CN113862201A

CN113862201A - Microbacterium for degrading aflatoxin B1 and application thereof

Info

Publication number: CN113862201A
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: 2021-12-31
Anticipated expiration: 2041-11-11
Also published as: CN113862201B

Abstract

The invention discloses a microbacterium for degrading aflatoxin B1 and application thereof. The strain is Microbacterium proteoliticum B204, and the preservation number of the Guangdong province microorganism strain preservation center is GDMCC NO: 62022. the Microbacterium proteoliticum B204 has obvious effect of degrading aflatoxin B1, and is low in preparation cost, simple in process and convenient for large-scale production.

Description

Microbacterium for degrading aflatoxin B1 and application thereof

Technical Field

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

Background

Aspergillus flavus (Aspergillus flauvs) is a common pathogenic bacterium in many important crops and animals worldwide. Aflatoxin B1(AFB1), a secondary metabolite produced by aspergillus flavus, is one of the most toxic and carcinogenic natural compounds found to date. Aspergillus flavus can infect many important crops, such as peanuts, corns, cotton and the like, and can pollute the crops before and after harvesting, thereby causing huge economic loss to agricultural production all over the world. According to the statistics of the food and agriculture organization of the United nations, huge economic losses are caused to agriculture. AFB1 was detected in many processed products such as soy sauce and aquatic feed which were sampled because aflatoxin B1 has relatively stable physicochemical properties and is difficult to degrade, and once the contaminated feed is eaten by poultry and livestock, AFB1 will be hydroxylated and metabolized in the animal body to form derivative AFM1 which is basically similar to AFB1 in toxicity and carcinogenicity, and a part of derivative of AFB1 will be discharged with urine and milk, while a large part will appear in milk products and meat products.

In conclusion, aflatoxin B1 brings different degrees of harm to agricultural production and human health, and a microbial inoculum for degrading aflatoxin B1 is urgently needed to be developed.

Disclosure of Invention

Therefore, the invention provides a microbacterium for degrading aflatoxin B1 and application thereof.

In order to achieve the above purpose, the invention provides the following technical scheme:

in one aspect, the present invention provides a strain, which is Microbacterium proteoliticum B204, and is characterized in that the accession number of the Guangdong province collection center for microbial cultures is GDMCC NO: 62022.

according to another aspect of the present invention, there is provided a microbial preparation, wherein the active ingredient is the above-mentioned strain and/or a metabolite thereof.

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

In one embodiment of the invention, the 16S rDNA sequence of the strain of Microbacterium proteoliticum B204 is as shown in SEQ ID No:1 is shown.

In one embodiment of the invention, the strain of Microbacterium proteoliticum B204 has a degradation rate of 87% to aflatoxin B1.

In one embodiment of the invention, the strain of Microbacterium proteolyticum B204 is screened and cultured in coumarin selection medium.

In one embodiment of the invention, the preparation method of the legumain selective medium comprises the following steps: weighing KH₂PO₄0.25g，NH₄NO₃ 1g，MgSO₄ 0.001g，FeSO₄0.001g of agar and 20g of agar, and adding 500ml of deionized water and sterilizing to obtain the product.

Bacterial preservation description: the Microbacterium proteoliticum B204 is preserved in Guangdong province microorganism strain preservation center at 11/01/2021, with the preservation addresses: the Guangzhou city Pieli Zhongluo No. 100 large yard No. 59 building No.5, the preservation number is GDMCC NO: 62022.

the invention has the following advantages:

the test proves that: the Microbacterium proteoliticum B204 has obvious effect of degrading aflatoxin B1, and is low in preparation cost, simple in process and convenient for large-scale 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 should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.

FIG. 1 is a photograph showing the morphology of a Microbacterium proteolyticum B204 colony provided in the present invention 1;

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

FIG. 3 is a graph showing the effect of temperature on the degradation of AFB1 by Microbacterium proteolyticum B204 according to the present invention;

FIG. 4 is a graph showing the effect of pH on the degradation of AFB1 by Microbacterium proteolyticum B204 according to the present invention;

FIG. 5 is a graph showing the effect of the inoculum size provided by the present invention on the degradation of AFB1 by Microbacterium proteolyticum B204;

FIG. 6 is a graph showing the effect of the rotational speed provided by the present invention on the degradation of AFB1 by Microbacterium proteolyticum B204;

FIG. 7 is a bar graph showing the effect of Microbacterium proteolyticum B204 on detoxification of different samples.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. 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 isolation and characterization of Microbacterium proteoliticum B204

1. Preparation of prescreening culture medium

Coumarin selection medium: weighing KH₂PO₄ 0.25g，NH₄NO₃ 1g，MgSO₄ 0.001g，FeSO₄0.001g of agar and 20g of agar are added into 500ml of deionized water and then put into a high-pressure steam sterilization pot, the setting procedure is 121 ℃, and the temperature is 20 DEGAnd min, after the sterilization is finished, placing the mixture in a super clean bench to be opened, adding the weighed coumarin into a primary screening culture medium, and slightly shaking until the coumarin is completely dissolved. Meanwhile, LB liquid culture medium and LB solid culture medium required by the experiment are configured to be used as activation and purification culture medium.

