CN107164280B - Vomitoxin degrading bacterium and application thereof - Google Patents
Vomitoxin degrading bacterium and application thereof Download PDFInfo
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Abstract
The invention discloses a vomitoxin degrading bacterium and application thereof. The degrading bacteria are obtained by selecting soil polluted by DON in paddy rice planting farmland in Yangjiang city, Guangdong province, culturing the soil by using a DON-containing enrichment medium, performing acclimation culture on an inorganic salt culture medium by using DON as a unique carbon source, performing pressure screening, separating and purifying. The microbial strain collection is named as Enterobacter cloacae (Enterobacter cloacae) WD601, is preserved in Guangdong province microbial strain collection center of Guangdong province microbial research institute of No. 59 building, No. 5 building, Guangzhou Miao 100 institute of Middleya Zhou, Guangzhou city in 2017 and in 02 month, and the preservation number is as follows: GDMCC No: 60194. the strain grows and breeds by taking DON as a unique carbon source and energy under an aerobic condition, and is rapidly degraded, the stability of degraded DON is good, the DON degradation rate can reach more than 50% after passage of 5-8 generations in an optimized culture medium.
Description
Technical Field
The invention relates to the technical field of biodegradation, and particularly relates to a vomitoxin degrading bacterium and application thereof.
Background
Vomitoxin (DON), also known as deoxynivalenol, is a B-type trichothecene toxin mainly produced by fusarium graminearum and fusarium flavum, and has serious pollution to grains. DON is known to cause a range of diseases in humans and animals, such as emesis, food refusal, diarrhea, esophageal perforation, and malabsorption, among which pigs are most susceptible to vomitoxin; meanwhile, DON can make the cell which is rapidly increased and in a division state to die, and high-concentration DON can inhibit the proliferation of animal immune cells and is easy to cause abortion of animals in gestation period or has teratogenic effect on embryos.
The DON has the highest detection rate in cereal mycotoxin, corresponding standards are set in different regions, for example, the safety standard of the DON in food is 1mg/kg as specified by FDA, and the DON allowable limit of products such as wheat and the like is not more than 1mg/kg as specified in GB2715-2005 and GB2761-2011 in China. At present, the mycotoxin pollution has actual and serious negative effects on food safety and agricultural product export in China, and how to effectively detoxify the mycotoxin pollution becomes a great social problem to be solved urgently.
At present, the detoxification method of the mildewed grains and feeds at home and abroad mainly comprises a physical adsorption method, a chemical detoxification method, a biological detoxification method and the like. The physical adsorption method has reversible DON adsorption and poor efficiency, and is easy to adsorb trace nutrient elements, thereby causing the loss of nutrient substances in cereals and feeds; chemical detoxification easily leaves some potential toxic substances, and causes secondary pollution to grains and feeds, so that the DON traditional detoxification method has certain limitation in practical application; the biological detoxification method is characterized in that DON is acted by enzyme released by microorganisms and is converted into a metabolite with lower toxicity, and the microbial degradation has the advantages of low cost, high efficiency, less nutrient loss, no secondary pollution, good ecological restoration and the like, so that great attention is paid.
The DON degrading bacteria reported in the early period are mostly derived from animal intestinal contents, and most anaerobic bacteria are separated from intestinal tracts and bovine rumens. Although the anaerobic flora can act on the DON and has a good degradation effect, the separation and purification of the strains are extremely difficult, and the strict and harsh reaction conditions are not suitable for large-scale DON biological detoxification; and most of the reported strains of facultative anaerobes are mixed flora, and the microbial strains for decomposing DON are not subjected to pure strain separation and identification. Therefore, a need exists for a pure aerobic or facultative anaerobic strain capable of efficiently degrading DON, thereby driving further development and application of DON biodegradation technology in grains and feeds.
Disclosure of Invention
In order to overcome the defects and shortcomings in the existing DON microbial degradation, the invention aims to provide a vomitoxin degrading bacterium. The bacterium is vomitoxin degrading bacterium which is screened and separated from soil polluted by DON, can grow in an inorganic salt culture medium by taking the DON as a unique carbon source under aerobic conditions, and can efficiently degrade the DON.
Another object of the present invention is to provide the use of the above-mentioned vomitoxin-degrading bacterium.
