CN114574398A

CN114574398A - Simultaneous degradation of AFB1Zen-blended acinetobacter Y1 in hospital and application thereof

Info

Publication number: CN114574398A
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: 2022-06-03
Anticipated expiration: 2042-03-29
Also published as: CN114574398B

Abstract

The invention discloses a method for simultaneously degrading AFB₁And ZEN, acinetobacter hospital Y1, under the classification name: acinetobacter Acinetobacter nosocomialis in hospital, Acinetobacter Acinetobacter nosocomialis Y1 in strain hospital are preserved in China general microbiological culture Collection center with the preservation numbers: CGMCC No.23794, the preservation time is as follows: 12/11/2021, the address of the depository is: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences. The invention also discloses a culture method and application of the acinetobacter Y1 in hospitals. The strain can degrade AFB simultaneously₁And ZEN, the cytotoxicity of degradation products is obviously lower than that of AFB₁And the toxicity of ZEN itself, and the degradation products themselves do not produce other inhibitory substances.

Description

Simultaneous degradation of AFB1Zen-blended acinetobacter Y1 in hospital and application thereof

Technical Field

The invention belongs to the field of biotechnologyInvolving the simultaneous degradation of AFB₁And ZEN, Acinetobacter hospital Y1 and uses thereof.

Background

Mycotoxins are toxic secondary metabolites produced by fungi, and according to the statistics of the world's Food and Agriculture Organization (FAO), about 25% of grains are contaminated with mycotoxins to varying degrees every year worldwide, and 2% of grains lose nutritional and economic value due to mycotoxin contamination, resulting in billions of dollars of economic loss. The most common mycotoxin in food is aflatoxin B₁(AFB₁) Zearalenone and vomitoxin, etc.

AFB₁The compound is a kind of secondary metabolite mainly produced by aspergillus flavus, aspergillus parasiticus and other aspergillus, and is also a naturally produced compound with the strongest carcinogenicity known at present; up to 28% of the world's primary hepatocellular carcinoma is caused by AFs, and hepatitis b carriers are more susceptible to AFs-induced liver cancer; AFs can also cause symptoms such as acute toxicity, decreased immunity, slow growth in children, etc.

Zearalenone (ZEN), also known as F-2 toxin, is a mycotoxin produced by fusarium such as fusarium graminearum, fusarium flavum, etc. and released into the environment. ZEN has estrogen-like action, presents chronic toxicity, mainly affects reproductive system, destroys reproductive function of animals and generates great harm to animal health. ZEN is a fusarium toxin that is the most polluting worldwide and its presence is detected in foodstuffs and processing by-products all over the world, e.g. europe, africa, asia, north and south america. The pollution of the grain by the ZEN will threaten the safety of the feed and the breeding industry. According to investigations, the feed in most countries receives varying degrees of pollution of ZEN. According to survey, the detection rate of ZEN in the complete feed in 2019 reaches 58 percent, and the maximum value is 5791 mu g/kg. Therefore, ZEN pollution brings great influence and huge economic loss to the breeding industry, and human health is indirectly endangered.

At present, the detoxification method of mycotoxin 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 due to its safetyThe comprehensiveness, specificity and high efficiency are gradually regarded as important. Some reports of the current AFB pairs at home and abroad₁Or ZEN respectively, including Bacillus, Pseudomonas putida, Pseudomonas aeruginosa, Rhodococcus and Enterobacter, which can convert AFB₁Or ZEN, but with concomitant degradation of AFB₁And ZEN have not been reported.

Disclosure of Invention

An object of the present invention is to solve at least the above problems and/or disadvantages and to provide at least the advantages described hereinafter.

The invention obtains a strain capable of efficiently degrading AFB (active carbon B) from hot spring water sample by screening₁And ZEN, and the strain identification and degradation mechanism analysis of the microorganisms are carried out.

The invention also aims to provide a strain for simultaneously degrading AFB₁And ZEN's hospital acinetobacter Y1.

Another object of the present invention is to provide a method for culturing Acinetobacter hospital Y1.

