specification ^-2 、10 ^-3 、10 ^-4 、10 ^-5 And l0 ^-6 Is a diluent of (a). Then split intomu.L 10 was separately aspirated ^-4 、10 ^-5 And l0 ^-6 Uniformly coating bacterial liquid on the surface of a coumarin selective medium by using a coater, then placing the plate in a constant temperature incubator at 30 ℃ for culturing for 1-21 days, checking whether new bacterial colonies grow in the culture dish every 24 hours, selecting single bacterial colonies with good growth, inoculating the single bacterial colonies to the new culture medium, repeatedly purifying for more than 3 times, and preserving bacterial strains in an ultralow temperature refrigerator after expanding culture.

3. Compound screen for degrading AFB1 strain

The strain to be tested is selected, activated for several times on an activation purification medium, inoculated in 15mL of liquid medium and cultured for 12h at the temperature of 30 ℃ at 180 r/min. In an ultra-clean workbench, 945mL of fermentation medium is taken out, a 15mL sterile clean centrifuge tube is added, 50 mu L of bacterial liquid is added, 5 mu L of AFB1 standard substance (10 mu g/mL) is added, the centrifuge tube is placed in a shaking table at 30 ℃ and 200 r/mm for 12h of incubation in a dark place, centrifugal treatment is carried out at 8000 r/mm and 20min, the collected supernatant is concentrated by a concentrator and re-dissolved by 1mL acetonitrile, and the supernatant is filtered by a filter membrane of 0.22 mu m before on-machine detection. The peak value of each component was detected by HPLC, whereby the degradation rate of the strain of Microbacterium Microbacterium proteolyticum B to AFB1 was calculated.

AFB1 degradation (%) = [ (blank AFB1 content-treated AFB1 content) ]/blank AFB1 content x 100%.

HPLC detection conditions: the chromatographic column uses C18 (250 mm×4.6.0.5 m, waters) from Waters, mobile phase A is ultrapure water, mobile phase B is a mixture of methanol and acetonitrile=1:1; the flow rate is 0.6mL/min; selecting a fluorescence detector for detection, wherein the excitation wavelength is 350nm, and the detection wavelength is 450nm; the sample loading was 10. Mu.L.

Drawing a standard curve: the prepared standard solutions were diluted into solutions of 100, 70, 50, 10 and 1. Mu.g/mL, respectively, and after analysis by an instrument, regression curves were made with the abscissa as the standard concentration and the ordinate as the peak area.

4. Identification of Microbacterium Microbacterium proteolyticum B204

(1) Cell morphology and physiological and biochemical feature detection of strain

As shown in FIG. 1, the micro-bacillus Microbacterium proteolyticum B microscope shows slender and irregular rod-shaped cells, the micro-bacillus Microbacterium proteolyticum B microscope shows milky yellow colonies on NA culture medium, the edges are neat, the colonies are wet, the micro-bacillus is gram-positive bacteria, the optimal growth temperature is 30 ℃, the optimal pH=7, the optimal NaCl concentration is 0.5mg/mL, and the bacteria grow better when the soluble starch is used as a carbon source and the soybean protein or yeast extract is used as a nitrogen source.

(2) Microbacterium Microbacterium proteolyticum B204 16S rDNA sequence determination and homology analysis

Genome extraction was performed according to the instructions of the purchased genome extraction kit (Shanghai Ind). 16SrDNA amplification and sequencing: PCR amplification of bacterial DNA Universal primers were chosen to amplify the 16SrDNA sequence. The amplified nucleotide sequence is shown as SEQ ID NO. 1, the B204 gene sequence is obtained by sequence comparison and sequencing, the sequence is compared with the sequence in the NCBI database by using BLAST algorithm, the compared sequence is constructed into a phylogenetic tree by adopting an adjacent method in software MEGA 6, and the scale 0.002 represents the frequency replacement of each nucleic acid site. As shown in FIG. 2, the homology of B204 with 16S rDNA of Microbacterium sp was confirmed, and B204 was determined to be Microbacterium sp based on the genetic distance, morphology and characteristics of the strain, and the obtained strain B204 was stored.

