CN107164280A - One plant of vomitoxin degradation bacteria and its application - Google Patents
One plant of vomitoxin degradation bacteria and its application Download PDFInfo
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Abstract
The present invention discloses one plant of vomitoxin degradation bacteria and its application.The degradation bacteria is the Rice Cropping farmland DON contaminated soils selected from Yangjiang city, by the enriched medium culture containing DON, then is obtained using DON as the minimal medium of sole carbon source domestication culture, pressure screening, separation, purifying.Entitled enterobacter cloacae (Enterobacter cloacae) WD601, Guangdong Province's Culture Collection of 5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100 Guangdong Microbes Inst, deposit number were preserved on 06 02nd, 2017：GDMCC No：60194.The bacterium carries out growth and breeding under aerobic condition using DON as sole carbon source and the energy, while by its fast degradation, degraded DON's has good stability, and passes on for 5~8 generations in Optimal Medium, and DON degradation rates can reach more than 50%.
Description
Technical field
The present invention relates to biodegradation technique field, and in particular to one plant of vomitoxin degradation bacteria and its application.
Background technology
Vomitoxin (deoxynivalenol, DON), also known as deoxynivalenol, mainly by cereal reaping hook
The B class trichothecene toxin that bacterium and fusarium culmorum are produced, pollutes very serious to cereal.Known DON is in human and animal
In cause a series of diseases, such as vomiting, food refusal, diarrhoea, esophageal perforation and the bad symptom of nutrient absorption, wherein pig to vomiting
Toxin is most sensitive；Meanwhile, DON can make rapid development, the Apoptosis in splitting status, and high concentration DON can not only suppress
Animal immune cell is bred, and also easily causes the miscarriage of gestational period animal or has teratogenesis to embryo.
Formulate in respective standard, such as FDA regulation food DON recall rate highests in cereal mycotoxin, different regions
DON safety standard is 1mg/kg, and China provides the product DON such as wheat permission in GB2715-2005 and GB2761-2011
Limitation is no more than 1mg/kg.At present, such mycotoxin pollution constitutes actual, tight to China's food security and the export of farm produce
Negatively affect again, how effective detoxification becomes Social Events urgently to be resolved hurrily.
At present, domestic and international cereal and feed mold poison-removing method mainly has physisorphtion, chemical detoxication method and biology
Detoxicity method etc..Physisorphtion adsorbs reversible and inefficient to DON, also easily adsorbs micronutrient element, causes cereal and feed
Middle nutriment is lost in；Chemical detoxication easily remains some potentially toxic materials, causes secondary pollution to cereal and feed, therefore
DON tradition poison-removing methods have certain limitation in actual applications；Biological detoxication method is the enzyme effect by Microbiological release
In DON, and the metabolite of more hypotoxicity is translated into, because microbial degradation has low cost, efficiency high, nutriment
Loss is few, the advantages of non-secondary pollution and good ecological recovery and by vast concern.
The DON degradation bacterias of reporting largely derive from animal intestinal tract content, to be isolated in enteron aisle and bovine rumen
The anaerobic species come are in the majority.Although anaerobic bacteria flora can act on DON and with preferable degradation effect, strain separating is pure
Change extremely difficult and rigorous and harsh reaction condition and be not appropriate for large-scale DON biological detoxications；And several plants reported are simultaneous
Property anaerobic bacteria be largely mixed bacterial, decomposing D ON microorganism fungus kind is not subjected to Pure strain separation and identification.Therefore,
Need a kind of the good of energy efficient degradation DON badly to support or the purebred bacterium of amphimicrobian, so as to drive DON biodegradations skill in cereal and feed
The further development and application of art.
The content of the invention
In order to overcome the shortcoming and deficiency in existing DON microbial degradations, it is an object of the invention to provide one plant of vomiting
Toxin degradation bacteria.The bacterium be from by DON contaminated soils screening separation, can be using DON as sole carbon source under aerobic condition
In minimal medium growth and can efficient degradation DON vomitoxin degradation bacteria.