It is another object of the present invention to provide a method for simultaneously degrading AFB₁And ZEN.

It is yet another object of the present invention to provide a method for degrading food contaminated with mycotoxins.

Therefore, the technical scheme provided by the invention is as follows:

simultaneous degradation of AFB₁And ZEN acinetobacter Y1 hospital, acinetobacter Y1 hospital, classification name: acinetobacter Acinetobacter nosocomialis in hospital, Acinetobacter Acinetobacter nosocomialis Y1 in strain hospital are preserved in China general microbiological culture Collection center with the preservation numbers: CGMCC No.23794, the preservation time is as follows: 12/11/2021, the address of the depository is: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.

The culture method of acinetobacter Y1 in hospital comprises the following steps:

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

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

Preferably, in the method for culturing acinetobacter hospital Y1, the acinetobacter hospital Y1 is inoculated into LB culture medium in an inoculation amount of 1%, and the fermentation broth is obtained after culturing for 24 hours under the conditions of 28 ℃ and 180 rpm.

Simultaneous degradation of AFB₁And ZEN, comprising the steps of:

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

2) simultaneously degrading AFB by using the fermentation liquor of the acinetobacter hospital Y1 in the step 1)₁And ZEN.

Preferably, the simultaneous degradation of AFB₁And ZEN, step 2), said fermentation broth of Acinetobacter hospital Y1 degrades AFB₁And ZEN, the degradation temperature is kept at 28-60 ℃, and the degradation time is kept at 12-72 hours.

Preferably, the simultaneous degradation of AFB₁And ZEN, step 2), the optimum degradation pH of fermentation broth of Acinetobacter hospital Y1 for ZEN is 6-10 for AFB₁The optimal degradation pH of (1) is 5-10.

Preferably, the simultaneous degradation of AFB₁And ZEN, step 2), Mn is added to the fermentation broth of Acinetobacter hospital Y1 while degrading ZEN²⁺、Cu²⁺Or K⁺；

When degrading AFB₁When Cu is added to the fermentation liquor of Acinetobacter hospital Y1²⁺Or K⁺。

The degradation method of the grain polluted by the mycotoxin comprises the following steps:

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

then adding the fermentation liquor into the AFB₁And ZEN toxin contaminated grain, and continuing the cultivation at a certain temperature for a period of time, said contaminated grainIt is eaten in the middle.

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

the fermentation liquor is obtained by culturing acinetobacter hospital Y1 for 24 hours at the temperature of 28 ℃ and the rotating speed of 180 rpm.

The invention at least comprises the following beneficial effects:

the acinetobacter hospital Y1 can degrade ZEN and AFB simultaneously₁. And, for ZEN and AFB₁The cytotoxicity of the degradation products is significantly lower than that of the degradation products. Especially ZEN degradation products, which do not cause any inhibitory effect on Vibrio fischeri per se. The acinetobacter Y1 in hospitals can be used for ZEN and AFB in grains in practical application₁The degradation efficiency is higher, and the method can be used in practical production 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 the optimization of growth medium for Acinetobacter hospital Y1 in the example of the present invention.

FIG. 2 is a diagram showing the pH optimization of Acinetobacter hospital Y1 for growth in the examples of the present invention.

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

FIG. 4 shows the degradation of ZEN and AFB by acinetobacter Y1 in hospital in the embodiment of the invention₁The optimum temperature profile of (2).

FIG. 5 shows that Acinetobacter hospital Y1 degrades ZEN and AFB in the embodiment of the invention₁The optimal time chart of (2).

FIG. 6 shows that Acinetobacter hospital Y1 degrades ZEN and AFB in the embodiment of the invention₁Optimum pH map of (1).

FIG. 7 shows that Acinetobacter hospital Y1 degrades ZEN and AFB in the embodiment of the invention₁The highest concentration profile of (c).

FIG. 8 shows that Acinetobacter hospital Y1 degrades ZEN and AFB in the embodiment of the invention₁Activity of (2)Material distribution analytical graph.

