CN114574398B

CN114574398B - Simultaneous degradation of AFB 1 Acinetobacter nosocomial Y1 and application thereof

Info

Publication number: CN114574398B
Application number: CN202210319116.6A
Authority: CN
Inventors: 邢福国; 杨博磊; 阿德戈克·维克多·托辛; 李奕霏
Original assignee: Institute of Food Science and Technology of CAAS
Current assignee: Institute of Food Science and Technology of CAAS
Priority date: 2022-03-29
Filing date: 2022-03-29
Publication date: 2023-11-28
Anticipated expiration: 2042-03-29
Also published as: CN114574398A

Abstract

The invention discloses a method for simultaneously degrading AFB ₁ And ZEN, acinetobacter nosocomial Y1, classified under the name: acinetobacter nosocomial Acinetobacter nosocomialis, acinetobacter nosocomial strain Acinetobacter nosocomialis Y1, was deposited in China general microbiological culture Collection center, with accession number: CGMCC No.23794, the preservation time is: 2021, 11 and 12, the deposit unit addresses are: beijing, chaoyang area, north Chen Xili No. 1, 3, china academy of sciences, microbiological institute. The invention also discloses a culture method and application of the acinetobacter in hospitals Y1. The strain of the invention can degrade AFB simultaneously ₁ And ZEN, the cytotoxicity of the degradation products was significantly lower than AFB ₁ And ZEN itself, and the degradation products themselves do not produce other inhibitory substances.

Description

Simultaneous degradation of AFB 1 Acinetobacter nosocomial Y1 and application thereof

Technical Field

The invention belongs to the field of biotechnology, and relates to simultaneous degradation of AFB ₁ And ZEN's Acinetobacter in hospital Y1 and its use.

Background

Mycotoxins are toxic secondary metabolites produced by fungi, and according to world Food and Agricultural Organization (FAO) statistics, about 25% of grains are contaminated with mycotoxins to different extents every year worldwide, and 2% of grains lose nutritional and economic values due to mycotoxin contamination, resulting in hundreds of billions of dollars of economyLoss. The most common mycotoxins in foodstuffs are aflatoxin B ₁ (AFB ₁ ) Zearalenone and vomitoxin, etc.

AFB ₁ The secondary metabolites mainly produced by aspergillus such as aspergillus flavus and aspergillus parasiticus are also naturally-produced compounds with the strongest known carcinogenicity at present; up to 28% of primary hepatocellular carcinoma worldwide is caused by AFs, with hepatitis b carriers being more susceptible to AFs-induced liver cancer; AFs can also cause symptoms such as acute poisoning, immune decline, slow growth in children, etc.

Zearalenone (ZEN), also known as F-2 toxin, is a mycotoxin produced by fusarium such as fusarium graminearum and released into the environment. ZEN has estrogen-like effect, is shown as chronic toxicity, mainly affects the reproductive system, damages the reproductive function of animals and generates great harm to animal health. ZEN is one of the most widely contaminated types of fusarium toxins in the world, and the presence of ZEN is detected in food and processing byproducts throughout the world, such as europe, africa, asia, north america, and south america. The pollution of the grains by ZEN will probably threaten the safety of the feed and the breeding industry. The feed in most countries was investigated for various degrees of ZEN contamination. The investigation shows that the detection rate of ZEN in 2019 complete feed reaches 58%, and the highest value is 5791 mug/kg. Therefore, the ZEN pollution has great influence and great economic loss on the breeding industry, and can indirectly endanger human health.

At present, the detoxification method of mycotoxins mainly comprises a physical detoxification method, a chemical detoxification method and a biological detoxification method. However, physical and chemical detoxification has many disadvantages and limitations, and biological detoxification is increasingly paid attention to safety, specificity and high efficiency. At present, some AFB is reported at home and abroad ₁ Or ZEN has degradation effect on microorganisms including Bacillus, pseudomonas putida, pseudomonas aeruginosa, rhodococcus and Enterobacter, etc., which can be used for AFB ₁ Or ZEN is transformed or degraded, but at the same time AFB is degraded ₁ And ZEN microorganisms have not been reported.

Disclosure of Invention

It is an object of the present invention to address at least the above problems and/or disadvantages and to provide at least the advantages described below.