Stock AFB 1: 1mg of AFB1 standard (C)₁₇H₁₂O₆Purity is more than or equal to 98 percent) is dissolved in chromatographic grade acetonitrile, the volumetric flask is fixed 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 to a reagent bottle to be sealed and then is stored at minus 20 ℃ in a dark place.

2. Screening of AFB 1-degrading strains

Primary screening of strains: weighing 1g of cow dung, soil, yeast, 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, vortexing the sample for 3min, and mixing uniformly to obtain 10^-1Diluting the diluted solution to 10 in turn according to the experimental operating specification^-2、10^-3、10^-4、10^-5And l0^-6The diluent (2). Then, suck 10 of 100. mu.L each^-4、10^-5And l0^-6The diluted solution is prepared by uniformly coating the bacterial liquid on the surface of a coumarin selective culture medium by using a coater, then inversely placing the plate in a 30 ℃ constant-temperature incubator for culturing for 1-21 days, checking whether a new bacterial colony grows in the culture dish every 24 hours, selecting a single bacterial colony which grows well, inoculating the single bacterial colony to the new culture medium, repeatedly purifying for more than 3 times, and storing the bacterial strain in an ultra-low temperature refrigerator after amplification culture.

3. Rescreening for degrading AFB1 strain

The strain to be tested is picked up and activated for a plurality of times on an activation purification culture medium, and then the strain is inoculated in 15mL of liquid culture medium and cultured for 12 hours at the temperature of 30 ℃ at 180 r/min. In a clean bench, 945mL of fermentation medium is taken out and added into a 15mL sterile clean centrifuge tube, then 50 μ L of bacterial liquid is added, finally 5 μ L of AFB1 standard substance (10 μ g/mL) is added, the centrifuge tube is placed in a shaking table under the conditions of 30 ℃ and 200r/mim and incubated for 12h in the dark, the centrifuge treatment is carried out under the conditions of 8000r/mim and 20min, the collected supernatant is concentrated by a concentrator and then re-dissolved by 1mL of acetonitrile, and the supernatant is filtered by a 0.22 μm filter membrane before being tested on a machine. The peak value of each component is detected by HPLC, so that the degradation rate of the Microbacterium proteolyticum B204 strain to AFB1 is calculated.

The degradation rate (%) of AFB1 was [ (content of blank AFB 1-content of treated AFB1) ]/content of blank AFB1 × 100%.

HPLC detection conditions: the chromatographic column adopts Waters C18(250mm multiplied by 4.60.5 m, Waters), the mobile phase A is ultrapure water, and the mobile phase B is a mixture of methanol and acetonitrile which are 1: 1; the flow rate is 0.6 mL/min; selecting a fluorescence detector for detection, wherein the excitation wavelength is 350nm, and the detection wavelength is 450 nm; the amount of sample was 10. mu.L.

Drawing a standard curve: diluting the prepared standard solution into solutions of 100, 70, 50, 10 and 1 mu g/mL respectively, analyzing by an instrument, and making a regression curve by taking the abscissa as the concentration of the standard substance and the ordinate as the peak area.

4. Identification of Microbacterium proteoliticum B204

(1) Detection of cell morphology and physiological and biochemical characteristics of strain

As shown in FIG. 1, the cells of Microbacterium proteolyticum B204 under microscope are slender and irregular rod-shaped, and are creamy yellow colonies on NA culture medium, with regular edges, wet colonies, and gram-positive bacteria, the optimum growth temperature is 30 ℃, the optimum pH is 7, the optimum NaCl concentration is 0.5mg/mL, soluble starch is used as carbon source, and soybean protein or yeast extract powder is used as nitrogen source for better bacterial growth.

(2) Microbacterium proteolyticum B20416S rDNA sequence determination and homology analysis

Genome extraction was performed according to the instructions of the purchased genome extraction kit (Shanghai Biotech). 16SrDNA amplification and sequencing: PCR amplification of bacterial DNA universal primers were selected to amplify the 16SrDNA sequence. The amplified nucleotide sequence is shown as SEQ ID NO 1, a B204 gene sequence is obtained by sequence alignment and sequencing, the sequence is aligned with the sequence in an NCBI database by using a BLAST algorithm, the aligned sequence is constructed into a phylogenetic evolution tree by using an adjacency method in software MEGA 6, and a ruler 0.002 shows the frequency substitution of each nucleic acid site. As shown in fig. 2, B204 is known to have 16S rDNA homology with Microbacterium (Microbacterium sp.) and B204 is determined to be Microbacterium (Microbacterium sp.) according to genetic distance, morphology and characteristics of the strain, and the obtained strain B204 is deposited.