FIG. 9 shows that metal ions degrade ZEN and AFB by acinetobacter hospital Y1 in the embodiment of the invention₁Influence graph of (c).

FIG. 10 shows the degradation of ZEN and AFB by different substances to Acinetobacter in Hospital Y1 in an embodiment of the present invention₁Influence graph of (c).

FIG. 11 shows ZEN and AFB in an example of the present invention₁And the effect of the degradation products on the activity of luminous vibrio fischeri.

FIG. 12 shows the comparison of acinetobacter Y1 in hospital on ZEN and AFB in grains in the embodiment of the invention₁And (4) degradation effect.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

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 present invention provides a biodegradable AFB₁Acinetobacter Y1 hospital of ZEN, acinetobacter Y1 of this hospital, under the classification designation: acinetobacter Acinetobacter nosocomialis in hospital, Acinetobacter Acinetobacter nosocomialis Y1 in strain hospital are preserved in China general microbiological culture Collection center with the preservation numbers: CGMCC No.23794, preservation time is as follows: 12/11/2021, the address of the depository is: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.

In some embodiments of the present invention, preferably, in the method for culturing acinetobacter hospital Y1, acinetobacter hospital Y1 is inoculated into LB medium for culture, wherein the pH of the medium is 5-10, and the culture 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 for 24h at 28 ℃ and 180rpm to obtain the fermentation liquid.

Simultaneous degradation of AFB₁And ZEN, comprising the steps of:

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

In some embodiments of the invention, preferably, in step 2), the fermentation broth of acinetobacter hospital Y1 degrades AFB₁And ZEN, the degradation temperature is kept at 28-60 ℃, and the degradation time is kept at 12-72 hours. More preferably, the optimal degradation temperature is 60 ℃. Moreover, the acinetobacter Y1 in hospital can completely degrade ZEN with 2 mug/mL in 48 hours and AFB with 2 mug/mL in 72 hours₁。

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

In some of the embodiments of the present invention, it is preferable that Mn is added to the fermentation broth of acinetobacter hospital Y1 when degrading ZEN in step 2)²⁺、Cu²⁺Or K⁺(ii) a More preferably, Mn²⁺、Cu²⁺、K⁺The concentration of (A) is 2 mmol/L;

when degrading AFB₁When Cu is added to the fermentation liquor of Acinetobacter hospital Y1²⁺Or K⁺. More preferably, Cu²⁺、 K⁺The concentration of (2) was 2 mmol/L.

When degrading AFB₁And when ZEN is adopted, SDS is not added.

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

the Acinetobacter hospital Y1 according to claim 1 is fermented and cultured to a fermentation liquid with OD value of 0.4-1.0,

then fermentingLiquid addition is AFB₁And ZEN toxin contaminated grain, and continuing the cultivation at a certain temperature for a period of time, said contaminated grain. Preferably, the culture is carried out at 50 ℃ for 72 hours with stirring every 12 hours.

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

the fermentation liquor is obtained by culturing acinetobacter Y1 in hospital at 28 ℃ and 180rpm for 24 h.

In order to make the technical solution of the present invention better understood by those skilled in the art, the following examples are now provided for illustration:

example 1

Screening of fungaltoxin degrading bacteria

Collecting samples: the experimental sample is collected from a Changbai mountain hot spring with the water temperature of 96 ℃.

Screening degrading bacteria: adding 10mL of hot spring water into 90mL of LB culture medium, culturing at 37 deg.C and 180rpm for 24h, adding 10mL of bacterial liquid, and adding ZEN and AFB with final concentration of 2ppm₁Culturing at 37 deg.C and 180rpm for 72 hr, and placing the rest bacteria liquid in 4 deg.C refrigerator for use. 10mL of LB medium was added with ZEN and AFB to a final concentration of 2ppm₁As a control group. After 72h of culture, ZEN and AFB in experimental and control groups were examined₁And (4) determining the degradation effect.