The invention screens out a strain of AFB which can be efficiently degraded simultaneously from hot spring water sample ₁ And ZEN, and the strain identification and degradation mechanism analysis of the microorganisms are carried out.

It is a further object of the present invention to provide a method for simultaneously degrading AFB ₁ And ZEN's Acinetobacter in Hospital Y1.

Another object of the present invention is to provide a method for culturing Acinetobacter in a hospital.

It is another object of the present invention to provide for simultaneous degradation of AFB ₁ And the method of ZEN.

It is still another object of the present invention to provide a method for degrading mycotoxin contaminated foodstuffs.

For this purpose, the technical scheme provided by the invention is as follows:

simultaneous degradation of AFB ₁ And ZEN, acinetobacter nosocomial Y1, which is designated by its classification: acinetobacter nosocomial Acinetobacter nosocomialis, acinetobacter nosocomial strain Acinetobacter nosocomialis Y1, was deposited in China general microbiological culture Collection center, with accession number: CGMCC No.23794, the preservation time is: 2021, 11 and 12, the deposit unit addresses are: beijing, chaoyang area, north Chen Xili No. 1, 3, china academy of sciences, microbiological institute.

The culture method of the Acinetobacter in the hospital comprises the following steps:

inoculating the acinetobacter in hospital Y1 into an LB culture medium or an NB culture medium for culture to obtain fermentation liquor.

Preferably, in the method for culturing the acinetobacter in hospital Y1, the acinetobacter in hospital Y1 is inoculated in LB medium for culturing, the pH of the medium is 5-10, and the culturing temperature is 28-45 ℃.

Preferably, in the method for culturing Acinetobacter nosocomial Y1, the Acinetobacter nosocomial Y1 is inoculated into LB medium according to an inoculum size of 1%, and cultured for 24 hours at a temperature of 28 ℃ and a rotation speed of 180rpm to obtain the fermentation broth.

Simultaneous degradation of AFB ₁ And a method of ZEN comprising the steps of:

1) Culturing the acinetobacter in hospital Y1 to obtain fermentation liquor;

2) Simultaneous degradation of AFB with the fermentation broth of Acinetobacter nosocomial Y1 as described in step 1) ₁ And ZEN.

Preferably, said simultaneous degradation of AFB ₁ And ZEN, step 2) wherein the fermentation broth of Acinetobacter nosocomial Y1 degrades AFB ₁ And ZEN, maintaining the degradation temperature at 28-60deg.C for 12-72 hr.

Preferably, said simultaneous degradation of AFB ₁ And ZEN, step 2) wherein the fermentation broth of Acinetobacter nosocomial Y1 has an optimal degradation pH of 6-10 for ZEN and for AFB ₁ The optimal degradation pH of (2) is 5-10.

Preferably, said simultaneous degradation of AFB ₁ And ZEN, step 2) when degrading ZEN, mn is added to the fermentation broth of Acinetobacter in the hospital Y1 ²⁺ 、Cu ²⁺ Or K ⁺ ；

When degrading AFB ₁ At the time, cu is added into the fermentation broth of Acinetobacter in the hospital Y1 ²⁺ Or K ⁺ 。

The degradation method of the mycotoxin contaminated grain comprises the following steps:

fermenting and culturing the acinetobacter in hospital Y1 to a fermentation liquor with an OD value of 0.4-1.0,

after that, the fermentation liquor is added into the AFB ₁ And ZEN toxin contaminated grain, and continuing to cultivate at a certain temperature for a period of time, wherein the contaminated grain is subjected to the treatment of the ZEN toxin.

Preferably, in the degradation method of the mycotoxin polluted grain, the volume-mass ratio of the fermentation liquor to the polluted grain is 1:3,

the fermentation broth is obtained by culturing Acinetobacter in a hospital at 28 ℃ and 180 rpm.

The invention at least comprises the following beneficial effects:

the Acinetobacter in hospital Y1 can degrade ZEN and AFB simultaneously ₁ . And, for ZEN and AFB ₁ The cytotoxicity of the degradation products is significantly lower than that of the degradation products themselves. Particularly ZEN degradation products, which do not themselves have any inhibitory effect on vibrio freudenreichii. The Acinetobacter in hospital Y1 of the invention is applied to ZEN and AFB in grains in actual practice ₁ The degradation efficiency of the polymer is also higher, and the polymer can be used in practical production and application.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a diagram showing optimization of growth medium of Acinetobacter nosocomial Y1 in an embodiment of the invention.