Another object of the present invention is to provide the application of above-mentioned vomitoxin degradation bacteria.
The purpose of the present invention is achieved through the following technical solutions：
The present invention provides one plant of vomitoxin degradation bacteria, and strain is named as enterobacter cloacae (Enterobacter
Cloacae) WD601, is the Rice Cropping farmland DON contaminated soils selected from Yangjiang city, is trained by the enrichment containing DON
Base culture is supported, then is obtained using DON as the minimal medium of sole carbon source domestication culture, pressure screening, separation, purifying.
Described enterobacter cloacae (Enterobacter cloacae) WD601 preservation information：Depositary institution：Guangdong Province
Culture Collection (GDMCC), preservation date：06 month 2017 02 day, preservation address：Xianlie Middle Road, Guangzhou City 100
Number 5 building, the building of compound the 59th Guangdong Microbes Inst, deposit number：GDMCC No：60194.
Described WD601 16S rDNA and enterobacter cloacae Enterobacter cloacae homology is 99%.
The 16S rDNA sequences such as SEQ ID NO of the bacterial strain：Shown in 1.
Present invention also offers the degraded reagent of the vomitoxin containing described vomitoxin degradation bacteria.
Present invention also offers application of the described vomitoxin degradation bacteria in terms of vomitoxin of degrading.
Present invention also offers application of the described vomitoxin degraded reagent in terms of vomitoxin of degrading.
Further, application of the described vomitoxin degradation bacteria in terms of vomitoxin of degrading, comprises the following steps：
Enterobacter cloacae (Enterobacter cloacae) WD601 is seeded to containing 40~60 μ g/ with 10% inoculum concentration
In mL DON minimal medium, 72~168h is cultivated at 37 DEG C.
Described minimal medium is：0.5g/L(NH4)2SO4, 0.2g/L MgSO4·7H2O, 0.05g/L CaCl2,
2.44g/L Na2HPO4, and 1.52g/L KH2PO4, adjust the 15~20min that sterilized at pH to 7.0,121 DEG C.
Using minimal medium as negative control, the positive is used as using the minimal medium that contains 40,60 μ g/mL DON
Control.
The present invention has the following advantages and effect relative to prior art：
(1) isolated and purified in the present invention and obtain the microorganism fungus kind enterobacter cloacae good to DON degradation properties
(Enterobacter cloacae) WD601 bacterial strains, it is numerous using DON as sole carbon source and energy progress growth under aerobic condition
Grow, while by its fast degradation, degradation rate is up to more than 50%.The enterobacter cloacae (Enterobacter cloacae) of the present invention
WD601 bacterial strains can grow in minimal medium, and screening separating and purifying technology is simple.
(2) enterobacter cloacae (Enterobacter cloacae) WD601 strains for degrading DON of the invention stability is good
It is good, passed on for 5~8 generations in Optimal Medium, DON degradation rates can reach more than 50%.In basic inorganic salt culture medium,
When DON initial concentrations are 40mg/L, 60mg/L, DON degradation rates are respectively 21.47% and 40.40% after 37 DEG C of culture 7d.
Brief description of the drawings
Fig. 1 is enterobacter cloacae (Enterobacter cloacae) WD601 bacterial strain colonial morphology figures that the present invention is provided；
Wherein, a：Colonial morphology on flat board；b：Microscope hypothallus form；c：ESEM hypothallus form (20,000 ×).
Fig. 2 is the 16S rDNA fragments for enterobacter cloacae (Enterobacter cloacae) WD601 that the present invention is provided
PCR amplifications.
Fig. 3 is the systematic evolution tree for enterobacter cloacae (Enterobacter cloacae) WD601 that the present invention is provided.
Fig. 4 is the canonical plotting obtained by offer HPLC detection DON standard items of the invention.