The bacterial liquid with degradation effect is diluted to different concentrations (10) by sterile water^-1、10^-2、10^-3、10^-4、10^-5、10^-6、 10^-7、10^-8) Respectively coating 100 mu L of diluent on LB solid culture medium, culturing for 24-48h in an incubator at 37 ℃, selecting a culture dish capable of selecting a monoclonal, inoculating the monoclonal on the culture dish into the LB culture medium, culturing for 24h at 37 ℃ and 180rpm, and then adding ZEN and AFB with the final concentration of 2ppm₁The 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 2ppm₁. HPLC detection of monoclonal pairs ZEN and AFB₁The degradation effect of (2) is selectedThe strain with the best solution effect.

Example 2

Identification of mycotoxin-degrading bacteria

And (3) strain identification: the 16S rDNA universal primer was used for amplification, and the amplification result was determined by 1% agarose gel electrophoresis. And (3) determining and analyzing the 16S rDNA gene sequence of the strain, and obtaining the recognized standard sequence data which is homologous with the 16S rDNA of the strain from a GenBank database by using Blast comparison at NCBI according to a sequencing result. Thereby determining that ZEN and AFB can be degraded simultaneously₁The microorganism is Acinetobacter hospital Y1(Acinetobacter nosocomialis Y1), the 16S rDNA sequence is shown in SEQ ID NO:3, and the microorganism can degrade AFB simultaneously₁And ZEN, acinetobacter hospital Y1, which is classified under the name: acinetobacter Acinetobacter nosocomialis in hospital, Acinetobacter Acinetobacter nosocomialis Y1 in strain hospital are preserved in China general microbiological culture Collection center with the preservation numbers: CGMCC No.23794, preservation time is as follows: 12/11/2021, the address of the depository is: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.

PCR reaction system and reaction program

PCR reaction system

The 16S rDNA universal primer sequence 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 hospital acinetobacter Y116S rDNA is shown in SEQ ID NO: 3:

TGGCGACGCTTACCTGCAGTCGAGCGGGGGAAGGTAGCTTGCTACTGGACCTAGCG GCGGACGGGTGAGTAATGCTTAGGAATCTGCCTATTAGTGGGGGACAACATCTCGAA AGGGATGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGATCTTCGGACCTTG CGCTAATAGATGAGCCTAAGTCGGATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAG GCGACGATCTGTAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACG GCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCT GATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTATGGTTGTAAAGCACTTTAAGCG AGGAGGAGGCTACTTTAGATAATACCTAGAGATAGTGGACGTTACTCGCAGAATAAG CACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCG GATTTACTGGGCGTAAAGCGCGCGTAGGCGGCTAATTAAGTCAAATGTGAAATCCCC GAGCTTAACTTGGGAATTGCATTCGATACTGGTTAGCTAGAGTGTGGGAGAGGATGG TAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGATGGCGA AGGCAGCCATCTGGCCTAACACTGACGCTGAGGTGCGAAAGCATGGGGAGCAAACA GGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGTCTACTAGCCGTTGGGGCCTT TGAGGCTTTAGTGGCGCAGCTAACGCGATAAGTAGACCGCCTGGGGAGTACGGTCGC AAGACTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGT TTAATTCGATGCAACGCGAAGAACCTTACCTGGCCTTGACATAGTAAGAACTTTCCA GAGATGGATAGGTGCCTTCGGGAACTTACATACAGGTGCTGCATGGCTGTCGTCAGC TCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTTTCCTTATTTG CCAGCGAGTAATGTCGGGAACTTTAAGGATACTGCCAGTGACAAACTGGAGGAAGG CGGGGACGACGTCAAGTCATCATGGCCCTTACGGCCAGGGCTACACACGTGCTACAA TGGTCGGTACAAAGGGTTGCTACACAGCGATGTGATGCTAATCTCAAAAAGCCGATC GTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCG CGGATCAGAATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACA CCATGGGAGTTTGTTGCACCAGAAGTAGCTAGCCTAACTGCAAAGAGGGCGGTACC ACGAGTACC