(3) API ZYM experiment

The API reagent strips are various, a proper reagent strip can be selected according to the experimental requirement, and the API20NE can determine the carbon source utilized by microorganisms; API20E can measure various carbohydrate substances of microbial fermentation/oxidation; the API ZYM can determine various enzymatic characteristics. The physiological and biochemical characteristics cannot be independently used as the basis of microorganism classification, and the integral judgment needs to be carried out by combining genotype characteristics, chemical component characteristics and the like.

The culture of single pure colonies was picked from the plates of the isolates using 5mL of 0.85% saline without impurities, the solutions were mixed well, the turbidity was between McFarland No.5 and No.6, the bacterial suspensions were filled with different tubes or sections with pipettes according to the instructions of API ZYM reagent strips, and after 4h A, B solution was added to interpret the experimental results. As shown in Table 1, the ZYM test results of the strain Microbacterium proteolyticum B.

TABLE 1

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Example 2 Effect of different temperatures on degradation of aflatoxin B1 by Microbacterium Microbacterium proteolyticum B204

Purified Microbacterium Microbacterium proteolyticum B is inoculated into 15mL of LB medium, 1.5 mu L of AFB1 (1 mg/mL) is added, the initial pH is 7 at different temperatures (14 ℃,20 ℃, 25 ℃,30 ℃, 35 ℃, 40 ℃,45 ℃, 50 ℃), the rotation speed of a shaking table is 180r/min, fermentation is carried out for 12 hours, meanwhile, 1.5 mu L of AFB1 is added into 15mL of LB medium as a blank control, and degradation amount of the AFB1 is measured after the culture is carried out for 12 hours at different temperatures respectively.

The results are shown in FIG. 3 as the effect of temperature on the degradation of AFB1 by strain B204. From 10 ℃ to 30 ℃, the degradation of the microbacterium Microbacterium proteolyticum B to the AFB1 is gradually increased, and the maximum degradation rate of the AFB1 is reached at 30 ℃.

Example 3 Effect of different pH on degradation of aflatoxin B1 by Microbacterium Microbacterium proteolyticum B204

The purified bacillus deproteinized bacteria are inoculated into 15mL of LB culture medium, then 1.5 mu LAFB1 (1 mg/mL) is added, and the degradation amount of AFB1 is measured after culturing for 12 hours at different initial pH values (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0) at a culture temperature of 30 ℃ and a shaking table rotation speed of 180r/min by adding 1.5 mu L of AFB1 into sterile 15mL of LB culture medium as a blank control.

As shown in FIG. 4, in order to show the effect of pH on the degradation of AFB1 by strain B204, the degradation rate was very low at pH 4.0, and the degradation rate of microbacterium Microbacterium proteolyticum B was gradually increased from pH 5.0 to 7.0, and the degradation effect of microbacterium Microbacterium proteolyticum B204 on AFB1 was remarkable at pH7.0, and the degradation ability of AFB1 was relatively decreased as the pH was further increased.

Example 4 Effect of different inoculum size on degradation of aflatoxin B1 by Microbacterium Microbacterium proteolyticum B204

Inoculating bacillus proteolyticus with different inoculum sizes (2%, 4%, 6%, 8%, 10%) into 15mL LB culture medium, adding 5 μL AFB1 (1 mg/mL), fermenting at 30deg.C under pH7 and shaking table rotation speed 180r/min for 12 hr while adding 1.5 μL AFBI as blank control into sterile 15mL LB culture medium, culturing at different initial pH values for 12 hr, and measuring degradation amount of AFB 1.

The results of the experiment are shown in FIG. 5, which shows the effect of inoculum size on the degradation of AFB1 by strain B204. The inoculation amount affects the effect of the microbacterium Microbacterium proteolyticum B and 204 bacteria on degrading the AFB1, the degradation effect is continuously increased along with the increase of the inoculation amount, the effect of the microbacterium Microbacterium proteolyticum B and 204 on degrading the AFB1 is best under the inoculation amount of 8 percent, and the degradation rate of the microbacterium Microbacterium proteolyticum B and 204 on the AFB1 is reduced along with the increase of the inoculation amount after the inoculation amount exceeds 8 percent.