The purpose of the invention is realized by the following technical scheme:
the invention provides a vomitoxin degrading bacterium, which is named enterobacter cloacae (Enterobactererccloacae) WD601 and is obtained by selecting soil polluted by DON in rice planting farmland in Yangjiang city, Guangdong province, culturing the soil by using a DON-containing enrichment medium, and then performing acclimation culture, pressure screening, separation and purification on an inorganic salt medium with DON as a unique carbon source.
The deposit information of Enterobacter cloacae (Enterobacter cloacae) WD 601: the preservation unit: guangdong province culture Collection (GDMCC), preservation date: year 2017, 06, 02 month, deposit address: the microbial research institute of Guangzhou province, No. 59 building, No. 5 building, Guangdong province, of the Zhonglu-Jieli, Guangzhou city, the preservation number: GDMCC No: 60194.
the WD601 has 99% homology of 16S rDNA with Enterobacter cloacae. The 16S rDNA sequence of the strain is shown as SEQ ID NO: 1 is shown.
The invention also provides a vomitoxin degrading reagent containing the vomitoxin degrading bacterium.
The invention also provides application of the vomitoxin degrading bacteria in degrading vomitoxin.
The invention also provides application of the vomitoxin degrading reagent in degrading vomitoxin.
Further, the application of the vomitoxin degrading bacteria in degrading the vomitoxin comprises the following steps:
enterobacter cloacae (Enterobacter cloacae) WD601 was inoculated into an inorganic salt medium containing DON at 40 to 60. mu.g/mL in an inoculum size of 10%, and cultured at 37 ℃ for 72 to 168 hours.
The inorganic salt culture medium comprises: 0.5g/L (NH)4)2SO4，0.2g/L MgSO4·7H2O，0.05g/L CaCl2，2.44g/L Na2HPO4And 1.52g/L KH2PO4Adjusting the pH value to 7.0, and sterilizing at 121 ℃ for 15-20 min.
Inorganic salt medium was used as negative control, and inorganic salt medium containing 40, 60. mu.g/mL DON was used as positive control.
Compared with the prior art, the invention has the following advantages and effects:
(1) in the invention, a microorganism strain Enterobacter cloacae WD601 strain with good DON degradation performance is obtained by separation and purification, DON is taken as a unique carbon source and energy source to grow and reproduce under aerobic conditions, and the DON is rapidly degraded at the same time, and the degradation rate is more than 50%. The Enterobacter cloacae WD601 strain can grow in an inorganic salt culture medium, and the screening, separating and purifying technology is simple.
(2) The Enterobacter cloacae (Enterobacter cloacae) WD601 strain has good DON degradation stability, and can be subcultured for 5-8 generations in an optimized culture medium, and the DON degradation rate can reach more than 50%. In a basic inorganic salt culture medium, when the initial DON concentration is 40mg/L and 60mg/L, the DON degradation rate after 7 days of culture at 37 ℃ is 21.47 percent and 40.40 percent respectively.
Drawings
FIG. 1 is a colony morphology of Enterobacter cloacae (Enterobacter cloacae) WD601 strain provided by the present invention; wherein, a: colony morphology on the plate; b: the shape of the thallus under a microscope; c: cell morphology (20,000X) under scanning electron microscope.
FIG. 2 shows the result of PCR amplification of 16S rDNA fragment of Enterobacter cloacae WD601 according to the present invention.
FIG. 3 is a phylogenetic tree of Enterobacter cloacae (Enterobacter cloacae) WD601 provided by the present invention.
FIG. 4 is a standard curve plot of DON standards detected by HPLC as provided by the present invention.
FIG. 5 shows the DON content before and after the HPLC detection reaction of Enterobacter cloacae WD601 provided by the present invention; wherein, a: negative control with inorganic salt medium without DON; b: a positive control with an initial DON concentration of 60 μ g/mL; c: when the initial concentration of DON is 60 mug/mL, the degradation rate is achieved after 3d of reaction; d: degradation rate after 7d of reaction when the initial concentration of DON was 60. mu.g/mL.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1 screening, isolation and purification of Enterobacter cloacae WD601
(1) Enrichment of strains
After the soil sample is sieved by a 40-mesh sieve, 1g of the soil sample is respectively taken and mixed with 3mL of physiological saline. 100 μ L of the suspension was added to 5mL of enrichment medium containing 10 μ g/mL DON, and three different temperatures were set: cultured at 25 ℃, 30 ℃, 37 ℃ and 180r/min for 72 h.