example 3

Growth condition optimization of mycotoxin degrading bacteria

Acinetobacter hospital Y1 was inoculated into LB medium, cultured at 37 ℃ and 180rpm for 24 hours, then inoculated with 1% inoculum size into LB (purchased from oxoid Co., Ltd.), NB (purchased from Beijing Solebao science and technology Co., Ltd.) and MM (purchased from Haibo Biotechnology Co., Ltd.) medium, cultured at 37 ℃ and 180rpm for 24 hours, and absorbance of the bacteria was detected by ultraviolet spectrophotometer to determine the optimal growth medium for the bacteria. The final results showed that the LB medium was the optimal growth medium for Y1, as shown in FIG. 1.

Inoculating acinetobacter Y1 in hospital into LB culture medium, culturing at 37 deg.C and 180rpm for 24h, respectively inoculating with 1% inoculum size into LB culture medium with pH of 2-10, culturing at 37 deg.C and 180rpm for 24h, and detecting absorbance of bacteria by ultraviolet spectrophotometer. The final results showed that Hospital Acinetobacter Y1 can grow normally in the pH range 5-10 (FIG. 2)

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

Example 4

Optimization of degradation conditions of mycotoxin degrading bacteria

Inoculating Acinetobacter hospital Y1 into LB medium at 1% inoculum size, culturing at 28 deg.C and 180rpm for 24 hr, and adding ZEN and AFB into multiple groups of culture solution to obtain final concentration of 2 μ g/mL₁Degrading the mixture for 72 hours at the conditions of 28 ℃, 37 ℃, 45 ℃, 55 ℃, 60 ℃ and 180rpm respectively, and then detecting ZEN and AFB by HPLC₁The optimum degradation temperature was determined to be 60 deg.c (fig. 4).

Inoculating Y1 into LB medium at 1% inoculum size, culturing at 28 deg.C and 180rpm for 24h, adding ZEN and AFB at final concentration of 2 μ g/mL₁And degrading at 60 ℃ and 180 rpm. Sampling every 6h or 12h, and then detecting ZEN and AFB by HPLC₁Finally, the determination that the acinetobacter Y1 in hospital can completely degrade 2 mug/mL ZEN within 48 hours and completely degrade 2 mug/mL AFB within 72 hours₁(FIG. 5).

Inoculating Acinetobacter hospital Y1 into LB culture medium with pH value of 2-10 according to 1% inoculum size, culturing at 28 deg.C and 180rpm for 24 hr,ZEN and AFB were added to a final concentration of 2. mu.g/mL, respectively₁Degrading for 72h at 60 ℃ and 180 rpm. Then detecting ZEN and AFB by HPLC₁Finally determining the optimum degradation pH of the acinetobacter Y1 in hospital to ZEN to be 6-10 for AFB₁The optimal degradation pH of (2) was 5-10 (FIG. 6).

Inoculating Acinetobacter hospital Y1 into LB medium at 1% inoculum size, culturing at 28 deg.C and 180rpm for 24h, adding ZEN and AFB at final concentrations of 5, 10, 20, 30, 40 and 50 μ g/mL₁Degrading for 72h at 60 ℃ and 180 rpm. Then detecting ZEN and AFB by HPLC₁The residual quantity of (2) confirms that the acinetobacter Y1 in hospital can completely degrade 20 mu g/mL ZEN and 5 mu g/mL AFB within 72h₁(FIG. 7).

Respectively inoculating acinetobacter Y1 in hospital into LB culture medium according to 1% inoculum size, culturing at 28 deg.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 centrifuged sediment is acinetobacter Y1 cells in hospital, and after being washed for 3 times by sterile PBS, the cells are redissolved by PBS with the same volume to obtain acinetobacter Y1 cell suspension in hospital; half volume of Acinetobacter hospital Y1 cell suspension was taken for cell disruption, and then centrifuged at 1000rpm for 10min, and the supernatant was taken and passed through a 0.22 μm filter to obtain the cell content. Acinetobacter hospital Y1 bacterial solution was used as a control group. ZEN and AFB were added to a final concentration of 2. mu.g/mL, respectively₁Degrading for 72h at 60 ℃ and 180 rpm. Then detecting ZEN and AFB by HPLC₁Determining the pair ZEN and AFB in Acinetobacter Y1 in hospital₁The active substance responsible for the degradation was mainly distributed in the supernatant after fermentation of Acinetobacter Y1 in hospital (fig. 8).