Fig. 5 is the HPLC detection reactions for enterobacter cloacae (Enterobacter cloacae) WD601 that the present invention is provided
Front and rear DON contents；Wherein, a：With the negative control of the minimal medium without DON；b：DON initial concentrations are 60 μ g/mL's
Positive control；c：When DON initial concentrations are 60 μ g/mL, degradation rate after reaction 3d；d：When DON initial concentrations are 60 μ g/mL
When, react degradation rate after 7d.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.
Embodiment 1, Enterobacter cloacae WD601 screening, separation and purifying
(1) enrichment of bacterial strain
Pedotheque is crossed after 40 mesh sieves, takes 1g to be mixed with 3mL physiological saline respectively.100 μ L suspension are taken, 5mL is added to
In enriched medium containing 10 μ g/mL DON, three kinds of different temperature are set：25 DEG C, 30 DEG C, 37 DEG C, and in 180r/min
Lower culture 72h.
The enriched medium containing DON is：10g peptones are weighed, 1g NaCl, 1g glucose is dissolved in 1000mL
In sterile distilled water, the 15min that sterilized at pH to 7.0,121 DEG C is adjusted.
(2) rubbing method bacterium
Supernatant is abandoned after drawing the pregnant solution that 100 μ L cultivate 3d, 3,000r/min centrifugation 5min respectively, contains 20 μ g/ with 100 μ L
ML DON inorganic salt liquids culture medium suspends precipitation, is coated on flat board, respectively at cultivating 24h at 25 DEG C, 30 DEG C, 37 DEG C.
Described minimal medium is：Weigh 0.5g (NH4)2SO4, 0.2g MgSO4·7H2O, 0.05g CaCl2,
2.44g Na2HPO4, and 1.52g KH2PO4, it is dissolved in 1000mL sterile distilled waters, adjusts and sterilized at pH to 7.0,121 DEG C
15min。
(3) plate streaking bacterium
After after medium sterilization, DON standard liquids are crossed after 0.22 μm of miillpore filter and added in culture medium, make DON dense eventually
Spend for 20 μ g/mL.Picking single bacterium colony is inoculated in minimal medium, respectively at 25 DEG C, 30 DEG C, 37 DEG C of incubator culture 24h.
Picking individual colonies, line and 24h are cultivated on minimal medium again, so rule to purify bacterial strain repeatedly.
The fresh single bacterium colony for selecting 18~24h of culture carries out Gram's staining, micro- Microscopic observation strain physiology and appearance.Will
Single bacterium colony abandons supernatant after being seeded to incubated overnight in minimal medium, 5,000r/min centrifugation 5min, with appropriate distilled water
Suspension thalline, 5,000r/min centrifugation 5min, repeats aforesaid operations once, abandons and 2mL sterile distilled water suspension thallines are used after supernatant,
Freeze-drying obtains bacterium powder, is observed with ESEM.
On minimal medium, strain growth can only be by the use of DON as unique carbon source, based on this, can be by
The DON bacterial strain of degrading is separated from flat board.By strain pregnant solution in the DON inorganic salts solid cultures containing 20 μ g/mL
Cultivated on base after 24h, the colony growth in 37 DEG C of culture medium is most fast, picking individual colonies carry out line purifying.By repeatedly
Line purifying one plant of White strain has been filtered out from the paddy soil of Guangdong Yangjiang, the bacterial strain on basic inorganic salt culture medium
As shown in Figure 1a, bacterium colony is rounded for growing state, and diameter is in 1mm or so, and neat in edge, surface wettability is glossy, in canescence.