The enrichment medium containing DON is as follows: 10g of peptone, 1g of NaCl and 1g of glucose were weighed, dissolved in 1000mL of sterile distilled water, pH was adjusted to 7.0, and sterilized at 121 ℃ for 15 min.
(2) Screening of strains by coating
Respectively sucking 100 μ L of the enriched solution, centrifuging at 3,000r/min for 5min, discarding the supernatant, suspending and precipitating with 100 μ L of DON inorganic salt liquid culture medium containing 20 μ g/mL, spreading on a flat plate, and culturing at 25 deg.C, 30 deg.C, and 37 deg.C for 24 hr.
The inorganic salt culture medium comprises: 0.5g (NH) was weighed4)2SO4，0.2g MgSO4·7H2O，0.05g CaCl2，2.44g Na2HPO4And 1.52g KH2PO4Dissolving in 1000mL sterile distilled water, adjusting pH to 7.0, and sterilizing at 121 deg.C for 15 min.
(3) Screening strains by plate streaking
And after the culture medium is sterilized, adding the DON standard solution into the culture medium after passing through a 0.22 mu m microporous filter membrane so that the DON final concentration is 20 mu g/mL. Single colonies were picked and inoculated in mineral salts medium and cultured in incubators at 25 deg.C, 30 deg.C, 37 deg.C for 24 h. Single colonies were picked again, streaked on mineral salts medium for 24h, and streaking was repeated in this manner to purify the strain.
Selecting and culturing a fresh single colony for 18-24 h, performing gram staining, and observing the physiological form of the strain under a microscope. Inoculating a single colony into an inorganic salt culture medium for overnight culture, centrifuging at 5,000r/min for 5min, then discarding the supernatant, suspending the thallus with a proper amount of distilled water, centrifuging at 5,000r/min for 5min, repeating the operation once, discarding the supernatant, then suspending the thallus with 2mL of sterile distilled water, freeze-drying to obtain bacterial powder, and observing by using a scanning electron microscope.
On the basis that the strain grows on an inorganic salt medium and can only utilize DON as a unique carbon source, the strain capable of degrading DON can be separated from the plate. Culturing the strain enrichment solution on a DON inorganic salt solid culture medium containing 20 mug/mL for 24h, then selecting a single colony for streak purification, wherein the colony in the culture medium at 37 ℃ grows fastest. A white strain is screened from the rice soil of Guangdong Yangjiang through repeated streaking and purification, the growth condition of the strain on a basic inorganic salt culture medium is shown in figure 1a, the colony is circular, the diameter is about 1mm, the edge is neat, the surface is moist and glossy, and the colony is grey white. The shape of the bacterial cells was observed under a microscope by gram staining, and the bacterial cells were gram-negative bacteria and were short rods, as shown in FIG. 1 b. After freeze-drying the centrifuged cells, the dried cell powder was observed by SEM and the cells were in the form of short rods with irregular length and round ends and a size of 0.5X 1-3 μm at a magnification of 20,000 (FIG. 1 c). The strain can grow by taking DON as a unique carbon source, and the DON degrading capability is shown. This strain was named WD601, inoculated into a DON-containing liquid medium, cultured and tested for its degradability.
Example 2 identification of isolated strains
(1) Colony morphology identification
Selecting a fresh single colony of WD601 cultured for 18-24 h for gram staining, and observing the physiological form of the strain under a microscope. Inoculating a single colony into an inorganic salt culture medium for overnight culture, centrifuging at 5,000r/min for 5min, then discarding the supernatant, suspending the thallus with a proper amount of distilled water, centrifuging at 5,000r/min for 5min, repeating the operation once, discarding the supernatant, then suspending the thallus with 2mL of sterile distilled water, freeze-drying to obtain bacterial powder, and observing by using a scanning electron microscope.
The morphological characteristics of the WD601 provided by the present invention are as follows: after the strain is cultured on an LB plate at 37 ℃ for 24h, a colony is circular as shown in figure 1a, the diameter is about 1mm, the edge is neat, and the surface is moist, glossy and grey-white. The shape of the bacterial cells was observed under a microscope by gram staining, and the bacterial cells were gram-negative bacteria and were short rods, as shown in FIG. 1 b. After freeze-drying the centrifuged cells, the dried cell powder was observed by SEM and the cells were in the form of short rods with irregular length and round ends and a size of 0.5X 1-3 μm at a magnification of 20,000 (FIG. 1 c).