Inoculating Acinetobacter hospital Y1 to Mn solutions each containing Mn at a final concentration of 2mmol/L in an amount of 1% respectively²⁺、 Ca²⁺、Mg²⁺、Na⁺、Fe³⁺、Cu²⁺、Li²⁺、K⁺、Fe²⁺、Zn²⁺The LB medium of (1), cultured at 28 ℃ and 180rpm for 24 hours, and ZEN and AFB were added to the respective final concentrations of 2. mu.g/mL₁Degrading for 72h at 60 ℃ and 180 rpm. Then detecting ZEN and AFB by HPLC₁To finally determine Mn²⁺、Cu²⁺And K⁺Can obviously enhance the degradation capability of Y1 to ZEN, and Cu²⁺And K⁺Can obviously enhance the AFB to acinetobacter Y1 in hospitals₁The degradation ability of (1) (FIG. 9).

Respectively inoculating acinetobacter Y1 in hospital into LB culture medium according to 1% inoculum size, culturing at 28 deg.C and 180rpm for 24h, centrifuging at 1000rpm for 10min, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain sterile supernatant; adding SDS, protease K, EDTA and boiled water into the supernatant respectively, and adding ZEN and AFB with final concentration of 2 μ g/mL respectively₁Degrading for 72h at 60 ℃ and 180 rpm. Then detecting ZEN and AFB by HPLC₁The result shows that SDS can obviously inhibit Y1 supernatant from degrading ZEN and AFB₁Degradation of ZEN and AFB by acinetobacter Y1 in hospital at high temperature₁The capacity had no significant effect (fig. 10).

Example 5

Cytotoxicity assay for mycotoxin degradation products

Inoculating Acinetobacter acinetobacter Y1 in hospital into LB culture medium according to 1% inoculum size, culturing at 28 deg.C and 180rpm for 48h, centrifuging at 1000rpm for 10min, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain sterile supernatant. ZEN and AFB were added to the mixture at a final concentration of 50. mu.g/mL₁Degrading for 72h at 60 ℃ and 180 rpm. Then detecting ZEN and AFB by HPLC₁To ensure ZEN and AFB₁Is completely degraded to obtain ZEN and AFB₁Degradation product solution. Control groups were LB supplemented with ZEN and AFB to a final concentration of 50. mu.g/mL₁And sterile supernatant of acinetobacter hospital Y1. Vibrio fischeri was cultured in 2216E liquid medium (purchased from Beijing Solebao technologies Co., Ltd.) at 28 ℃ and 180rpm for 24 hours, and then degradation product solutions and control solutions of equal volumes were added, respectively, and cultured at 28 ℃ and 180rpm for 72 hours. Then, the luminescence property of Vibrio fischeri was detected by a spectrophotometer (detection wavelength: 550nm), and the Vibrio fischeri cell viability was calculated. As shown in the results of FIG. 11, the inhibition rate of ZEN to Vibrio fischeri is 32%, while the ZNE degradation product has no inhibition effect on Vibrio fischeri; AFB₁The inhibition ratio of Vibrio fischeri was 31% while AFB₁The inhibition rate of the degradation product on the vibrio fischeri is 8 percent. It can be seen that ZEN and AFB₁The cytotoxicity of the degradation products is significantly lower than that of the degradation products. Especially ZEN degradation products, which do not cause any inhibitory effect on Vibrio fischeri per se.