FIG. 2 is a graph showing the pH optimization of growth of Acinetobacter nosocomial Y1 in the example of the present invention.

FIG. 3 is a graph showing the growth temperature optimization of Acinetobacter nosocomial Y1 in the example of the present invention.

FIG. 4 shows the degradation of ZEN and AFB by Acinetobacter in hospital Y1 in the examples of the invention ₁ Is a temperature optimum map of (a).

FIG. 5 shows the degradation of ZEN and AFB by Acinetobacter in hospital Y1 in the examples of the invention ₁ Is a time-optimal diagram of (a).

FIG. 6 shows the degradation of ZEN and AFB by Acinetobacter in hospital Y1 in the examples of the invention ₁ Is a pH optimum map of (3).

FIG. 7 shows degradation of ZEN and AFB by Acinetobacter in a hospital Y1 in an embodiment of the invention ₁ Is the highest concentration plot of (2).

FIG. 8 shows degradation of ZEN and AFB by Acinetobacter in a hospital Y1 in an embodiment of the invention ₁ Is an active material distribution analysis chart of (a).

FIG. 9 shows degradation of ZEN and AFB by Acinetobacter nosocomial Y1 with metal ions in an embodiment of the invention ₁ Is a graph of the influence of (1).

FIG. 10 shows the degradation of ZEN and AFB by Acinetobacter nosocomial Y1 by different substances in the examples of the present invention ₁ Is a graph of the influence of (1).

FIG. 11 shows ZEN and AFB in an embodiment of the invention ₁ And its degradation products to luminous feret arcGraph of bacterial activity effects.

FIG. 12 shows the effect of Acinetobacter nosocomial Y1 on ZEN and AFB in grains according to an embodiment of the invention ₁ Degradation effect.

Detailed Description

The present invention is described in further detail below with reference to the drawings to enable those skilled in the art to practice the invention by referring to the description.

It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

The invention provides a method capable of degrading AFB ₁ Acinetobacter nosocomial Y1 of ZEN, which Acinetobacter nosocomial Y1, is designated by its classification: acinetobacter nosocomial Acinetobacter nosocomialis, acinetobacter nosocomial strain Acinetobacter nosocomialis Y1, was deposited in China general microbiological culture Collection center, with accession number: CGMCC No.23794, the preservation time is: 2021, 11 and 12, the deposit unit addresses are: beijing, chaoyang area, north Chen Xili No. 1, 3, china academy of sciences, microbiological institute.

In some embodiments of the present invention, preferably, in the method for culturing Acinetobacter nosocomial Y1, the Acinetobacter nosocomial Y1 is inoculated in LB medium for culturing, wherein the pH of the medium is 5-10, and the culturing temperature is 28-45 ℃.

In some embodiments of the present invention, preferably, the acinetobacter hospital Y1 is inoculated into LB medium at an inoculum size of 1%, and cultured at a temperature of 28℃and a rotation speed of 180rpm for 24 hours to obtain the fermentation broth.

1) Culturing the acinetobacter in hospital Y1 to obtain fermentation liquor;

2) In the steps of1) The fermentation liquid of the acinetobacter in hospital Y1 can degrade AFB at the same time ₁ And ZEN.

In some embodiments of the invention, preferably, in step 2), the fermentation broth of acinetobacter hospital Y1 degrades AFB ₁ And ZEN, maintaining the degradation temperature at 28-60deg.C for 12-72 hr. More preferably, the optimal degradation temperature is 60 ℃. In addition, acinetobacter in hospital Y1 can completely degrade 2 mug/mL ZEN in 48h and completely degrade 2 mug/mL AFB in 72h ₁ 。

In some embodiments of the present invention, preferably, in step 2), the fermentation broth of Acinetobacter nosocomial Y1 has an optimal degradation pH of 6-10 for ZEN and an optimal degradation pH for AFB ₁ The optimal degradation pH of (2) is 5-10.

In some embodiments of the present invention, preferably, mn is added to the fermentation broth of Acinetobacter nosocomial Y1 in step 2) when ZEN is degraded ²⁺ 、Cu ²⁺ Or K ⁺ The method comprises the steps of carrying out a first treatment on the surface of the More preferably Mn ²⁺ 、Cu ²⁺ 、K ⁺ Is 2mmol/L;

when degrading AFB ₁ At the time, cu is added into the fermentation broth of Acinetobacter in the hospital Y1 ²⁺ Or K ⁺ . More preferably, cu ²⁺ 、 K ⁺ The concentration of (C) was 2mmol/L.