(2) Analysis of biochemical Properties
The white bacterial strain is subjected to physiological and biochemical identification by referring to a common bacterial strain identification method in 'handbook of bacteria system identification'.
Oxidative fermentation of glucose and other sugar alcohols: the WD601 fresh single colony cultured for 18-24 h is punctured and inoculated in Huffson and Liffson two's culture medium, and is covered with sterilized vaseline paraffin oil with the thickness of about 0.5-1 cm to isolate air, and meanwhile, an experimental group without oil sealing is designed to verify the oxidation fermentation type of the strain. After incubation at room temperature for 1, 2, 3, 7, 14d, the color change was observed. And respectively replacing glucose with lactose, maltose, mannose, arabinose, fructose, xylose and sucrose to detect the sugar alcohol utilization condition of the strain.
The Huffson two-culture medium comprises: weighing 2g of peptone, 5g of NaCl, 10g of glucose and 0.2g of K2HPO4The mixture was dissolved in 1000mL of sterile distilled water, 3mL of 1% bromothymol blue indicator was added, and the mixture was sterilized at 115 ℃ for 20 min. In sugar alcohol fermentation experiments, glucose was replaced with 1% of sugar alcohol to be tested.
Methyl red and V.P determination: inoculating a WD601 single colony in a culture solution, culturing for 2-6 days at a proper temperature, adding a drop of methyl red reagent into a culture medium, and making the reagent become red positive; the culture solution and 40% NaOH are mixed in equal amount, a little creatine is added, and the V.P experiment is positive if red color appears within 10 min.
The methyl red and V.P culture medium is: 5g of peptone, 5g of glucose, 5g K g of glucose were weighed2HPO4Dissolved in 1000mL of sterile distilled water, and sterilized at 115 ℃ for 20 min.
Starch hydrolysis: and (3) dibbling a fresh single colony on a starch culture medium, culturing for 2-5 days at a proper temperature to form obvious colonies, dropwise adding an iodine solution on a flat plate, and observing whether a non-discoloring transparent ring exists around the colony.
The starch culture medium is as follows: adding 0.2% soluble starch into meat peptone, and sterilizing at 121 deg.C for 20 min.
Oxidase and catalase: taking a fresh single colony cultured for 18-24 h, smearing the single colony on filter paper wetted by 1% dimethyl p-phenylenediamine hydrochloride water solution, and observing color change within 10 s; fresh single colony is smeared on the drop of 10% H2O2On the slide glass, the presence or absence of bubble generation was observed.
And (3) detecting L-tryptophan dehydrogenase and urease: inoculating a fresh single colony for culturing for 24h, and if the culture solution turns yellow to red, then urease exists; 2-4 drops of the culture solution are mixed with a ferric trichloride solution (33%), and the color change is observed, if the color is reddish brown, L-tryptophan dehydrogenase exists.
The detection culture medium for the L-tryptophan dehydrogenase and the urease comprises: weighing 3g L-tryptophan, 5g NaCl and 1gK2HPO4，1g KH2PO410mL of 95% ethanol was added, dissolved in 1000mL of distilled water, and sterilized at 121 ℃ for 20 min. Dissolving urea in 100mL of water, filtering and sterilizing, and adding the sterilized culture medium into the culture medium.
Gelatin liquefaction and hydrogen sulfide experiments: taking a fresh single colony for puncture inoculation, culturing for 1, 3 and 7 days at a proper temperature, observing the color change of the culture medium, and turning black to be positive by hydrogen sulfide; and observing whether the gelatin is liquefied.
The hydrogen sulfide detection culture medium comprises: weighing 7.5g of beef extract, 10g of peptone, 5g of NaCl and 120g of gelatin, dissolving in 1000mL of sterile distilled water, and sterilizing at 121 ℃ for 20 min. 5mL of 10% FeCl is added after the culture medium is sterilized2(filtration sterilization).