Example 5 Effect of different rotational speeds on degradation of aflatoxin B1 by Microbacterium Microbacterium proteolyticum B204

Purified microbacterium Microbacterium proteolyticum B is inoculated into 15mL of LB culture medium, 1.5 mu L of AFB1 (1 mg/mL) is added, the culture temperature is 30 ℃, the initial pH is 7 at different initial rotation speeds (120 r/min, 150r/min, 180r/min, 200r/min, 220r/min and 250 r/min), the fermentation is carried out for 12 hours, meanwhile, 1.5 mu L of AFB1 is added to 15mL of LB culture medium as a blank control, and the degradation amount of the AFB1 is measured after the culture is carried out for 12 hours at different rotation speeds.

The experimental results are shown in FIG. 6, which shows the effect of rotational speed on the degradation of AFB1 by strain B204. The rotational speed influences the effect of the microbacterium Microbacterium proteolyticum B and 204 bacteria on degrading the AFB1 to a certain extent, the degradation effect is continuously increased along with the increase of the rotational speed, the effect of the microbacterium Microbacterium proteolyticum B and 204 on degrading the AFB1 is best under the condition of 200r/min rotational speed, and the degradation rate of the B204 on the AFB1 is reduced along with the increase of the inoculum size after the rotational speed exceeds 200 r/min.

Example 6 use of Strain B204 in biological detoxification

After the microbacterium Microbacterium proteolyticum B is activated twice, single colony is selected and placed in 50mL of sterilized NB, placed in a shaking table at 30 ℃ and 200r/min, and cultured overnight for 12 hours, and OD value is measured to be 0.6.

Accurately weighing samples of corn, peanut and cheese, each 10.0+ -0.03 g, respectively laying samples with numbers of YM204, HS204 and RL204 in a 90mm culture dish, drawing 9 regions on the culture dish cover with black Mark pen, and each region about 1cm ² Size, 1mL of AFB1 standard solution (concentration: 100. Mu.g/mL) was taken, and 100. Mu.L of AFB1 standard solution (concentration: 100. Mu.g/mL) was added to each region. The same conditions as above were used as a blank.

Taking 450 mu L of microbacterium Microbacterium proteolyticum B culture solution, adding different culture plates according to the number, adding the 9 areas for 10 times, covering a cover, placing in a constant temperature incubator at 30 ℃ for culturing for 16 hours, and repeating the operation of adding the culture solution every 3 hours.

After the culture is finished, adding 10mL of organic solvent acetonitrile into 10g of sample for 2 times, completely transferring into a 50mL centrifuge tube, putting into a centrifuge after ultrasonic oscillation for 30min, and setting a centrifugal adjusting piece as follows: the temperature was 25℃and the rotational speed was 8000r/min for 10min, the pipette was used to aspirate the supernatant from the centrifuge tube and the collected organic layer solution was concentrated in a 10mL centrifuge tube. The extracted and collected solution was placed in a concentrator, concentrated at 45℃for 2 hours, then 1mL of acetonitrile was added to obtain AFB1 acetonitrile solution, and the solution was filtered with a 0.22 μm filter membrane and placed in a liquid phase vial. HPLC detection conditions: the chromatographic column uses Waters C18 (250 mm. Times.4.6.0.5 m, waters), mobile phase A is ultrapure water, mobile phase B is methanol: acetonitrile = 1:1 mixture; the flow rate is 0.6mL/min; selecting a fluorescence detector for detection, wherein the excitation wavelength is 350nm, and the detection wavelength is 450nm; the sample loading was 10. Mu.L.

As shown in FIG. 7, the degradation effect of corn is best, peanut is the most, and finally cheese is obtained, so that the effect of degrading AFB1 is better when the sample particles are smaller, which is probably due to the fact that bacterial liquid is difficult to uniformly spray when the sample particles are larger, and the effect of degrading AFB1 is reduced.

The optimal degradation conditions of the microbacterium Microbacterium proteolyticum B on the aflatoxin B1 are as follows: the optimal culture condition is pH=6.7, naCl=0.48 g/L, the temperature=31.5 ℃, the soluble starch is 1%, the soybean protein is 0.5%, the degradation rate of aflatoxin B1 h in fermentation liquor is 87%, and the degradation rate of aflatoxin B1 h on the corn surface is 81%.