(3) API ZYM experiment

The API reagent strips are various, and can be selected according to the experiment requirement, and the API20NE can be used for measuring the carbon source utilized by the microorganism; API20E can measure various carbohydrates fermented/oxidized by microorganisms; API ZYM can determine various enzymatic characteristics. Physiological and biochemical characteristics cannot be used alone as the basis for microorganism classification, and the overall judgment needs to be performed by combining genotype characteristics, chemical composition characteristics and the like.

Using 5mL of 0.85% physiological saline without impurities, single pure colony cultured from the plate of the isolate was picked up into physiological saline to mix the solutions uniformly, the turbidity was between McFarland No.5 and No.6, the bacterial suspension was filled into different tubes or portions with a pipette according to the API ZYM reagent strip instructions, and after 4h A, B solution was added to interpret the experimental results. As shown in Table 1, ZYM test results of the strain Microbacterium proteolyticum B204.

TABLE 1

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

Inoculating purified Microbacterium proteolyticum B204 into 15mL LB culture medium, adding 1.5 μ L AFB1(1mg/mL), fermenting at different temperatures (14 deg.C, 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C) with initial pH of 7 and shaker rotation speed of 180r/min for 12h while adding 1.5 μ L AFB1 into sterile 15mL LB culture medium as blank control, and respectively culturing at different temperatures for 12h to measure AFB1 degradation amount.

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 Microbacterium proteolyticum B204 shows a gradually rising state on the degradation of AFB1, and the maximum degradation rate of AFB1 is reached at 30 ℃.

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

The purified bacillus proteoliticus is inoculated into 15mL of LB culture medium, then 1.5 mu of LAFB1(1mg/mL) is added, the degradation amount of AFB1 is measured after the culture is respectively carried out for 12h under different initial pH values by using different initial pH values (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0) and the shaking table rotating speed of 180r/min at the culture temperature of 30 ℃ and fermenting for 12h and using sterile 15mL of LB culture medium and 1.5 mu of AFB1 as blank controls.

The test result is shown in fig. 4, which shows that the pH has an influence on the degradation of AFB1 by strain B204, the degradation rate is very low at pH 4.0, the degradation rate of Microbacterium proteoliticum B204 gradually increases from pH 5.0 to pH7.0, the degradation effect of Microbacterium proteoliticum B204 on AFB1 is significant at pH7.0, and the degradation capability on AFB1 is relatively reduced as the pH continues to increase.

Example 4 Effect of different inoculum sizes on the degradation of Aflatoxin B1 by Microbacterium proteolyticum B204

Inoculating bacillus proteoliticus with different inoculum sizes (2%, 4%, 6%, 8%, 10%) into 15mL LB medium, adding 5 μ L of AFB1(1mg/mL), fermenting for 12h at 30 ℃, pH7, and shaking table rotating speed of 180r/min, and measuring degradation of AFB1 after culturing for 12h under different initial pH values respectively by using sterile 15mL LB medium and 1.5 μ L of AFBI as blank control.

The results of the experiment are shown in fig. 5, which is the effect of the inoculum size on the degradation of AFB1 by strain B204. The effect of the Microbacterium proteolyticum B204 on degrading AFB1 is influenced by the inoculation amount, the degradation effect is continuously increased along with the increase of the inoculation amount, under the inoculation amount of 8%, the effect of the Microbacterium proteolyticum B204 on degrading AFB1 is best, and after the inoculation amount exceeds 8%, the degradation rate of the Microbacterium proteolyticum B204 on AFB1 is reduced along with the increase of the inoculation amount.

Example 5 Effect of different rotation speeds on the degradation of Aflatoxin B1 by Microbacterium proteolyticum B204

The purified Microbacterium proteolyticum B204 is inoculated into 15mL LB culture medium, then 1.5 μ L AFB1(1mg/mL) is added, and after the culture is carried out for 12h by using different initial rotation speeds (120r/min, 150r/min, 180r/min, 200r/min, 220r/min and 250r/min), the degradation amount of AFB1 is measured after the culture is carried out for 12h respectively at different rotation speeds by using sterile 15mL LB culture medium and 1.5 μ L AFB1 as blank controls at the culture temperature of 30 ℃ and the initial pH of 7.