The physiological and biochemical results of the strains are shown in table 1, sugar alcohol fermentation experiments are the most main and basic experiments for identifying bacteria, and are particularly important for identifying bacteria in enterobacteriaceae, and experiments show that WD601 is a fermentation type strain, can utilize glucose, lactose, maltose, mannitol, fructose, xylose, sucrose and the like, and cannot utilize arabinose. M.R the experiment is mainly for testing the ability of the strain to produce a large amount of acidic products such as lactic acid, succinic acid, acetic acid and formic acid by using glucose, the experimental result is negative, which indicates that the WD601 strain may be a gas-producing strain. V.P, the test is positive, which shows that the strain decomposes glucose into pyruvic acid, further decarboxylates to generate acetyl methyl ethanol, and finally reacts with NaOH to generate red compound. WD601 strain can also hydrolyze starch and act on semi-solid gelatin to produce liquid amino acids, produce catalase and urease, and do not produce oxidase and L-tryptophan dehydrogenase. The experimental results were essentially consistent with the biochemical characteristics of enterobacteriaceae, and it was preliminarily determined that WD601 strain belongs to the family enterobacteriaceae.
TABLE 1 physio-biochemical Properties of WD601
| Item | Results | Item | Results |
| Oxidation-fermentation (O/F) | Fermentation type | Starch hydrolysis | + |
| Glucose | + | V.P experiment | + |
| Lactose | + | M.R experiment | - |
| Maltose | + | Hydrolysis of gelatin | + |
| Mannitol | + | Hydrogen sulfide | - |
| Arabinose | - | Oxidase enzyme | - |
| Fructose | + | Contact enzyme | + |
| Xylose | + | Urease | + |
| Sucrose | + | L-tryptophan dehydrogenase | _ |
Note: "+" indicates a positive result. "-" indicates negative results.
(3)16S rDNA Gene sequence determination
Extracting the whole genome of the strain culture solution by adopting a genome extraction kit, and designing an upstream primer 27F:5 '-AGAGTTTGATCMTGGCTCAG-3' and downstream primer 1492R: 5 '-TACGGYTACCTTGTTACGACTT-3' to perform PCR amplification reaction, wherein the reaction system is as follows: 2.5. mu.L of genomic DNA, 2.5. mu.L of 10 XBuffer (with Mg)2+) mu.L of dNTPs (10mol/L), 0.2. mu.L of enzyme (5U/. mu.L), and 0.5. mu.L of each of the upstream and downstream primers (10mol/L), and sterile distilled water was added to the mixture to make 25. mu.L. The PCR reaction conditions were as follows: pre-denaturation at 94 ℃ for 4min, denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 45s, extension at 72 ℃ for 60s, extension at 72 ℃ for 10min after 30 cycles, and detection of the amplification product by 1% agarose gel electrophoresis. The amplification product was purified using a PCR purification kit and sent to Meiji Biotechnology Ltd for sequencing. And submitting the sequencing result to GenBank for BLAST comparison, and selecting a strain with higher homology for evolutionary tree analysis by adopting MEGA software.
The above data were determined in triplicate and averaged, the test results are expressed in x + -s, statistical analysis and processing of the experimental data was performed using Origin 85, and sequence homology was analyzed using GenBank and MEGA7.0.
Extracting bacterial DNA in a fresh WD601 culture solution by using a bacterial DNA extraction kit, performing PCR amplification according to the PCR system in the above by using the genomic DNA of a strain as a template, and finding that a specific band appears at about 1500bp of a product by 1% agarose gel electrophoresis, wherein the specific band is shown in figure 2. Sequencing the purified PCR product, outputting a peak diagram, submitting sequence results to GenBank for BLAST comparison, finding that the strain has higher homology with Enterobacteriaceae (Enterobacteriaceae), selecting 16 strains with more than 98 percent of sequence homology, constructing an evolutionary tree by MEGA7.0.25 software, and obtaining an evolutionary tree result shown in figure 3, wherein the WD601 strain is in the same main branch with Enterobacter (Enterobacter) and Western (Cedecetadavasae), but has closer genetic relationship with Enterobacter cloacae (Enterobacter cloacae) of the Enterobacter from genetic distance analysis, so that the strain WD601 is identified as the Enterobacter and accords with WD601 physiological and biochemical experiment results.
The invention finally identifies the strain to the genus, and the result shows that the strain is identified as Enterobacter cloacae WD601, and the homology with Enterobacter cloacae is 99%. In the present invention, the obtained strain was named Enterobacter cloacae (Enterobacter cloacae) WD 601.