When degrading AFB ₁ In both cases, the addition of SDS was avoided.

The invention also provides a degradation method of the mycotoxin polluted grain, which comprises the following steps:

after that, the fermentation liquor is added into the AFB ₁ And ZEN toxin contaminated grain, and continuing to cultivate at a certain temperature for a period of time, wherein the contaminated grain is subjected to the treatment of the ZEN toxin. Preferably, the culture is carried out at 50℃for 72 hours with stirring every 12 hours.

In the above scheme, preferably, the volume-mass ratio of the fermentation liquor to the polluted grain is 1:3,

For a better understanding of the technical solution of the present invention, the following examples are now provided for illustration:

example 1

Mycotoxin degrading bacteria screening

Sample collection: the experimental sample is collected in the Changbai mountain hot spring, and the temperature of the hot spring is 96 ℃.

Screening degrading bacteria: adding 10mL of hot spring water into 90mL of LB medium, culturing at 37deg.C and 180rpm for 24 hr, collecting 10mL of bacterial liquid, and adding ZEN and AFB with final concentrations of 2ppm ₁ Culturing at 37deg.C and 180rpm for 72 hr, and placing the rest bacterial liquid in a refrigerator at 4deg.C. 10mL of LB medium was added with ZEN and AFB at a final concentration of 2ppm ₁ As a control group. After 72h incubation, ZEN and AFB were detected in the experimental and control groups ₁ And determining the content and the degradation effect.

Diluting the bacterial liquid with degradation effect to different concentrations by using sterile water (10 ^-1 、10 ^-2 、10 ^-3 、10 ^-4 、10 ^-5 、10 ^-6 、 10 ^-7 、10 ^-8 ) Respectively coating 100 μL of diluent on LB solid medium, culturing at 37deg.C in incubator for 24-48 hr, selecting culture dish capable of selecting monoclonal, inoculating the monoclonal on culture dish to LB medium, culturing at 37deg.C and 180rpm for 24 hr, and adding ZEN and AFB with final concentration of 2ppm ₁ The cells were cultured at 37℃and 180rpm for 72 hours. The control group was LB medium plus ZEN and AFB at a final concentration of 2ppm ₁ . HPLC detection of monoclonal pairs ZEN and AFB ₁ And (3) selecting the strain with the best degradation effect.

Example 2

Identification of mycotoxin degrading bacteria

And (3) strain identification: amplification results were determined by electrophoresis on a 1% agarose gel using a 16S rDNA universal primer for amplification. The sequence of the strain 16S rDNA gene is determined and analyzed, sequencing results are aligned at NCBI by Blast, and the number of recognized standard sequences homologous to the strain 16S rDNA is obtained from GenBank databaseAccording to the above. Thereby determining that ZEN and AFB can be degraded simultaneously ₁ The microorganism of (2) is Acinetobacter nosocomial Y1 (Acinetobacter nosocomialis Y1), the 16S rDNA sequencing is shown as SEQ ID NO. 3, and the microorganism can simultaneously degrade AFB ₁ And ZEN, acinetobacter nosocomial Y1, classified under the name: acinetobacter nosocomial Acinetobacter nosocomialis, acinetobacter nosocomial strain Acinetobacter nosocomialis Y1, was deposited in China general microbiological culture Collection center, with accession number: CGMCC No.23794, the preservation time is: 2021, 11 and 12, the deposit unit addresses are: beijing, chaoyang area, north Chen Xili No. 1, 3, china academy of sciences, microbiological institute.