The results of the experiment are shown in fig. 6, which is the effect of rotational speed on the degradation of AFB1 by strain B204. The rotating speed influences the effect of the Microbacterium proteolyticum B204 bacteria on degrading AFB1 to a certain extent, the degradation effect is continuously increased along with the increase of the rotating speed, the effect of the Microbacterium proteolyticum B204 on degrading AFB1 is best under the condition of the rotating speed of 200r/min, and the degradation rate of B204 on AFB1 is reduced along with the increase of the inoculation amount after the rotating speed exceeds 200 r/min.

Example 6 application of Strain B204 in biological detoxification

After the Microbacterium proteoliticum B204 is activated twice, a single colony is picked and placed in 50mL sterilized NB, the NB is placed in a shaker at 30 ℃ and 200r/min, and cultured overnight for 12 hours, and the OD value is measured to be 0.6.

Accurately weighing corn, peanut and cheese samples, each 10.0 + -0.03 g, numbering YM204, HS204 and RL204 respectively, spreading in 90mm culture dish, drawing on the culture dish cover with black mark pen to divide into 9 regions, each region is about 1cm²The size of the solution was 1mL of AFB1 standard solution (100. mu.g/mL), and each region was added100 μ L of AFB1 standard solution (concentration 100 μ g/mL). The same conditions were taken for the samples as blank control.

Adding 450 μ L of Microbacterium proteolyticum B204 culture solution into different culture plates according to the above number, adding into the 9 regions 10 times, covering with a cover, culturing in 30 deg.C incubator for 16h, and repeating the above culture solution adding operation every 3 h.

After the culture is finished, 10mL organic solvent acetonitrile is added into a 10g sample for 2 times, the sample is completely transferred into a 50mL centrifugal tube, the sample is placed into a centrifugal machine after ultrasonic oscillation is carried out for 30min, and a centrifugal adjusting piece is set as follows: the temperature is 25 ℃, the rotating speed is 8000r/min, the time is 10min, a pipette gun absorbs supernatant liquid in the centrifuge tube, and the collected organic layer solution is concentrated in a 10mL centrifuge tube. The solution collected by extraction was placed in a concentrator and concentrated at 45 ℃ for 2h, after which 1mL of acetonitrile was added to give a solution of AFB1 acetonitrile which was filtered through a 0.22 μm filter into a liquid phase vial. HPLC detection conditions: the chromatographic column was C18 from Waters (250 mm. times. 4.60.5 m, Waters), the mobile phase A was ultrapure water, the mobile phase B was methanol: acetonitrile 1:1 mixture; the flow rate is 0.6 mL/min; selecting a fluorescence detector for detection, wherein the excitation wavelength is 350nm, and the detection wavelength is 450 nm; the amount of sample was 10. mu.L.

As shown in FIG. 7, the degradation effect of corn is the best, the peanut is the second time, and finally cheese is the last time, it can be seen that the degradation effect of AFB1 is better when the sample particles are smaller, which is probably because the bacterial liquid is difficult to spray uniformly when the sample particles are larger, and the degradation effect of AFB1 is reduced.

The optimal degradation conditions of the Microbacterium proteoliticum B204 on the aflatoxin B1 are as follows: the optimum culture condition is that the pH is 6.7, the NaCl is 0.48g/L, the temperature is 31.5 ℃, the soluble starch is 1 percent, the soybean protein is 0.5 percent, the degradation rate of the aflatoxin B124 h in the fermentation broth is 87 percent, and the degradation rate of the aflatoxin B124 h on the surface of the corn is 81 percent.

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Beijing university of Industrial and commercial

<120> Microbacterium for degrading aflatoxin B1 and application thereof

<130> GG20834948A

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1402

<212> DNA

<213> Artificial Sequence

<400> 1

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. The strain is Microbacterium proteoliticum B204, and the preservation number of the Guangdong province microorganism strain preservation center is GDMCC NO: 62022.

2. a bacterial preparation, characterized in that the active ingredient is the strain of claim 1 and/or a metabolite thereof.

3. The use of the strain of claim 1 or the microbial agent 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 the product for food detoxification.

4. The use according to claim 3,

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

5. The use according to claim 3,

the degradation rate of the strain in the Microbacterium proteoliticum B204 to the aflatoxin B1 is 87%.

6. The use according to claim 3,

the strain in the Microbacterium proteoliticum B204 is screened and cultured by adopting a coumarin selective medium.

7. The use according to claim 6,

the preparation method of the legumin selection culture medium comprises the following steps: weighing KH₂PO₄ 0.25g，NH₄NO₃ 1g，MgSO₄ 0.001g，FeSO₄0.001g of agar and 20g of agar, and adding 500ml of deionized water and sterilizing to obtain the product.