(4) Strain preservation
Preparing the strain culture into 30% glycerol tube, and storing in-80 deg.C refrigerator for use. The strain is preserved in Guangdong province microbial culture collection center (GDMCC) in 2017, 6 and 2 days, the center is located in Guangdong province microbial research institute of No. 59 building, No. 5 building, Guangdong province, Mieli Zhouyu No. 100, Guangzhou city, the strain number is GDMCC No: 60194.
example 3 use of Enterobacter cloacae (Enterobacter cloacae) WD601 to degrade DON
(1) Degradation rate of DON by HPLC
After 1mL of culture solution is filtered through a 0.22mL microporous filter membrane, the content of DON in the degraded culture medium is detected by HPLC with an ultraviolet detector. The conditions for detecting DON by high performance liquid chromatography are as follows: a column (4.6X 150mm, 5 μm) from Agilent ZORBAX SB-C18 was used, and the mobile phase used methanol/water (20/80V/V). The column temperature during the test was 35 ℃, the flow rate was 0.9mL/min, and the wavelength of the detector was 218 nm. The DON degradation rate was calculated using the following formula:
(2) and (3) drawing a DON standard curve: DON stock solution (1mg/mL) is taken and diluted to 4, 10, 20, 50 and 100 mu L/mL working solution respectively, 20 mu L of sample is injected, and the average value is obtained by repeating the measurement for three times. And drawing a DON standard curve according to the relation between the concentration and the peak area.
Respectively preparing DON working solution with concentration of 4, 10, 20, 50 and 100 μ g/mL, measuring DON concentration and peak area relation standard curve, as shown in FIG. 4, and correlation coefficient R2＝0.9999。
(3) DON degradation reaction
Enterobacter cloacae (Enterobacter cloacae) WD601 was inoculated into 2mL of liquid medium containing 40. mu.g/mL of LDON and cultured for 3 days and 7 days, respectively, to amplify the DON concentration (60. mu.g/mL) to repeat the experiment. Inorganic salt medium was used as a negative control, and inorganic salt medium containing 40. mu.g/mL and 60. mu.g/mL DON was used as a positive control.
The DON degradation capability of WD601 is detected by HPLC, the result is shown in Table 2, after a single WD601 colony is inoculated to DON with the final concentration of 40 and 60 mu g/mL for reaction for 3d and 7d, the DON concentration is reduced to different degrees, which indicates that WD601 has the capability of obviously degrading DON. As shown in Table 2, the time of DON peak was about 16.20min (FIG. 5b) in the case of the negative control of inorganic salt medium without DON (FIG. 5a), and the degradation rate was 7.1% after 3d (FIG. 5c) and 40.40% after 7d (FIG. 5d) at the initial DON concentration of 60. mu.g/mL.
TABLE 2 HPLC determination of DON degradation of the strains under different reaction conditions
(4) The Enterobacter cloacae WD601 is prepared into a liquid or solid vomitoxin degradation reagent containing the strain after optimized expansion culture, and the reagent also has a degradation effect. The degradation reagent is used for DON degradation experiments by adopting the same method as the step (3), and the degradation efficiency reaches more than 50%.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> university of southern China's science
<120> vomitoxin degrading bacterium and application thereof
<130>1
<160>3
<170>PatentIn version 3.5
<210>1
<211>1448
<212>DNA
<213>Artificial Sequence
<220>
<223> 16S rDNA sequence of Enterobacter cloacae (Enterobacter cloacae) WD601
<400>1
ccctcgggat acacacgtgg tagcgccctc ccgaaggtta agctacctac ttcttttgca 60
acccactccc atggtgtgac gggcggtgtg tacaaggccc gggaacgtat tcaccgtagc 120
attctgatct acgattacta gcgattccga cttcatggag tcgagttgca gactccaatc 180
cggactacga cgcactttat gaggtccgct tgctctcgcg aggtcgcttc tctttgtatg 240
cgccattgta gcacgtgtgt agccctactc gtaagggcca tgatgacttg acgtcatccc 300