Observed under the microscope by Gram's staining, as shown in Figure 1 b, the bacterial strain is Gram-negative bacteria, form to thalli morphology
In rod-short.After the thalline freeze-drying after centrifugation, dry bacterium powder is observed with SEM, 20,000 × multiple
Under, thalline is in differ in length to arrange irregular rod-short, two ends blunt circle, and size is at 0.5 × 1~3 μm (Fig. 1 c).The bacterial strain energy
It is enough to be grown using DON as unique carbon source, illustrate there may be degraded DON ability.It is WD601 by the Strain Designation,
And be seeded to its degradation capability is cultivated and determined in the fluid nutrient medium containing DON.
The identification of embodiment 2, isolated strains
(1) colonial morphology is identified
The fresh single bacterium colony for selecting 18~24h of culture WD601 carries out Gram's staining, micro- Microscopic observation strain physiology
Form.Single bacterium colony is seeded to incubated overnight in minimal medium, supernatant is abandoned after 5,000r/min centrifugation 5min, with appropriate
Distilled water suspension thalline, 5,000r/min centrifugation 5min, repeats aforesaid operations once, abandons outstanding with 2mL sterile distilled waters after supernatant
Floating thalline, freeze-drying obtains bacterium powder, observed with ESEM.
The morphological feature for the WD601 that the present invention is provided is as follows：Bacterial strain is cultivated after 24h for 37 DEG C on LB flat boards, such as Fig. 1 a institutes
Show that bacterium colony is rounded, diameter is in 1mm or so, and neat in edge, surface wettability is glossy, in canescence.Existed by Gram's staining
Observed under microscope, as shown in Figure 1 b, the bacterial strain is Gram-negative bacteria to thalli morphology, and form is in rod-short.Will centrifugation
After thalline freeze-drying afterwards, dry bacterium powder is observed with SEM, 20,000 × multiple under, thalline be in length not
Deng the rod-short that arrangement is irregular, two ends blunt circle, size is at 0.5 × 1~3 μm (Fig. 1 c).
(2) Analysis of Biochemical Characteristics
Reference《Bacterial system identification handbook》Common bacterial strain authentication method carries out Physiology and biochemistry identification to White strain.
The oxidative fermentation of glucose and other glycitols：The fresh single bacterium colony percutaneous puncture-inoculations of 18~24h WD601 will have been cultivated
In stopping with the gloomy Er Shi culture mediums of sharp husband, with the vaseline paraffin oil seal cover of sterilizing, about 0.5~1cm is thick, to completely cut off air, together
When design do not do oil sealing experimental group verify bacterial strain oxidative fermentation type.After incubated at room temperature 1,2,3,7,14d, observation color becomes
Change.Glucose is replaced with lactose, maltose, mannose, arabinose, fructose, xylose and sucrose respectively, the sugar alcohol of bacterial strain is detected
Class utilization power.
It is described to stop and the sharp gloomy Er Shi culture mediums of husband are：Weigh 2g peptones, 5g NaCl, 10g glucose, 0.2g
K2HPO4, it is dissolved in 1000mL sterile distilled waters, the bromthymol blue indicator 3mL of addition 1%, sterilize 20min at 115 DEG C.
During glycitols fermenting experiment, glucose is replaced with tested 1% sugar alcohol.
Methyl red and V.P are determined：Inoculation WD601 single bacteriums are fallen within nutrient solution, and thermophilic 2~6d of culture adds in the medium
Enter a drop methyl red reagent, redden as the positive；Nutrient solution and 40% NaOH mixed in equal amounts are taken, is added in a little creatine, 10min
Occur red then positive for V.P experiments.
Described methyl red and V.P culture mediums be：Weigh 5g peptones, 5g glucose, 5g K2HPO4, it is dissolved in 1000mL
In sterile distilled water, sterilize 20min at 115 DEG C.
Starch Hydrolysis：Take fresh single bacterium colony dibbling on starch culture-medium, thermophilic 2~5d of culture is formed after obvious bacterium colony,
Iodine solution is added dropwise on flat board, whether observation periphery of bacterial colonies has non-discoloring transparent circle.
Described starch culture-medium is：Added in gravy peptone 0.2% soluble starch, sterilize at 121 DEG C 20min.