Example 6

Application of acinetobacter Y1 in hospital in food polluted by mycotoxin

Acinetobacter hospital Y1 was inoculated in LB medium at 1% inoculum size, and cultured at 28 ℃ and 180rpm for 24 hours. Respectively adding the bacterial liquid and the grain polluted by the mycotoxin according to the proportion of 1:3 (bacterial liquid: grain), culturing for 72 hours at the temperature of 50 ℃, and stirring once every 12 hours. The control group is food contaminated by LB culture medium and mycotoxin. After the culture is finished, the mycotoxin in the grain of the control group and the grain of the treatment group are respectively extracted by using an immunoaffinity column, and the content of the mycotoxin is detected. As shown in FIG. 12 (degradation rate is (control toxin content-treatment toxin content) × 100%/control toxin content), Acinetobacter hospital Y1 was used for AFB in grain₁The degradation efficiency is high, the degradation rate reaches 67%, the degradation efficiency of the ZEN in the grains is poor, but the degradation rate also reaches 41%.

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

While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

SEQUENCE LISTING

<110> agricultural product processing institute of Chinese academy of agricultural sciences

<120> acinetobacter hospital Y1 capable of simultaneously degrading AFB1 and ZEN and application thereof

<130> 2021

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> Artificial Synthesis

<400> 1

agagtttgat cctggctcag 20

<210> 2

<211> 22

<212> DNA

<213> Artificial Synthesis

<400> 2

tacggctacc ttgttacgac tt 22

<210> 3

<211> 1429

<212> DNA

<213> Acinetobacter nosocomialis of Hospital

<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 AFB₁And ZEN acinetobacter Y1 hospital, acinetobacter Y1 hospital, classification name: acinetobacter Acinetobacter nosocomialis in hospital, Acinetobacter Acinetobacter nosocomialis Y1 in strain hospital are preserved in China general microbiological culture Collection center with the preservation numbers: CGMCC No.23794, preservation time is as follows: 12/11/2021, the address of the depository is: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.

2. The culture method of acinetobacter Y1 in hospital is characterized by comprising the following steps:

the Acinetobacter hospital Y1 according to claim 1 is inoculated into LB medium or NB medium and cultured to obtain a fermentation liquid.

3. The method for culturing Acinetobacter hospital Y1 according to claim 2, wherein said Acinetobacter hospital Y1 is inoculated into LB medium for culture at a pH of 5-10 and a culture temperature of 28-45 ℃.

4. The method for culturing Acinetobacter hospital Y1 according to claim 2, wherein said Acinetobacter hospital Y1 is inoculated into LB medium at an inoculum size of 1%, and cultured at 28 ℃ and 180rpm for 24 hours to obtain said fermentation broth.

5. Simultaneous degradation of AFB₁And ZEN, characterized by the steps of:

1) culturing acinetobacter hospital Y1 according to claim 1 to obtain a fermentation broth;

6. Simultaneous degradation of AFB as claimed in claim 5₁And ZEN, characterized in that, in the step 2), the fermentation liquid of the acinetobacter hospital Y1 degrades AFB₁And ZEN, the degradation temperature is kept at 28-60 ℃, and the degradation time is kept at 12-72 hours.

7. Simultaneous degradation of AFB according to claim 5₁And ZEN, characterized in that, in the step 2), the pH of the fermentation liquid of Acinetobacter hospital Y1 for optimum degradation of ZEN is 6-10, and for AFB₁The optimum degradation pH of (2) is 5-10.

8. Simultaneous degradation of AFB as claimed in claim 5₁And ZEN, characterized in that in the step 2), Mn is added to the fermentation liquor of acinetobacter hospital Y1 when degrading ZEN²⁺、Cu²⁺Or K⁺；

9. The degradation method of the food polluted by the mycotoxin is characterized by comprising the following steps:

then adding the fermentation liquor into the AFB₁And ZEN toxin contaminated grain, and continuing the cultivation at a certain temperature for a period of time, said contaminated grain.

10. The method of degrading mycotoxin contaminated foodstuff according to claim 9, wherein the volume to mass ratio of the fermentation broth to the contaminated foodstuff is 1:3,