caccttcctc cagtttatca ctggcagtct cctttgagtt cccggcctaa ccgctggcaa 360
caaaggataa gggttgcgct cgttgcggga cttaacccaa catttcacaa cacgagctga 420
cgacagccat gcagcacctg tctcagagtt cccgaaggca ccaatccatc tctggaaagt 480
tctctggatg tcaagagtag gtaaggttct tcgcgttgca tcgaattaaa ccacatgctc 540
caccgcttgt gcgggccccc gtcaattcat ttgagtttta accttgcggc cgtactcccc 600
aggcggtcga cttaacgcgt tagctccgga agccacgcct caagggcaca acctccaagt 660
cgacatcgtt tacggcgtgg actaccaggg tatctaatcc tgtttgctcc ccacgctttc 720
gcacctgagc gtcagtcttt gtccaggggg ccgccttcgc caccggtatt cctccagatc 780
tctacgcatt tcaccgctac acctggaatt ctacccccct ctacaagact ctagcctgcc 840
agtttcgaat gcagttccca ggttgagccc ggggatttca catccgactt gacagaccgc 900
ctgcgtgcgc tttacgccca gtaattccga ttaacgcttg caccctccgt attaccgcgg 960
ctgctggcac ggagttagcc ggtgcttctt ctgcgggtaa cgtcaatcgc tgaggttatt1020
aacctcaacg ccttcctccc cgctgaaagt actttacaac ccgaaggcct tcttcataca 1080
cgcggcatgg ctgcatcagg cttgcgccca ttgtgcaata ttccccactg ctgcctcccg 1140
taggagtctg gaccgtgtct cagttccagt gtggctggtc atcctctcag accagctagg 1200
gatcgtcgcc taggtgagcc attaccccac ctactagcta atcccatctg ggcacatctg 1260
atggcaagag gcccgaaggt ccccctcttt ggtcttgcga cgttatgcgg tattagctac 1320
cgtttccagt agttatcccc ctccatcagg cagtttccca gacattactc acccgtccgc 1380
cgctcgccgg caaagtagca agctactctc cgctgccgct cgactgcatg tgtagcctgc 1440
gccattgc 1448
<210>2
<211>20
<212>DNA
<213>Artificial Sequence
<220>
<223> upstream primer 27F
<400>2
agagtttgat cmtggctcag 20
<210>3
<211>22
<212>DNA
<213>Artificial Sequence
<220>
<223> downstream primer 1492R
<400>3
tacggytacc ttgttacgac tt 22
Claims (5)
1. A vomitoxin degrading bacterium is characterized in that: named Enterobacter cloacae: (Enterobacter cloacae) WD601 was deposited at 2017, No. 06, No. 02, at the Guangzhou province microbial strain collection center of the institute of microorganisms of Guangzhou, No. 59, building, 5, Zhonglu, Mingzhou, with the following collection numbers: GDMCC No: 60194.
2. a vomitoxin degrading agent characterized by: comprising the vomitoxin-degrading bacterium according to claim 1.
3. Use of the vomitoxin-degrading bacterium of claim 1 for non-disease treatment purposes in degrading vomitoxin.
4. Use of the vomitoxin degrading agent of claim 2 for non-disease therapeutic purposes in degrading vomitoxin.
5. Use according to claim 3, characterized in that:
the vomitoxin-degrading bacterium of claim 1, which is inoculated in an amount of 10% to an inorganic salt medium containing 40 to 60 μ g/mL of vomitoxin, and cultured at 37 ℃ for 72 to 168 hours.
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| CN111548940B (en) * | 2020-04-23 | 2023-02-10 | 华东理工大学 | Method for screening and producing biosurfactant facultative anaerobes |
| CN113699077A (en) * | 2021-09-08 | 2021-11-26 | 河南工业大学 | Microbial degradation method of vomitoxin |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103243047A (en) * | 2013-05-09 | 2013-08-14 | 中国农业大学 | Bacillus subtilis capable of effectively degrading vomitoxin and application of bacillus subtilis |
| WO2016044956A1 (en) * | 2014-09-26 | 2016-03-31 | University Of Guelph | Novel bacterial endophyte with antifungal activity |
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2017
- 2017-06-30 CN CN201710522809.4A patent/CN107164280B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103243047A (en) * | 2013-05-09 | 2013-08-14 | 中国农业大学 | Bacillus subtilis capable of effectively degrading vomitoxin and application of bacillus subtilis |
| WO2016044956A1 (en) * | 2014-09-26 | 2016-03-31 | University Of Guelph | Novel bacterial endophyte with antifungal activity |
Non-Patent Citations (2)
| Title |
|---|
| IDENTIFICATION AND CHARACTERIZATION OF DEOXYNIVALENOL-DEGRADING BACTERIAL STRAIN BACILLUS CIRCULANS C1-5-9;Long, M等;《FRESENIUS ENVIRONMENTAL BULLETIN》;20160731;第25卷(第7期);第2427-2433页 * |
| 生物降解呕吐毒素的肠杆菌筛选与鉴定;李晓凤等;《现代食品科技》;20170620;第33卷(第6期);第125-131页 * |
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