PCR reaction system and reaction program

PCR reaction system

The sequence of the 16S rDNA universal primer is as follows:

Primer-16SF：27F：AGAGTTTGATCCTGGCTCAG SEQ ID NO:1

Primer-16SR：1492R：TACGGCTACCTTGTTACGACTT SEQ ID NO:2

PCR reaction procedure

The sequencing result of the Acinetobacter in hospital Y1 16S rDNA is shown as SEQ ID NO: 3:

TGGCGACGCTTACCTGCAGTCGAGCGGGGGAAGGTAGCTTGCTACTGGACCTAGCG GCGGACGGGTGAGTAATGCTTAGGAATCTGCCTATTAGTGGGGGACAACATCTCGAAAGGGATGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGATCTTCGGACCTTG CGCTAATAGATGAGCCTAAGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGATCTGTAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACG GCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTATGGTTGTAAAGCACTTTAAGCG AGGAGGAGGCTACTTTAGATAATACCTAGAGATAGTGGACGTTACTCGCAGAATAAG CACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGATTTACTGGGCGTAAAGCGCGCGTAGGCGGCTAATTAAGTCAAATGTGAAATCCCC GAGCTTAACTTGGGAATTGCATTCGATACTGGTTAGCTAGAGTGTGGGAGAGGATGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGATGGCGA AGGCAGCCATCTGGCCTAACACTGACGCTGAGGTGCGAAAGCATGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGTCTACTAGCCGTTGGGGCCTT TGAGGCTTTAGTGGCGCAGCTAACGCGATAAGTAGACCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGT TTAATTCGATGCAACGCGAAGAACCTTACCTGGCCTTGACATAGTAAGAACTTTCCAGAGATGGATAGGTGCCTTCGGGAACTTACATACAGGTGCTGCATGGCTGTCGTCAGC TCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTTTCCTTATTTGCCAGCGAGTAATGTCGGGAACTTTAAGGATACTGCCAGTGACAAACTGGAGGAAGG CGGGGACGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAA TGGTCGGTACAAAGGGTTGCTACACAGCGATGTGATGCTAATCTCAAAAAGCCGATCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCG CGGATCAGAATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTTGTTGCACCAGAAGTAGCTAGCCTAACTGCAAAGAGGGCGGTACC ACGAGTACC

example 3

Mycotoxin degrading bacteria growth condition optimization

Acinetobacter in hospital Y1 was inoculated into LB medium, cultured at 37℃at 180rpm for 24 hours, then inoculated into LB (purchased from oxoid Co., ltd.), NB (purchased from Beijing Soy Bao technology Co., ltd.) and MM (purchased from Haibo biotechnology Co., ltd.) medium in an inoculum size of 1%, respectively, cultured at 37℃at 180rpm for 24 hours, and the absorbance of the bacteria was measured by an ultraviolet spectrophotometer to determine the optimum growth medium for the bacteria. The final results showed that LB medium was the most suitable growth medium for Y1, as shown in FIG. 1.

Acinetobacter in hospital Y1 was inoculated into LB medium, cultured at 37℃at 180rpm for 24 hours, then inoculated into LB medium with pH values of 2-10 respectively in an inoculum size of 1%, cultured at 37℃at 180rpm for 24 hours, and the absorbance of the bacteria was detected by an ultraviolet spectrophotometer. The final results showed that Acinetobacter nosocomial Y1 can grow normally in the pH range of 5-10 (FIG. 2)

Acinetobacter in hospital Y1 was inoculated into LB medium, cultured at 37℃at 180rpm for 24 hours, then inoculated into LB medium at 1% of the inoculum size, cultured at 28℃at 37℃at 45℃at 55℃at 60℃at 180rpm for 24 hours, and the absorbance of the bacteria was examined by ultraviolet spectrophotometer. The final results showed that Acinetobacter nosocomial Y1 can grow normally in the temperature range of 28-45 ℃ (FIG. 3).

Example 4

Mycotoxin degrading bacteria degradation condition optimization

Inoculating Acinetobacter nosocomial Y1 into LB medium at 1% inoculum size, culturing at 28deg.C and 180rpm for 24 hr, and adding ZEN and AFB with final concentration of 2 μg/mL into multiple groups of culture solutions ₁ Degrading at 28deg.C, 37deg.C, 45deg.C, 55deg.C and 60deg.C and 180rpm for 72 hr, respectively, and detecting ZEN and AFB by HPLC ₁ The optimum degradation temperature was determined to be 60 c (fig. 4).

Inoculating Y1 into LB medium at 1% inoculum size, culturing at 28deg.C and 180rpm for 24 hr, adding ZEN and AFB with final concentration of 2 μg/mL, respectively ₁ Degradation occurs at 60℃and 180 rpm. Sampling every 6 hr or 12 hr, and detecting ZEN and AFB by HPLC ₁ Finally, the residual quantity of the acinetobacter in hospital Y1 can be completely degraded by 2 mug/mL ZEN in 48 hours and completely degraded by 2 mug/mL AFB in 72 hours ₁ (FIG. 5).