Oxidizing ferment and catalase：The fresh single bacterium colony for having cultivated 18~24h is taken, is applied to by 1% hydrochloride base to benzene
On the filter paper of two amine aqueous solutions wetting, observation color change in 10s；Fresh single bacterium colony is taken to be applied to have dripped and have 10%H2O2Glass
On piece, observation has bubble-free generation.
L-Trp dehydrogenase is detected with urase：Fresh single bacterium colony culture 24h is inoculated with, if nutrient solution is red by xanthochromia, is had
Urase is present；Take 2~4 drop nutrient solutions to be mixed with liquor ferri trichloridi (33%), observe color change, be in such as bronzing, then have
L-Trp dehydrogenase is present.
Described L-Trp dehydrogenase and urase detection culture medium are：Weigh 3g L-Trps, 5g NaCl, 1g
K2HPO4, 1g KH2PO4, 10mL 95% ethanol is added, is dissolved in 1000mL distilled water, sterilize 20min at 121 DEG C.Separately will
Urea is dissolved in 100mL water, filtration sterilization, after being added to after medium sterilization in culture medium.
Gelatin liquefaction and hydrogen sulfide experiment：Fresh single bacterium colony percutaneous puncture-inoculation is taken, in cultivating 1,3,7d under thermophilic, observation is cultivated
Base color change, blackening is that hydrogen sulfide is positive；Observe whether gelatin liquefies simultaneously.
Described sulfurated hydrogen detection culture medium is：7.5g beef extracts are weighed, 10g peptones, 5g NaCl, 120g gelatin is molten
In 1000mL sterile distilled waters, sterilize 20min at 121 DEG C.After the FeCl that 5mL 10% is added after medium sterilization2(filtering
Sterilizing).
The Physiology and biochemistry of bacterial strain the results are shown in Table 1, and glycitols fermenting experiment is the experiment for identifying that bacterium is main and most basic,
Identification particularly to enterobacteriaceae lactobacteriaceae is particularly important, and experiment shows, WD601 is fermented type bacterial strain, it is possible to use glucose,
Lactose, maltose, mannitol, fructose, xylose and sucrose etc., it is impossible to utilize arabinose.M.R is tested primarily to examining bacterium
Plant and utilize glucose generation lactic acid, the ability of a large amount of acid products such as butanedioic acid, acetic acid and formic acid, experimental result is negative, table
Bright WD601 bacterial strains may be one plant of aerogenesis type bacterial strain.V.P experiments are the positive, and it is pyruvic acid by breakdown of glucose to show bacterial strain, is entered
And decarboxylation generation acetonyl ethanol, finally with NaOH reaction generation red compounds.WD601 bacterial strains can also hydrolysis starch and
Semisolid gelatin generation Liquid amino acid is acted on, catalase and urase can be produced, oxidizing ferment is not produced and L-Trp is de-
Hydrogen enzyme.Experimental result accords with the biochemical character of enterobacteria substantially, primarily determines that WD601 bacterial strains belong to enterobacteriaceae.
The WD601 of table 1 physio-biochemical characteristics
| Project | As a result | Project | As a result |
| Oxidation-fermentation (O/F) | Fermented type | Starch Hydrolysis | + |
| Glucose | + | V.P is tested | + |
| Lactose | + | M.R is tested | - |
| Maltose | + | Gelatin hydrolysis | + |
| Mannitol | + | Hydrogen sulfide | - |
| Arabinose | - | Oxidizing ferment | - |
| Fructose | + | Catalase | + |
| Xylose | + | Urase | + |
| Sucrose | + | L-Trp dehydrogenase | _ |
Note："+" represents that result is the positive."-" represents that result is feminine gender.