While the invention has been described in detail in the foregoing general description and specific examples, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

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Sequence listing

<110> university of Beijing Industrial and commercial university

<120> microbacterium for degrading aflatoxin B1 and use thereof

<130> GG20834948A

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1402

<212> DNA

<213> Artificial Sequence

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cttaccatgc aagtcgaacg gtgaagccaa gcttgcttgg tggatcagtg gcgaacgggt 60

gagtaacacg tgagcaacct gccctggact ctgggataag cgctggaaac ggcgtctaat 120

actggatatg agctctcatc gcatggtggg ggttggaaag attttttggt ctgggatggg 180

ctcgcggcct atcagcttgt tggtgaggta atggctcacc aaggcgtcga cgggtagccg 240

gcctgagagg gtgaccggcc acactgggac tgagacacgg cccagactcc tacgggaggc 300

agcagtgggg aatattgcac aatgggcgga agcctgatgc agcaacgccg cgtgagggat 360

gacggccttc gggttgtaaa cctcttttag cagggaagaa gcgagagtga cggtacctgc 420

agaaaaagcg ccggctaact acgtgccagc agccgcggta atacgtaggg cgcaagcgtt 480

atccggaatt attgggcgta aagagctcgt aggcggtttg tcgcgtctgc tgtgaaatcc 540

cgaggctcaa cctcgggcct gcagtgggta cgggcagact agagtgcggt aggggagatt 600

ggaattcctg gtgtagcggt ggaatgcgca gatatcagga ggaacaccga tggcgaaggc 660

agatctctgg gccgtaactg acgctgagga gcgaaagggt ggggagcaaa caggcttaga 720

taccctggta gtccaccccg taaacgttgg gaactagttg tggggaccat tccacggttt 780

ccgtgacgca gctaacgcat taagttcccc gcctggggag tacggccgca aggctaaaac 840

tcaaaggaat tgacggggac ccgcacaagc ggcggagcat gcggattaat tcgatgcaac 900

gcgaagaacc ttaccaaggc ttgacataca ccagaacatc gtagaaatac gggactcttt 960

ggacactggt gaacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt 1020

aagtcccgca acgagcgcaa ccctcgttct atgttgccag cacgtaatgg tgggaactca 1080

tgggatactg ccggggtcaa ctcggaggaa ggtggggatg acgtcaaatc atcatgcccc 1140

ttatgtcttg ggcttcacgc atgctacaat ggccggtaca aagggctgca ataccgtgag 1200

gtggagcgaa tcccaaaaag ccggtcccag ttcggattga ggtctgcaac tcgacctcat 1260

gaagtcggag tcgctagtaa tcgcagatca gcaacgctgc ggtgaatacg ttcccgggtc 1320

ttgtacacac cgcccgtcaa gtcatgaaag tcggtaacac ctgaagccgg tggcctaacc 1380

cttgtggagg gagccgtcga ag 1402

Claims

1. A strain, characterized in that the strain is MicrobacteriumMicrobacterium proteolyticumB204, which is deposited under the accession number GDMCC NO:62022.

2. a microbial agent comprising the strain of claim 1 as an active ingredient.

3. Use of the strain of claim 1 or the microbial inoculum of claim 2 in (A1) or (A2) or (A3) as follows:

(A1) Degrading aflatoxin B1;

(A2) Preparing a product for degrading aflatoxin B1;

(A3) Preparing a product for detoxification of foods.

4. The use according to claim 3, wherein,

the Microbacterium speciesMicrobacterium proteolyticumThe 16S rDNA sequence of the strain in B204 is shown in SEQ ID No: 1.

5. The use according to claim 3, wherein,

the Microbacterium speciesMicrobacterium proteolyticumThe degradation rate of the strain B204 on aflatoxin B1 is 87%.

6. The use according to claim 3, wherein,

the Microbacterium speciesMicrobacterium proteolyticumAnd B204, screening and culturing the strain by adopting a coumarin selective medium.

7. The use according to claim 6, wherein,

the preparation method of the coumarin selective medium comprises the following steps: weighing KH ₂ PO ₄ 0.25g，NH ₄ NO ₃ 1g， MgSO ₄ 0.001g，FeSO ₄ 0.001g,20g agar, and adding 500ml deionized water, and sterilizing.