Inoculating Acinetobacter in Hospital Y1 into LB culture medium with pH value of 2-10 respectively at 1%, culturing at 28deg.C and 180rpm for 24 hr, and adding ZEN and AFB with final concentration of 2 μg/mL respectively ₁ Degradation is carried out at 60℃and 180rpm for 72h. Then HPLC detection of ZEN and AFB ₁ Finally, the optimal degradation pH of the Acinetobacter in the hospital Y1 to ZEN is determined to be 6-10, and the AFB is determined ₁ The optimal degradation pH of (C) was 5-10 (FIG. 6).

Inoculating Acinetobacter nosocomial Y1 into LB medium at 1% inoculum size, culturing at 28deg.C and 180rpm for 24 hr, adding ZEN and AFB with final concentrations of 5, 10, 20, 30, 40 and 50 μg/mL respectively ₁ At 60 ℃ and 1Degradation for 72h at 80 rpm. Then HPLC detection of ZEN and AFB ₁ Is determined to be capable of completely degrading 20 mug/mL ZEN and 5 mug/mL AFB in 72 hours ₁ (FIG. 7).

Inoculating Acinetobacter nosocomial Y1 into LB culture medium according to 1% inoculum size, culturing at 28deg.C and 180rpm for 24h, centrifuging at 1000rpm for 10min, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain sterile supernatant; the centrifugal sediment is Acinetobacter in hospital Y1 cells, after 3 times of washing with sterile PBS, the cells are redissolved with PBS with the same volume to obtain Acinetobacter in hospital Y1 cell suspension; half of the volume of the Acinetobacter nosocomial Y1 cell suspension was taken for cell disruption, and then centrifuged at 1000rpm for 10min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain the cell content. Acinetobacter in hospital Y1 bacterial liquid is used as a control group. ZEN and AFB were added at a final concentration of 2. Mu.g/mL, respectively ₁ Degradation is carried out at 60℃and 180rpm for 72h. Then HPLC detection of ZEN and AFB ₁ Determining the residual amount of the bacteria in Acinetobacter in Hospital Y1 for ZEN and AFB ₁ The active substances acting as degradants are mainly distributed in the supernatant after fermentation of Acinetobacter in the hospital (FIG. 8).

Acinetobacter nosocomial Y1 was inoculated to a culture medium containing Mn at a final concentration of 2mmol/L, respectively, at an inoculum size of 1% ²⁺ 、 Ca ²⁺ 、Mg ²⁺ 、Na ⁺ 、Fe ³⁺ 、Cu ²⁺ 、Li ²⁺ 、K ⁺ 、Fe ²⁺ 、Zn ²⁺ Culturing at 28deg.C and 180rpm for 24 hr, adding ZEN and AFB with final concentration of 2 μg/mL, respectively ₁ Degradation is carried out at 60℃and 180rpm for 72h. Then HPLC detection of ZEN and AFB ₁ Residual amount of (C) and finally determine Mn ²⁺ 、Cu ²⁺ And K ⁺ Can obviously enhance the degradation capability of Y1 to ZEN, and Cu ²⁺ And K ⁺ Can obviously enhance the AFB of the Acinetobacter in the hospital Y1 ₁ Is shown (FIG. 9).

Inoculating Acinetobacter nosocomial Y1 into LB culture medium according to 1% inoculum size, culturing at 28deg.C and 180rpm for 24h, centrifuging at 1000rpm for 10min, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain sterile supernatant; SDS, protease K, EDTA and EDTA are added to the supernatant, respectivelyWater bath treatment, adding ZEN and AFB with final concentration of 2 μg/mL ₁ Degradation is carried out at 60℃and 180rpm for 72h. Then HPLC detection of ZEN and AFB ₁ The result shows that SDS can obviously inhibit Y1 supernatant from degrading ZEN and AFB ₁ Degradation of ZEN and AFB by Acinetobacter in hospitals Y1 at high temperature ₁ The capacity had no significant effect (fig. 10).