(3) 16S rDNA gene sequencings
The full-length genome of strain cultured solution, design sense primer 27F are extracted using genome extraction agent box：5′-
AGAGTTTGATCMTGGCTCAG-3 ' and anti-sense primer 1492R：5 '-TACGGYTACCTTGTTACGACTT-3 ', enter performing PCR expansion
Increase reaction, reaction system is：2.5 μ L genomic DNA, 2.5 μ L 10 × Buffer (with Mg2+), 1 μ L dNTPs
(10mol/L), 0.2 μ L enzyme (5U/ μ L), each 0.5 μ L of upstream and downstream primer (10mol/L), addition sterile distilled water to 25 μ L.
PCR reaction conditions are as follows：94 DEG C of pre-degenerations 4min, 94 DEG C of denaturation 45s, 55 DEG C of annealing 45s, 72 DEG C of extension 60s, after 30 circulations
Continue to extend 10min in 72 DEG C, amplified production is detected by 1% agarose gel electrophoresis.Amplified production is pure with PCR
Change kit to be purified, and deliver to U.S. lucky biological Co., Ltd and be sequenced.Sequencing result is submitted in GenBank and carried out
BLAST is compared, and is chosen the higher bacterial strain of homology and is carried out phylogenetic analysis using MEGA softwares.
Data above, which is repeated three times, is averaged, and test result is represented with x ± s, utilizes 85 pairs of experiment numbers of Origin
According to statistical analysis and processing is carried out, sequence homology is analyzed using GenBank and MEGA 7.0.
The DNA of bacteria in fresh WD601 nutrient solutions is extracted using DNA of bacteria extraction agent box, with the base of bacterial strain
Because group DNA is as template, enters performing PCR amplification according to the PCR system in above-mentioned, product is found by 1% agarose gel electrophoresis
Occurs a specific band at about 1500bp, as shown in Figure 2.PCR primer after purification is sequenced, peak figure is exported,
Sequence results are committed to GenBank and carry out BLAST comparisons, the bacterial strain and enterobacteriaceae (Enterobacteriaceae) is found
With higher homology, bacterial strain of the wherein 16 plants sequence homologies more than 98% is chosen, passes through MEGA7.0.25 software structures
Chadogram is built, chadogram result is shown in Fig. 3, as a result show WD601 bacterial strains and Enterobacter (Enterobacter) and western western bacterium
Belong to (Cedeceadavisae) and be in same main split, but from the enterobacter cloacae of Genetic Distance Analysis and Enterobacter
Therefore (Enterobacter cloacae) affiliation closer to, enterobacteria being accredited as by bacterial strain WD601, this life with WD601
Reason biochemical test result is consistent.
As a result final identification bacterial strain of the invention shows, this plant of bacterium is identified as enterobacter cloacae to category
(Enterobacter cloacae) WD601, the homology with enterobacter cloacae Enterobacter cloacae is 99%.This
It is enterobacter cloacae (Enterobacter cloacae) WD601 by obtained Strain Designation in invention.
(4) culture presevation
30% glycerol tube is made in microbial strain culture, preservation in -80 DEG C of refrigerators is placed on, it is stand-by.Above-mentioned bacterial strains in
On June 2nd, 2017 is preserved in Guangdong Province's Culture Collection (GDMCC), and this is centrally located at Guangzhou, Guangdong martyr
5 building, the building of compound the 59th of Road 100 Guangdong Microbes Inst, culture presevation number is GDMCC No：60194.
The application of embodiment 3, enterobacter cloacae (Enterobacter cloacae) WD601 degradeds DON
(1) HPLC surveys DON degradation rates
1mL nutrient solutions are taken to cross after 0.22mL miillpore filters, with the HPLC detection degraded wild Oryza species with UV-detector
Middle DON contents.High performance liquid chromatography detection DON condition is：Using Agilent ZORBAX SB-C18 chromatographic column (4.6 ×
150mm, 5 μm), mobile phase uses methanol/water for 20/80 (V/V).Column temperature during test is 35 DEG C, and flow is 0.9mL/min,
The wavelength of detector is 218nm.DON degradation rates are calculated using following equation：
(2) drafting of DON standard curves：DON storing solutions (1mg/mL) are taken to be diluted to 4,10,20,50,100 μ L/mL respectively
Working solution, the μ L of sample introduction 20 are repeated three times and average.DON standard curves are drawn according to concentration and peak area relation.
The DON working solutions that concentration is 4,10,20,50 and 100 μ g/mL are respectively configured, DON concentration and peak area relation is measured
Standard curve, as shown in figure 4, coefficient R2=0.9999.
(3) DON degradation reactions
Enterobacter cloacae (Enterobacter cloacae) WD601 is taken, liquid of the 2mL containing 40 μ g/mLDON is inoculated in and trains
Support and cultivate 3d and 7d in base respectively, expand DON concentration (60 μ g/mL) and repeat to test.Using minimal medium as negative control,
Positive control is used as using the minimal medium that contains 40 μ g/mL and 60 μ g/mL DON.
WD601 DON degradation capabilities are detected using HPLC, 2 are the results are shown in Table, inoculation WD601 single bacteriums drop down onto final concentration difference
After 40 and 60 μ g/mL DON reactions 3d and 7d, there is different degrees of decline in DON concentration, shows that WD601 has obvious
Degraded DON ability.HPLC results as shown in table 2, negative control (Fig. 5 a), DON are used as using the minimal medium without DON
Appearance time be about 16.20min (Fig. 5 b), when DON initial concentrations be 60 μ g/mL when, reaction 3d after degradation rate be 7.1%
40.40% (Fig. 5 d) is up to after (Fig. 5 c), 7d.
Bacterial strain DON degrades under the conditions of the HPLC of table 2 surveys differential responses
(4) enterobacter cloacae (Enterobacter cloacae) WD601 optimizations are expanded and liquid is made after culture or solid
The degraded reagent of the vomitoxin containing the bacterial strain of state, the reagent is likewise supplied with degradation effect.The degraded reagent is used and step
(3) identical method carries out DON degradation experiments, and degradation efficiency reaches more than 50%.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
SEQUENCE LISTING
<110>South China Science ＆ Engineering University
<120>One plant of vomitoxin degradation bacteria and its application
<130> 1
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1448
<212> DNA
<213> Artificial Sequence
<220>
<223>Enterobacter cloacae（Enterobacter cloacae）WD601 16S rDNA sequences
<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 tgaggttatt 1020
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>Sense primer 27F
<400> 2
agagtttgat cmtggctcag 20
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223>Anti-sense primer 1492R
<400> 3
tacggytacc ttgttacgac tt 22
Claims (5)
1. one plant of vomitoxin degradation bacteria, it is characterised in that：Entitled enterobacter cloacae (Enterobacter cloacae)
WD601,5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100 Guangdong Province's microbe research was preserved on 06 02nd, 2017
Guangdong Province's Culture Collection, deposit number：GDMCC No：60194.
The reagent 2. a kind of vomitoxin is degraded, it is characterised in that：Contain the vomitoxin degradation bacteria described in claim 1.
3. application of the vomitoxin degradation bacteria in terms of vomitoxin of degrading described in claim 1.
4. application of the vomitoxin degraded reagent in terms of vomitoxin of degrading described in claim 2.
5. application according to claim 3, it is characterised in that：
The vomitoxin degradation bacteria described in claim 1 is seeded into the inorganic salts containing 40~60 μ g/mL with 10% inoculum concentration to train
Support in base, 72~168h is cultivated at 37 DEG C.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111548940A (en) * | 2020-04-23 | 2020-08-18 | 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 |
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| 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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| 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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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111548940A (en) * | 2020-04-23 | 2020-08-18 | 华东理工大学 | Method for screening and producing biosurfactant facultative anaerobes |
| 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 |
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| CN107164280B (en) | 2020-02-18 |
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