Example 5

Cytotoxicity assay for mycotoxin degradation products

Acinetobacter nosocomial Y1 was inoculated into LB medium at 1% inoculum size, cultured at 28℃and 180rpm for 48h, centrifuged at 1000rpm for 10min, and the supernatant was filtered through a 0.22 μm filter to obtain a sterile supernatant. ZEN and AFB were added at a final concentration of 50. Mu.g/mL, respectively ₁ Degradation is carried out at 60℃and 180rpm for 72h. Then HPLC detection of ZEN and AFB ₁ Residual amount of ZEN and AFB is ensured ₁ Is completely degraded to obtain ZEN and AFB ₁ Degradation product solution. The control group was LB-added ZEN and AFB at a final concentration of 50. Mu.g/mL, respectively ₁ And sterile supernatant of Acinetobacter in hospital Y1. Vibrio freudenreichii was cultured in 2216E liquid medium (available from Beijing Soy Bao technology Co., ltd.) at 28℃and 180rpm for 24 hours, and then an equal volume of the degradation product solution and the control group solution were added, respectively, and cultured at 28℃and 180rpm for 72 hours. Then, the luminescence property of Vibrio freudenreichii was detected by a spectrophotometer (detection wavelength: 550 nm), and the cell viability of Vibrio freudenreichii was calculated. As shown in the results of FIG. 11, the inhibition ratio of ZEN to Vibrio freudenreichii is 32%, while ZNE degradation products have no inhibitory effect on Vibrio freudenreichii; AFB (alpha-fetoprotein) ₁ The inhibition rate of Vibrio fischeri was 31%, while AFB ₁ The inhibition rate of the degradation product to the Vibrio fischeri is 8 percent. From this, ZEN and AFB can be seen ₁ The cytotoxicity of the degradation products is significantly lower than that of the degradation products themselves. Particularly ZEN degradation products, which do not themselves have any inhibitory effect on vibrio freudenreichii.

Example 6

Application of Acinetobacter in hospital Y1 in grain polluted by mycotoxin

Inoculating Acinetobacter nosocomial Y1 into LB medium at 1% inoculum size, respectively, at 28deg.C and 180rpmCulturing for 24 hours. According to the proportion of 1:3 (bacterial liquid: grain), bacterial liquid and mycotoxin are respectively added to pollute grain, and the grain is cultivated for 72 hours at 50 ℃ and stirred every 12 hours. The control group is LB culture medium plus mycotoxin contaminated grain. After the culture is finished, mycotoxins in the grains of the control group and the treated group are respectively extracted by using an immunoaffinity column, and the content of the mycotoxins is detected. As shown in FIG. 12 (degradation rate is (control toxin content-treatment toxin content) ×100%/control toxin content), acinetobacter nosocomial Y1 was used for AFB in grain ₁ The degradation efficiency of the catalyst is higher, the degradation rate reaches 67%, the degradation efficiency of ZEN in grains is poorer, but the degradation rate also reaches 41%.

The number of modules and the scale of processing described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the present invention will be readily apparent to those skilled in the art.

Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.

SEQUENCE LISTING

<110> institute of agricultural products, national academy of agricultural sciences

<120> Acinetobacter in hospital Y1 for simultaneous degradation of AFB1 and ZEN and application thereof

<130> 2021

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> Synthesis

<400> 1

agagtttgat cctggctcag 20

<210> 2

<211> 22

<212> DNA

<213> Synthesis

<400> 2

tacggctacc ttgttacgac tt 22

<210> 3

<211> 1429

<212> DNA

<213> Acinetobacter nosocomial Acinetobacter nosocomialis

<400> 3

tggcgacgct tacctgcagt cgagcggggg aaggtagctt gctactggac ctagcggcgg 60

acgggtgagt aatgcttagg aatctgccta ttagtggggg acaacatctc gaaagggatg 120

ctaataccgc atacgtccta cgggagaaag caggggatct tcggaccttg cgctaataga 180

tgagcctaag tcggattagc tagttggtgg ggtaaaggcc taccaaggcg acgatctgta 240

gcgggtctga gaggatgatc cgccacactg ggactgagac acggcccaga ctcctacggg 300

aggcagcagt ggggaatatt ggacaatggg cgcaagcctg atccagccat gccgcgtgtg 360

tgaagaaggc cttatggttg taaagcactt taagcgagga ggaggctact ttagataata 420

cctagagata gtggacgtta ctcgcagaat aagcaccggc taactctgtg ccagcagccg 480

cggtaataca gagggtgcaa gcgttaatcg gatttactgg gcgtaaagcg cgcgtaggcg 540

gctaattaag tcaaatgtga aatccccgag cttaacttgg gaattgcatt cgatactggt 600

tagctagagt gtgggagagg atggtagaat tccaggtgta gcggtgaaat gcgtagagat 660

ctggaggaat accgatggcg aaggcagcca tctggcctaa cactgacgct gaggtgcgaa 720

agcatgggga gcaaacagga ttagataccc tggtagtcca tgccgtaaac gatgtctact 780

agccgttggg gcctttgagg ctttagtggc gcagctaacg cgataagtag accgcctggg 840

gagtacggtc gcaagactaa aactcaaatg aattgacggg ggcccgcaca agcggtggag 900

catgtggttt aattcgatgc aacgcgaaga accttacctg gccttgacat agtaagaact 960

ttccagagat ggataggtgc cttcgggaac ttacatacag gtgctgcatg gctgtcgtca 1020

gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttt tccttatttg 1080

ccagcgagta atgtcgggaa ctttaaggat actgccagtg acaaactgga ggaaggcggg 1140

gacgacgtca agtcatcatg gcccttacgg ccagggctac acacgtgcta caatggtcgg 1200

tacaaagggt tgctacacag cgatgtgatg ctaatctcaa aaagccgatc gtagtccgga 1260

ttggagtctg caactcgact ccatgaagtc ggaatcgcta gtaatcgcgg atcagaatgc 1320

cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccatgg gagtttgttg 1380

caccagaagt agctagccta actgcaaaga gggcggtacc acgagtacc 1429

Claims

1. Simultaneous degradation of AFB ₁ And ZEN Acinetobacter in hospitalAcinetobacter nosocomialis) Y1, wherein the Acinetobacter in hospital Y1 is preserved in China general microbiological culture Collection center with the accession number: CGMCC No.23794, the preservation time is: 2021, 11 and 12, the deposit unit addresses are: beijing, chaoyang area, north Chen Xili No. 1, 3, china academy of sciences, microbiological institute.

2. The culture method of the Acinetobacter in the hospital is characterized by comprising the following steps of:

inoculating the acinetobacter hospital Y1 according to claim 1 into LB culture medium or NB culture medium for culture to obtain fermentation broth;

inoculating the acinetobacter in hospital Y1 into LB culture medium for culture, wherein the pH of the culture medium is 5-10, and the culture temperature is 28-45 ℃.

3. The method for culturing Acinetobacter nosocomial Y1 according to claim 2, wherein said Acinetobacter nosocomial Y1 is inoculated into LB medium at an inoculum size of 1%, and cultured at a temperature of 28℃and a rotation speed of 180rpm for 24h to obtain said fermentation broth.

4. Simultaneous degradation of AFB ₁ And a method of ZEN comprising the steps of:

1) Culturing Acinetobacter nosocomial Y1 according to claim 1 to obtain a fermentation broth;

2) Simultaneous degradation of AFB with the fermentation broth of Acinetobacter nosocomial Y1 as described in step 1) ₁ And ZEN, the fermentation broth of Acinetobacter in Hospital Y1 degrading AFB ₁ And ZEN, maintaining the degradation temperature at 28-60deg.C for 12-72 hr, and optimally degrading ZEN with fermentation broth of Acinetobacter in hospital at pH 6-10 for AFB ₁ The optimal degradation pH of (2) is 5-10.

5. The simultaneous degradation of AFB as in claim 4 ₁ And ZEN method, characterized in that in step 2), mn is added to the fermentation broth of Acinetobacter in the hospital Y1 ²⁺ 、Cu ²⁺ Or K ⁺ 。

6. A method for degrading contaminated mycotoxins in foodstuffs, comprising the steps of:

fermenting and culturing the Acinetobacter nosocomial Y1 according to claim 1 until the OD value is 0.4-1.0 to obtain fermentation liquor,

after that, the fermentation liquor is added into the AFB ₁ And ZEN toxin contaminated grain and continued to incubate at 50 ℃ for 72 hours.

7. The method for degrading contaminated mycotoxins in grain of claim 6, wherein the ratio of the volume to mass of the fermentation broth to the contaminated grain is 1:3,

the fermentation broth is obtained by culturing 24h of Acinetobacter in a hospital at 28 ℃ and 180 rpm.